Protein Kinase C βII Plays an Essential Role in Dendritic Cell Differentiation and Autoregulates Its Own Expression^*

Dendritic cells are professional antigen-presenting cells of hematopoietic origin that play a central role in the initiation and regulation of the adaptive immune responses (1–6). Unlike most hematopoietic cells that arise by classical divergent hematopoiesis, human DC can arise from a variety of different immediate precursors. These include CD34⁺ and CD34⁺CD86⁺ hematopoietic progenitor cells (HPC)¹ (7, 8), myeloid progenitors (CFU-M/DC and CFU-DC) (9, 10), peripheral blood monocytes (11–13), and immature neutrophils (14) in what could be considered “convergent” hematopoiesis. In addition to this range of normal progenitors, myeloid leukemia blasts arrested in all stages of differentiation can be driven to undergo DC differentiation (15). On top of this complexity of progenitors, the process of differentiation itself can give rise to DC that are quite different in both their phenotype and function (10), and manipulation of specific signal transduction pathways during differentiation can yield DC with markedly altered function (5, 16). Conversely, exogenous stimuli can inhibit progenitor to DC differentiation in both normal (i.e. wound healing) and pathologic (tumors) settings, resulting in suppression of adaptive immune responses (17).

The intracellular signaling pathways that regulate progenitor to DC differentiation are only beginning to be defined. It has been well established that specific membrane receptors (GM-CSF, IL-4, TNF-α, FLT3L, CD40, etc.) can be activated in vitro by their exogenous ligands to initiate DC differentiation (7, 11, 18). Downstream of these membrane proximal events, signal transduction involving STAT3 (19, 20), intracellular calcium flux (21–24), specific NFκB family members (25), and Notch family members (26) has been implicated. However, how the various pathways are arrayed in relationship to each other has not been well characterized. We have shown previously that protein kinase C (PKC) activation is necessary and sufficient to drive human CD34⁺ HPC→DC differentiation (8). It is a likely downstream component of cytokine-mediated differentiation, as PKC inhibitors preferentially block DC differentiation in human CD34⁺ HPC driven by GM-CSF + TNF-α (27). PKC has been shown to be a downstream component of signaling through the GM-CSF receptor (28) and CD40 (29–31) in other cell types, and these receptors are also centrally involved in DC differentiation. Consistent with a role for PKC in DC lineage commitment, we and others have found that direct activation of PKC signaling by the 2,3-diacylglycerol (DAG) analogs (including phorbol 12-myristate 13-acetate (PMA) and bryostatin-1) in normal CD34⁺ HPC (8, 32, 33) and myeloid leukemic blasts (27, 34, 35) specifically generates DC in the absence of cell proliferation. Of particular interest is the CD34⁺CD86⁺ myeloid leukemia cell line KG1, which we and others have found can be induced to undergo differentiation to fully functional dendritic cells that express DC-specific receptor/chemokines (DC-CK1, DC-STAMP) and have the unique DC ability to cross-present antigens to activate T cells (27, 36). More importantly, a spontaneously arising differentiation-resistant daughter subline KG1a has been derived from KG1, and the two cell lines differ in both PKC activity and isoform expression (37).

PKC comprise a family of 12 serine/threonine protein kinases clustered into three isoform families as follows: classical cPKC (α, βI, βII (alternatively spliced variants of the PKC-β gene), and γ) that are activated by DAG and Ca²⁺, novel PKCs (δ, ϵ, θ, and η) that are activated by DAG without Ca²⁺, and atypical (ζ and ι) that do not require DAG and Ca²⁺ (38). It is clear from a wide range of cell types that the different PKCs are not functionally redundant, as specific isoforms modulate specific biological processes. Adapter proteins that bind specific activated PKC isoforms and substrates, such as RACK-1, may further refine the biological effect of PKC activation (39). Relevant to DC differentiation is the finding that PKC-β associates with RACK-1 and the common β chain of the GM-CSF/IL-3/IL-5 receptor, potentially linking cytokine receptor signaling to PKC isoform activation (28). Although a role has been demonstrated for both specific PKC isoforms (40–42) and PKC signal strength (43) in regulating hematopoiesis, involvement of specific PKC isoforms in DC differentiation has not been described previously. We have now extended our previous studies to examine the role and regulation of specific PKC isoforms in DC differentiation.

MATERIALS AND METHODS

Cells and Culture—K562, KG1, and KG1a were obtained from American Type Culture Collection (Manassas, VA) and cultured as described previously (27, 34). CD34⁺ bone marrow hematopoietic progenitor cells were isolated from organ donor bone marrow under protocols approved by the University of Miami Institutional Review Board, and purified by positive selection as described previously (8). Monocytes were enriched from the mononuclear cells of normal donors by plastic adherence (13). Progenitor cells were differentiated to DC with PMA (10 ng/ml, Sigma) + TNF-α (10 ng/ml, R & D Systems, Minneapolis, MN) for 5–7 days (27, 34) or with GM-CSF (1000 units/ml) and IL-4 (1000 units/ml, both from R & D Systems) for 12 days, and TNF-α (10 ng/ml) for the last 4 days (45). For all differentiation cultures, 50% media changes (with fresh differentiating agents) were performed every 3rd day. Where indicated, cells were pretreated with the PKC inhibitor bisindolylmaleimide I (5 μm). Cell viability was assessed by trypan blue dye exclusion.

