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Originally published In Press as doi:10.1074/jbc.M106640200 on December 31, 2001

J. Biol. Chem., Vol. 277, Issue 10, 7726-7735, March 8, 2002
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Activation of the p38 Mitogen-activated Protein Kinase Mediates the Suppressive Effects of Type I Interferons and Transforming Growth Factor-beta on Normal Hematopoiesis*

Amit VermaDagger , Dilip K. DebDagger , Antonella SassanoDagger , Shahab UddinDagger , John Varga§, Amittha WickremaDagger , and Leonidas C. PlataniasDagger

From the Dagger  Section of Hematology-Oncology and § Section of Rheumatology, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60607

Received for publication, July 16, 2001, and in revised form, December 28, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Type I interferons (IFNs) are potent regulators of normal hematopoiesis in vitro and in vivo, but the mechanisms by which they suppress hematopoietic progenitor cell growth and differentiation are not known. In the present study we provide evidence that IFNalpha and IFNbeta induce phosphorylation of the p38 mitogen-activated protein (Map) kinase in CD34+-derived primitive human hematopoietic progenitors. Such type I IFN-inducible phosphorylation of p38 results in activation of the catalytic domain of the kinase and sequential activation of the MAPK-activated protein kinase-2 (MapKapK-2 kinase), indicating the existence of a signaling cascade, activated downstream of p38 in hematopoietic progenitors. Our data indicate that activation of this signaling cascade by the type I IFN receptor is essential for the generation of the suppressive effects of type I IFNs on normal hematopoiesis. This is shown by studies demonstrating that pharmacological inhibitors of p38 reverse the growth inhibitory effects of IFNalpha and IFNbeta on myeloid (colony-forming granulocytic-macrophage) and eythroid (burst-forming unit-erythroid) progenitor colony formation. In a similar manner, transforming growth factor beta , which also exhibits inhibitory effects on normal hematopoiesis, activates p38 and MapKapK-2 in human hematopoietic progenitors, whereas pharmacological inhibitors of p38 reverse its suppressive activities on both myeloid and erythroid colony formation. In further studies, we demonstrate that the primary mechanism by which the p38 Map kinase pathway mediates hematopoietic suppression is regulation of cell cycle progression and is unrelated to induction of apoptosis. Altogether, these findings establish that the p38 Map kinase pathway is a common effector for type I IFN and transforming growth factor beta  signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytokines play important roles in the regulation of normal hematopoiesis, and a balance between the actions of hematopoietic growth factors and myelosuppressive factors is required for optimal production of cells of different hematopoietic lineages. Several previous studies (1-10) have established that type I interferons (IFNs)1 are potent regulators of normal hematopoiesis in vitro and in vivo. Despite the well documented effects of interferons as negative regulators of hematopoiesis, the mechanisms by which such effects occur remain unknown. Interferons exhibit negative regulatory effects on hematopoietic progenitor colony formation in clonogenic assays in methylcellulose. The negative effects of IFNs are exerted on progenitor cells of all hematopoietic lineages, including early and late erythroid (BFU-E and CFU-E), myeloid (CFU-GM), and megakaryocytic progenitors (CFU-MK) (1-10). Other reports (11) have also shown that interferons inhibit the growth of progenitors derived from CD34+CD38- cells, a primitive cell population, indicating that their inhibitory effects occur at a very early level of stem cell differentiation or the stem cells themselves.

The mechanisms by which type I interferons (IFNalpha , -beta , and -omega ) transduce signals have been elucidated to a great extent over the last few years. All type I IFNs bind to a common receptor, the type I IFN receptor. The binding of interferons to their common receptor results in activation of two tyrosine kinases of the Janus family, Tyk-2 and Jak-1, that are constitutively associated with the different receptor subunits (reviewed in Refs. 12-14). Activation of these tyrosine kinases results in phosphorylation of several signaling elements and activation of multiple downstream cellular pathways, including the Stat pathway (reviewed in Refs. 12-14), the Crk pathway (15-17), and the IRS-PI 3'-kinase cascade (18-21). Type I IFN-dependent transcription of target genes is regulated by the Stat pathway. During engagement of the type I interferon receptor, activated Jak kinases induce tyrosine phosphorylation of Stat proteins, which results in formation of several different Stat complexes. The IFNalpha -tyrosine-phosphorylated forms of Stat1 and Stat2 associate with IRF-9 to form the mature interferon-stimulated gene factor-3 complex. This complex translocates to the nucleus of cells and binds to interferon-stimulated response elements (ISREs) in the promoters of interferon-stimulated genes to initiate gene transcription (12-14). Stat 1:1 homodimers, Stat 3:3 homodimers, Stat 1:3 heterodimers, Stat 5:5 homodimers, and CrkL:Stat5 heterodimers are also formed during engagement of the type I IFN receptor and move to the nucleus where they bind to GAS regulatory elements in the promoters of IFN-activated genes (12-14, 16, 22). Thus, signaling specificity via the IFNalpha -activated Jak/Stat pathway is established by the formation of multiple different complexes that activate distinct regulatory elements in the promoters of IFN-regulated genes.

