Activation of Protein Kinase Cη by Type I Interferons*

Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKCη does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFNα on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKCη as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis.

Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKC does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFN␣ on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKC as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis.
Type I interferons (IFNs) 2 exhibit important biological effects, including antiviral properties and regulation of normal and malignant cell growth (1)(2)(3)(4)(5). Inducible or constitutive production of IFNs appears to be a key component of cellular defense mechanisms against viral infections and the immunosurveillance against cancer (1)(2)(3)(4)(5). These cytokines exhibit important regulatory effects on cell cycle progression, gene transcription, and mRNA translation (6 -11). Beyond their relevance in the regulation of innate responses, the important biological effects of IFNs have led to extensive clinical-transla-tional work over the years that resulted in their introduction in clinical medicine as antiviral and antitumor agents, although they are also used in clinical neurology for the treatment of multiple sclerosis (4,12,13).
One of the most important biological activities of IFNs is their ability to act as regulators of normal hematopoietic progenitor cell growth and to control normal hematopoiesis. It has been known for the last 3 decades that type I IFNs are potent suppressors of hematopoietic progenitor cell growth in vitro (14 -18), and such effects may account for the development of pancytopenias that patients receiving IFN treatment frequently develop. IFNs inhibit the growth of all different classes of normal bone marrow-derived hematopoietic precursors, including progenitors of myeloid (CFU-GM), erythroid (CFU-E and BFU-E) and megakaryocytic (CFU-MK) lineages (14 -20). The effects of IFNs have been also shown to occur on cell populations that contain bone marrow cells at an early precursor stage (CD34ϩCD38Ϫ) (20), underscoring the ability of IFNs to regulate both early and late stages of hematopoietic development.
Over the years, the mechanisms of type I IFN signaling have been extensively studied and defined in a variety of cellular systems and backgrounds. Clearly, engagements of Jak kinases and Stat proteins are events of critical importance in the generation of the biological properties of IFNs (2)(3)(4)(5)(6)(7)(8). The activation of Jaks occurs at the receptor level, followed by direct activation of interacting Stats, providing a mechanism of rapid turnover of signals from the cell surface to the nucleus (2)(3)(4)(5)(6)(7)(8). Type I IFNs also activate p38 mitogen-activated protein kinase signaling pathways that complement the function of Jak-Stat pathways and are required for optimal transcriptional activation of IFN-regulated genes (21)(22)(23)(24). In addition, there is accumulating evidence that type I IFNs activate the Akt/mammalian target of rapamycin signaling pathway and its downstream effectors (25)(26)(27)(28) and that such activation is required for the initiation of mRNA translation of interferon-stimulated genes (28). Members of the PKC family (␦, ⑀, and ) have been shown previously to be activated and play roles in the generation of type I and/or type II IFN responses (29 -33). However, much remains to be defined regarding the overall contribution of the PKC family to the generation of IFN responses as well as the specific roles of distinct isoforms in IFN signaling.
In this study we provide the first evidence for engagement of PKC, a member of the novel subgroup of PKC isotypes in type I IFN signaling. Our data establish that this PKC isoform is phosphorylated/activated by IFN␣ or IFN␤ treatment of sensitive cells. We also show that engagement of PKC by the type I IFN receptor regulates IFN␣-dependent G 0 /G 1 cell cycle arrest and plays an essential role in the generation of the suppressive effects of type I IFNs on normal and leukemic myeloid (CFU-GM) progenitors. Altogether, our findings implicate PKC as a novel member of the PKC family with an important role in the generation of IFN responses and define a unique and specific role for this PKC isoform in IFN-mediated control of myelopoiesis.

