Input:
CD40 signaling in professional antigen-presenting cells, including B cells, macrophages, and dendritic cells, is critical for the efficient activation of humoral and cell-mediated immune responses [1], [2], [3]. CD40 signaling is activated in a T cell-dependent manner, as the ligand for CD40, CD154, is expressed primarily by activated T cells. CD40 engagement leads to the activation of various signaling molecules, including stress-activated protein kinases and the transcription factor NF-kappaB, which upregulate the expression of cytokines and other factors that promote immune responses. The mechanism by which CD40 induces these signaling pathways has not been completely defined. The cytoplasmic domain of CD40 does not appear to have intrinsic enzymatic activity, but is able to mediate signaling through the recruitment of several intracellular proteins. Members of the TNF receptor-associated factor (TRAF) family, including TRAF1, TRAF2, TRAF3, and TRAF6, appear to be particularly important for the initiation and regulation of CD40 signaling [4]. These proteins function in part as adaptor molecules, binding to the cytoplasmic tail of CD40 and recruiting other proteins to the receptor-associated complex. Some of the TRAFs also function as E3 ubiquitin ligases, and this enzymatic activity may contribute to signal propagation and regulation. Among the multiple TRAFs that associate with CD40, TRAF3 can function as a negative regulator of signaling, while TRAF2 and TRAF6 promote the activation of downstream signaling pathways [4].
We recently demonstrated that HOIL-1L interacting protein (HOIP), a ubiquitin ligase that can catalyze the assembly of linear polyubiquitin chains [5], is recruited to CD40 in a TRAF2-dependent manner following engagement of CD40 by agonistic antibody [6]. These and other findings led us to hypothesize that HOIP functions downstream of TRAF2 in the CD40 signaling pathway and that HOIP is necessary for the activation of NF-kappaB and possibly other signaling molecules. To test this hypothesis, we employed somatic cell gene targeting to ablate expression of HOIP in a mouse B cell line that has proven to be a useful model for B cell CD40 signaling [7], [8], [9]. We found that the CD40-induced upregulation of CD80 (a costimulatory molecule for T cells) was defective in HOIP-deficient cells. Similarly, the CD40 and IL-4 driven production of germline transcripts from the immunoglobulin epsilon heavy chain locus, an event that precedes immunoglobulin gene rearrangement and isotype switching, was defective in the absence of HOIP. We also found that the CD40-mediated activation of NF-kappaB and the stress-activated protein kinase c-Jun kinase (JNK) was defective in HOIP-deficient cells. Consistent with impaired NF-kappaB activation, association of the IkappaB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement.

engagement

recruitment

cytoplasmic domain

binding

cytoplasmic tail

binding

cytoplasmic tail

binding

cytoplasmic tail

binding

cytoplasmic tail

associate

associate

associate

recruited

HOIL-1L interacting protein

engagement

association

engagement

expressed

expression

HOIP

ablate

expression

defective

induced

deficient

HOIP

absence

HOIP

deficient

HOIP

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following

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Input:
Generation of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP
In our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-kappaB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells.
To enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector).

recruited

HOIP

interaction

HOIP

recruitment

HOIP

expression

HOIP

expression

HOIP

deficient

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disrupt

HOIP

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disrupted

disrupted

deficient

HOIP

deficient

HOIP

disruption

deficient

HOIP

deficient

HOIP

restoring

expression

reconstituted

HOIP

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Input:
HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription
Engagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.
CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLepsilon) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLepsilon transcription in parental A20.2J cells as expected. In contrast, only low levels of GLepsilon expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLepsilon expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.

Engagement

engagement

expression

expression

GLepsilon

expression

GLepsilon

reduced

upregulation

deficient

deficient

HOIP

defect

upregulation

deficient

deficient

HOIP

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deficient

HOIP

required

HOIP

mediated

mediated

upregulation

mediated

activation

upregulation

activation

transcription

upregulates

Engagement

expression

upregulation

induced

upregulation

engagement

reconstituted

HOIP

stimulated

upregulation

upregulation

upregulation

reconstituted

HOIP

upregulation

due

defect

disruption

induced

transcription

induced

expression

reconstituted

HOIP

transcription

germline epsilon

production

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transcription

GLepsilon


Input:
HOIP mediates CD40-stimulated NF-kappaB and JNK activation
The results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-kappaB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-kappaB pathway is initiated with the phosphorylation of IkappaB proteins by the IkappaB kinase complex (IKK). In resting cells, IkappaB proteins are responsible for sequestering NF-kappaB subunits in the cytoplasm. Phosphorylation by the IKK complex targets IkappaB proteins for ubiquitination and degradation, allowing NF-kappaB to enter the nucleus and activate gene expression. CD40-mediated phosphorylation and degradation of IkappaBalpha in HOIP-deficient cells was dramatically impaired relative to that observed in parental A20.2J cells (Fig. 4). We also assayed activation of the stress-activated protein kinase JNK in response to CD40 engagement (Fig. 4). CD40-mediated JNK activation in HOIP-deficient cells was impaired as measured by phosphorylation of Thr183 and Tyr185 in JNK. CD40-induced activation of NF-kappaB and JNK in HOIP-reconstituted cells was normal, demonstrating that the defects observed in gene-deficient cells were due to the absence of HOIP expression.

associated

engagement

expressed

expression

HOIP

sequestering

NF-kappaB subunits

deficient

HOIP

impaired

mediated

deficient

HOIP

phosphorylation

IkappaBalpha

mediated

phosphorylation

mediated

degradation

reconstituted

HOIP

degradation

IkappaBalpha

responsible

sequestering


Input:
HOIP is necessary for association of the IKK complex with CD40
The marked defects in CD40-mediated cell activation and signaling displayed by HOIP-deficient cells suggested that HOIP mediates recruitment of critical components of the CD40 signaling apparatus to the receptor. Previously, we demonstrated that HOIP is recruited to the CD40 signaling complex in a TRAF2-dependent manner, suggesting that HOIP functions downstream of TRAF2 [6]. Studies by others suggest that the TRAF2-associated proteins cIAP1 and cIAP2 play a role in the recruitment of HOIP to TNFR1 and CD40 [14]. Therefore, we determined whether the association of HOIP with CD40 in A20.2J cells was altered by treatment of cells with an inhibitor of cIAP activity, a membrane-permeable peptide derived from the apoptosis regulator SMAC [15]. We found that pretreatment of cells with the SMAC peptide dramatically reduced the amount of cIAP1 associated with the CD40 signaling complex in cells stimulated with anti-CD40 antibody-coated beads (Fig. 5A). SMAC peptide treatment also resulted in a slight but reproducible decrease in the amount of the major HOIP form recovered by CD40 immunoprecipitation, along with an apparent increase in higher molecular weight species recognized by anti-HOIP antibody. In contrast, treatment with the SMAC peptide did not alter the amount or molecular weight of HOIP present in cell lysates. These data suggest that SMAC peptide treatment specifically alters the characteristics of CD40-associated HOIP rather than the entire cellular pool of this protein. Together, these results support the idea that the cIAP proteins influence the recruitment and post-translational modification state of CD40-associated HOIP.
To test the possibility that HOIP is responsible for the recruitment of other critical signaling proteins to CD40, we immunoprecipitated CD40 signaling complexes from HOIP-deficient and HOIP-reconstituted cells (Fig. 5B). As described previously, CD40 signaling complexes immunoprecipitated from A20.2J cells contained HOIP as well as TRAF2, TRAF3, IKKalpha/beta, and IKKgamma [6]. The amounts of TRAF2, TRAF3, and cIAP1 in CD40 immunoprecipitates from HOIP-deficient cells were similar to those from parental A20.2J cells, indicating that HOIP is not required for the association of these proteins with CD40. In contrast, IKKalpha/beta and IKKgamma were not detectable in CD40 immunoprecipitates recovered from HOIP-deficient cells (Fig. 5B,C). The amounts of IKKalpha/beta and IKKgamma in CD40 immunoprecipitates from HOIP-reconstituted cells were similar to those detected in samples prepared from parental cells, demonstrating that the defects in IKK recruitment we observed were due specifically to the absence of HOIP expression. These data indicate that HOIP is required for recruitment of the NF-kappaB-activating complex to the CD40 signaling complex.
As shown here and in our previous study [6], IKKgamma present in CD40 immunoprecipitates appears to have a higher molecular weight than that found in cell lysates. To confirm that this higher molecular weight species was indeed IKKgamma, we generated A20.2J cell lines containing a stably integrated retroviral vector that encoded an epitope-tagged version of mouse IKKgamma. Cell lysates and CD40 immunoprecipitates prepared from these cells were analyzed by Western blotting for the epitope tag (Fig. 5C). This analysis produced a pattern of bands that was essentially the same as that obtained using an antibody specific for native IKKgamma (compare panels B and C in Fig. 5). Moreover, analysis of cells expressing epitope tagged-IKKgamma confirmed that recruitment of IKKgamma to CD40 requires HOIP. To determine what was responsible for the increased molecular weight of IKKgamma in CD40 immunoprecipitates, the protein samples were incubated with lambda phosphatase, which removes phosphates attached to tyrosine, threonine, or serine residues in proteins. This treatment reduced much of CD40-associated IKKgamma to an apparent molecular weight similar to that of the protein in cell lysates (Fig. 5C). However, at least one band of significantly higher molecular weight remained after phosphatase treatment, suggesting that CD40-associated IKKgamma is subject to at least one other modification in addition to phosphorylation. Overall, these data demonstrate that HOIP is required for the association of IKKgamma with CD40, and suggest that this event is coupled to post-translational modifications of IKKgamma.

association

recruited

HOIP

associated

associated

recruitment

HOIP

recruitment

HOIP

association

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associated

recovered

HOIP

recognized

HOIP

associated

HOIP

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HOIP

associated

HOIP

recruitment

association

association

association

recruitment

IKKgamma

associated

IKKgamma

associated

IKKgamma

association

IKKgamma

expression

HOIP

expressing

IKKgamma

deficient

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reduced

associated

decrease

HOIP

deficient

HOIP

deficient

HOIP

absence

expression

phosphorylation

IKKgamma

necessary

HOIP

association

resulted

decrease

resulted

increase

increase

HOIP

reconstituted

HOIP

required

HOIP

association

reconstituted

HOIP

requires

recruitment

HOIP

required

HOIP

association

coupled

association

post-translational modifications

post-translational modification

HOIP

post-translational modifications

IKKgamma

dependent

recruited

functions downstream

HOIP

role

recruitment

role

recruitment

altered

association

alter

HOIP

alters

HOIP

influence

cIAP

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post-translational modification