KG1a was transfected by electroporation with pcDNA 3.1 (neo vector control), pEGFP-C1 (GFP control), PKC-α-GFP, PKC-βII-GFP, or PKC-γ-GFP (Living Color Vectors; all vectors were from Clontech) and selected for G418 resistance. For PKC-βII-GFP, single cell clones with high or low expression were generated.

Flow Cytometry—Adherent and nonadherent cells were harvested using 3 mm EDTA and stained as reported previously (27) using the following monoclonal antibodies: HLA-ABC and HLA-DR (both from VMRD Inc., Pullman, WA), CD11c, CD14, CD34, CD40, CD80, CD83, and CD86 (all from Immunotech, Westbrook, ME). Appropriate isotype-matched antibodies were used as controls. 10,000 live cells were analyzed on a Coulter XL flow cytometer (Coulter, Hialeah, FL) using the software supplied by the manufacturer.

For MHC class I and II intracellular staining, cells were cultured in media alone or with γ-interferon (250 units/ml,R&D Systems) for 72 h. Cells were then permeabilized using Cytofix/Cytoperm solution (BD Biosciences), as per the manufacturer's instructions, and stained for MHC class I and class II expression as above for flow cytometry.

T Cell Proliferation—Allogeneic T cell proliferation was performed as described previously (27). Progenitors were differentiated as indicated and γ-irradiated (3,000 rads). Normal donor resting allogeneic T cells were purified (>90% CD3⁺) as described previously (46) and co-cultured at various ratios with differentiated DC for 72 h. 1 μCi/well [methyl-³H]thymidine was added for the final 18 h of culture, and incorporation was measured using the Beta Plate scintillation counting system (Wallac Inc., Gaithersburg, MD). Where indicated, T cells alone stimulated with PMA (10 ng/ml) and ionomycin (100 ng/ml) were used as positive controls. The data are presented as the mean of triplicate wells ± S.D.

Confocal Fluorescent Microscopy—CD34⁺ HPC, monocytes, K562, or KG1 were treated as indicated, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in 1% bovine serum albumin, and stained with antibodies against the specific PKC isoforms indicated (anti-PKC-α (clone c-20), PKC-βI (c-16), PKC-βII (c-18), and PKC-γ (c-19), Santa Cruz Biotechnology, Santa Cruz, CA). Cells were then stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) together with Texas Red-X phalloidin and TO Pro-3. Cells were imaged by confocal microscopy (Zeiss LSM-510). Images of typical mid-cell sections are shown.

Western Blot—Western blots were performed as described previously (34). Briefly, protein levels quantitated in cell lysates by using the Micro BCA reagent kit (Pierce). Equal amounts of protein were separated by SDS-PAGE (4% stacking, 10% resolving), electroblotted to nitrocellulose, and probed with antibodies against Rel B (clone c-19), PKC-α (H-7), PKC-βI (E-3), PKC-βII (F-7), and actin (c-2) (all from Santa Cruz Biotechnology) or PKC-γ (Zymed Laboratories Inc.). The proteins were visualized by chemiluminescent detection (ECL, Amersham Biosciences).

Electromobility Shift Assay (EMSA)—EMSAs were done for NFκB family members as described previously (27). Briefly, equal amounts of protein from nuclear lysates were incubated with ³²P-labeled primer containing consensus NFκB-binding sites (GAT CCA ACG GCA GGG GAA TTC CCC TCT CCT TA) and separated on 4% polyacrylamide gels. For supershift assays, samples were first incubated with anti-Rel B antibody (Santa Cruz Biotechnology). Samples were visualized by autoradiography.

Northern Blot—Northern blot analysis was performed as described previously (47). Briefly, total RNA was isolated (RNeasy, Qiagen, Valencia, CA) and equalized by serial dilution and ethidium bromide visualization, separated on 1% formaldehyde/agarose gel, transferred to nylon membranes, and hybridized against PKC-βII-specific or glyceraldehyde-3-phosphate dehydrogenase probes.