MATERIALS AND METHODS
Cells and Reagents-The CMLderived lymphoblastoid crisis KT1 cell line, the multiple myeloma U266 cell line, and the acute myelomonocytic U937 cell line were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and antibiotics. The osteosarcoma U20S cell line was grown in McCoy's media supplemented with 10% fetal bovine serum and antibiotics. Primary human CD34ϩ progenitor cells were either purchased from Stem Cell Technologies (Vancouver, British Columbia, Canada) or obtained from the bone marrow of normal donors after obtaining informed consent approved by the Institutional Review Board of Northwestern University. Bone marrow mononuclear cells were isolated using Histopaque (Sigma), and CD34ϩ cells were further purified using indirect positive selection (Miltenyi, Bergisch Gladbach, Germany), as in our previous studies (19,34). Recombinant human IFN␣ was obtained from Hoffmann-La Roche. Recombinant IFN␤ was obtained from Biogen Idec. An antibody against the phosphorylated form of PKC on Ser-674 was purchased from Upstate Biotechnology, Inc. (Billerica, MA); an antibody against PKC was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and an antibody against GAPDH was obtained from Chemicon (Billerica, MA). PKC and PKC peptide inhibitors were purchased from Calbiochem. U937 cells were transfected by nucleofection following the manufacturer's protocol (Amaxa AG, Cologne, Germany). A constitutively active PKC mutant (36) was provided by Dr. Gottfried Baier (Innsbruck Medical University, Innsbruck, Austria) and was used in overexpression experiments.
Cell Lysis and Immunoblotting-Cells were serumstarved, stimulated with 1 ϫ 10 4 IU/ml of the indicated IFN for the indicated times, and subsequently lysed in phosphorylation buffer as described previously (19,37). Immunoprecipitation and immunoblotting using an enhanced chemilu-FIGURE 1. Type I IFN-dependent phosphorylation and activation of PKC. A, KT1 cells were serum-starved overnight and treated with IFN␣ for the indicated times. Cells were lysed, and lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-PKC or anti-GAPDH antibodies, as indicated. B, U266 cells were serum-starved overnight and treated with IFN␣ for the indicated times. Cells were lysed and lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-PKC or anti-GAPDH antibodies, as indicated. C, KT1 cells were serum-starved overnight and treated with IFN␤ for the indicated times. Cells were lysed, and lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-PKC or anti-GAPDH antibodies, as indicated. D, U266 cells were serum-starved overnight and treated with IFN␤ for the indicated times. Cells were lysed, and lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-PKC or anti-GAPDH antibodies, as indicated. FIGURE 2. Type I IFN-dependent activation of PKC. A, KT1 cells were serum-starved overnight, treated with IFN␣ for 20 min, and lysed in phosphorylation lysis buffer. Cell lysates were immunoprecipitated (IP) with an anti-PKC antibody or control nonimmune rabbit immunoglobulin (RIgG) as indicated and subjected to in vitro kinase assays, using histone H1 as an exogenous substrate. Immunoprecipitated proteins were resolved by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot from the kinase assay was subsequently immunoblotted with an anti-PKC antibody to control for loading. B, KT1 cells were serumstarved overnight, treated with IFN␤ for 20 min, and lysed in phosphorylation lysis buffer. Cell lysates were immunoprecipitated with an anti-PKC antibody or control nonimmune rabbit immunoglobulin as indicated and subjected to in vitro kinase assays, using histone H1 as an exogenous substrate. Immunoprecipitated proteins were resolved by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot from the kinase assay was subsequently immunoblotted with an anti-PKC antibody to control for loading. minescence (ECL) method were performed as in previous studies (19,37).
Evaluation of Erythroid Differentiation-Human primary erythroid progenitor cells were enriched by in vitro culture of CD34ϩ cells isolated from normal bone marrows or obtained commercially from Stem Cell Technologies. After CD34ϩ cell isolation, differentiating erythroid progenitors were obtained by culturing cells for 4 -14 days in medium with 15% fetal bovine serum, 15% human AB serum, 10 ng/ml interleukin-3, 2 units/ml erythropoietin, and 50 ng/ml stem cell factor (19,35). At the indicated time points, an aliquot of cells was removed from culture, washed with phosphate-buffered saline, and stained with glycophorin A and CD71 or the appropriate antibody controls (BD Biosciences) prior to flow cytometric analysis.