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Input:
Our results indicate that the protein HOIP is critical for CD40-induced signals that regulate B cell function. Our data show that HOIP-dependent cellular responses include CD40-mediated upregulation of CD80 expression and synthesis of germline RNA transcripts from the immunoglobulin heavy chain locus, two events that are important for T-cell-dependent antibody-mediated immune responses. At the molecular level, our data indicate that HOIP functions downstream of TRAF2 in the CD40 signaling pathway and that HOIP has a key role in promoting the recruitment of the IKK complex to CD40. Consistent with this, CD40-induced activation of NF-kappaB is dependent on the presence of HOIP. In addition, our data show that HOIP facilitates the activation of JNK in response to CD40 engagement. Together, our findings provide support for the conclusion that HOIP is a key component of the CD40 signaling pathway. Given the importance of CD40 signaling in both cellular and humoral immune responses, our results indicate that HOIP has a critical role in the regulation of the immune system.
The functional properties of HOIP have only been partially characterized. Initial studies showed that HOIP and the related protein HOIL-1 are components of a large (~600 kDa) protein complex capable of synthesizing linear polyubiquitin chains [5]. Subsequent studies showed that a HOIP-containing complex can interact with IKKgamma and facilitate activation of NF-kappaB via the canonical pathway [16]. These data, considered together with ours, suggest that a HOIP-containing complex mediates recruitment of IKKgamma to the CD40 signaling complex. In addition, CD40-associated HOIP could play a role in activating IKKgamma after its recruitment to the signaling complex [16]. The higher molecular weight forms of IKKgamma we observed in CD40 immunoprecipitates would be consistent with the presence of post-transcriptional modifications including phosphorylation and ubiquitination, which have been suggested to reduce or enhance IKKgamma activity, respectively [16], [17].
The mechanisms by which HOIP mediates recruitment of the IKK complex to CD40 and by which HOIP is recruited to CD40 remain unclear. A previous study indicates that HOIP may mediate direct contacts with the IKK complex [16], suggesting that it functions as an adaptor for the recruitment of the IKK complex to CD40. However, the ubiquitin ligase activity of HOIP suggests that it is more than a simple adapter molecule. As IKKgamma appears capable of binding linear polyubiquitin [18], [19], it is possible that HOIP directs formation of linear polyubiquitin chains on a CD40-associated factor, and it is these chains that serve to recruit IKKgamma to the CD40 signaling complex. The molecular interactions necessary for recruitment of HOIP itself to the CD40 signaling complex also remain to be fully characterized. We previously showed that the recruitment of HOIP to the signaling complex is TRAF2-dependent [8]. Potentially, TRAF2 and HOIP directly interact, but it is possible that the ubiquitin ligase activity of TRAF2 [20] (or TRAF2-associated proteins, such as the cIAPs [21]) generates K63-linked polyubiquitin chains to which HOIP can bind and thus associate with the CD40 signaling complex [22].
While our previous work indicated a potentially important link between TRAF2 and HOIP in CD40 signaling, the signals and functions tested here are dependent upon TRAF6 as well as TRAF2. In previous experiments with TRAF-deficient A20.2J cells, we found that the activation of NF-kappaB by CD40 could be mediated by either TRAF2 or TRAF6, while activation of JNK by CD40 was largely dependent on TRAF6 alone [8]. HOIP deficiency compromises the CD40-mediated activation of both NF-kappaB and JNK, indicating that signals mediated by both TRAF2 and TRAF6 likely pass through HOIP. Our previous work also demonstrated that the CD40-mediated activation of NF-kappaB and JNK, while TRAF6-dependent, was not compromised by the disruption of the binding site for TRAF6 in the cytoplasmic domain of CD40 or deletion of the receptor binding domain (the TRAF-C domain) in TRAF6 [8]. These observations indicate that TRAF6 need not directly bind CD40 in order to mediate certain signals, suggesting the assembly of a signaling complex not directly associated with the receptor. If such a complex exists, our results indicate that the absence of HOIP compromises its function as well.
Although the experiments presented here focus on CD40, our results and those of other groups [16], [22] support the possibility that HOIP is important in many signaling pathways in which TRAF2 or TRAF6 are involved, including those associated with various members of the TNF receptor superfamily and the Toll-like receptors. The potential importance of HOIP in immune function and TNFR family signaling is further supported by the recent discovery that HOIP interacts with a protein known as SHARPIN (SHANK-associated RH domain interacting protein in postsynaptic density), which appears capable of working together with HOIP and HOIL to mediate the assembly of linear polyubiquitin [14], [23], [24]. Interestingly, mice with a spontaneous mutation in the gene encoding SHARPIN (chronic proliferative dermatitis (cpdm) mice) exhibit chronic inflammation of the skin and internal organs, defective development of secondary lymphoid tissue, and defects in the production of switched immunoglobulin isotypes [17], [25]. The apparently intimate functional link between SHARPIN and HOIP strongly suggests that at least part of the cpdm phenotype stems from defects in the regulation or function of HOIP.

recruitment

engagement

interact

IKKgamma

recruitment

IKKgamma

associated

HOIP

recruitment

HOIP

recruitment

recruited

HOIP

recruitment

binding

IKKgamma

recruit

IKKgamma

recruitment

HOIP

recruitment

HOIP

interact

HOIP

bind

HOIP

associate

HOIP

bind

interacts

HOIP

SHARPIN

link

SHARPIN

HOIP

expression

reduce

phosphorylation

IKKgamma

reduce

ubiquitination

IKKgamma

deficiency

HOIP

disruption

binding site

deletion

receptor binding domain

absence

HOIP

defects

regulation

phosphorylation

IKKgamma

mediated

upregulation

upregulation

expression

promoting

recruitment

mediates

recruitment

activating

IKKgamma

enhance

phosphorylation

IKKgamma

enhance

ubiquitination

IKKgamma

mediates

HOIP

recruitment

functions as an adaptor

HOIP

recruitment

serve

recruit

necessary

recruitment

dependent

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mediated

downstream

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promoting

role

HOIP

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dependent

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HOIP

ubiquitination

IKKgamma


Input:
Cell lines
The mouse B cell line A20.2J has been previously described [26], [27]. Cells were grown in RPMI 1640, 10% FCS, 10 microM 2-ME, 2 mM L-glutamine, and antibiotics. HI-5 insect cells (Invitrogen) expressing CD154 have been described [28].

expressing


Input:
Antibodies
Mouse anti-birch profilin antibody (4A6 [29]), rat anti-mouse CD40 (1C10 [30]) and a rat isotype control antibody (mAb72, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) were isolated from hybridoma supernatants. FITC-labeled anti-mouse CD80 and an isotype control Ab were from eBiosciences. Rabbit anti-HOIP Ab [6] was the kind gift of Dr. Betty Eipper (University of Connecticut Health Center, Farmington, Connecticut). Goat anti-rat IgG, and HRP-labeled secondary Abs were from Jackson ImmunoResearch Laboratories, Inc. All other antibodies used were described previously [6].


Input:
Somatic cell gene targeting
The generation of HOIP-deficient cells was accomplished using a homologous recombination approach described previously [7], [8]. Segments of Rnf31 gene sequence used in the targeting construct (Fig. 1A) were amplified by PCR from A20.2J genomic DNA. The oligonucleotide primers used to generate the 5' flank (1224 bp) were 5'-ttttctagagcggtggcttaagtgaccc-3' and 5'-tattctagatgcagcatctgagaaagcaagc-3'. The 3' flank (6032 bp) primers were 5'-aaaaccggtgtatgcttctttacgggagaaaaatattag-3' and 5'-tataccggtatgaagccaaaggaacactgagag-3'. Restriction endonuclease sites in the oligonucleotide primers allowed insertion of the PCR products into the targeting vector. A20.2J cells were transfected (by electroporation [7]) with the targeting construct and subcloned in medium containing 600 microg/ml G418 sulfate. Homologous recombination in G418-resistant clones was detected by PCR of genomic DNA, as described [7]. Oligonucleotide primers used for screening were 5'-cttcctgatctcagctttaccgtcac-3' (homologous to genomic sequence; approximate position noted in Fig. 1) and 5'-caatccatcttgttcagccat-3' (homologous to sequence in NeoR). Clones in which one copy of Rnf31 had been disrupted were transiently transfected with an expression plasmid encoding Cre, in order to remove NeoR. G418-sensitive subclones were subjected to a second round of targeting to disrupt the remaining copy of Rnf31. G418-resistant clones were tested for homologous recombination by PCR and HOIP protein expression by Western blot.

expression

HOIP

deficient

HOIP

disrupted


Input:
Retroviral transduction
A retroviral vector (pMIP) was used for the stable transduction of cells with HOIP and IKKgamma cDNA constructs. pMIP was constructed by replacing the IRES and GFP encoding sequences in pMIG [31] with the IRES and puromycin resistance gene from pIRESpuro2 (Clontech). FLAG-tagged mouse HOIP cDNA was inserted into the multiple cloning site of pMIP. A pMIP construct encoding mouse IKKgamma with an amino terminal birch profilin peptide (BP)-tag was also prepared. Retroviral particles were generated by transient transfection of 293T cells with pMIP, pMIP-HOIP, or pMIP-IKKgamma and the packaging vector pCL-Eco [32]. Two days after transfection, culture supernatants were harvested and filtered. A20.2J or HOIP-deficient cells were added to 24-well plates (2.5x105 cells/well) with 1 ml virus-containing supernatant and 8 microg/ml polybrene. Plates were centrifuged at 500 x g for 2 hrs at room temperature. Cells were cultured in B cell culture medium for 48 hrs, after which the transduced cells were selected in 3 microg/ml puromycin.

deficient

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Input:
CD80 upregulation and flow cytometry
Cells (5x104 in 2 ml) were cultured in 24-well plates for 3 days with 5 microg/ml anti-CD40 or an isotype control antibody. Cells were stained for flow cytometry with FITC-anti-CD80 or an appropriate control antibody as described [28]. Data acquired with a FACScan flow cytometer (BD Biosciences) were analyzed with WinMDI 2.8 (Scripps Research Institute, San Diego, CA).

upregulation


Input:
GLepsilon transcript assay
The CD40-simulated activation of GLepsilon transcription was evaluated as previously reported [33]. Briefly, 1x106 cells were stimulated overnight with anti-CD40 antibody (10 microg/ml) or an isotype control antibody, with or without 500 U/ml mouse IL-4 (BD Biosciences). RNA was isolated using Trizol (Invitrogen), and reverse-transcribed (Superscript III kit, Invitrogen). Quantitative PCR for GLepsilon and Hprt1 was performed using SYBR GREEN master mix (Applied Biosystems), and an Applied Biosystems 7900HT Fast Real-Time PCR instrument. Expression of GLepsilon in each sample was normalized to the expression of Hprt1.