PKC Kinase Assay—PKC kinase activity levels were determined using the SignaTECT Assay System (Promega), according to manufacturer instructions. Briefly, 7 × 10⁶ cells each of KG1, KG1a, KG1a 3.1 (vector alone), and KG1a E9 (PKC-βII transfected) were homogenized in 25 mm Tris buffer (pH 7.4) containing 0.5 mm EDTA, 0.5 mm EGTA, 10 mm β-mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 50 μg/ml phenylmethylsulfonyl fluoride and purified through a DEAE column in a 5-ml final elute volume. A 5-μl aliquot of each sample was incubated in a buffer containing calcium, peptide target, and [α-³²P]ATP with and without phospholipids for 5 min at 30 °C, when the reaction was stopped with 7.5 m guanidine-HCl buffer. Each reaction was spotted onto binding paper and quantified by scintillation counting. PKC activities in picomoles of ATP/min were determined as indicated in the kit protocol, normalized to their respective protein content, and expressed relative to the activity of the undiluted (1:1) KG1a cell lysates.

PKC-β Promoter Analysis—The BAC clone RP11-548B6 (Children's Hospital Oakland-BACPAC Resources, Oakland, CA) was digested with SacI and XbaI, and the resulting 1.7-kb DNA fragment containing the PKC-β promoter was cloned into the same corresponding sites in the pBlueScript SK+ vector. The new clone was incubated with NotI and blunted before further digestion with SacI to yield a 1.2-kb fragment (spanning the region from +65 to -1092 relative to the start of transcription) that was cloned into the SacI and SmaI sites of the pGL3-basic vector (Promega, Madison, WI) to yield the reporter construct in forward orientation (pPKC-Forward). The promoter in reverse orientation (pPKC-Reverse) was generated after ligation of the 1.2-kb fragment with the pGL3-basic vector digested previously with KpnI, blunted, and cut again with SacI. The identity and orientation of each clone were confirmed by restriction enzyme digest and DNA sequencing. KG1a-PKC-βII-GFP (E9) cells and KG1a-neo cells were incubated for 24 h at a concentration of 0.25 × 10⁶ and 0.5 × 10⁶ cells/ml, respectively, before electroporation with the Nucleofector system (Amaxa Biosystems, Cologne, Germany) using the T-01 program. Briefly, 10 × 10⁶ were resuspended in 100 μl of reagent V and mixed with 10 μg of the reporter constructs plus 100 ng of the pRL-CMV plasmid (Promega) encoding Renilla luciferase to normalize for transfection efficiency. Cells were transfected also with the pDsRed2-C1 plasmid (BD Biosciences) plus pRL-CMV to normalize the Renilla luciferase expression levels between both cell lines. Twenty four hours after transfection, cells were harvested, lysed, and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

RESULTS

Blockade of PKC Activation Inhibits Cytokine-mediated Monocyte→DC Differentiation—We have shown previously that that the PKC-specific inhibitor bisindolylmaleimide I (bis) could inhibit cytokine-driven DC differentiation in primary CD34⁺ HPC (27). To assess whether this was unique to CD34⁺ HPC or represented a common role for PKC in downstream cytokine signal transduction, we first examined whether bisindolylmaleimide I could inhibit cytokine-mediated generation of dendritic cells from primary human monocytes in vitro. We evaluated the effect on three DC characteristics as follows: morphology, immunophenotype, and the ability to activate allogeneic T cells. Morphologically, undifferentiated monocytes were loosely adherent round cells (Fig. 1A, left panel), whereas monocytes cultured with GM-CSF + IL-4 + TNF-α differentiated into clusters of DC (48) (center panel). Inhibition with bis resulted in the loss of these clusters (Fig. 1A, right panel), with a predominance of adherent spindle-shaped cells. Bis also blocked cytokine-mediated up-regulation of characteristic DC markers (MHC I and II, CD11c, CD40, CD80, CD86, and CD83) while preventing the complete down-regulation of the monocyte marker CD14 (Fig. 1B). The immunophenotype of these bis-treated monocytes appeared to be that of activated monocytes. Functionally, inhibition of PKC activation generated cells that were significantly less able to induce allogeneic T cell proliferation at all T cell:APC ratios tested (especially at the higher ratios) but were more immunostimulatory than undifferentiated monocytes (Fig. 1C). The latter observation would also be consistent with the bis-treated cells being activated monocytes. Together, these data suggest that PKC activation is also a downstream component of cytokine receptor-mediated signaling of DC differentiation from monocytes.

PKC-βII Is Specifically Activated by DC Differentiation Signals—To determine which PKC isoform(s) were being activated during the initiation of DC differentiation, we took advantage of the fact that PKC activation results in translocation of the enzyme from the cytoplasm to the plasma membrane (49) and can be directly visualized by confocal microscopy. We focused on the DAG/Ca²⁺-dependent cPKC family (α, βI, βII, and γ), as we have found evidence for involvement of calcium in PMA-mediated DC differentiation (27, 34). In three different progenitors that we have shown previously differentiated to DC in response to PMA (primary CD34⁺ HPC, KG1, and K562), all four PKC isoforms are cytoplasmic when cells were unstimulated (media, Fig. 2A). Within 5–30 min of PMA stimulation, only the PKC-βII isoform translocated to the plasma membrane in all three cell types. Translocation of PKC-βII was also seen when primary monocytes were differentiated with GM-CSF + IL-4 for 24 h (Fig. 2B). This translocation took somewhat longer to detect compared with PMA treatment but is consistent with the slower kinetics of DC differentiation we see with cytokines versus PKC agonists. These findings indicate there is immediate/early activation of PKC during DC differentiation and that PKC-βII may specifically mediate this lineage commitment.

