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Cell Growth & Differentiation Vol. 11, 573-580, November 2000
© 2000 American Association for Cancer Research

Articles

Erythropoietin Induction of Tissue Inhibitors of Metalloproteinase-1 Expression and Secretion Is Mediated by Mitogen-activated Protein Kinase and Phosphatidylinositol 3-kinase Pathways1

Zahra Kadri, Emmanuelle Petitfrère, Cédric Boudot, Jean-Marc Freyssinier, Serge Fichelson, Patrick Mayeux, Hervé Emonard, William Hornebeck, Bernard Haye and Claudine Billat2

Laboratoire de Biochimie, Centre National de la Recherche Scientifique Formation de Recherche en Evolution 2260, Institut Fédératif de Recherche 53 Biomolécules, UFR Sciences Exactes et Naturelles, UFR de Medecine, Université de Reims Champagne-Ardenne, F51687 Reims Cedex 2, France [Z. K., E. P., C. B., H. E., W. H., B. H., C. B.], Laboratoire d’ hématopoïèse, Site Transfusionnel Cochin [J-M. F., S. F.], Institut Cochin de Génétique Moléculaire, Institut National de la Santé et de la Recherche Médicale U363, Université René Descartes [P. M.], Hôpital Cochin, F57014 Paris, France

Abstract

In the present study, we demonstrate that erythropoietin (Epo) induces the expression and the release of tissue inhibitors of metalloproteinase-1 (TIMP-1) in a time- and dose-dependent manner in Epo-dependent cell line UT-7 cells and in normal human erythroid progenitor cells from cord blood (CD36+) and required de novo protein synthesis. TIMP-1 was not expressed in the absence of Epo. Inhibition of the mitogen-activated protein kinase pathway by the specific inhibitors PD98059 and U0126 and of phosphatidylinositol 3-kinase by LY294002, strongly inhibited Epo-induced TIMP-1 expression and secretion. In the absence of Epo, both latent and active forms of matrix metalloproteinase-9 (MMP-9) were secreted into media. Upon Epo stimulation, MMP-9 and pro-MMP-9 secretion was inhibited in a dose-dependent manner parallel to TIMP-1 induction. The addition of PD98059, U0126, and LY294002 in the presence of Epo restored MMP-9 production in UT-7 and CD36+ cells. Our findings strongly suggest an inversely coordinated regulation of the TIMP-1 gene and MMP-9 production by Epo via mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

Introduction

Epo3 exerts its effects by binding to a cell surface receptor. Epo-binding induces a rapid phosphorylation of JAK2 tyrosine kinase. JAK2 activation triggers a rapid but transient tyrosine phosphorylation of the Epo-receptor and of many intracellular signaling molecules leading to the growth and differentiation of erythroid cells (1, 2, 3) . Several intracellular pathways are activated by Epo such as the Ras/extracellular signal-regulated kinase pathway (4 , 5) , which is thought to be an important regulator of mitogenic activity (6) , and the p38 MAP kinase cascade (7 , 8) . PI 3-kinase is also activated by Epo through several pathways; a direct association between PI 3-kinase and the Epo-receptor has been shown (9, 10, 11, 12) . Alternatively, associations between PI 3-kinase and the protein adapters insulin receptor substrate-2 and Grb2-associated binder-1 have been described in UT-7 cells (13 , 14) . It has also been reported that PI 3-kinase could be activated by binding to Vav (15) . STAT5 is known to be activated by a wide variety of cytokines, and several experiments suggest that STAT5 was involved in cell proliferation in hematopoietic cell lines (16 , 17) .