Expression

GLepsilon

expression

simulated

activation

activation

transcription

transcription

GLepsilon


Input:
NF-kappaB and JNK activation assays
For activation of CD40 signaling, 5x104 HI5 insect cells or HI5 insect cells expressing mouse CD154 (CD40 ligand) were added to 1x106 B cells. Cells were centrifuged for 1 minute at 400 x g (to promote contact between ligand cells and B cells) then incubated at 37degreesC for times indicated in Fig. 4. Following stimulation, cells were chilled on ice for 2 minutes. Cell pellets were dissolved in 2X SDS-PAGE loading buffer, sonicated, and heated for 5 minutes at 95degreesC. Total cell lysates (1x105 cell equivalents per lane) were fractionated by SDS-PAGE and blotted to polyvinylidine fluoride (PVDF) membranes. Membranes were probed with antibodies as indicated in Fig. 4. Chemiluminescent detection (Pierce Biotechnology) was used for the visualization of bands on Western blots. Images of blots were recorded with a low-light imaging system (LAS4000, Fuji Medical Systems) and on X-ray film.

expressing


Input:
Immunoprecipitations
Immunoprecipitation of CD40 was performed by activated receptor capture, as previously described [6]. Briefly, 3x107 cells (A20.2J and derivatives) were incubated for 60 minutes at room temperature with 10 microl magnetic protein G beads (Dynal) pre-coated with 10 microg goat anti-rat IgG (Jackson), and 10 microg anti-CD40 (1C10) or an isotype control antibody (mAb72). Cells/beads were then pelleted by centrifugation and lysed in buffer containing 1% Triton X100. Beads were washed with lysis buffer to remove unbound material. In some experiments, material associated with the beads was dephosphorylated with lambda phosphatase (New England BioLabs) as per manufacturer's instructions. Beads were resuspended in 2X SDS-PAGE sample buffer and heated for 5 minutes at 95degreesC. Material eluted from the beads was fractionated by SDS-PAGE and transferred to PVDF membranes for Western blotting. In some experiments, cells were cultured for 6 hrs with 25 microM antennapedia-linked SMAC-N7 peptide (Calbiochem) or an appropriate volume of the solvent used for the peptide (DMSO). After incubation, cells (in peptide- or DMSO-containing medium) were stimulated with antibody-coated beads as outlined above.


Input:
HOIP gene targeting.
(A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP-/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD.

expression

HOIP

expression

HOIP

deficient

HOIP

decrease

expression

disruption

following

decrease

disruption


Input:
CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.
Cells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment.

expression

defective

mediated

deficient

HOIP

deficient

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reversed

defective

defective

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mediated

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Input:
GLepsilon transcription is defective in CD40-stimulated HOIP-deficient cells.
A20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLepsilon transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.

defective

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deficient

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deficient

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GLepsilon

transcripts


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CD40-induced activation of NF-kappaB and JNK is defective in HOIP-deficient cells.
CD40-mediated signaling was defective in HOIP-deficient cells (HOIP-/- + pMIP), but intact in parental cells transduced with an empty retroviral vector (A20.2J + pMIP) and in HOIP-deficient cells transduced with a retroviral vector encoding HOIP (HOIP-/- + HOIP). Cells were activated with CD154-expressing insect cells for the times indicated. As a negative control, cells were stimulated with insect cells lacking CD154 (5 minute time point only). Western blots of whole-cell lysates were probed with the indicated antibodies. Phospho-IkappaBalpha (pIkappaBalpha) and phospho-JNK (pJNK) blots were stripped and reprobed for total IkappaBalpha and total JNK, respectively (anti-JNK antibodies recognize p46 and p54 isoforms). IkappaBalpha blots were also reprobed for actin to demonstrate equal lane loading. Molecular weights are indicated at right. Similar results were obtained in a second experiment and in two additional experiments with a second HOIP-deficient clone.

expressing

deficient

HOIP

deficient

HOIP

deficient

HOIP

lacking

deficient

HOIP


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IKK recruitment to the CD40 signaling complex is defective in HOIP-deficient cells.
(A) SMAC peptide treatment reduces recruitment of cIAP1 to CD40 and may modify HOIP recruitment. A20.2J cells were incubated for six hours with membrane-permeable SMAC-N7 peptide or 1.5% DMSO (solvent used for the peptide). Following the incubation, cell lysates were prepared, fractionated by SDS-PAGE, and evaluated by Western blot (lanes 1 and 2). Cells incubated with DMSO or SMAC-N7 were also stimulated with magnetic beads coated with anti-CD40 or an isotype control antibody. Immunoprecipitated (IP) material bound to the beads was loaded in lanes 3-5. Samples of the cell lysates after immunoprecipitation appear in lanes 6-8. Western blots were probed with antibodies specific for cIAP1, TRAF2, TRAF3, and HOIP (approximate molecular weights indicated on left). Similar results were obtained in two additional experiments. (B) CD40 was isolated by immunoprecipitation (as in (A)) from A20.2J cells and HOIP-deficient cells transduced with an empty retroviral vector (pMIP) or a retroviral vector encoding HOIP. Material immunoprecipitated with an isotype control antibody (isotype) or anti-CD40 antibody was examined by Western blotting for CD40, TRAF2, TRAF3, cIAP1, HOIP, IKKalpha/beta, and IKKgamma (right panels). HOIP expression was required for coprecipitation of IKK proteins with CD40. Cell lysates from unstimulated cells are shown in the left panels. Similar results were obtained in a second experiment and in two experiments with a second HOIP-deficient clone. (C) To further evaluate HOIP-dependent recruitment of IKKgamma to CD40, A20.2J cells or HOIP-deficient (HOIP-/-) A20.2J cells were transduced with an empty retroviral vector or a retroviral construct encoding BP epitope-tagged IKKgamma (noted in the figure as pMIP and IKKgamma, respectively). Lysates (lanes 1-3) and immunoprecipitation (IP) samples (lanes 4-11) from the cell lines were fractionated by SDS-PAGE and evaluated by Western blotting with antibodies to the BP tag (IKKgamma, upper panel) and TRAF2. The anti-CD40 IP sample in lane 11 was treated with lambda phosphatase; the sample in lane 10 was mock-treated. Protein samples (minus those treated with phosphatase) were also fractionated on a separate gel (lower acrylamide concentration) for the evaluation of TRAF3 and HOIP (bottom two panels). Similar results were obtained in two additional experiments.

recruitment

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HOIP

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IKKgamma

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HOIP

reduces

recruitment

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HOIP

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HOIP

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HOIP

modify

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M-CSF Induces Monocyte Survival by Activating NF-kappaB p65 Phosphorylation at Ser276 via Protein Kinase C
Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-kappaB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-kappaB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCalpha/beta inhibitor Go-6976, overexpression of dominant negative PKCalpha constructs and PKCalpha siRNA reduced NF-kappaB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-kappaBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCalpha as important upstream kinases for M-CSF-induced NF-kappaB transcriptional activation, NF-kappaB-regulated gene expression, NF-kappaB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-kappaB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-kappaB activation in human mononuclear phagocytes.