Restoration of DC Differentiation in KG1a Transfected with PKC Isoforms—We and others have shown that the human CD34⁺CD86⁺CD14^- myeloid leukemia cell line KG1, which has the same phenotype as primary CD34⁺CD86⁺ human DC progenitors (50), similarly undergoes differentiation in response to cytokines or PMA to dendritic cells that have DC-specific molecular expression (e.g. DC-CK1 and DC-STAMP) and function (e.g. cross-presentation of antigen) (27, 35, 36, 51–55). KG1a is a less differentiated cell line spontaneously derived from KG1 and does not differentiate in response to phorbol esters (27, 56, 57). KG1a has less overall PKC activity and lower expression of βI and βII compared with KG1 (27, 37). Given our demonstration that PKC-βII activation occurs during KG1 differentiation, we next asked if transfection of specific PKC isoforms into KG1a could restore the ability of these cells to differentiate to DC. As seen in Fig. 3A, KG1a was stably transfected with specific functional PKC isoform-GFP fusion constructs (58). Analysis of bulk cultures of the KG1a-PKC-βII transfectants revealed both high and low expressing clones. Single cell cloning resulted in isolation of each type of cell. Confocal imaging for GFP demonstrated cytoplasmic localization for PKC-α-GFP, PKC-βII-GFP, and PKC-γ-GFP in the unstimulated KG1a transfectants (Fig. 3B). PKC-α-GFP also appears as punctate cytosolic accumulations in unstimulated cells, which have been reported previously in NIH-3T3 cells (59). The cytosolic localization in unstimulated cells demonstrated that the majority of the transfected enzyme was not constitutively activated. Following PMA treatment, only PKC-βII-GFP underwent significant translocation to the plasma membrane. In addition to the plasma membrane, PKC-βII-GFP also translocated elongated subcellular structures that appeared to be contiguous with the plasma membrane on stacked views (Fig. 3B, arrow). These results are consistent with our findings with the endogenous PKC isoforms and indicate that the different PKC-GFP isoforms transfected into KG1a behave in a similar fashion to the endogenous PKC isoforms in KG1 (as well as in primary CD34⁺ HPC and monocytes).

We next assessed the ability of PKC-transfected KG1a to undergo DC lineage commitment in response to PMA. We have shown previously in KG1 and CD34⁺ HPC that this is characterized by development of typical DC morphology with extended branching dendrites, decreased proliferation and increased cell death, acquisition of DC surface markers (MHC I, II, CD40, CD80, CD86, and CD83), up-regulation of the NFκB family member Rel B, and the ability to induce allogeneic T cell proliferation.

Morphology—Recent studies have demonstrated the importance of the extended dendrite morphology in the ability DC to activated T cells (60). We have found previously that in contrast to the clusters of cells with short dendrites induced by cytokine combinations, PMA-induced DC differentiation from primary CD34⁺ HPC or KG1 results in more tightly adherent individual cells with longer processes (either spindle shaped or branching) (27) as seen in Fig. 4A for KG1 (8). Both unstimulated (media) vector control KG1a and PKC-transfected cells were morphologically round and loosely adherent (Fig. 4A). Treatment with PMA had little effect on vector control KG1a morphology. However, similar to the parental KG1 line, PMA stimulation induced development of tightly adherent cells with extended cytoplasmic processes in the PKC-βII-GFP transfected cells. Although the KG1a PKC-α-GFP and PKC-γ-GFP cells also became adherent with some spindle-shaped cells, many of the cells were adherent round cells with very large vacuoles that is not a typical DC morphology.

Differentiation-induced Cell Proliferation Arrest and Death—We have found previously that PMA-induced DC differentiation is associated with arrest of cell proliferation and induction of cell death (20–50%) in all the progenitors we have examined. Compared with KG1, the proliferation of KG1a was largely unaffected by PMA treatment (Fig. 4B). Although the KG1a-PKC transfectants have a higher basal rate of proliferation than either KG1 or KG1a, PKC-GFP transfection restored the ability of PMA to inhibit cell proliferation down to levels equivalent to PMA-treated KG1. Similarly, PMA induced progressively more cell death over time in KG1 but had no effect on KG1a (Fig. 4C). However, transfection of either PKC-α-GFP, PKC-βII-GFP, or PKC-γ-GFP restored the ability of PMA to induce cell death in the transfected KG1a to levels equal to or greater than KG1.