RNA Isolation and PCR-Real time RT-PCR was performed as in our previous studies (27,28). RNA was isolated using a standard methodology (Qiagen, Hilden, Germany) and used as substrate for reverse transcription reactions. Quantitative real time PCR (Applied Biosystems, Foster City, CA) was then used to measure the relative expression of indicated mRNA transcripts with normalization to GAPDH. PCR was performed under the following conditions: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles at 95°C for 15 s followed by 1 min at 60°C.
Hematopoietic Cell Progenitor Assays-Bone marrow from normal donors and bone marrow or peripheral blood from CML patients was collected after obtaining consent approved by the Institutional Review Board of Northwestern University. Mononuclear cells were isolated using Histopaque (Sigma) separation, and CD34ϩ cells were further purified using indirect positive selection (Miltenyi, Bergisch Gladbach, Germany). Primary human CD34ϩ progenitor cells were also purchased from Stem Cell Technologies (Vancouver, British Columbia, Canada). CD34ϩ cells were then transfected using transfection reagent purchased from Mirus (Madison, WI) with control siRNA or siRNAs targeting specific PKC isoforms. Two different siRNA targeting PKC (Ambion ID777 and ID778), as well as siRNA targeting PKC␣ siRNA, PKC␤ siRNA, PKC siRNA, and control siRNA were purchased from Ambion (Foster City, CA).
Growth of erythroid or myeloid progenitors was subsequently determined in clonogenic assays in methylcellulose, as in our previous studies (34,38,39). In some experiments progenitor FIGURE 3. Lack of regulatory effects of PKC on type I IFN-induced gene transcription. A, U2OS cells were transfected with a ␤-galactosidase expression vector and an ISRE-luciferase plasmid. Forty eight hours after transfection, triplicate cultures were preincubated for 1 h in the presence or absence of a PKC-specific peptide inhibitor. Subsequently, the cells were incubated for 6 h in the presence or absence of IFN␣, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of three experiments. B, U2OS cells were transfected and treated with PKC-specific peptide inhibitor as in A. Subsequently, cells were incubated for 6 h in the presence or absence of IFN␤, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of six experiments. C, U2OS cells were transfected with a ␤-galactosidase expression vector, an ISRE-luciferase plasmid, and either an empty vector plasmid or a constitutively active PKC-A/E construct. Forty eight hours after transfection, triplicate cultures were incubated for 6 h in the presence or absence of IFN␣, as indicated, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control empty vector-transfected untreated cells and represent means Ϯ S.E. of three experiments. D, U2OS cells were transfected with a ␤-galactosidase expression vector and an ISRE-luciferase plasmid. Forty eight hours after transfection, triplicate cultures were preincubated for 1 h in the presence or absence of a PKC-specific peptide inhibitor. Subsequently, the cells were incubated for 6 h in the presence or absence of IFN␤, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of five experiments. E, U2OS cells were transfected with a ␤-galactosidase expression vector and an 8ϫ GAS-luciferase plasmid. Forty eight hours after transfection, triplicate cultures were preincubated for 1 h in the presence or absence of a PKC-specific peptide inhibitor. Subsequently, the cells were incubated for 6 h in the presence or absence of IFN␣, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of three experiments. F, U2OS cells were transfected with a ␤-galactosidase expression vector and an 8ϫ GAS-luciferase plasmid. Forty eight hours after transfection, triplicate cultures were preincubated for 1 h in the presence or absence of a PKC-specific peptide inhibitor. Subsequently, the cells were incubated for 6 h in the presence or absence of IFN␤, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of five experiments. G, U2OS cells were transfected with a ␤-galactosidase expression vector and an 8ϫ GAS-luciferase plasmid. Forty eight hours after transfection, triplicate cultures were preincubated for 1 h in the presence or absence of a PKC-specific peptide inhibitor. Subsequently, the cells were incubated for 6 h in the presence or absence of IFN␤, and luciferase activity was measured. Data are expressed as fold increase in luciferase activity over control untreated cells and represent means Ϯ S.E. of six experiments. colonies from methylcellulose cultures were plucked and used for RT-PCR analysis.