TIMPs are secreted as multifunctional proteins that can inhibit specifically the catalytic activity of MMPs, thus controlling extracellular matrix homeostasis (18) . Presently, four mammalian TIMPs have been identified: TIMP-1 to TIMP-4 (19, 20, 21, 22) . TIMP-1 is a 28.5-kDa glycoprotein that is secreted in a soluble form by different cell types and forms a 1:1 complex with MMP-9 (23) . Besides acting as proteinase inhibitors, TIMP-1 and TIMP-2 may also modulate cell growth and were first identified as having erythroid-potentiating-activity. Subsequently, TIMP-1 was found to be able to stimulate the growth and differentiation of murine erythroid precursors (24) and of K-562 and ELM-I-1-3 erythroleukemia cell lines (25, 26, 27) . TIMP-1 has been also shown to act as a serum mitogen on a wide array of cultured cells (26 , 28 , 29) . In addition, TIMP-1 has been described to promote the survival of cells and to suppress apoptosis in B cells (30) or in human breast epithelial cells (31) . The effect of TIMP-1 on cell growth seems to involve functions that are distinct from those imparting MMP inhibition (32) .

TIMP-1 expression is regulated by a variety of cytokines or growth factors and other soluble factors in hematopoietic cell lines (33 , 34) , and its expression was shown to be controlled by several DNA response elements that respond to variations in the level and activity of the transcription factors AP-1 and Ets transcriptional regulatory proteins (35) . Furthermore, STAT3 can bind AP-1/Ets sequences of the TIMP-1 promoter then contributing to transcription by OSM (36 , 37) as c-Fos and Jun (38) . Recently, a new cis-acting element named upstream TIMP-1 element-1 has been shown to be essential for transcriptional activation of the human TIMP-1 promoter (39) .

Different signaling pathways have been shown to be involved in the regulation of the expression of MMPs. A cross-talk between the MAP kinase and JAK-STAT signaling pathways has been shown to be required to achieve maximal induction of the OSM-response element encompassing the AP-1 and STAT binding sites leading to activation of MMP-1 gene expression by OSM (37) . Recently, inhibition of PI 3-kinase, MAP kinase (MEK1), or p38 MAP kinase by specific inhibitors was shown to strongly promote fibronectininduced MMP-2 and MMP-9 in T-lymphocytes (40) .

In this report, using a human leukemic Epo-dependent cell line, UT-7, and normal human erythroid progenitor cells from cord blood, we examined the Epo-regulation of TIMP-1 expression and secretion. We showed that Epo induced both TIMP-1 mRNA accumulation and TIMP-1 secretion and repressed MMP-9 secretion. The MEK inhibitors (PD98059 and U0126) and the PI 3-kinase inhibitor (LY294002) were found to inhibit Epo-induced TIMP-1 expression and secretion. As TIMP-1 disappeared, MMP-9 was recovered in culture medium.

Results

Epo Induces TIMP-1 mRNA Accumulation.
To investigate responsiveness of the TIMP-1 gene to Epo, UT-7 cells were cultured with or without Epo, and TIMP-1 mRNA levels were estimated at different times of culture. As compared with control, Epo was found to increase TIMP-1 mRNA by 4 h before reaching a steady state after 10 h that lasted for 48 h (Fig. 1A)Citation . TIMP-1 mRNA was also induced by Epo in CD36+ cells after 10 h of culture. Induction of TIMP-1 mRNA transcript expression was dose-dependent, and maximal stimulation was obtained with 1 unit/ml of Epo (Fig. 1B)Citation . To determine whether Epo induction of TIMP-1 mRNA requires de novo protein synthesis, we used CHX, an inhibitor of protein synthesis. Then, cells were treated with CHX (500 µM) for 4 h with or without Epo. Treatment of cells with CHX and Epo completely inhibited induction of TIMP-1 mRNA (Fig. 2)Citation .



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  		Fig. 1. Epo induces TIMP-1 mRNA in a time and dose-dependent fashion. A, UT-7 cells were treated with Epo (2 units/ml) from 0 to 48 h and CD36+ cells from 0 to 10 h. B, UT-7 cells were treated for 10 h with different Epo concentrations. RNA were extracted and probed by Northern analysis for TIMP-1 mRNA. Ethidium bromide staining of 28S is shown in the top panel to indicate equivalent loading of RNA samples. UT-7 TIMP-1 mRNA levels were quantified by densitometry as described in "Materials and Methods" and were expressed as the mean ± SEM of three separate experiments.