Surface Antigen Immunophenotype—KG1 expressed high levels of MHC I and II and low levels of CD86 in media alone and up-regulated CD80 and CD83 upon PMA stimulation (27). In comparison, unstimulated vector control KG1a in media expressed only MHC I, and PMA treatment down-regulated this expression without inducing other DC markers (Fig. 5A). Most unexpectedly, all three PKC isoform-transfected KG1a had down-regulated MHC I and up-regulated CD40 and CD86 in media alone. Conversely, unlike KG1 none of the unstimulated transfectants expressed surface MHC II, although it could be detected intracellularly (below). Constitutive expression was also seen for CD80 except for the low expressing βII clone E11 and CD83 (except for PKC-γ-GFP). These findings suggest that some PKC signaling is occurring in the transfected KG1a in the absence of exogenously added PMA. Stimulation with PMA results in variable up-regulation of MHC I in all the transfectants, MHC II (PKC-γ-GFP, PKC-βII-GFP (slight)), CD86 (PKC-α-GFP, PKC-γ-GFP), and CD83 (PKC-α-GFP). Conversely, PMA treatment down-regulated CD80, CD86, and CD83 in transfectants that had high level constitutive expression of these markers. We have found previously that PMA treatment can down-regulate constitutive expression of MHC I, MHC II, CD80, and CD86 in DC progenitors (27, 34). This may represent a complex relationship between PKC signal strength and expression of these markers, which is also suggested by the immunophenotypic differences between the PKC-βII high (E9) and low (E11) expressing clones.

To determine why there was little MHC I and II surface expression in the KG1a-PKC transfectants, we examined whether they were present intracellularly and could be induced by γ-interferon stimulation. As seen in Fig. 5B (upper panels), KG1, KG1a-neo, and KG1a-PKC-βII (E9) cells all had low constitutive expression of intracellular MHC I and II. γ-IFN treatment induced expression of both in KG1, whereas there was less (MHC I) to no effect (MHC II) in KG1a-neo cells. In contrast to the control neo cells, KG1a-PKC-βII (E9) regained the ability to up-regulate both intracellular MHC I and II in response to γ-IFN. Although this up-regulation resulted in increased MHC I surface expression, there was virtually no increase in MHC II surface expression (Fig. 5B, lower panels). We have also found that that the addition of TNF-α (as a maturation factor) to γ-IFN treatment does not alter MHC I or II expression compared with γ-IFN alone, nor does the addition of γ-IFN during PMA-induced differentiation alter MHC I or II expression compared with γ-IFN alone (not shown). These findings suggest that although γ-IFN-inducible MHC II gene expression is reconstituted by PKC-βII transfection, MHC II transport to the cell surface remains impaired. We have also found that compared with KG1, there is low expression of the chaperone protein Erp57 (which is involved in peptide loading onto MHC I complexes) in KG1a that is not increased in KG1a-PKC-βII cells (data not shown), which may explain the low basal levels of MHC I surface expression in these cells.

Rel B Expression—Immediate/early up-regulation of expression of the NFκB family member Rel B is an essential event for both DC differentiation (61) and function (62). It is the inducible nature of Rel B that is important, as it has been shown that constitutive expression of Rel family members will actually inhibit DC differentiation (63). Although KG1 have low levels of Rel B that was rapidly induced following PKC activation, KG1a had higher levels of constitutive expression that was not inducible (Fig. 6A). Transfection of PKC-α, -βII, or -γ down-regulated Rel B expression in unstimulated cells, which was then inducible by PMA stimulation. The restoration of a KG1-like pattern of inducible Rel B protein expression was also reflected in the regulation of nuclear Rel B capable of binding NFκB DNA motifs as detected by EMSA (Fig. 6B). Most surprisingly, the total NFκB DNA binding activity was down-regulated in the unstimulated KG1a-PKC-βII and was inducible by PMA, suggesting a common mechanism of NFκB regulation that is not specific for Rel B.

T Cell Proliferation—Functionally, restoration of the ability of KG1a to undergo DC differentiation by transfected PKC isoforms should be reflected in the re-acquisition of T cell stimulatory capacity. As seen in Fig. 7A (left panel), undifferentiated KG1, KG1a, or KG1a transfectants did not induce allogeneic T cell proliferation. The fact that the undifferentiated PKC-βII E9 clone expresses MHC I, MHC II (slight), CD40, CD80, and CD86 and yet still did not elicit T cell proliferation suggests factors other than antigen presentation receptors and co-stimulatory ligands are necessary to elicit T cell activation. KG1 differentiated with PMA, and more so with addition of the maturation signal TNF-α, can stimulate T cell proliferation. By comparison, PMA + TNF-α-treated KG1a, KG1a vector control, and KG1a-GFP do not acquire allostimulatory capacity. These controls also demonstrate that PMA carryover from the differentiation cultures does not account for the T cell proliferation seen in the co-cultures. In contrast, PMA-induced differentiation of all three PKC-transfected KG1a resulted in the ability to drive significant T cell proliferation. This was significantly greater than undifferentiated monocytes (Fig. 7A, right panel, using the same donor T cells), and was equivalent to monocyte-derived DC.