In Vitro Kinase Assays-Immune complex assays to detect the kinase activity of specified proteins were performed as in our previous studies (29). Briefly, cells were serum-starved, stimulated with 1 ϫ 10 4 IU/ml of the indicated IFNs and then immunoprecipitated overnight with an anti-PKC antibody or rabbit IgG as a control. Immunoprecipitates were then washed three times with phosphorylation lysis buffer and twice with kinase buffer (25 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM dithiothreitol, and 20 M ATP). Immunoprecipitated proteins were resuspended in 30 l of kinase buffer to which 5 g of histone H1 and 10 Ci of [␥-32 P]ATP were added. The reaction was allowed to proceed for 20 min at room temperature prior to termination following the addition of SDS-sample buffer. Proteins were then analyzed by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot was subsequently immunoblotted with an anti-PKC antibody.
Luciferase Assays-Cells were transfected with a ␤-galactosidase expression vector and either an ISRE luciferase construct (22) or a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8ϫ-GAS) (40), using the Superfect transfection reagent in accordance with the manufacturer's recommended procedure (Qiagen). The ISREluciferase construct was previously provided by Dr. Richard Pine (Public Health Research Institute, New York). The 8ϫ-GAS construct was previously provided by Dr. Christopher Glass (University of California, San Diego). Forty eight hours after transfection, triplicate cultures were either left untreated or treated with PKC or PKC peptide inhibitors (Calbiochem) for 60 min. Following inhibitor incubation, triplicate cultures were then either left untreated or treated with 5 ϫ 10 3 units/ml IFN␣ or IFN␤ as indicated, and luciferase activity was meas-ured as in our previous studies (22,29). In addition, in some experiments U2OS cells were also transfected with a ␤-galactosidase expression vector, an ISRE luciferase construct (22), and either an empty vector plasmid or a constitutively active PKC mutant provided by Dr. Gottfried Baier (Innsbruck Medical University, Innsbruck, Austria) (36).
Evaluation of Apoptosis and Cell Cycle-Cells were transfected with the indicated plasmid constructs via nucleofection (Amaxa, Cologne, Germany), according to the manufacturer's instructions. The evaluation of apoptosis was assessed by annexin V-propidium iodide staining using an apoptosis detection kit (Pharmingen), as in previous studies (41,42). The evaluation of cell cycle was assessed by propidium iodide staining and flow cytometric analysis. Briefly, the cells were synchronized by serum starvation for 24 h, and then re-plated in media containing serum, pretreated with PKC or PKC peptide inhibitor (Calbiochem) for 60 min, followed by treatment with 2500 -3000 units/ml of IFN␣ for 24 h. Cells were then harvested, washed in cold phosphate-buffered saline, fixed with ice-cold ethanol, and incubated for 20 min with propidium iodide (Sigma) prior to flow cytometric analysis.
Antiviral Assays-The antiviral effects of human IFN␣ and IFN␤ were determined as in our previous studies (27), using EMCV as the challenge virus.

RESULTS
In initial studies, we sought to determine whether PKC is phosphorylated in response to treatment of cells with different type I IFNs. We examined the effects of IFN␣ on the phosphorylation of PKC in IFN-sensitive hematopoietic cell lines. KT1 or U266 cells were incubated in the presence or absence of IFN␣ for different times, and cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKC against Ser-674. IFN␣ induced strong phosphorylation of PKC in both cell lines studied (Fig. 1, A  and B). Similarly, treatment of KT1 or U266 cells with another type I IFN, IFN␤, also resulted in strong phosphorylation of PKC (Fig. 1, C and D). To directly determine whether the kinase domain of PKC is activated in a type I IFN-dependent manner, experiments were performed in which lysates from IFN␣or IFN␤-treated cells were immunoprecipitated with an anti-PKC antibody and subjected to in vitro kinase assays using histone H1 as an exogenous substrate. As shown in Fig. 2, treatment of cells with either IFN␣ (Fig. 2A) or IFN␤ (Fig. 2B) resulted in PKC kinase activity, indicating that the kinase domain of this PKC isoform is activated during its engagement by the type I IFN receptor.