In addition to the Stat pathway, type I IFNs activate members of the Map family of kinases, including Erk kinases (23) and the p38 Map kinase (24-26). We and others (24-26) have shown recently that activation of p38 is required for transcriptional activation of IFN-sensitive genes. In addition, our studies have demonstrated that such transcriptional regulation of IFN-sensitive genes is unrelated to effects on DNA binding of Stat complexes or serine phosphorylation of Stats (26), apparently involving a Stat-independent nuclear mechanism. Thus, coordination of the functions of the IFN-activated Stat and p38 pathways is necessary for full transcriptional activation in response to interferons (24-26).

In the present study, we determined whether the p38 Map kinase pathway is engaged in type I IFN signaling in primary human hematopoietic progenitors and whether its function is required for the generation of the suppressive effects of interferons on normal hematopoiesis. Our data demonstrate that p38 and its downstream effector, MapKapK-2, are rapidly activated by IFNalpha or IFNbeta treatment of enriched primary human progenitor cells. Pharmacological inhibition of p38 activation reverses the type I IFN-dependent inhibition of hematopoietic progenitor colony formation, demonstrating that the function of this pathway is essential for the generation of the suppressive effects of type I IFNs on hematopoiesis. We also demonstrate that TGF-beta , another potent inhibitor of normal hematopoiesis (27-32), also activates p38 in progenitor cells and that pharmacological inhibitors of p38 reverse its suppressive effects on progenitor colony formation, indicating a critical role for this pathway in mediating myelosuppressive signals in human bone marrow cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytokines and Antibodies-- Antibodies against the phosphorylated forms of p38 and Erk were obtained from Cell Signaling Technology (Beverly, MA) and were used for immunoblotting. A polyclonal antibody against p38 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody against Erk2 was obtained from Transduction Laboratories (Lexington, KY). A polyclonal antibody against MapKap kinase-2 was obtained from Upstate Biotechnology Inc. Human recombinant IFNalpha 2 was provided by Hoffmann-La Roche. Human recombinant consensus IFNalpha was provided by Amgen Inc. Human recombinant IFNbeta was provided by Biogen. The p38 Map kinase inhibitors SB203580 and SB202190 and the Mek kinase inhibitor PD098059 were purchased from Calbiochem.

Cell Lysis and Immunoblotting-- Bone marrow aspirate or peripheral blood specimens were obtained from normal donors after obtaining informed consent, as approved by the Institutional Review Board of the University of Illinois, Chicago. Human primary erythroid progenitors were isolated and enriched as described previously (33) or from positively selected CD34+ cells, obtained from the bone marrows or peripheral blood of normal healthy volunteers. Briefly, mononuclear cells were separated by Ficoll-Paque, and CD34+ cells were obtained by positive immunomagnetic bead selection, using Macs columns (Miltenyi Biotech). The cells were passed through the columns twice to obtain a purity of 90-95% CD34+ cells, which was confirmed by flow cytometry using fluorescein isothiocyanate-conjugated anti-CD34 antibodies. The CD34+ cells were cultured in a medium (IMDM) containing 15% fetal calf serum, 15% human AB serum, 500 units/ml penicillin, 40 µg/ml streptomycin, 10 ng/ml interleukin-3, 2 units/ml erythropoietin, 50 ng/ml stem cell factor, and 50 ng/ml insulin-like growth factor. Prior to activation by IFNalpha or TGF-beta , day 7 cells (CFU-E) were washed twice with IMDM. The cells were then stimulated with IFNalpha or IFNbeta or TGF-beta for the indicated times and lysed in phosphorylation lysis buffer, as previously described (18-22). Immunoprecipitations and immunoblotting, using an ECL method, were performed essentially as described previously (18-22).

MapKapK-2 Kinase Assays-- These assays were performed as described previously (24). Briefly, cells were treated with IFNalpha for the indicated times and lysed in phosphorylation lysis buffer. Total cell lysates were then immunoprecipitated with an antibody against MapKap kinase-2, and immunoprecipitated proteins were washed three times in phosphorylation lysis buffer and two times in kinase buffer (25 mM Hepes, pH 7.4, 25 mM MgCl2, 25 mM beta -glycerophosphate, 100 µM sodium orthovanadate, 2 mM dithiothreitol, 20 µM ATP) and resuspended in 30 µl of kinase buffer containing 5 µg of Hsp-25 protein (StressGen Laboratories) and 25 µCi of [gamma -32P]ATP. The reaction was incubated for 30 min at room temperature and was terminated by the addition of SDS sample buffer. Proteins were subsequently analyzed by SDS-PAGE, and the phosphorylated form of Hsp-25 was detected by autoradiography.