We next assessed whether the degree of PKC-βII expression affects the ability to induce allo-T cell proliferation. As seen in Fig. 7B, the high expressing βII E9 clone consistently induced greater T cell activation than the low expressing E11 clone, implying that the strength of the PKC signal may affect the immunogenicity of the resulting DC. Altogether, these data demonstrate that transfection with PKC-α-GFP, PKC-βII-GFP, or PKC-γ-GFP can reconstitute the ability of KG1a to undergo DC lineage commitment as seen for KG1.

PKC Induction of Endogenous PKC Isoform Expression— Because we have consistently observed activation of PKC-βII and not other cPKC isoforms during DC differentiation, we were surprised by the ability of the α and γ isoforms to also restore aspects of DC differentiation in the KG1a cells. We sought to understand the underlying mechanism by first comparing the relative levels of endogenous PKC versus PKC-GFP expression in the transfected KG1a. Because of the GFP tag, we can distinguish transfected PKC from endogenous PKC by molecular weight on Western blots. Most unexpectedly, we found relatively low levels of transfected PKC-GFP protein expression (Fig. 8A). Except for the high expressing PKC-βII-GFP clone E9, the transfected PKC-GFP proteins were only detectable upon long exposures. Remarkable, however, was the up-regulation of all the endogenous cPKCs in each of the KG1a transfectants. This is most marked for PKC-βII and demonstrates that although un-transfected KG1a did not express much PKC-βII, the ability to up-regulate this expression was still intact. These findings suggested that the transfected PKC-GFPs were not up-regulating PKC activity by their own overexpression but rather by inducing endogenous PKC expression. This would also explain why PKC-α or -γ transfection can restore the ability of KG1a to differentiate to DC, namely via up-regulation of endogenous PKC-βII.

How do the transfected PKC-GFPs induce endogenous PKC-βII expression? Because PKC-βI and -βII are alternatively spliced variants from the PKC-β gene that can be generated in a regulated fashion (64), we first examined if increased PKC-βII expression was because of a switch of splicing from βI to βII. KG1a expressed very little PKC-βI to begin with (Fig. 8B), making it unlikely that changes in splicing account for the increased βII expression. The fact that both PKC-βI and -βII protein were induced in the PKC-transfected KG1a suggests that expression of the PKC-β gene is up-regulated. Consistent with this, PKC-β mRNA expression was significantly up-regulated in the KG1a transfectants (Fig. 8C). Because PKC enzymes are degraded following activation (65), we determine whether the induced endogenous PKC-βII could be activated by following protein levels during PMA treatment. As seen in Fig. 8D, PKC-βII was degraded during PMA stimulation in KG1, KG1a, and all the PKC-transfected KG1a. This indicates that the induced endogenous PKC-βII can be activated and is likely to be signaling. Together, our data indicate that the transfected PKC-GFP are up-regulating endogenous PKC isoform gene expression (in particular PKC-βII), and this confers on KG1a the ability to undergo DC lineage commitment. These findings also suggest that regulation of PKC-βII expression may in turn regulate the ability of progenitors to undergo DC differentiation.

Autoregulation of PKC Promoter Activity—The ability of the transfected PKC-GFP to up-regulate endogenous PKC-βII mRNA expression suggested that this is happening at the transcriptional or post-transcriptional (e.g. mRNA stabilization) level. Previous studies have demonstrated a phorbol ester-inducible site in the PKC-β promoter (66, 67), suggesting that PKC activity may positively autoregulate its own gene expression. The relationship of the enzyme activation constant, K_act = (active enzyme)/(inactive enzyme), predicts that under steady state conditions, an increase in the total concentration of PKC enzyme (by transfection or induction of endogenous expression) will result in an increase in the number of activated PKC molecules. We first measured total PKC kinase activity under steady state conditions. As seen in Fig. 9A, unstimulated KG1 had more kinase activity than KG1a or KG1a-neo, whereas KG1a-βII (E9) had significantly more than both. To determine whether this increased PKC kinase activity resulted in increased PKC-β promoter activity, we cloned a 1.2-kb fragment of the human PKC promoter containing the previously reported PMA-inducible site (-11 to +43 (66)) into a luciferase reporter construct (pPKC-Forward). There was significantly higher luciferase activity when pPKC-Forward (but not the reverse orientation and empty vector negative controls) was transfected into E9 compared with the KG1a-neo control transfectants, demonstrating a 5-fold increase in promoter activity under unstimulated conditions (Fig. 9B). Finally, if an increase in constitutive PKC activity is inducing PKC-β expression, inhibition of this kinase activity would result in decreased expression. Consistent with this, unstimulated PKC-βII (E9) transfectants cultured with the PKC inhibitor bis I undergo a loss of both PKC-βII protein and mRNA expression (Fig. 9C). Together, these data indicate that constitutive PKC kinase activity can positively autoregulate PKC-β gene expression by PKC-responsive elements in the promoter.