Previous work has shown that another member of the PKC family of isoforms, PKC␦, plays an important role in IFN␣-dependent transcriptional regulation (20). To determine whether PKC also regulates type IFN-dependent transcription, luciferase promoter assays were performed to determine the effects of PKC inhibition on IFN-dependent transcriptional activity via ISRE or GAS elements. U2OS cells were transfected with either ISRE or 8ϫ-GAS luciferase constructs and pretreated with a PKC-specific peptide inhibitor prior to treatment with either IFN␣ or IFN␤. Luciferase activity was then measured and normalized to ␤-galactosidase activity. Inhibition of PKC activity had no significant effects on IFN␣-inducible luciferase activity for ISRE elements (Fig. 3A). Similarly, although there was some minimal decrease in IFN␤-inducible luciferase activity in the presence of the PKC peptide inhibited, there was still clear inducible IFN␤-dependent transcription (Fig. 3B), suggesting that PKC activity is not essential for type I IFN-dependent transcriptional activation via ISRE elements. Consistent with this, overexpression of a constitutively active PKC mutant did not result in enhanced transcription via ISRE elements (Fig.  3C). Pretreatment of cells with an inhibitor against an atypical PKC isoform, PKC (used as a control), had also no significant effects on type I IFN-dependent transcriptional activation via ISRE elements (Fig. 3D). Similarly, inhibition of either PKC (Fig. 3, E and F) or PKC (Fig. 3G) activities had no significant effects on type I IFN-dependent transcription via GAS elements.
Because IFN-induced transcription is strongly associated with generation of IFN-dependent antiviral responses, we also assessed the effects of PKC inhibition on antiviral activity. U2OS cells were pretreated with PKC pseudo-substrate inhibitor and then challenged with EMCV. As shown in Fig. 4, both IFN␣ and IFN␤ protected U2OS cells from the cytopathic effects of EMCV in a dose-dependent manner, but inhibition of PKC activity did not reverse such IFN-induced antiviral protection (Fig. 4, A and B). Thus, in contrast to two other members of the group of novel PKC isoforms (␦ and ) whose activities are required for type I IFN-dependent gene transcription (29,30), PKC does not regulate transcriptional activation of interferon-stimulated genes or mediate induction of type I IFNantiviral responses.
It is well established from previous work that type I IFN treatment induces G 0 /G 1 cell cycle arrest in sensitive cells (43,44), including KT1 cells (44), in which we demonstrated IFN␣ and IFN␤ phosphorylation/activation of PKC. To examine whether PKC plays a role in the generation of type I IFN-dependent growth inhibitory responses, experiments were performed in which the requirement of PKC in the induction of IFN-dependent G 0 /G 1 arrest was examined. KT1 cells, synchronized by serum starvation, were treated with either a PKC inhibitor or a PKC inhibitor, used as control. Both inhibitors were peptide pseudosubstrates specific for PKC or PKC, respectively. The cells were then treated with IFN␣ for 24 h prior to flow cytometric analysis to evaluate cell cycle progression. As expected (44), IFN␣ treatment resulted in a substantial increase in the percentage of cells that were arrested in the G 0 /G 1 phase of the cell cycle compared with control-treated cells (Fig. 5A). However, such an increase was attenuated in cells pretreated with the PKC pseudosubstrate inhibitor (Fig.  5A). On the other hand, treatment of cells with the PKC pseudosubstrate inhibitor did not have any significant effects on such IFN-induced G 0 /G 1 arrest (Fig. 5B), suggesting a specific role for PKC in the process.