 


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  		Fig. 2. Effect of protein synthesis inhibition on Epo-induced TIMP-1. UT-7 cells were starved for 16 h and left untreated (control), treated with Epo, treated with CHX (500 µM) alone or treated with Epo with CHX for 4 h. RNA was prepared and hybridized with a TIMP-1 cDNA probe as described in "Materials and Methods." Equal RNA application was shown as an ethidium-bromide stained gel.

 
Epo Induces TIMP-1 Secretion.
To examine whether TIMP-1 mRNA was translated into the corresponding protein, Western immunoblot analysis was performed on culture media from UT-7 and CD36+ cells. The time-dependent induction of TIMP-1 expression by Epo was also observed at the protein level with a strong induction at 48 h of culture. TIMP-1 secretion in normal erythroid progenitor cell media was also induced by Epo (2 units/ml) in a time-dependent manner (Fig. 3A)Citation . TIMP-1 was not detected without Epo. No TIMP-2 was detected in UT-7 and CD36+ culture media after 24, 48, or 72 h of culture without or with Epo (2 units/ml; not shown). As shown in Fig. 3BCitation , Epo-induced TIMP-1 secretion was also dose-dependent, and a maximum was obtained after the addition of 1 unit/ml of Epo to the culture medium of UT-7 or CD36+ cells.



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  		Fig. 3. Epo-induced TIMP-1 secretion in UT-7 and CD36+ cells. A, UT-7 cells ({square}) or CD36+ cells ({blacksquare}) were cultured for different times (0-72 h) in the presence of Epo (2 units/ml) or not. B, UT-7 ({square}) and CD36+ cells ({blacksquare}) were cultured for 24 h with different Epo concentrations. Media were analyzed by Western blotting with anti-TIMP-1 antibodies. Signals were quantified as described in "Materials and Methods." The results from one representative experiment are shown.

 
MEK and PI 3-kinase Inhibitors Down-Regulate Epo-induced TIMP-1 Expression.
MEK and PI 3-kinase inhibitors were added to UT-7 and CD36+ cells in the presence of Epo (2 units/ml) for 48 h. To determine the concentration to be used for further experiments, their effects were assessed on Epo-induced cell proliferation and differentiation. IC50 was obtained for Epo-induced CD36+ cell proliferation with 10 µM of PD98059 and 1 µM of U0126. These concentrations inhibit Epo-induced UT-7 cell proliferation by 25 and 42%, respectively. IC50 was obtained for Epo-induced CD36+ cell proliferation with 20 µM LY294002, a selective inhibitor of PI 3-kinase. This concentration also inhibited Epo-induced UT-7 cell proliferation by 70% (Fig. 4A)Citation . Epo-induced cell differentiation of CD36+ and UT-7 cells was not affected by the addition of PD98059, U0126, or LY294002 to the culture medium at the indicated concentrations nor on apoptosis (not shown). Specificity of inhibitors was tested on Epo-induced p44/42 MAP kinase phosphorylation in UT-7 and CD36+ cells because wortmannin, another PI 3-kinase inhibitor, was described previously to inhibit Epo-induced MAP kinase activation (41) . Fig. 4BCitation shows that LY294002 had no effect on p44/42 MAP kinase phosphorylation, indicating that PI 3-kinase acts independently of the MAP kinase pathway. Inhibition of the Epo-induced phosphorylation of p44/42 MAP kinase by U0126 and PD98059 confirmed the results obtained on Epo-induced proliferation. Then, Fig. 5Citation shows that TIMP-1 expression induced by Epo was significantly down-regulated by LY294002, strongly inhibited by PD98059, and almost completely inhibited by U0126 in UT-7 and CD36+ cells. The same results were obtained after 24 and 48 h of culture (not shown). These results suggest that Epo induces TIMP-1 gene expression through MAP kinase (p44/p42) and PI 3-kinase pathways.