Rac1 Activation Assays-- The activation of Rac1 by IFNalpha was determined using a methodology described recently (26). Briefly, the pGEX-4T3 construct encoding for the GTPase binding domain of human PAK1 (26) was expressed in Escherichia coli as a GST fusion protein (GST-PBD). The cells were treated with the indicated IFNs and lysed in phosphorylation lysis buffer. In some experiments the cells were starved for 2 h prior to interferon treatment. Cell lysates were incubated with 5 µg of GST-PBD, and bound proteins were separated by SDS-PAGE and immunoblotted with a monoclonal antibody against Rac1 to detect GTP-bound Rac1.

Hematopoietic Progenitor Cell Assays-- Bone marrow aspirates were obtained from normal donors after obtaining informed consent, as approved by the Institutional Review Board of the University of Illinois. The effects of IFNalpha , IFNbeta , and TGF-beta on hematopoietic progenitor colony formation were determined by clonogenic assays in methylcellulose, essentially as described previously (34). Briefly, bone marrow mononuclear cells were separated by Ficoll-Paque sedimentation, and cells were cultured with the indicated cytokines in a methylcellulose mixture containing hematopoietic growth factors (66), in the presence or absence of SB203580 (5 or 10 µM), SB202190 (5 or 10 µM), or PD098059 (2 or 10 µM). Colony-forming units granulocyte-macrocytic (CFU-GM) and burst-forming units-erythroid (BFU-E) were scored on day 14 of culture.

Evaluation of Apoptosis-- Isolated CD34+ bone marrow cells were grown in IMDM supplemented with 30% fetal calf serum, 10 ng/ml interleukin-3, 2 IU/ml recombinant human erythropoietin, 20 ng/ml granulocyte-colony-stimulating factor, and 50 ng/ml stem cell factor. The cells were exposed to IFNalpha (1000 units/ml) or TGF-beta (20 ng/ml), in the presence or absence of SB203580 (10 µM), as indicated. The percentage of apoptotic cells were determined at various time points by flow cytometry after staining with fluorescein-conjugated annexin-V and propidium iodide, as described previously (35).

Cell Cycle Analysis-- Isolated CD34+ bone marrow cells were grown in IMDM supplemented with 30% fetal calf serum, 10 ng/ml interleukin-3, 2 IU/ml recombinant human erythropoietin, 20 ng/ml granulocyte-colony-stimulating factor, and 50 ng/ml stem cell factor. The cells were exposed to IFNalpha (1000 units/ml) or TGF-beta (20 ng/ml), in the presence or absence of SB203580 (10 µM), as indicated. The cells were subsequently fixed in 70% ethanol, and cell cycle stage analysis was done by measuring DNA content by flow cytometry after staining with propidium iodide, as described previously (35).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We sought to determine whether the p38 Map kinase pathway is activated in response to type I IFN treatment of human hematopoietic progenitors. We initially performed studies with purified human eythroid progenitors, at the CFU-E stage of differentiation. The hematopoietic progenitor cells were incubated for different times in the presence or absence of IFNalpha , and after cell lysis, total lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. As shown in Fig. 1, IFNalpha treatment induced strong phosphorylation of the p38 Map kinase in human progenitors (Fig. 1, A and B). In a similar manner, when progenitor cells were treated with IFNbeta , p38 was rapidly phosphorylated (Fig. 1, C and D), strongly suggesting that in addition to IFNalpha this kinase is involved in signaling for all different type I IFNs in primitive hematopoietic cells. In parallel studies, we determined whether type I IFN treatment induces Erk kinase activation in enriched progenitors. CD34+-derived erythroid progenitors were treated with IFNalpha ; the cells were lysed, and cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Erk-specific antibody. IFNalpha treatment induced phosphorylation of Erk2 in primary progenitors (Fig. 1, E and F), indicating that this member of the Erk family of Map kinases is also engaged in type I IFN signaling in primary hematopoietic cells.


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  		Fig. 1.   Type I IFN-dependent activation of Map kinases and MapKapK-2 in primary human hematopoietic progenitors. A, purified erythroid progenitors at the CFU-E level of differentiation were incubated in the presence or absence of IFNalpha , for the indicated times in minutes. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. B, the blot shown in A was stripped and re-probed with an antibody against p38, to control for protein loading. C, purified human erythroid progenitors were incubated in the presence or absence of IFNbeta for the indicated times in minutes. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. D, the blot shown in C was stripped and re-probed with an antibody against p38, to control for protein loading. E, enriched human hematopoietic progenitors were treated with IFNalpha for 60 min, as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Erk antibody. F, the blot shown in E was stripped and re-probed with an antibody against Erk-2. G, enriched human progenitors were incubated in the presence or absence of IFNalpha for the indicated times in minutes at 37 °C. Total cell lysates were immunoprecipitated (IP) with an anti-MapKapK-2 antibody, and immunoprecipitated proteins were subjected to an in vitro kinase assay, using Hsp-25 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated proteins were detected by autoradiography.