As our data suggested a selective role for PKC in the induction of cell cycle arrest, we pursued further studies aimed to determine the role of this kinase in the generation of the regulatory effects of IFN␣ on normal hematopoiesis. Human bone marrow-derived CD34ϩ progenitors were transfected with PKC siRNA or control siRNA, and the effects of PKC knockdown on hematopoietic progenitor colony formation were assessed by clonogenic assays in methylcellulose (Fig. 6A). PKC knockdown using the PKC-specific siRNA (Fig. 6, B and C) had no significant effects on erythroid progenitor (BFU-E) colony growth and did not reverse generation of the inhibitory effects of IFN␣ on erythroid progenitors (Fig. 6A). On the other hand, there was a substantial and consistent increase in the number of CFU-GM colonies from bone marrow-derived CD34ϩ cells in which PKC was knocked down (Fig. 6A). Importantly, PKC knockdown reversed the growth inhibitory effects of IFN␣-dependent suppression of CFU-GM colony formation. Such reversal of the effects of IFN␣ was complete and statistically significant, even when the base-line increase in CFU-GM colonies caused by PKC siRNA was considered (Fig. 6A). Similar results (Fig. 6D) were obtained when a different siRNA targeting PKC (Fig. 6E) was used. To confirm the specificity of these findings, additional experiments were performed in which different PKC isoforms (PKC␣, PKC␤, and PKC) were knocked down using specific siRNAs. In contrast to what we observed in the case of PKC (Fig. 6, A and D), inhibition of expression of these isoforms in CD34ϩ progenitors did not reverse the suppressive effects of IFN␣ on myeloid CFU-GM colony formation (Fig. 7, A-F).
Thus, in normal hematopoietic progenitor cells, inhibition of PKC expression results in a lineage-selective attenuation of IFN␣-induced colony suppression, establishing an important and specific role for this kinase in the generation of the effects of IFN␣ on hematopoiesis.
As our data demonstrated a requirement for PKC in the generation of myelosuppressive effects of IFN␣ on myeloid but not erythroid progenitors, we sought to determine whether the lack of requirement for PKC in the generation of suppressive effects on the erythroid lineage correlates with decreased expression of the kinase during erythropoiesis. Consistent with our previous studies (35), CD34ϩ progenitor cells cultured in vitro in the presence of a cytokine mixture (35) were driven toward erythroid differentiation, as determined by flow cytometric analysis using double labeling for glycophorin A and CD71 (Fig. 8A). Relative PKC mRNA expression steadily decreased throughout erythroid development, and its expression in day 14 erythroid progenitors was clearly less than the expression levels seen in early erythroid progenitor cells (Fig.  8B), suggesting that relative PKC expression is down-regulated during erythropoiesis. Thus, the specific effects of PKC on myeloid but not erythroid progenitors correlate with downregulation of PKC expression during erythropoiesis, further suggesting a selective role for this kinase in the control of human myelopoiesis.
Beyond being a suppressor of normal human myeloid progenitor growth, IFN␣ has potent inhibitory effects against chronic myelogenous leukemia CFU-GM progenitors (4,39) and has major clinical activity in the treatment of CML patients. To determine whether PKC plays a role in the generation of the antileukemic effects of IFN␣ on leukemic CFU-GM progenitors, the effects of PKC knockdown were examined, using samples from different CML patients. The growth-suppressive effects of IFN␣ on CML-derived CFU-GM colonies were attenuated in CML progenitor cells transfected with siRNA targeting PKC but not control siRNA (Fig. 9, A-E). Thus, in addition to its regulatory effects on normal myelopoiesis and its requirement for the induction of growth suppression by IFNs, PKC plays a critical role in the generation of antileukemic responses in chronic myeloid leukemia cells.