overexpression

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beta

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Monocytes are produced in the bone marrow and circulate in blood for 24-48 hours [1]. In the absence of serum, monocytes die via apoptosis [1], [2]. Macrophage colony-stimulating factor (M-CSF) stimulates mononuclear phagocyte survival and differentiation [3]. Importantly, M-CSF-mediated cell survival and activation is associated with a variety of human diseases, including atherosclerosis, transplant vascular sclerosis and breast cancer metastasis [4], [5], [6].
We previously identified that NF-kappaB activation is important in M-CSF-induced monocyte survival [7]. In addition to its role in mononuclear phagocyte survival, the transcription factor NF-kappaB regulates numerous genes that play important roles in cellular signaling, stress response, cell growth, survival, differentiation and inflammation [8]. There are five members in the NF-kappaB family: RelA/p65, p50, p52, c-Rel and RelB. The most common activating complex is the p50/p65 heterodimer, driven by the activation domain in the NF-kappaB p65 subunit. NF-kappaB p65 regulates important developmental processes [9], [10]. Mice lacking NF-kappaB p65 die in utero and have extensive liver damage via enhanced apoptosis [9]. Embryonic macrophages from NF-kappaB p65 null mice are susceptible to TNFalpha-induced apoptosis which is rescued by overexpressing the NF-kappaB p65 subunit [10]. Moreover, inhibiting NF-kappaB induces cell death in many cell types and cytokine-independent survival is mediated by constitutively active NF-kappaB in murine macrophages [11].
In monocytes and macrophages, NF-kappaB is an important transcriptional factor for expression of cytokines and cell surface receptors [12]. However, unlike resting T-cells, NF-kappaB is constitutively present in the nuclei of primary monocytes and monocytic cell lines in the absence of exogenous stimuli as demonstrated by mobility shift analysis [13]. Similarly, constitutively active NF-kappaB was observed in human alveolar macrophages [14].
In the classic/canonical pathway, the NF-kappaB p50/p65 complex is sequestered in the cytosol by IkappaBalpha [15]. Upon stimulation by cytokines or UV radiation, IkappaBalpha is phosphorylated, ubiquitinated, and degraded, releasing NF-kappaB p50/p65 to translocate to the nucleus and transactivate target genes. After its release from IkappaBalpha, NF-kappaB p65 can undergo post-translational modification to activate gene transcription. The role of NF-kappaB p65 phosphorylation on NF-kappaB transcriptional activity varies by stimulus, time of stimulation and cell type [16]. Previous research shows that phosphorylation of NF-kappaB p65 at Ser276, Ser529 or Ser536 plays an important role in regulating transcriptional activity of NF-kappaB [17]. In TNFalpha-treated murine fibroblasts, Ser276 of NF-kappaB p65 is phosphorylated by MSK1 to enhance NF-kappaB transcriptional activity [18]. In macrophages treated with endotoxin, NF-kappaB transcription activity is associated with phosphorylation on Ser276 and Ser536 that is regulated through protein kinase A (PKA) and IKKbeta respectively[16], [19]. In human T cells, NF-kappaB p65 is constitutively phosphorylated on Ser536 to facilitate NF-kappaB p65 nuclear translocation following cellular stimulation [20]. Accumulating evidence reveals that NF-kappaB p65 phosphorylation at Ser276 is crucial for its transcriptional activity. Upon nuclear translocation, phosphorylation of Ser276 on NF-kappaB p65 by PKA recruits the transcription co-activator, p300 to potentiate NF-kappaB-regulated gene transcription [21]. However, other studies show that PKA inhibits NF-kappaB-regulated gene expression by stabilizing IkappaBalpha [22], [23]. Interestingly, the serine/threonine kinase Pim-1 directly phosphorylates NF-kappaB p65 at Ser276 by stabilizing to prevent ubiquitin-proteasome degradation [24]. Several other phosphorylation sites are also described to enhance NF-kappaB gene transactivation [25].
Here, we investigate the role of protein kinase C (PKC) in M-CSF-stimulated NF-kappaB activation. PKC proteins are multifunctional kinases that differ in structure, function and co-factor requirement [26]. PKCs are involved in diverse cell responses, including cell growth, survival, differentiation and development [27]. The 12 closely related enzymes of the PKC family are divided into three classes: conventional (cPKCs: alpha betaI betaII and gamma require Ca2+ and diacylglycerol (DAG); novel (nPKCs: delta, epsilon, eta, theta and mu) require DAG; and atypical (aPKCs: xi, iota and lambda) require neither Ca2+ nor DAG. Monocytes and macrophages predominantly express conventional PKC isoforms (PKCalpha PKCbetaI and PKCbetaII) and novel PKCs (PKCdelta and PKCepsilon). Conventional PKCs regulate differentiation of the human promyelocytic leukemia cell line HL60 to macrophages [28]. PKCalpha induces IL-1alpha, iNOS and TNFalpha mRNA production after lipopolysaccharide (LPS) exposure [29]. In addition, accumulating evidence suggests that conventional PKCs like PKCalpha have anti-apoptotic functions. For example, PKCalpha is overexpressed in a variety of tumor cells including gliomas, liver, and lung [30], [31]. In epithelial cells, inhibition of PKCalpha induces PKCdelta-dependent apoptosis [31]. Interestingly, in human monocytes and premonocytic THP-1 leukemia cells, novel PKCs like PKCdelta have the opposite effect on cell survival, Voss et al showed that PKCdelta directly phosphorylates caspase-3 and promotes etoposide-induced apoptosis [32]. Moreover, knockout mouse studies suggest that another novel PKC, PKCepsilon is critically involved in early LPS-mediated signaling in activated macrophages [33].
Previously, we reported that M-CSF promotes monocyte survival through the activation of the PI3-K/AKT pathway [3]. In addition to AKT activation, M-CSF stimulates PKC in human monocytes and increases NF-kappaB DNA binding [34]. However, whether PKC and/or NF-kappaB activation is critical in M-CSF-stimulated mononuclear phagocyte survival and/or differentiation is unclear. In other cells, PKC plays an important role in NF-kappaB activation and cell survival [35], however the specific mechanisms of this activation and the biological effects on cellular phenotype are not known. Therefore, we focused at understanding the role of PKC in the regulation of NF-kappaB activation and M-CSF-induced monocyte survival.
Here we demonstrates that M-CSF upregulated the NF-kappaB transcription and cell survival in human and mouse macrophages. This activity was reduced by the conventional, but not novel, PKC inhibitors, dominant negative PKCalpha constructs or PKCalpha siRNA. Conventional PKC regulated NF-kappaB-regulated gene expression and phosphorylation of Ser276 of NF-kappaB p65 occurred in an M-CSF-dependent fashion correlating with its maximal transcriptional activity. Furthermore, PKCalpha-regulated phosphorylation of Ser276 of NF-kappaB p65 plays an important role in regulating its activity in mononuclear phagocytes and murine embryonic fibroblasts.

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PKCalpha

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IkappaBalpha

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PKCdelta

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Upon

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Upon

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stabilizing

IkappaBalpha

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PKCalpha

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overexpressed

PKCalpha

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post-translational modification

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phosphorylation

IKKbeta

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production

production

iNOS

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TNFalpha


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M-CSF Induces NF-kappaB Transcriptional Activity in Human Monocyte-Derived Macrophages (MDMs) and Mouse Macrophage Cell Line, RAW 264.7
To determine if M-CSF induced NF-kappaB DNA binding in human macrophages, we performed EMSA analysis on nuclear lysates from M-CSF-treated MDMs. Similar to previous reports [11], nuclear NF-kappaB constitutively bound DNA in non-stimulated monocytes (Figure 1A). Interestingly, adding M-CSF did not alter NF-kappaB DNA binding by EMSA. In contrast, after transiently transfecting human MDMs with pNF-kappaB-SEAP constructs containing four NF-kappaB consensus binding sequences, M-CSF treatment of the transfected cells resulted in a 2.3-fold increase in SEAP release in the culture media compared to PBS (vehicle)-treated transfected MDMs (Figure 1B). As a control, the pTAL-SEAP construct lacking NF-kappaB binding sites was used. Cells transfected with the pTAL-SEAP construct did not produce SEAP in the absence or presence of M-CSF (Figure 1B).
We next investigated whether M-CSF induced NF-kappaB activity in the mouse macrophage cell line, RAW 264.7. RAW 264.7 cells were transfected with either the NF-kappaB-SEAP reporter or control pTAL-SEAP construct. As shown in Figure 1C, M-CSF treatment of RAW 264.7 cells increased NF-kappaB reporter activity by 2.5-fold over that of non-treated cells. Together, our data demonstrate that M-CSF induced NF-kappaB transcriptional activity in macrophages.

increased

activity

activity

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PKC Inhibition Reduces NF-kappaB Activity in Human MDMs and RAW 264.7 Cells
Since NF-kappaB is activated by PKC in several cell types [36], we next determined if M-CSF-induced NF-kappaB transcriptional activity was dependent on PKC activation and if calcium, a co-factor for conventional PKC isoform activation, was important. In addition, because of the number of conventional PKCs existing within mononuclear cells, pharmacological inhibitors of PKC family activation was used to determine the relationship of PKC activation to M-CSF-induced cellular survival. MDMs were transfected with the pNF-kappaB-SEAP reporter and then treated with the general PKC inhibitor, Ro-31-8220; the conventional PKCalpha/beta inhibitor Go-6976; or the intracellular calcium blocker BAPTA/AM. Ro-31-8220 significantly suppressed M-CSF-induced NF-kappaB activity compared to cells treated with M-CSF alone (Figure 2A). In addition, Go-6976 and BAPTA/AM also blocked NF-kappaB activity in M-CSF-treated MDMs. Trypan blue exclusion analysis did not indicate cell death suggesting that this suppression was not due to non-specific toxicity. These data indicate that M-CSF mediated NF-kappaB activation through calcium-dependent conventional PKC activation.
We next investigated whether PKC inhibition affected NF-kappaB activity in the mouse macrophage cell line, RAW 264.7. Similar to MDMs, PKC inhibitors Ro-31-8220, Go-6976 and BAPTA/AM reduced M-CSF-induced NF-kappaB activity in a dose-dependent manner in RAW 264.7 macrophages (Figure 2B). To ensure that PKC specifically regulated NF-kappaB p65 and not the closely related family member, c-Rel, we co-transfected c-Rel and NF-kappaB-SEAP constructs into the Raw 264.7 cell line and measured NF-kappaB activity in response to M-CSF. There was no increased NF-kappaB activity in cells expressing c-Rel (Figure S1). These observations indicate that PKC functioned upstream of NF-kappaB p65 in MDMs and RAW 264.7 cells.

expressing

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inhibitor

PKCalpha

inhibitor

beta

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c-Rel

functioned


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NF-kappaB and PKC(s) Mediate Human MDM Survival in Response to M-CSF Stimulation
Since PKC and NF-kappaB are critical in cell survival [37], [38], we hypothesized that M-CSF promoted cell survival through PKC in human MDMs. To detect apoptosis, the expression of cleaved caspase-3, a marker of apoptosis, was analyzed in the presence or absence of M-CSF and PKC inhibitors. In MDMs pretreated with PKC inhibitors (Ro-31-8220, Go-6976, or BAPTA/AM) and stimulated with M-CSF, cleaved caspase-3 was elevated to levels of cells treated with vehicle alone (Figure 3A). In contrast, cells incubated with M-CSF and vehicle had less cleaved caspase-3 than cells incubated with vehicle alone or M-CSF with PKC inhibitors. These data supported the hypothesis that M-CSF-induced NF-kappaB activity was regulated by conventional PKCs, not novel PKCs.
To confirm that PKC inhibition decreased cell survival, cells were treated with M-CSF in the absence or presence of PKC inhibitors and then also examined for apoptosis by Annexin V/PI staining. PKC inhibitors in the presence of M-CSF reduced the number of Annexin V/PI negative MDMs compared to cells treated with M-CSF alone (Figure 3B). These observations further suggested that MDM survival is promoted by M-CSF and critically involves conventional PKCs and NF-kappaB activity.