Previous studies (36, 37) have shown that a downstream effector of p38 is the MapKapK-2 kinase, which is activated in response to stress and growth factors as well as in response to type I IFN treatment of human cell lines (24). We examined whether IFNalpha treatment results in activation of this kinase in expanded CD34+ bone marrow-derived hematopoietic progenitor cells. Lysates from IFNalpha -treated or untreated cells were immunoprecipitated with a specific antibody against MapKap kinase-2, and in vitro kinase assays were performed on the immunoprecipitates, using Hsp-25 as an exogenous substrate. Fig. 1G shows that this downstream effector of the p38 Map kinase is activated in an IFNalpha -dependent manner. Such an activation is detectable after 20 min of IFNalpha treatment and is very strong after 60 min of incubation with IFNalpha . Thus, treatment of primary hematopoietic progenitors with IFNalpha results in phosphorylation and activation of MapKapK-2, indicating that this kinase is a downstream effector of p38 in human hematopoietic progenitors and may participate in the generation of the biological effects of type I IFNs in hematopoietic cells.

As our data established that both the p38 and Erk2 are activated in primary human hematopoietic progenitor cells, we sought to obtain information on the role that these Map kinases play in type I IFN-dependent suppression of normal hematopoietic cell progenitor growth. We performed experiments in which bone marrow mononuclear cells were cultured in methylcellulose with IFNalpha , in the presence or absence of specific inhibitors for the p38 pathway (SB203580 and SB202190) or the Erk pathway (PD98059). As shown in Fig. 2A, addition of SB203580 had no significant effects on normal colony formation for myeloid (CFU-GM) or erythroid (BFU-E) progenitors, suggesting that the p38 pathway does not mediate signals required for growth or differentiation of hematopoietic progenitors (Fig. 2A). As expected, IFNalpha inhibited colony formation for both myeloid (CFU-GM) and erythroid (BFU-E) progenitors (Fig. 2, A-C). Concomitant treatment of cells with SB203580, used at doses of either 5 or 10 µM, reversed the growth inhibitory effects of IFNalpha on both CFU-GM and BFU-E progenitors (Fig. 2A), indicating that activation of p38 is essential for the induction of the inhibitory effects of IFNalpha on human hematopoietic cells. Similarly, the suppressive effects of IFNalpha on CFU-GM and BFU-E colony formation were also reversible when the bone marrow mononuclear cells were cultured in methylcellulose in the presence of another p38-specific inhibitor, SB202190 (Fig. 2B). On the other hand, PD98059, a specific Mek kinase inhibitor which blocks Erk but not p38 activation, had no effect (Fig. 3C), indicating that activation of the Erk pathway is not required for the generation of the suppressive effects of IFNalpha on normal hematopoiesis.


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  		Fig. 2.   IFNalpha inhibits the growth of human bone marrow-derived hematopoietic progenitors in a p38-dependent manner. A, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of IFNalpha (IU/ml) and the p38 Map kinase inhibitor SB203580 (µM). The data are expressed as percent control of CFU-GM or BFU-E colony numbers for untreated cells. Means ± S.E. of 4 independent experiments for each condition are shown. SB indicates SB203580. B, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of SB202190 (5 µM) and the indicated doses of IFNalpha (IU/ml). The data are expressed as percent control of CFU-GM or BFU-E colony numbers for untreated cells. Means ± S.E. of 4 independent experiments for each condition are shown. SB indicates SB202190. C, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of MEK1 kinase inhibitor PD098059 (µM) and IFNalpha (IU/ml). The data are expressed as percent control of CFU-GM or BFU-E colony numbers for untreated cells. Means ± S.E. of 4 independent experiments for each condition are shown. PD indicates PD098059.


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  		Fig. 3.   p38 is required for the induction of the suppressive effects of IFNbeta on normal hematopoietic progenitors. A, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of IFNbeta (IU/ml) and SB203580 (µM). Means ± S.E. of 4 independent experiments for each condition are shown. SB indicates SB203580. B, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of MEK1 kinase inhibitor PD098059 (µM) and IFNbeta (IU/ml). The data are expressed as percent control of CFU-GM or BFU-E colony numbers for untreated cells. Means ± S.E. of 4 independent experiments for each condition are shown. PD indicates PD098059.