To further evaluate the mechanisms via which PKC mediates growth inhibitory effects in hematopoietic cells, we determined the effects of overexpression of a constitutively active PKC mutant on myeloid colony formation, as well as apoptosis. Overexpression of a constitutively active PKC mutant resulted in a statistically significant suppression of formation of myeloid (CFU-GM) but not erythroid (BFU-E) colonies in clonogenic assays in methylcellulose (Fig. 10A). Notably, overex-pression of active PKC in a myeloid cell line did not induce apoptosis (Fig. 10, B and C), suggesting that its suppressive effects on myeloid progenitor growth result from effects on cell cycle progression and not induction of apoptosis.
Over the years there has been extensive accumulating evidence that different members of the PKC family participate in the induction of diverse functional and biochemical responses, depending on the cellular context, the triggering stimulus, and the isoform involved. Different PKC isotypes have been shown to play important roles in the regulation of cell survival and apoptosis, as well as gene transcription and differentiation (45)(46)(47)(48)(49)(50)(51). Importantly, several PKC isoforms have been implicated in the regulation of different aspects of normal hematopoiesis (reviewed in Refs. 48,52) and have been found to be involved in signaling for various hematopoietic growth factors, including erythropoietin, thrombopoietin, and stem cell factor (48,(52)(53)(54)(55)(56). Beyond their roles in various signaling pathways in normal cells, it is now well established that certain members of the PKC family are abnormally activated in different types of malignant cells, including leukemic cells (45, 58 -63). In fact, as the PKC family of kinases plays an important role in the pathophysiology of various solid tumors and hematologic malignancies, there are emerging efforts toward the development of new pharmacological agents or other means to target members of the PKC family in the treatment of cancer (64 -66). The diversity of PKC signaling and the wide spectrum of biological functions regulated by this family of proteins underscores the need to better define the specific functions of distinct isotypes in different cellular systems. Remarkably, different PKC isoforms can regulate opposing cellular functions, depending on the context of their engagement and activation. For example, PKC␦ mediates primarily pro-apoptotic responses (67,68), but it can also mediate antiapoptotic effects (69). In contrast, PKC␣ mediates mitogenic signals (70 -72), although generation of anti-tumor responses is positively controlled by PKC␦ (68) and negatively by PKC␣ (73). It is likely that a balance between the function of different PKC isoforms is required for optimal con-trol of several biological responses, but the precise contribution of different PKC isoforms in such systems remains to be defined.
There has been accumulating evidence over the last few years implicating PKCs in type I and II IFN signaling (29 -33, 74, 75). A particularly interesting finding has been the identification of PKC␦ as a kinase that regulates phosphorylation of Stat1 on serine 727 in response to both type I (29,31) and type II IFNs (75). These data have established a critical role for this PKC isoform in IFN signaling, via its ability to regulate optimal transcription of IFNsensitive genes that participate in induction of the biological effects of IFNs. Notably, the function of this PKC isoform in IFN signaling exhibits some cell type specificity, as there is evidence that at least one additional PKC isoform, PKC⑀ (33,75), also functions as a serine kinase for Stat1 in response to IFN␥ in different cell types. Thus, it is possible that other PKC isoforms or non-PKC serine kinases (76) compensate for the serine kinase activity of PKC␦ in certain cell types, highlighting the significance of the existence of the multiple distinct isoforms of the PKC family.
In this study, we examined the activation and functional relevance of PKC, another novel member of the PKC family of proteins, in the generation of IFN responses. Our data provide the first evidence that this PKC isoform is rapidly activated in response to IFN␣ or IFN␤ treatment of cells. Interestingly, in experiments to define its role in IFN-generated cellular responses, we found that engagement of PKC does not regulate type I IFN-dependent transcription. Thus, in contrast to the roles that two other isoforms, PKC␦ (29) and PKC (30), play in the regulation of type I IFN-dependent transcriptional activation via ISRE or GAS elements, PKC does not exhibit such function in IFN signaling. Our data suggest that PKC functions at a post-transcriptional level and has effects on IFNdependent cell cycle arrest.