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  		Fig. 4. Effects of MEK and PI 3-kinase inhibitors on the proliferation and on p44/42 phosphorylations induced by Epo. LY294002 (20 µM), PD98059 (10 µM), and U0126 (1 µM) were added to the culture medium for 48 h in the presence of Epo (2 units/ml). A, cell number was determined, and results were expressed as a percentage of stimulation (Epo = 100%) and are the mean ± SEM of three separate experiments ({square}) UT-7 cells, ({blacksquare}) CD36+ cells. B, cellular extracts from 0.75 x 106 UT-7 cells and from 2 x 106 CD36+ cells were analyzed by Western blot with anti-phosphoMAPK (p44/42) and anti-MAPK antibodies.

 


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  		Fig. 5. TIMP-1 mRNA expression in response to MEK and PI 3-kinase inhibitors. RNA were prepared from UT-7 and CD36+ cells cultured for 10 h without Epo or with Epo (2 units/ml) in the presence of U0126 (1 µM), PD98059 (10 µM), or LY294002 (20 µM). RNA was hybridized with a cDNA TIMP-1 probe and subsequently with a GAPDH probe. Equal RNA application was shown as an ethidium-bromide-stained gel.

 
MEK and PI 3-kinase Inhibitors Down-Regulate Epo-induced TIMP-1 Secretion.
To examine the effects of PD98059, U0126, and LY294002 on TIMP-1 secretion, Western blots were performed on culture media from UT-7 and CD36+ cells cultured for 48 h in the presence of Epo (2 units/ml) and different kinase inhibitors. Fig. 6ACitation shows that the MEK inhibitors PD98059 and U0126 inhibited TIMP-1 secretion in UT-7 cells and CD36+ cells in the same manner. LY294002 was shown to inhibit TIMP-1 secretion induced by Epo by ~50% in UT-7 cells, and by ~90% (Epo = 100%) in CD36+ cells. Fig. 6BCitation shows that other growth factors such as GM-CSF and SCF induce TIMP-1 secretion in culture media from UT-7 cells. The MEK inhibitors PD98059 and U0126 and the PI 3-kinase inhibitor LY294002 were shown to inhibit TIMP-1 secretion induced by SCF.



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  		Fig. 6. Effects of MEK and PI 3-kinase inhibitors on TIMP-1 secretion induced by Epo. A, UT-7 cells ({square}), or CD36+ cells ({blacksquare}) were grown under Epo (2 units/ml) for 48 h in the presence of PD98059 (10 µM), U0126 (1 µM), or LY294002 (20 µM). Media obtained after 48 h of culture were subjected to Western blot analysis using anti-TIMP-1 antibodies. Signal was quantified as described in "Materials and Methods." Results were expressed as a percentage of stimulation, (Epo = 100%) and are the mean ± SEM of three separate experiments. B, UT-7 cells were cultured for 48 h without growth factor (control) or with GM-CSF (2.5 ng/ml), or SCF (50 ng/ml) alone or in the presence of PD98059 (10 µM), U0126 (1 µM), or LY294002 (20 µM). Conditioned media were then treated by Western blot as described above. Rh-TIMP-1 (10 ng) was used as standard.

 
Epo Regulates MMP-9 Secretion via MAP Kinase and PI 3-kinase Pathways.
Since MMP-9 was described as the main protease target of TIMP-1, we investigated whether MMP-9 secretion could be also regulated by Epo. To this end, media from UT-7 cells cultured without or with different Epo concentrations for 24 h were analyzed by gelatin zymography. Fig. 7ACitation shows that after 24 h of culture without Epo, two secreted gelatinases of 84 and 92 kDa could be evidenced on zymograms. The 92-kDa protein was identified by Western-blot as the latent form of MMP-9. With Epo (2 units/ml), no pro-MMP-9 was detected in culture medium. A faint band (84 kDa) may represent the active MMP-9 form. When Epo was present in the culture medium, the amounts of secreted MMP-9 and pro-MMP-9 decreased as Epo concentrations increased and were totally inhibited by 0.1 unit/ml and 1 unit/ml of Epo for MMP-9 and pro-MMP-9, respectively (Fig. 7B)Citation . This effect was dose-dependent. MMP-9 was also found in media of CD36+ cells cultured without Epo for 24 h (not shown). The influence of MEK and PI 3-kinase inhibitors on MMP-9 production by UT-7 and CD36+ cells cultured in presence of Epo was further analyzed. Fig. 8Citation indicated that addition of PD98059, U0126, and LY294002 for 48 h restored MMP-9 secretion from cells. These results strongly suggest that Epo inhibits MMP-9 secretion via MAP kinase and PI 3-kinase pathways.