IFNbeta is another member of the family of type I IFNs, which has also been shown previously to suppress hematopoietic progenitor colony formation (7, 9). This cytokine also binds to the type I IFN receptor to initiate activation of signaling cascades, which ultimately regulate p38 activation (12-14). As our data established that IFNbeta activates p38 in primitive human hematopoietic cells, we determined whether pharmacological inhibition of p38 activation reverses its suppressive effects on progenitor colony formation. As expected, addition of IFNbeta to the methylcellulose cultures, at doses of 100 IU/ml or 1000 IU/ml, suppressed CFU-GM and BFU-E colony formation (Fig. 3, A and B), but concomitant treatment with SB203580 reversed such effects (Fig. 3A). On the other hand, similar to our findings in the experiments in which IFNalpha was used, concomitant treatment with PD98059 did not reverse the IFNbeta -dependent suppression of hematopoietic cell progenitor growth (Fig. 3B). Altogether, these experiments established that the function of the p38 pathway is essential for type I IFN-dependent suppression of normal hematopoiesis, indicating that this pathway is a common effector for all type I IFNs, mediating hematopoietic suppressive signals.

As our data established that type I IFNs require the p38 pathway to mediate their growth inhibitory effects, we considered the possibility that p38 may function as a common pathway for the generation of the effects of other myelosuppressive cytokines as well. TGF-beta is a well known potent inhibitor of hematopoiesis (27-31) that has been shown to activate the p38 kinase pathway in other systems (38, 39). We determined whether TGF-beta induces activation of the p38 in primary hematopoietic progenitor cells, and if so, whether p38 activation is essential for TGF-beta -induced growth inhibitory effects. Fig. 4, A and B, shows that p38 is phosphorylated/activated in a TGF-beta -dependent manner in enriched progenitor cells. Interestingly, in contrast to our findings with type I IFNs, we failed to detect TGF-beta -inducible activation of Erk2 (Fig. 4, C and D), indicating that the Erk pathway is selectively activated during IFNalpha but not TGF-beta stimulation of human progenitors. In subsequent studies we sought to determine whether, as in the case of type I IFNs, TGF-beta induces activation of the downstream effector of p38, MapKapK-2 kinase. Enriched human hematopoietic progenitors were incubated in the presence or absence of TGF-beta , and cell lysates were immunoprecipitated with an antibody against MapKapK-2, and in vitro kinase assays were carried out on the immunoprecipitates using Hsp-25 as an exogenous substrate. As shown in Fig. 4, E and F, treatment of the progenitor cells with TGF-beta resulted in strong activation of MapKapK-2. Pretreatment of cells with the p38-specific inhibitor SB203580 blocked the activation of MapKapK-2, indicating that such an activation is p38-dependent. SB203580 is a specific p38 inhibitor that acts by binding to the ATP site of the p38 molecule and abrogating its kinase activity (40-42). A previous report (43) has shown that high doses of SB203580 can also inhibit activation of the TGF-beta I and II receptors in vitro. This prompted us to perform studies to exclude the possibility that the inhibitory effects of SB203580 on the sequential p38/MapKapK-2 activation are due to blocking of the TGF-beta receptors. KG-1 cells, which are CD34+, were pretreated with SB203580, and the TGF-beta -dependent phosphorylation of p38 was determined by anti-phospho-p38 immunoblotting. Consistent with our findings using primary CD34+-derived primary progenitors, TGF-beta treatment resulted in strong phosphorylation of p38. On the other hand, SB203580 did not inhibit the phosphorylation of p38, which is regulated by upstream MKK activation, confirming that SB203580 does not block activation of TGF-beta receptors in intact cells (Fig. 5, A and B). Similarly, TGF-beta treatment resulted in strong phosphorylation of Mkk3/6 (Fig. 5, C and D) and activation of the upstream regulator Rac1 (Fig. 5E), and such inductions were not blocked by SB203580 (Fig. 5, C-E), further confirming that the inhibitory effects of SB203580 on MapKapK-2 activation result by selective blocking of the p38 kinase domain and not inhibition of the TGF-beta receptor.


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  		Fig. 4.   TGF-beta activates p38 and MapKapK-2, but not Erk, in CD34+ human hematopoietic progenitors. A, purified human hematopoietic progenitors were incubated for 60 min at 37 °C in the presence or absence of TGF-beta as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. B, the blot shown in A was stripped and re-probed with an antibody against p38 to control for protein loading. C, purified progenitors were treated with TGF-beta for 60 min, as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Erk antibody. D, the blot shown in C was stripped and re-probed with an antibody against Erk-2. E, purified human erythroid progenitors were incubated in the presence or absence of TGF-beta with and without SB203580 for 30 min at 37 °C. Total cell lysates were immunoprecipitated (IP) with an anti-MapKapK-2 antibody, and immunoprecipitated proteins were subjected to an in vitro kinase assay using Hsp-25 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated Hsp-25 was detected by autoradiography. F, the blot shown in E was then probed with an anti-MapKapK-2 antibody to control for protein loading.