less

cleaved

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Annexin V

expression

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elevated

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M-CSF-Induces PKCalpha Kinase Activity
Since our data suggested that conventional PKCs were involved in activating NF-kappaB in response to M-CSF in primary human macrophages, we next investigated whether the conventional PKC, PKCalpha was a downstream target of M-CSF in human MDMs. PKCalpha was immunoprecipitated from human MDMs at the indicated time points (Figure 4), and then a PKC kinase assay was performed using a fluorescein-tagged peptide as a substrate. As shown in Figure 4 (upper panel), the peptide substrate was maximally phosphorylated within 10 minutes of stimulation (1.8-fold over resting cells, lower panel), and then returned to basal levels by 15 minutes. This effect was not seen in M-CSF-stimulated monocytes when isogenic antibodies (IgG) were used for immunoprecipitation. Western blots of identical samples using an antibody recognizing PKCalpha demonstrated that equal amounts of PKCalpha were assayed (Figure 4, middle panel).

downstream target

PKCalpha


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M-CSF-Dependent Activation of PKC Does Not Regulate the Classical NF-kappaB p65 Activation Pathway
Canonical activation of NF-kappaB occurs via phosphorylation and degradation of IkappaBalpha leading to the release and nuclear translocation of the NF-kappaB p50/p65 heterodimer to transactivate target genes [37], [39]. Since conventional PKC activity was important in regulating M-CSF-induced NF-kappaB activation, we next investigated whether IkappaBalpha degradation was regulated by PKC. Cells were treated with cyclohexamide (CHX) to inhibit protein synthesis of IkappaBalpha, and its degradation was followed. As shown in Figure 5A, PKC inhibition with Ro-31-8220 did not alter M-CSF-induced IkappaBalpha degradation, suggesting that M-CSF-induced PKC activity augmented NF-kappaB transcriptional activity by an alternative pathway, like post-translational modification of NF-kappaB p65.

synthesis

IkappaBalpha

inhibit

synthesis

phosphorylation

IkappaBalpha

Activation

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degradation

degradation

IkappaBalpha

degradation

IkappaBalpha

degradation

IkappaBalpha

post-translational modification

Regulate

Activation

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post-translational modification


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M-CSF Induces Phosphorylation of NF-kappaB Ser276 in a PKC-dependent Fashion
Since M-CSF did not regulate NF-kappaB activation by influencing IkappaBalpha, we next sought to determine if M-CSF affected NF-kappaB p65 by post-translational mechanisms. Thus, we examined the phosphorylation of NF-kappaB p65 with specific phospho-NFkappaB p65 (Ser276 and Ser536) antibodies. M-CSF induced the phosphorylation of Ser276 but not Ser536 of NF-kappaB p65 in MDMs. Compared to vehicle, the general PKC inhibitor Ro-31-8220 reduced Ser276 phosphorylation, but not Ser536, phosphorylation in M-CSF-stimulated cells (Figure 5B). Furthermore, M-CSF-stimulated NF-kappaB p65 phosphorylation at residue Ser276 in RAW 264.7 cells was also PKC dependent (Figure 5C). These studies suggested that PKC(s) regulated Ser276 phosphorylation but not Ser536 in both human MDMs and mouse macrophages after M-CSF stimulation.
We next performed cellular fractionation to identify the cellular location of phosphorylated NF-kappaB p65 in Raw 264.7 cells. Non-phosphorylated NF-kappaB p65 was located in both cytosolic and nuclear fractions, but phosphorylated Ser276 and Ser536 NF-kappaB p65 was primarily located in nuclear fraction after M-CSF stimulation (Figure 5D). Notably, constitutive phosphorylation of Ser536 NF-kappaB p65 was found in these cells. Importantly, Ro-31-8220 reduced M-CSF-induced Ser276 phosphorylation of NF-kappaB p65 in both the cytosolic and nuclear fractions, while M-CSF-induced NF-kappaB p65 Ser536 phosphorylation was present in the nucleus regardless of PKC inhibition. These observations indicate that M-CSF-induced Ser276 and Ser536 are regulated differently by conventional PKC activation in mononuclear phagocytes. The purity of the cytosol and nuclear cell fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively (Figure 5D).

location

located

both cytosolic and nuclear fractions

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M-CSF-dependent PKC Regulates NF-kappaB-targeted Genes
NF-kappaB induces a number of downstream genes, including the IkappaB family. Among the IkappaB molecules, IkappaBalpha is highly induced by NF-kappaB activation [40]. Having shown that PKC regulated NF-kappaB activity in M-CSF-stimulated MDMs, we next determined whether inhibition of PKC activity decreased expression of NF-kappaB-regulated genes. We treated both MDMs and RAW 264.7 cells with the PKC inhibitor Ro-31-8220 for 30 minutes and then stimulated with M-CSF. IkappaBalpha gene was measured by qRT-PCR. As shown in Figures 6A and 6B, M-CSF enhanced IkappaBalpha gene expression and PKC inhibition by Ro-31-8220 decreased IkappaBalpha gene expression in both MDMs and RAW 264.7 cells (p<0.01), demonstrating that PKC affected NF-kappaB-regulated gene expression in macrophages.
To further define the role of PKC in mediating human MDM survival in response to M-CSF, we examined the expression of the anti-apoptotic gene BCL-xL, which is also regulated by NF-kappaB. As shown in Figure 6C, Ro-31-8220 reduced M-CSF-stimulated BCL-xL expression compared to cells treated with M-CSF and the vehicle control DMSO (p<0.05).

expression

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Identification of PKCalpha as the Upstream Activator of NF-kappaB in Myeloid Cells
Even though the PKC family consists of 10 members, finding that PKCalpha/beta inhibitors and intracellular calcium inhibitors reduced M-CSF-induced NF-kappaB activity, suggested PKCalpha was involved in NF-kappaB activation after M-CSF treatment. To confirm the role of PKCalpha in NF-kappaB activation in macrophages, constructs for either wildtype (WT)-PKCalpha or kinase-deficient (KD)-PKCalpha was co-transfected with the pNF-kappaB-SEAP reporter gene and SEAP secretion was measured. As shown in Figure 7A, MDMs co-transfected with pNF-kappaB-SEAP and WT-PKCalpha had a 1.8-fold increase in NF-kappaB transcriptional activity after M-CSF activation compared with NS cells (p = 0.05), similar to M-CSF-treated cells expressing only pNF-kappaB-SEAP. Transfecting human macrophages with the KD-PKCalpha construct significantly reduced M-CSF-induced NF-kappaB activity compared to WT-PKCalpha transfected cells (p = 0.016). Similarly, RAW 264.7 cells transfected with WT-PKCalpha had 2.5-fold more NF-kappaB transcriptional activity after M-CSF activation compared to unstimulated RAW 264.7 cells (NS) transfected with WT-PKCalpha (Figure 7B). Expression of the KD-PKCalpha construct into RAW 264.7 cells reduced M-CSF-induced NF-kappaB activity to 1.5-fold (p = 0.045) compared to cells transfected with WT PKCalpha.
Cell survival was also examined in MDMs expressing either WT-PKCalpha or KD-PKCalpha constructs by Annexin V-FITC and PI staining. As expected, M-CSF increased MDM survival as measured by the percent of Annexin V/PI negative cells. Similarly, expression of WT-PKCalpha protected cells from apoptosis. In contrast, expression of KD-PKCalpha decreased M-CSF-induced cell survival (p<0.01) (Figure 7C).
Next, we examined the effect of expressing the PKCalpha constructs on NF-kappaB phosphorylation. As shown in Figure 7D, expression of KD-PKCalpha in RAW 264.7 cells did not affect the constitutive phosphorylation at Ser536 of NF-kappaB p65, but attenuated the phosphorylation at Ser276. Expression of WT-PKCalpha did not effect the phosphorylation of either residue with or without M-CSF stimulation. These observations demonstrate that PKCalpha is important in M-CSF-regulated cell survival and NF-kappaB activation and likely regulated through phosphorylation of Ser276 of NF-kappaB p65.
To further validate the impact that PKCalpha played in M-CSF-induced NF-kappaB transcriptional activity, we next employed PKCalpha siRNA treatment of MDM or RAW cells. A pool of specific PKCalpha siRNA were transfected into MDM or Raw 264.7 cells in the presence or absence or M-CSF. Reducing native PKCalpha expression decreased M-CSF-induced NF-kappaB transcriptional activity in both MDM (Figure 7E) (p = 0.012) and Raw 264.7 cells (Figure 7F) (p = 0.01). We also examined cell survival of the MDMs by Annexin V-FITC and PI staining after PKCalpha siRNA transfection. As shown in Figure 7G, M-CSF-induced MDM survival was reduced in the cells transfected with PKCalpha siRNA compared with cells transfected with control siRNA (p = 0.047). In Figure 7H, we confirmed that PKCalpha siRNA transfection decreased PKCalpha protein expression in both MDM and Raw 264.7 cells. Our results indicated that PKCalpha regulated NF-kappaB activation and M-CSF-regulated cell survival.