DISCUSSION

Our findings support a model where quantitative and qualitative PKC-βII activation is a key component of the signal transduction pathways that induce DC differentiation and that regulation of this activation is in part through regulation of PKC-β gene expression. Specifically, these data indicate the following. 1) PKC is a critical downstream signaling component of cytokine-induced monocyte→DC differentiation, consistent with our previous findings in CD34⁺ HPC→DC differentiation (27). 2) Of the PKC isoforms, PKC-βII is the most centrally involved in DC differentiation. 3) Different aspects of DC differentiation are induced by differing degrees of PKC signal strength. 4) PKC signal strength is regulated (at least in part) by PKC-β gene expression, which involves a positive autoregulatory feedback loop.

The ability of the PKC inhibitor bisindolylmaleimide I to inhibit cytokine-driven monocyte (or CD34⁺ HPC) progenitor to DC differentiation indicates that PKC is a downstream component of these signaling pathways. It is not yet clear which cytokine receptor is triggering PKC activation, as these receptors typically signal via Janus tyrosine kinase/signal transducers and activators of transcription and TRAFs, whereas PKC has been best characterized to be activated by G-coupled proteins and phospholipase C. However, the PKC adapter protein RACK-1 associates constitutively with the common β chain of the GM-CSF/IL-3/IL-5 receptor, and PKC-βII rapidly associates with this complex upon receptor activation (28).

Involvement of PKC-βII in GM-CSF receptor signaling is consistent with our observation that this isoform is activated in CD34⁺ HPC by GM-CSF + TNF-α stimulation, and appears to be the cPKC isoform that is predominantly activated by PMA in three different DC progenitors. PKC-βII has also been implicated previously in myeloid differentiation (40), and our findings demonstrate a correlation between PKC-βII expression and the ability to undergo DC differentiation in KG1 and KG1a. The rapid activation of PKC-βII indicates that it is involved in the primary signaling rather than a later secondary response. Together, these data suggest that DC differentiation is specifically signaled through PKC-βII. However, we could not rule out the involvement of low level activation of the other cPKCs, activation of novel nPKC, and/or atypical PKC isoforms, and the formal possibility that PMA is acting through a non-kinase phorbol ester receptor (68) and that PKC-βII activation is an epiphenomenon.

To address these possibilities, we asked the seemingly straightforward question whether reconstitution of specific PKC isoform expression would reconstitute the ability of KG1a to undergo DC differentiation, providing strong evidence for a central role for that isoenzyme. The inability of KG1a to undergo differentiation (57) has been attributed to lower expression of PKC isoforms/activity (37). Confocal studies of the KG1a-PKC-GFP transfectants demonstrated that only PKC-βII-GFP was clearly activated by PMA, consistent with what we saw for the endogenous cPKC. Most surprisingly, however, each of the PKC-GFP isoforms could reconstitute the ability of KG1a to undergo DC differentiation as assessed by inhibition of cell proliferation and induction of cell death, costimulatory ligand expression, re-establishment of inducible Rel B expression, and acquisition of allostimulatory capability. Although PKC-GFP transfection did not restore the high level MHC II surface expression seen on KG1, it did restore γ-interferon inducibility as seen by intracellular staining. These findings suggest that although some aspects of MHC II expression are regulated by PKC signaling, transport/processing for surface expression is not. The transfected KG1a also down-regulated constitutive MHC I expression, which is likely due to the increased constitutive PKC activity in these cells (and is consistent with the observation that PMA-induced PKC activation down-regulates MHC I (e.g. Fig. 4C)).

The unexpected the expression of CD40, CD80, CD86, and CD83 on the unstimulated KG1a-PKC-GFP transfectants is also likely due to increased constitutive PKC activity that is seen in the kinase assays. Most interestingly, this is the same “semi-activated” phenotype of the pathogenic Langerhans cells in Langerhans cell histiocytosis (69). It is unclear whether the variation in surface marker expression between the PKC-α, -βII, and -γ transfectants is due to qualitative and/or quantitative differences between the clones. However, the difference in MHC I, MHC II, CD40, and CD80 expression between the low (E11) and high (E9) expresser of PKC-βII suggests that the degree of PKC signal strength plays a defining role in determining which DC characteristics are manifested. This is supported by the observation that even though unstimulated KG1a-PKC-GFP expresses MHC and costimulatory ligands, they are still unable to activated T cells unless PKC is fully activated by an exogenous PKC agonist. Why there is no direct correlation between MHC/costimulatory ligand expression and the ability to activate T cells is unclear, although we and others have seen the same phenomenon in other myeloid DC progenitors (15, 70). Recently, Benvenuti et al. (60) have reported that even though DC from Rac 1/2^-/- mice have the same degree of MHC/costimulatory ligand expression as WT DC, they are far less capable of priming naive T cells. This appears to be in part due to the inability of these knockout DC to form the DC membrane extensions that physically interact with the T cell. Similarly, we believe that factors responsive to PKC signaling are involved in these initial physical/adhesive interactions and may be playing an important role in the immunogenicity of the PKC transfectants.