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  		Fig. 5.   SB203580 does not inhibit activation of TGF-beta receptors and upstream regulators of the p38 pathway in intact cells. A, KG-1 cells were incubated in the presence or absence of SB203580 (10 µM) for 60 min and were subsequently treated for 60 min with TGF-beta , in the continuous presence or absence of SB203580. The cells were lysed, and total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38. B, the blot shown in A was stripped and re-probed with an anti-p38 antibody. C, KG-1 cells were incubated in the presence or absence of SB203580 (10 µM) for 60 min and were subsequently treated for 60 min with TGF-beta , in the continuous presence or absence of SB203580. The cells were lysed, and total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Mkk3/6 antibody. D, the blot shown in C was stripped and re-probed with an anti-Mkk3 antibody. E, KG-1 cells were incubated in the presence or absence of SB203580 (10 µM) for 60 min and were subsequently treated for the indicated times with TGF-beta , in the continuous presence or absence of SB203580. Cell lysates were bound to GST-PBD or control GST as indicated, and bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Rac1 antibody, to detect GTP-bound Rac1.

In subsequent studies to evaluate the effects of TGF-beta on hematopoietic progenitor colony formation, we found that addition of TGF-beta to the methylcellulose cultures, at doses of either 5 or 10 ng/ml, strongly suppressed CFU-GM and BFU- E colony formation (Fig. 6). Concomitant treatment with SB203580 reversed such an inhibition (Fig. 6A), whereas treatment of cells with the Mek kinase inhibitor PD98059 had no effect (Fig. 6B), suggesting that selective activation of the p38 pathway by the TGF-beta receptors mediates the suppressive effects of the cytokine on hematopoietic progenitors. Consistent with this, when another selective inhibitor of p38, SB202190, was used, similar results were obtained (Fig. 6C).


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  		Fig. 6.   TGF-beta inhibits the growth of human bone marrow-derived hematopoietic progenitors in a p38-dependent manner. A, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of SB203580 (µM) and TGF-beta (ng/ml). B, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of the indicated doses of the MEK1 kinase inhibitor PD 98059 (µM) and TGF-beta (ng/ml). C, bone marrow mononuclear cells were plated in a methylcellulose culture assay system, in the presence or absence of SB202190 (10 µM) and TGF-beta (ng/ml). The data are expressed as percent control of BFU-E or CFU-GM colony numbers for untreated cells. Means ± S.E. of 3 independent experiments for A and B and 2 independent experiments for C are shown. SB indicates SB203580, and PD indicates PD098059.

Altogether, our studies established a critical role for p38 in the generation of the growth inhibitory effects of IFNalpha and TGF-beta on normal hematopoiesis. To understand the mechanisms by which such activities occur, experiments were performed in which the effects of IFNalpha and TGF-beta on hematopoietic progenitor-cell cycle progression and induction of apoptosis were determined in the presence or absence of p38 inhibitors. Human CD34+ cells were cultured in suspension media with IFNalpha or TGF-beta for 5 days in the presence or absence of SB203580. Treatment with either IFNalpha (Fig. 7A) or TGF-beta (Fig. 7B) caused a G0/G1 arrest, consistent with previous reports (28, 58). Concomitant treatment of cells with SB203580 completely reversed the IFNalpha -induced cell cycle arrest (Fig. 7A), indicating that p38 activation is essential for this event. Similarly, the induction of TGF-beta -dependent cell cycle arrest was partially reversed by treatment with the p38 inhibitor (Fig. 7B). As other studies have shown that p38 mediates anti-apoptotic signals under certain conditions, we considered the possibility that IFNalpha and/or TGF-beta may be suppressing hematopoiesis by inducing apoptosis via a p38-dependent mechanism. To address this issue, experiments were performed in which cells were incubated with IFNalpha - or TGF-beta in the presence or absence of SB203580, and the induction of apoptosis was evaluated by annexin V staining. As shown in Fig. 8, neither IFNalpha nor TGF-beta induced apoptosis of CD34+ cells, whereas addition of SB203580 in the cultures had no effect. These data indicate that p38 does not mediate pro-apoptotic signals on primary hematopoietic progenitor cells and that the suppressive effects of IFNalpha or TGF-beta on hematopoiesis are unrelated to induction of apoptosis.