In experiments to determine the requirement of PKC activity in the generation of the myelosuppressive effects of IFN␣ on normal hematopoietic progenitors, we found that PKC activity is essential for the generation of the suppressive effects of IFNs on myeloid (CFU-GM) but not erythroid progenitors (BFU-E). Interestingly, PKC targeting also enhanced myeloid FIGURE 8. Decreased expression of PKC during erythroid differentiation. A, CD34ϩ progenitor cells were cultured for 14 days, as described under "Materials and Methods." Glycophorin A and CD71 (transferrin receptor) levels were monitored by flow cytometry at various time points during the maturation program. The panels on the left show the isotype controls, and the panels on the right show the expression levels for GlyA and CD71. B, CD34ϩ progenitor cells were cultured for 14 days. mRNA was isolated at the indicated time points, and quantitative RT-PCR using PKC-specific primers was used to determine PKC expression at different times. All samples were normalized to GAPDH. Data are expressed as relative expression, calculated as percent expression of day 3/4 values. Data represent means Ϯ S.E. of three experiments.
colony formation from normal bone marrows, suggesting a regulatory role for this PKC isoform on normal myelopoiesis but not erythropoiesis. This finding is of high interest as it relates to the cell type and context specificity of PKC-mediated biological outcomes. In hematopoietic cells, the specific functional roles of members of the PKC family remain largely unknown. There has been some evidence for selective expression of certain PKC isoforms in murine progenitor cell clones and human CD34ϩ progenitor cells (77). It also appears there is also some selectivity in the expression of distinct isoforms in CD34ϩ progenitors versus terminally differentiated hematopoietic cells (53). However, the functional implications of such expression remain to be defined. Our studies demonstrating down-regulation of PKC during progression of erythropoiesis and selective PKC functional responses on myeloid cells suggest a specific developmental and functional role of this isoform in normal human myelopoiesis. As it is likely that a balance between growth factors and hematopoietic suppressor cytokines is required for optimal regulation of normal hematopoiesis, the involvement of PKC in IFN␣ signaling in hematopoietic precursor cells further suggests an important role for this cytokine in the regulation of normal hematopoiesis.
Our studies also provide the first evidence that this PKC isoform is involved in the generation of the antileukemic effects of IFN␣ in chronic myeloid leukemia cells, as evidenced by the reversal of IFN␣-dependent growth inhibitory responses on CML-derived leukemic CFU-GM progenitors in vitro. Interestingly, as with several other PKC isoforms, the regulatory effects of PKC on tumorigenesis and malignant cell growth vary depending on the cellular context, and in some cases appear to be opposing. On the one hand, PKC-deficient mice display an exaggerated response to phorbol ester-induced tumor formation (79), suggesting that PKC plays a key role in suppressing tumorigenesis. Similarly, via its regulatory effects on cell cycle progression, PKC appears to promote growth inhibitory effects and/or cell differentiation in different systems (80 -82). On the other hand, it has been shown that PKC expression is associated with the development of resistance of Hodgkin lymphoma cell lines to chemotherapy-induced apoptosis (83) and promotes proliferation of glioblastoma cell lines via the extracellular signal-regulated kinase (ERK)/Elk-1 pathway (57). Moreover, PKC has been implicated in the up-regulation of multidrug-resistant associated genes in different types of tumors (78). Clarifying the determinants that account for the diversity of functional responses in response to this PKC isotype in malignant cells should provide important insights on the mechanisms of regulation of growth and survival of malignant cells. Our findings, establishing a novel and specific function for PKC in type IFN signaling, provide the first link between this PKC isoform and IFNs, cytokines that are potent inhibitors of malignant cell growth and key agents in the immunosurveillance against cancer. Further studies to identify spe- cific elements in normal myeloid or myeloid leukemia cells that may interact and be regulated by the IFN-activated form of PKC may ultimately lead to the identification of novel cellular targets for the development of new translational efforts in the treatment of malignancies.