co-transfected

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Reducing

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Input:
PKC is Essential in Regulating NF-kappaB Activity
M-CSF-dependent PKC activity facilitated NF-kappaB p65 phosphorylation at Ser276 but not Ser536. To confirm the role of PKC and p65 Ser276 phosphorylation on NF-kappaB activity, we co-transfected the NF-kappaB-SEAP construct with either WT NF-kappaB p65 or NF-kappaB p65 276S/A constructs in Raw 264.7 cells, then treated cells with the PKC inhibitor Ro-31-8220 in the absence or presences of M-CSF. As expected in cells transfected with vector control, inhibiting PKC reduced M-CSF-stimulated NF-kappaB activity compared to cells treated with M-CSF and the vehicle DMSO (p = 0.001) (Figure 8A). In contrast, expressing WT NF-kappaB p65 (p65 WT) increased NF-kappaB activity, while the general PKC inhibitor Ro-31-8220 decreased this NF-kappaB activity (p = 0.001). Notably, the introduction of NF-kappaB p65 276 S/A construct significantly reduced NF-kappaB activity compared with the WT NF-kappaB p65 construct (p = 0.005) and M-CSF treatment was unable to overcome this inhibition.
Furthermore, we co-transfected the NF-kappaB-SEAP construct along with either the WT NF-kappaB p65, NF-kappaB p65 276S/A or NF-kappaB p65 536S/A constructs into a NF-kappaB p65-/- murine fibroblast cell line and measured NF-kappaB activity. As predicted, expression of WT NF-kappaB p65 (p65 WT) in NF-kappaB p65-/- cell line constitutively activated NF-kappaB compared to cells transfected with vector control without any stimulation (Figure 8B). In comparison, transfecting the NF-kappaB p65 276S/A construct reduced NF-kappaB activity by 5-fold (p = 0.006, WT NF-kappaB p65 vs. NF-kappaB p65 276S/A), while expressing the NF-kappaB p65 536S/A construct increased NF-kappaB activity in the p65-/- cells to levels similar to WT NF-kappaB p65 transfected cells.
Since M-CSF-induced PKC activation regulated NF-kappaB activity via Ser276 residue of NF-kappaB p65 in primary human MDMs and RAW 264.7 cells, we next examined if this occurred in NF-kappaB p65-/- fibroblasts in response to a native stimulus for these cells, TNFalpha. NF-kappaB activity was measured in the WT NF-kappaB p65 or NF-kappaB p65 276S/A transfected cells treated with TNFalpha in the absence or presence of Ro-31-8220 (Figure 8C). As expected, TNFalpha increased NF-kappaB activity in NF-kappaB p65-/- cells expressing WT p65 and (p = 0.001) PKC inhibitors decreased NF-kappaB activity to the non-stimulated (NS) level (p = 0.007). Introducing NF-kappaB p65 276 S/A constructs significantly reduced NF-kappaB activity compared with the WT NF-kappaB p65 construct (p = 0.001). Notably, TNFalpha failed to increase NF-kappaB activity in the NF-kappaB p65-/- cells expressing NF-kappaB p65 276S/A constructs. These observations are similar to macrophages overexpressing the NF-kappaB p65 276S/A (Figure 8A). Our data demonstrate that Ser276 of NF-kappaB p65 is essential in regulating NF-kappaB activity and suggests that PKC regulates NF-kappaB activity by modulating the phosphorylation of NF-kappaB p65 at Ser276 residue.

co-transfected

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phosphorylation


Input:
NF-kappaB transcriptional activity is important in regulating intracellular signaling, stress response, proliferation, survival, differentiation and inflammation [8]. To regulate gene transcription, NF-kappaB p50/p65 heterodimers translocate to the nucleus from the cytoplasm, however, their translocation is not sufficient to activate gene transcription. Post-translational modification of NF-kappaB heterodimers such as phosphorylation or acetylation also contributes to its transcriptional activity [41], [42], [43]. Because NF-kappaB constitutively translocates to the nucleus in monocytic lineage [13] and activates gene transcription and survival in murine macrophages [11], we sought to determine how M-CSF affected NF-kappaB transcriptional activity in human MDMs and whether this activation regulated their survival. The present study demonstrated that M-CSF stimulated PKCalpha kinase upstream of NF-kappaB transcriptional activity in primary human MDMs and RAW 264.7 cells. We showed inhibition of conventional PKCs by PKC inhibitors, kinase deficient PKCalpha or PKCalpha siRNA blocked M-CSF-induced cell survival and NF-kappaB-regulated gene expression. This block correlated with a reduction in M-CSF-induced phosphorylation of NF-kappaB p65 at Ser276. Consistent with these findings the dominant negative PKCalpha constructs also inhibited NF-kappaB p65 phosphorylation at Ser276 but not at Ser536 resulting in reduced NF-kappaB transcriptional activation and M-CSF-induced MDM survival. A simplified proposed cartoon model in Figure 9 demonstrates that M-CSF induced monocytes survival is regulated by activating NF-kappaB p65 phosphorylation at Ser276 via PKC. Finally, in a NF-kappaB p65-/- fibroblast cell line, we confirmed that the Ser276 residue of NF-kappaB p65 is important and essential for PKC modulation of NF-kappaB activity. More compelling is that we observed a similar regulation between two different cell lineages in our study.
Post-translational modification of NF-kappaB p65 regulates its activating or repressing effects on gene expression. Among the seven reported putative NF-kappaB p65 phosphorylation sites, five increase nuclear translocation, DNA binding and NF-kappaB transcriptional activity [17]. Similar to our finding, Zhong et al. found that in response to LPS, NF-kappaB p65 is phosphorylated at the highly conserved Ser276 residue by the catalytic subunit of PKA [16]. In addition to PKA, Ser276 can be phosphorylated by MSK1 in the nucleus upon TNFalpha treatment [18]. Phosphorylation of NF-kappaB p65 at Ser536 occurs in response to many inflammatory stimuli as well as kinases, IKKalpha, IKKbeta and RSK1 [17], [18], [19], [20], [21]. Notably, in addition to phosphorylating Ser276 of NF-kappaB, our study revealed that M-CSF also modulated the phosphorylation of the NF-kappaB p65 subunit at Ser536, however, PKC inhibitors or kinase deficient PKCalpha constructs did not affect this phosphorylation event. Moreover, mutant constructs containing a point mutation at Ser536 of NF-kappaB p65 did not reduce NF-kappaB activity in cells lacking endogenous NF-kappaB p65, while the NF-kappaB p65 276S/A construct did reduce NF-kappaB activity in these cells. Therefore, specific post-translational modification of NF-kappaB p65 is likely to regulate transcriptional activation in response to specific stimuli [15], [43].
It is notable that M-CSF activated NF-kappaB p65 Ser276 phosphorylation and transcriptional activation in a PKCalpha-dependent manner. PKCalpha is a member of the conventional PKC family of protein kinases that are critical for cell growth, differentiation and cell death. PKCalpha primarily modulates anti-apoptotic and proliferation signals following cytokine treatment in various cell types [42]. Expression of PKCalpha attenuates apoptosis in many different cell types, while PKCalpha inhibition generally potentiates apoptosis [42]. In our studies using conventional PKC inhibitors and dominant negative PKCalpha constructs, PKCalpha was critical in M-CSF-induced macrophage survival by inducing Ser276 phosphorylation of NF-kappaB p65 in both primary human MDMs and Raw 264.7 cells. We also demonstrated PKC inhibition downregulated NF-kappaB-induction of the anti-apoptotic gene BCL-xL. Consistent with our finding, others reported NF-kappaB regulated the gene expression of many anti-apoptotic proteins including other BCL-2 family members and inhibitor of apoptosis (IAP) proteins which are regulators of apoptosis and function upstream of caspases [44]. It has been shown that phosphorylation of p65 at Ser276 prevents its degradation by ubiquitin-mediated proteolysis and promotes cell survival in HeLa cells [24]. In addition to BCL-xL expression as examined in this study, we are evaluating the possibility that PKCalpha-regulated NF-kappaB activity upregulates additional anti-apoptotic genes in an M-CSF-dependent fashion to modulate cell survival.
The use of inhibitors and siRNA in our study has not ruled out that other conventional PKCs might also be important in M-CSF-induced NF-kappaB activation and macrophage survival. In contrast to the actions of PKCalpha activation of other conventional PKC isoforms, like PKCbetaI/betaII, appear necessary for macrophage apoptosis. The PKCbetaI/betaII isoforms are expressed in apoptotic U937 myelomonocytic cells [45]. The human myeloid cell line HL-525 which is devoid of endogenous PKCbetaII is resistant to TNFalpha-induced apoptosis [46]. Re-expression of PKCbetaII in HL-525 cells restores their susceptibility to TNFalpha-induced apoptosis implying that PKCbetaII is pro-apoptotic and may be required to induce macrophage apoptosis.
Recent studies demonstrated that NF-kappaB transcriptional activity can be regulated by phosphorylation of the NF-kappaB p65 subunit in the absence of IkappaBalpha degradation [47]. By this pathway, phosphorylation of NF-kappaB regulates its interaction with other components of the basal transcriptional machinery without affecting its capability to bind DNA [21]. Since NF-kappaB can bind the promoters of numerous gene targets, post-translational modification of NF-kappaB p65 may affect its interaction with other proteins, refining the expression of gene targets. We did not find that PKC was involved in IkappaBalpha degradation or NF-kappaB p65 nuclear translocation induced by M-CSF, but that M-CSF-induced phosphorylation of NF-kappaB p65 at Ser276 was dramatically reduced by the conventional PKC inhibitor and kinase deficient PKCalpha and by siRNA towards PKCalpha. Current studies are underway to delineate the protein complexes formed by activated NF-kappaB and other transcriptional co-activators in response to M-CSF and PKC activation. Recently studies indicated that the non-canonical pathway was activated during human monocyte-macrophage differentiation [48]. During this process, expression of IKKalpha among other proteins, is elevated leading to increased p52 NF-kappaB (relB) expression through partial proteolytic degradation of the p100 NF-kappaB protein. Thus, it is possible that M-CSF regulates monocyte survival through both the canonical and non-canonical NF-kappaB pathways, which will be a focus in future research.
We previously showed that M-CSF reduced caspase-3 and -9 activity and prevented monocyte apoptosis [3]. This study reveals that treatment of monocytes with conventional PKC inhibitors, or overexpression of kinase-deficient PKCalpha blocked M-CSF-induced cell survival. Our data revealed that conventional PKCs are upstream of NF-kappaB activation in response to M-CSF and mediate post-translational activation of NF-kappaB p65 by phosphorylating Ser276 to regulate gene transcription and cell survival. Thus, our observation may provide insight in potential therapeutic targets for inflammatory diseases.