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  		Fig. 7.   Activation of p38 is required for induction of G0/G1 cell cycle arrest of CD34+ hematopoietic cells in response to IFNalpha and TGF-beta . A, isolated human bone marrow CD34+ cells were cultured for 5 days in the presence or absence of IFNalpha (1000 IU/ml) and SB203580 (10 µM). Cell cycle stage analysis was evaluated by measuring DNA content by flow cytometry, after staining with propidium iodide. Data are expressed as percentage change over the values for control-untreated CD34+ cells in the G0/G1 phase of the cell cycle. B, isolated human bone marrow CD34+ cells were cultured for 5 days in the presence or absence of TGF-beta (20 ng/ml) and SB203580 (10 µM). Cell cycle stage analysis was evaluated by measuring DNA content by flow cytometry, after staining with propidium iodide. Data are expressed as percentage change over the values for control-untreated CD34+ cells in the G0/G1 phase of the cell cycle.


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  		Fig. 8.   IFNalpha and TGF-beta do not induce apoptosis of human CD34+-derived hematopoietic progenitors. Isolated human bone marrow CD34+ cells were cultured in the presence and absence of 1000 IU/ml IFNalpha or 20 ng/ml TGF-beta , as indicated. Cells were analyzed for apoptosis by flow cytometry after staining with an antibody against annexin V. The data are expressed as % annexin-positive cells at days 2-4 of culture, as indicated. Data are expressed as means ± S.E. of 2 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well established that IFNalpha is a potent suppressor of normal hematopoietic progenitor cell growth in vitro, and this appears to be the mechanism by which this cytokine induces cytopenias when administered to humans in vivo (1-8). Similarly, previous studies (27-31) have established that TGF-beta inhibits the growth of normal bone marrow progenitor cells in vitro. Despite the well documented activities of these cytokines as hematopoietic suppressors, the mechanisms and the signaling cascades, whose activations are required for such effects, remain unknown. In the present study we demonstrate that type I IFNs (IFNalpha and IFNbeta ), as well as TGF-beta , induce activation of p38 in enriched erythroid progenitors, providing the first evidence for activation of the p38 Map kinase pathway in primitive hematopoietic cells. Most importantly, our data indicate that specific inhibitors of the p38 Map kinase reverse the suppressive effects of type I IFNs and TGF-beta on normal hematopoiesis. By using a similar approach, we established recently (59) that the function of p38 is essential for the generation of suppressive effects of IFNalpha on leukemic CFU-GM progenitors from patients with chronic myelogenous leukemia. SB203580 and SB202190 act by binding to the ATP site of the p38 molecule and abrogating its kinase activity. Mutagenesis studies and x-ray crystallographic structures of p38-inhibitor complexes have established the basis of their selectivity (40-42). Both SB203580 and SB202190 have similar target specificities, and in addition to inhibiting p38 (also called p38alpha ), they inhibit the p38beta 2 kinase isoform but not the p38gamma and p38delta isoforms of the same family (44-47). Thus, our data demonstrating reversal of the growth inhibitory effects of IFNalpha , IFNbeta , and TGF-beta by treatment with these pyridinyl imidazole compounds provide strong evidence for an important role of p38 (p38alpha ) and possibly p38beta 2 in the induction of the inhibitory effects of type I IFNs and TGF-beta on hematopoietic progenitor cell growth and differentiation.

Our data also demonstrate that although type I IFNs activate the Erk pathway in primary hematopoietic progenitors, TGF-beta does not. Most importantly, inhibition of Erk kinase activation by the PD98059 inhibitor does not affect the induction of the suppressive effects of type I IFNs and TGF-beta on normal hematopoietic progenitors. Previous studies (68, 69) have shown that Erk kinases can inhibit cell proliferation and/or stimulate cell differentiation in response to phorbol 12-myristate 13-acetate or retinoic acid in the HL-60 leukemic cell line. Thus, the Erk pathway may play a role in the generation of growth inhibitory and differentiation signals in response to selective stimuli in certain leukemic phenotypes. Nevertheless, our data clearly establish that in normal hematopoietic progenitors this pathway does not play a role in mediating the suppressive effects of type I IFNs and TGF-beta .