Varying degrees of PKC signaling is also supported by the confocal microscopy studies, where significant translocation of activated PKC-βII-GFP in the KG1a transfectants is only seen after PMA treatment. Further evidence for a differential effect of PKC signal strength is seen in the regulation of Rel B expression. These data and additional transcriptional run-off studies² suggest that the absence of PKC-βII activity (KG1a) results in constitutive/non-inducible Rel B expression; intermediate activity (unstimulated KG1 and KG1a-PKC-GFP) suppresses expression, and high PKC activity (PMA-stimulated KG1, KG1a-PKC-GFP) induces Rel B expression. Most interestingly, our EMSA studies suggest that the other NFκB families are similarly regulated. These data support a model where a continuous gradient of PKC activation elicits different aspects of DC differentiation and is similar to a model of differential lineage commitment based on the degree of PKC activation in other myeloid lineages (43). Although PKC signal strength has not been examined previously in DC differentiation, it has been shown that different degrees of CD40 signaling (which activates downstream PKC signaling in other cells (29, 30)) elicits different levels of DC migratory ability and cytokine secretion (71).

The tight association between PKC expression level and signaling activity is predicted by the steady state K_act relationship such that the increase in total PKC concentration in the KG1a transfectants results in an increase in the number of active PKC molecules. The ability of PKC expression levels to affect significantly PKC signaling is underscored by observations in a wide range of other cell types; the levels of mature enzyme are tightly regulated during development, and even modest changes in these levels can result in significant biological effects (66). It has also been suggested that other signaling pathways may work by regulating PKC expression, such as the ability of vitamin D₃ to drive HL-60 differentiation via up-regulation of PKC-β expression (72).

Although PKC protein expression and function has been shown to be tightly regulated by many post-transcription and post-translational mechanisms (65), regulation of PKC gene expression is largely uncharacterized. We have found that all three of the transfected cPKCs increase PKC expression, not through overexpression of the transfected cDNA (which is low for all the clones except βII-E9) but rather through significant induction of endogenous PKC-βII gene expression. Our promoter and PKC inhibitor studies suggest that the underlying mechanism involves a positive autoregulatory loop where increased constitutive PKC signaling activates a phorbol ester-inducible element in the PKC-β promoter, which has been reported to up-regulate gene expression 8–20-fold (66, 67, 73). We also see evidence that PKC-α and PKC-γ expression is similarly auto-induced (although to a much lesser extent than PKC-βII), and phorbol ester-inducible elements have also been described in the promoters of these genes (67, 74). Although not reported for hematopoietic cells, Liu et al. (44) have recently reported the same mechanism of autoregulation of steady state PKC-β gene expression in colonic epithelial cells, involving up-regulation of the PKC-β promoter by activated PKC and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-dependent signaling. Thus PKC autoregulation of its own expression may be a general characteristic of the cPKC isoforms in a variety of cell types.

Because of this interrelationship, however, we cannot definitively say that PKC-βII is the only cPKC involved in DC differentiation, given that endogenous α and γ are also up-regulated in the KG1a transfectants. However, there is reasonably strong evidence that PKC-βII is playing the major role. It is the only isoform that is consistently activated during DC differentiation in a number of different progenitors. It is only cPKC that has significantly different expression in KG1 versus KG1a. Differences in PKC-βII levels (e.g. E9 versus E11, which do not differ substantially in PKC-α or -γ expression) correlate with differences in DC surface markers. It would not, however, be surprising if there was some degree of functional redundancy between the cPKC isoforms.

Finally, the ability of KG1a to stably express endogenous PKC-βII through auto-induction raises the question how they lost this expression in the first place. One possibility is that the PKC-β promoter is also negatively regulated, and inhibitory elements have been described between -3000 and -690 of the 5′-untranslated region (which may not be included in our promoter construct) (67). If exogenous stimuli can inhibit PKC-β expression through these elements, this may provide a mechanism by which progenitor to DC differentiation can be inhibited in inflammatory settings where an adaptive immune response is unwanted (e.g. sterile trauma and wound healing). Although such a PKC-specific checkpoint has not been described, it has been well established that a number of cytokines (in particular those secreted by tumors) can inhibit the differentiation of myeloid progenitors to DC through signaling to yet undefined downstream targets (17).