interaction

Expression

PKCalpha

expression

BCL-xL

expressed

PKCbetaI

expressed

betaII

devoid

PKCbetaII

expression

PKCbetaII

expression

IKKalpha

expression

translocate

nucleus

translocate

nucleus

translocation

their

translocation

nuclear

kinase deficient

PKCalpha

reduction

induced

inhibited

dominant negative PKCalpha

phosphorylation

kinase deficient

PKCalpha

lacking

inhibition

PKCalpha

downregulated

induction

prevents

phosphorylation

by

absence

degradation

reduced

induced

kinase deficient

reduced

induced

siRNA

kinase deficient

PKCalpha

reduced

reduced

kinase-deficient

PKCalpha

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylated

phosphorylated

Phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylation

phosphorylating

stimulated

PKCalpha

induced

phosphorylation

activating

phosphorylation

in response to

phosphorylated

upon

phosphorylated

TNFalpha

in response to

Phosphorylation

activated

phosphorylation

inducing

PKCalpha

phosphorylation

induction

BCL-xL

by

degradation

mediated

mediated

ubiquitin

proteolysis

induced

translocation

induced

induced

elevated

expression

increased

expression

expression

through

increased

degradation

mediate

activation

activation

phosphorylating

degradation

proteolysis

degradation

IkappaBalpha

degradation

IkappaBalpha

degradation

Post-translational modification

post-translational modification

post-translational modification

modulated

phosphorylation

point mutation

dependent

activated

PKCalpha

affect

post-translational modification

interaction

involved

degradation

involved

induced


Input:
Materials
Recombinant human M-CSF was purchased from R&D Systems (Minneapolis, MN). Ro-31-8220, Go-6976, Rotterin, and BAPTA/AM were obtained from Calbiochem (San Diego, CA). Low endotoxin RPMI and X-vivo serum free medium were obtained from Lonza Walkersville Inc (Walkersville, MD). Fetal bovine serum (FBS, certified <0.06 endotoxin units/ml endotoxin levels) was purchased from Atlanta Biological (Lawrenceville, GA). All other cell culture reagents were obtained through Invitrogen (Carlsbad, CA). PKCalpha NF-kappaB p65, Lamin B, beta-actin, and GAPDH antibodies, anti-mouse and rabbit IgG-HRP conjugated antibodies, and PKCalpha siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-NFkappaB p65 (Ser276 or Ser536) antibodies were purchased from Cell Signaling Technology (Danvers, MA). DNA constructs pTAL-SEAP and pNF-kappaB-SEAP were obtained from Clontech (Mountian View, CA). Expression vectors containing CMV-p65 WT, CMV-p65 276S/A and CMV-p65 536S/A were cloned in pBa5e Puro vector or pFLAG-CMV-2 vector as described previously [49]. Kinase deficient CMV-PKCalpha (KD-PKCalpha) or wild type pCMV-PKCalpha (WT-PKCalpha) were generous gifts from Alexandra C. Newton (University of California, San Diego, CA). IkappaBalpha (NFKBIA) and GAPDH primers for real-time RT-PCR were obtained from SABiosciences (Frederick, MD). A pool of mouse or human PKCalpha specific siRNA were purchased from Santa Cruz Biotechnology (Santa cruz, CA).

Kinase deficient

PKCalpha

KD

PKCalpha


Input:
Cell Culture
The murine macrophage cell line RAW 264.7 was purchased from ATCC (Manassas, VA). RAW 264.7 cells were maintained in RPMI supplemented with 5% FBS and antibiotic-antimycotic (1000 U/ml penicillin, 1000 microg/ml streptomycin sulfate, and 250 ng/ml amphotericin B) at 37degreesC. Mouse embryonic fibroblasts (MEFs) cell line lacking specific NFkappaB signaling subunits NF-kappaB p65 (p65-/- cell line) [50] were cultured in DMEM medium supplemented with 10% FBS and antibiotic-antimycotic solution.


Input:
Purification of Peripheral Blood Monocytes and Monocyte-Derived Macrophages (MDMs)
Monocytes were isolated from source leukocyte packs obtained from the American Red Cross as described previously [51]. Monocytes used in real time PCR experiments and transfection experiments were purified by positive selection using CD14 Monocyte Isolation Kit from Miltenyi Biotech (Auburn, CA) (>90% pure). In some experiments, monocytes were isolated by clumping method (70% pure). To obtain monocyte-derived macrophages (MDMs), monocytes were cultured in RPMI-1640 medium containing 10% FBS, and 10 microg/ml polymyxin B and 20 ng/ml M-CSF.


Input:
Electrophoresis of the Mobility Shift Analysis (EMSA)
Nuclear extracts were isolated from MDMs by using a nuclear extraction kit according to the manufacturer's instruction from Active Motif (Carlsbad, CA). Briefly, MDMs were lysed in hypotonic lysis buffer (20 mM Hepes, pH 7.5; 5 mM NaF, 10 microM Na2MoO4 0.5% NP-40 and 0.1 mM EDTA), and then nuclei were resuspended in lysis buffer supplemented with 0.5 mM DTT and 0.2 mM PMSF. The NF-kappaB consensus oligonucleotides (sense: AGTTGAGGGGACTTTCCCAGGC; antisense: GCCTGGGAAAGTCCCCTCAACT) labeled with 32P by T4 polynucleokinase (Promega, Madison, WI) were incubated with nuclear extracts in binding buffer (10 mM Tris pH 7.6, 1 mM DTT, 0.5 mM EDTA, 2 microg polydI-dC and 10% Glycerol) at 30degreesC for 30 minutes. The free DNA and DNA-protein mixtures were resolved in 5% native polyacrylamide gels in 0.5x TBE buffer (45 mM Tris, 45 mM boric acid and 1 mM EDTA, pH 8.3) by electrophoresis. The gel was dried and subjected to autoradiography analysis.


Input:
Transient Transfection of RAW 264.7 Cells
RAW 264.7 cells were seeded in 12-well plates in RPMI medium containing 5% FBS one day prior to transfection, Secreted alkaline phosphatase (SEAP) reporter plasmids pTAL-SEAP or pNF-kappaB-SEAP were transfected into cells using Qiagene Effectene or Attractene transfection kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. For co-transfection studies, PKCalpha or NF-kappaB p65 constructs were mixed at a ratio of 5:1 with the reporter plasmid. For siRNA transfection, 100 nM of PKCalpha siRNA or control siRNA were used. Cells were incubated with the transfection reagents for 16-24 hours, washed and starved in RPMI without FBS for 4 hours. Samples were then stimulated with 100 ng/ml M-CSF for 4 hours at which point the medium was collected for the SEAP analysis. For inhibition studies, cells were pre-incubated with inhibitors for 30 minutes before M-CSF treatment. On average, we realized 30-40% transfection efficiency as confirmed by GFP expression in Raw 264.7 cells.

co-transfection

PKCalpha

co-transfection

expression

GFP


Input:
Transient Transfection of Primary Human Monocytes
Monocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20x106 cells/ml, and 2 microg of total plasmid DNA 100 nM of PKCalpha siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCalpha or NF-kappaB p65 constructs were mixed at a ratio of 5:1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4x105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF.


Input:
Secreted Alkaline Phosphatase (SEAP) Analysis
Secreted alkaline phosphatase activity was analyzed by GreatEscape SEAP kit according to the manufacturer's instruction. Briefly, medium was diluted in the dilution buffer and heated to 65degreesC to inactivate endogenous phosphatases then incubated with the substrate for 30 minutes. The chemiluminence signal was recorded using a luminometer.


Input:
Annexin V/PI Apoptosis Assay
Cell apoptosis assay was performed as described previously [51] using the Annexin V-FITC apoptosis detection kit (BD PharMingen, San Diego, CA). Briefly, human monocytes (5x106) were incubated with inhibitors for 30 minutes in X-vivo medium and restimulated with 100 ng/ml M-CSF overnight. The cells were removed from the culture dish using Accutase (eBioscience, San Diego, CA) and stained with Annexin V-FITC and PI and analyzed by flow cytometry (FACSCalibur; BD PharMingen). Annexin V-FITC and PI double negative cells were considered non-apoptotic cells for statistical analysis.


Input:
Western Blot Analysis
Cells were washed with PBS and resuspended in cell lysis buffer. (Cell Signaling Technology) and incubated for 10 minutes on ice then centrifuged to remove the insoluble fraction. The protein concentration was determined by the BCA protein assay (Bio-Rad, Hercules, CA). Cell lysates were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred onto nitrocellulose membranes and subjected to Western blotting. The immunoblotted proteins were detected by ECL reagent (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).


Input:
Analysis of PKCalpha Kinase Activity
Human MDMs were starved for 2 hours before stimulation with M-CSF (0-60 minutes), then washed with cold PBS and removed from the plates. The cells were resuspended in lysis buffer (20 mM Tris, 5 mM MgCl2, 1 mM EGTA, 20 mM beta-glycerol phosphate, 1 mM PMSF 2 microg/ml aprotinin and 2 microg/ml leupeptin, pH 7.5), sonicated and centrifuged to obtain whole cell lysates, which were then immunoprecipitated with anti-PKCalpha antibody and protein G-agarose (Invitrogen). Immune complexes were washed twice, and PKCalpha activity was analyzed by non-radioactive Peptag assay kit (Promega). Briefly, kinase buffer, activator, peptide protection solution and Peptag peptide were incubated with the immune complexes at 30degreesC for 30 minutes. Reactions were stopped by boiling for 10 minutes and samples were separated on 0.8% agarose gel. Phosphorylated peptide migrated toward the anode (+), while non-phosphorylated peptide migrated toward the cathode (-). Fluorescein-tagged peptides were visualized by UV light.


Input:
RNA Isolation and Quantitative Real-time PCR
MDMs or RAW 264.7 cells were serum-starved overnight or for 2 hours, respectively, prior to incubating with inhibitors for 30 minutes. The cells were restimulated with 100 ng/ml M-CSF and total mRNA was extracted from cells with Trizol (Invitrogen) and 1-2 microg of total RNA was used to synthesized cDNA using SuperScript III (Invitrogen). Quantitative real-time (qRT)-PCR was performed using SYBR Green Master Mix (Applied BioSystems, Carlsbad, CA). The reactions were performed using an ABI PRIZM 7700 machine with software Sequence Detector version 1.7 (Applied Biosystems). The target gene values were normalized to the values of GAPDH as a housekeeping gene and expressed as relative fold increase 2(-deltadeltaCt) over the non-stimulated samples (NS).