Type I IFNs and TGF-beta activate different signaling pathways. The type I IFN receptor activates Jak-Stat pathways to regulate gene transcription, whereas the TGF-beta receptor transduces signals via engagement of members of the Smad family of transcription factors (32, 61-63). It is of particular interest that, despite the fact that these cytokines activate different cascades to regulate the transcriptional machinery, both have as a common effector the p38 Map kinase pathway. In previous studies (24, 26), we have established that the function of p38 is required for transcriptional regulation via ISRE or GAS elements. As all interferon-stimulated genes have in their promoters ISRE and/or GAS elements, these studies have established that p38 activation is required for gene transcription of essentially all interferon-stimulated genes. The effects of p38 on IFNalpha -dependent gene transcription are unrelated to any effects on the activation of the Stat pathway, as p38 inhibition does not regulate tyrosine or serine phosphorylation of Stats and has no effects on DNA binding of Stat complexes (26). It is therefore possible that p38 mediates IFN-dependent antiproliferative effects by up-regulating IFN genes that mediate growth inhibitory responses. In fact, previous studies have established that several IFN-regulated genes mediate antiproliferative responses and/or exhibit tumor suppressor activity, including the PML gene (46), PKR (66, 67), and IRF-1 (65). Similarly, TGF-beta -induced activation of p38 and its downstream effector Tak-1 (63) plays an important role in TGF-beta transcriptional regulation, by activating AP-1 complexes via phosphorylation of c-Jun (64). Thus, the mechanisms by which p38 regulates TGF-beta -dependent hematopoietic suppression may also involve induction of transcription of genes that suppress cell growth.

In addition to its effects on gene regulation, activation of the p38 Map kinase pathway by IFNs and TGF-beta may have additional effects that mediate growth inhibition, such as regulation of signals that modify cell cycle progression in hematopoietic cells. An involvement of p38 and its upstream kinases MKK3 and MKK6 in the induction of G1/G0 cell cycle arrest has been documented previously (49, 50) and has been attributed to an antagonism of Erk-mediated expression of cyclin D1 (51). Another mechanism by which p38 may negatively regulate cell cycle progression is activation of the mitotic spindle assembly checkpoint pathway that monitors the correct formation of the spindle and attachment of kinetochores. In support of this, previous studies (52) have shown that treatment of cells with nocodozole, which disrupts the spindle, causes activation of p38 during the M-phase but not other phases of the cell cycle, whereas checkpoint activation can be suppressed by SB203580. There is also evidence that p38 and its upstream regulator, MKK6, are involved in the causation of a G2 arrest after ultraviolet or gamma -irradiation (53). In this case, the mechanism appears to be a p38-dependent inhibition of cdc25B activation, via phosphorylation of serines 309 and 361 (54). On the other hand, p38 apparently regulates cdc42-mediated G1 cell cycle arrest in response to serum starvation (55), indicating that p38 can regulate distinct phases of the cell cycle machinery in response to different stress stimuli.

p38 has been also implicated in the induction of apoptosis under certain conditions, but the generation of apoptotic effects appears to be very specific to cell type and context (56, 57). Our data clearly establish that IFNalpha and TGF-beta do not induce apoptosis of hematopoietic progenitors and that SB203580 treatment has no noticeable effects on the rate of apoptosis of hematopoietic cells. These findings are consistent with previous work that has shown that the antimitogenic activity of IFNalpha is primarily ascribed to G1 cell cycle arrest and not to induction of apoptosis (reviewed in Ref. 58). Although type I IFNs are known to induce G0/G1 arrest in other cellular types (58), our findings provide the first direct evidence that this occurs in human hematopoietic precursors. Our data demonstrating that p38 is required for induction of G0/G1 arrest of primitive progenitors and hematopoietic suppression in response to IFNalpha and TGF-beta provides evidence for a novel role of the p38 pathway in the regulation of normal hematopoiesis. It is possible that p38 functions as a common signaling mediator of hematopoietic suppression, providing a link between the pathways of different cytokines that inhibit normal hematopoietic progenitor growth. Future studies to define the validity of such an hypothesis are warranted and may have important clinical implications, because they could result in the development of clinical methodologies to protect the hematopoietic system during cytokine treatment for malignancies.

    ACKNOWLEDGEMENT

We thank Dr. Gary Bokoch for providing the pGEX construct for the production of the GST-PBD fusion protein.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA77816 and CA73381, by a Merit Review Grant from the Department of Veterans Affairs, and a grant from the American Cancer Society, Illinois Division (to L. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Section of Hematology-Oncology, the University of Illinois at Chicago, MBRB, MC-734, Rm. 3150, 900 S. Ashland Ave., Chicago, IL 60607-7173. Tel.: 312-355-0155; Fax: 312-413-7963; E-mail: Lplatani@uic.edu.

Published, JBC Papers in Press, December 31, 2001, DOI 10.1074/jbc.M106640200

    ABBREVIATIONS

The abbreviations used are: IFN, interferon; Stat, signal transducer and activator of transcription; Mapk, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; GST, glutathione S-transferase; CFU-E, colony-forming unit-erythroid; BFU-E, burst-forming unit-erythroid; TGF-beta , transforming growth factor beta ; ISREs, interferon-stimulated response elements; IMDM, Iscove's modified Dulbecco's medium; Erk, extracellular signal-regulated kinase; CFU-GM, colony-forming units granulocyte-macrocytic; Mek, Mapk/Erk kinase; GAS, interferon gamma -activated site.

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DISCUSSION
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