Input:
Statistical Analysis
Statistical comparisons were performed using analysis of variance testing (Minitab software, State Park, PA or SPSS16 software, SPSS Inc. Chicago, IL). Statistical significance was defined as p<=0.05.


Input:
Ethics Statement
All research involving human blood (Buffy coat) were ordered from American Red cross and have been approved by the Ohio State University review board (ARC IRB Protocol #2001-26).


Input:
M-CSF induces NF-kappaB transcriptional activity in macrophages.
(A) Nuclei were extracted from human MDMs treated without or with M-CSF for 15 or 30 minutes. NF-kappaB DNA binding activity was analyzed by EMSA Shown is a representative blot from three independent experiments. NS: non-stimulated. (B) Human MDMs transiently transfected with pTAL-SEAP or pNF-kappaB-SEAP constructs were treated with M-CSF in X-vivo medium and incubated for 6 hours before the collection of medium. NF-kappaB activity was analyzed by measuring the amount of SEAP secreted into the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. (C) RAW 264.7 cells were transiently transfected with pTAL-SEAP or pNF-kappaB-SEAP construct. Cells were serum starved for 4 hours prior to 2 hours stimulation with mouse recombinant M-CSF (100 ng/ml). Culture media was collected to measure SEAP production. Data are expressed mean +/- S.E.M, for three independent experiments.


Input:
Inhibition of PKC reduces NF-kappaB activity in M-CSF stimulated macrophages.
(A) Human MDMs transfected with pNF-kappaB-SEAP were pre-incubated with inhibitors; Ro-31-8220, Go-6976, or BAPTA/AM for 30 minutes prior to 6 hours M-CSF stimulation. (B) RAW 264.7 cells transfected with the pNF-kappaB-SEAP construct were pre-incubated with inhibitors for 30 minutes prior to 2 hours of stimulation with mouse recombinant M-CSF. The NF-kappaB activity was analyzed by measuring SEAP production in the medium and data are expressed as fold increase of SEAP activity over that in pTAL-SEAP transfected resting cells. The graph represents mean +/- S.E.M for three independent experiments. *The p-values of cells treated with inhibitors/M-CSF compared to vehicle/M-CSF were <=0.05.


Input:
Inhibition of PKC or NF-kappaB induces apoptosis in MDMs.
(A) MDMs were pre-incubated in RPMI medium containing inhibitors (Ro-31-8220: 5 microM, Go-6976: 5 microM, BAPTA/AM: 2.5 microM) for 30 minutes prior to the addition of M-CSF. As a control, untreated cells were incubated with dimethyl sulfoxide (DMSO). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody recognizing the active cleaved form of caspase-3. The blots were reblotted with beta-actin that served as a loading control. The ratio of active caspase-3 bands (17 kD and 19 kD) to beta-actin control was determined by densitometry analysis (bottom panel). Data represents the mean +/- S.E.M from two independent donors. (B) Apoptosis of the treated MDMs was also measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. The percentage of surviving cells (Annexin V/PI negative) for the cells treated with vehicle/M-CSF was arbitrarily set as 100. Data shown represent the mean +/- S.E.M from three independent experiments. *The p-values of inhibitors/M-CSF compared to vehicle/M-CSF were <=0.05.

negative

Annexin V

active


Input:
M-CSF activates PKCalpha in human MDMs.
MDMs treated with M-CSF (100 ng/ml) for varying amounts of time were lysed and immunoprecipitated using anti-PKC antibody or control IgG antibody. One half of the samples was used to analyze PKC kinase activity using a fluorescein tagged peptide and visualized by agarose gel electrophoresis (top panel), while the other half was subjected to Western blot analysis to confirm equal amounts of PKCalpha were immunoprecipitated from each sample (middle panel). The kinase assay was quantitated using Quantity One software (Bio-Rad) (bottom panel). Data represents the average fold increase of PKCalpha activity in non-stimulated samples compared to M-CSF-treated MDM +/- S.E.M for three independent experiments. NS: non-stimulated. * The p-values of M-CSF stimulated compared to non-stimulated were <=0.05.

activates

PKCalpha


Input:
M-CSF-induced PKC activity does not regulate IkappaBalpha degradation but regulates the phosphorylation of NF-kappaB p65 at Ser276.
(A) MDMs were pretreated with cycloheximide (CHX) in the absence or presence of Ro-31-8220 for 30 minutes prior to M-CSF stimulation for the indicated times. Whole cell lysates were subjected to Western blotting with anti-IkappaBalpha antibody. Data are representative of three independent experiments. (B) MDMs were pretreated with either vehicle or Ro-31-8220 for 30 minutes before the addition of M-CSF for 10 minutes. Whole cell lysates were resolved by SDS-PAGE and phospho-Ser276 or phospho-Ser536 of NF-kappaB p65 was detected using phospho-specific antibodies to either residue of NF-kappaB p65. (C) Whole cell lysates of RAW 264.7 cells treated with vehicle control or Ro-31-8220 in the absence or presence of M-CSF were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-kappaB p65 antibodies. (D) Cytosolic and nuclear fractions of RAW 264.7 were obtained from the treated cells and immunoblotted for phospho-NF-kappaB. The purity of the cytosolic and nuclear fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively. Shown are representative blots from three independent experiments.

phosphorylation

phospho

phospho

degradation

IkappaBalpha

regulate

degradation

regulates

phosphorylation


Input:
Inhibition of M-CSF-induced PKC reduces NF-kappaB-regulated genes in both MDMs and RAW 264.7 cells.
MDMs (A and C) and RAW 264.7 (B) cells were pretreated with Ro-31-8220 or solvent control DMSO for 30 minutes prior to M-CSF stimulation for the indicated times. Total RNA was isolated and converted to cDNA. Real-time RT PCR was performed using primers for IkappaBalpha, BCL-xl or GAPDH. Data are expressed as relative fold increase of IkappaBalpha or BCL-xl gene expression upon treatment over non-stimulated cells. Data represent the mean +/- S.E.M for three independent experiments.

expression

IkappaBalpha

expression

BCL-xl

increase

expression


Input:
PKCalpha regulates phosphorylation of NF-kappaB p65 at Ser276.
(A) MDM or (B) RAW 264.7 cells were transiently transfected with pNF-kappaB-SEAP along with either WT-PKCalpha or the kinase-deficient (KD)-PKCalpha construct at a 1:5 ratio. Cells were serum starved and stimulated with M-CSF and then SEAP secretion in the medium was measured. Data is from of three independent experiments. The p-value of cells transfected with KD compared to those transfected with WT was 0.05. (C) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by flow cytometry using Annexin V-FITC and propidium iodine (PI). (D) Whole cell lysates from the transfected RAW 264.7 cells were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-kappaB p65 antibodies. Blots were immunoblotted with PKCalpha to determine equal protein expression for the PKCalpha constructs. beta-actin served as a loading control. Shown is a representative blot from three independent experiments. (E) MDM or (F) RAW 264.7 cells were transiently transfected with a pNF-kappaB-SEAP along with either 100 nM PKCalpha siRNA or control siRNA for 20-24 hours. Cells were serum starved for 2-4 hours and stimulated with 100 ng/ml M-CSF for 6 hours for MDM or RAW 264.7 for 2 hours and then SEAP secretion in the medium was measured. Shown is data of three independent experiments. (G) MDMs were removed from the plate using accutase and apoptosis of MDMs was measured by Annexin V-FITC and propidium iodine (PI) staining and analyzed by flow cytometry. (H) Whole cell lysates from the transfected MDM and RAW 264.7 cells were subjected to Western blot analysis with PKCalpha antibody. beta-actin served as a loading control. Shown is a representative blot from at least three independent experiments.

transfected

PKCalpha

transfected

PKCalpha

expression

PKCalpha

transfected

PKCalpha siRNA

kinase-deficient

PKCalpha

phosphorylation

regulates

PKCalpha

phosphorylation


Input:
NF-kappaB p65 Ser276 is essential in regulating NF-kappaB activity.
(A) Raw 264.7 cell line was transiently transfected with pNF-kappaB-SEAP along with empty vector or plasmid encoding either NF-kappaB p65 WT or NF-kappaB p65 276S/A. The cells were transfected for 18-24 hours, serum starved for 4 hours, and then incubated with 10 microM of Ro-31-8220 for 30 minutes prior to treatment with 100 ng/ml of M-CSF for 2 hours and SEAP secretion in the medium was measured. (B) NF-kappaB p65-/- cell line was transiently transfected with pNF-kappaB-SEAP along with empty vector or plasmid encoding either NF-kappaB p65 WT, NF-kappaB p65 276S/A, or NF-kappaB p65 536S/A. The cells were cultured for 24 hours and then serum starved for 4 hours. Cells were then incubated in fresh DMEM medium for 2 hours and SEAP secretion in the medium was measured. The results shown are fold change over empty vector + pTAL-SEAP. (C) NF-kappaB p65-/- cell line was transiently transfected with pNF-kappaB-SEAP with either empty vector or plasmid encoding either NF-kappaB p65 WT or NF-kappaB p65 276S/A. The cells were transfected for 24 hours and serum starved for 4 hours, and then incubated with 10 microM of Ro-31-8220 for 30 minutes prior to treatment with 10 ng/ml of TNFalpha. The supernatant were collected after 2 hours of treatment and SEAP secretion in the medium was measured. The results shown are the fold change over empty vector + pNF-kappaB-SEAP. Data shown are mean +/- S.E.M for at least three independent experiments performed in duplicate.

transfected

transfected

transfected

transfected

transfected

transfected

transfected


Input: Proposed model for M-CSF-induced monocyte survival via PKC regulation by activating NF-kappaB p65 phosphorylation at Ser276.

phosphorylation

activating

phosphorylation


Input:
SHARPIN Is Essential for Cytokine Production, NF-kappaB Signaling, and Induction of Th1 Differentiation by Dendritic Cells
Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-kappaB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naive CD4+ T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.

secretion

secretion

GMCSF

mutations

Sharpin

deficient

SHARPIN

deficiency

SHARPIN

deficient

SHARPIN

reduced

deficient

transcription

reduced

deficient

secretion

loss

Sharpin

transcription

transcription

GMCSF