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  		Fig. 7. Epo dose-effect on MMP-9 production. A, UT-7 cells were grown in the presence of Epo (2 units/ml) for 24 h or not (control). One hundred and fifty µl of culture medium were concentrated and subjected to gelatin zymography and Western blot analysis using anti-pro-MMP-9 antibodies. Rh-pro-MMP-9 (300 ng) was used as standard (Std). B, UT-7 cells were cultured with or without different Epo concentrations for 24 h, and 150 µl of culture medium were concentrated and subjected to gelatin zymography. MMP-9 ({blacksquare}) and pro-MMP-9 ({square}) signals were quantified as described in "Materials and Methods." The results from one representative experiment are shown.

 


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  		Fig. 8. Effect of MEK and PI 3-kinase inhibitors on MMP-9 production. UT-7 cells (A) and CD36+ cells (B) were incubated with Epo alone (2 units/ml) or with specific inhibitors of MEK (PD98059, 10 µM; U0126, 1 µM) and with the PI 3-kinase inhibitor LY294002 (20 µM) for 48 h in the presence of Epo (2 units/ml). One hundred and fifty µl of culture medium were concentrated and subjected to gelatin zymography. MMP-9 ({blacksquare}) and pro-MMP-9 ({square}) signals were quantified as described in "Materials and Methods," and depicted results represent one representative experiment.

 
Discussion

Erythropoietin-induced TIMP-1 secretion was described previously in an Epo-responsive cell line ELM-I-1-3 (34) , but the regulation of TIMP-1 gene expression by Epo was not investigated. In this report we aimed to identify Epo-signaling elements that are involved in the regulation of TIMP-1 expression and secretion by erythroid cells. For that purpose, we used Epo-responsive cells: a human erythroleukemic Epo-dependent cell line, UT-7 cells, and normal erythroid progenitor cells from human cord blood. UT-7 cells were reported previously to express erythroid differentiation markers such as glycophorin-A in the presence of Epo (42) , and upon Epo stimulation. Epo-receptors, JAK2, and cytoplasmic proteins were tyrosine-phosphorylated (43) . CD36+ cells were also Epo-responsive cells, in as much as activation of STAT5 and tyrosine phosphorylation of the Eporeceptor and JAK2 were observed after Epo stimulation of these cells. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 days of secondary culture (44) .

All experiments were performed in a serum-free culture medium because FCS contains TIMPs and MMPs. Under these experimental conditions, Epo induces proliferation and differentiation of cells (not shown). No DNA fragmentation was noticeable when UT-7 cells were cultured for 3 days in the presence of Epo. In the absence of Epo, cells ceased to proliferate.

In this report, we clearly demonstrate that Epo induces TIMP-1 both at the mRNA and protein levels in a time- and dose-dependent manner. The increase in TIMP-1 mRNA following Epo addition requires a de novo protein synthesis. TIMP-1 mRNA translation in the corresponding protein was perfectly correlated.

By using specific kinase inhibitors, we showed that the MAP kinase pathway activated by Epo was involved in Epo-induced TIMP-1 expression and secretion in UT-7 cells and in CD36+ cells. Indeed, specific MEK inhibitors PD98059 and U0126 could inhibit TIMP-1 expression induced by Epo and secretion, an effect which correlated with Epo-induced cell proliferation but not with cell differentiation, because we found that cell proliferation was inhibited in the presence of PD98059 and U0126. Previous reports have shown that the MAP kinase pathway is a key regulator of cell proliferation in response to Epo (4 , 6) . Moreover, the activation of c-Jun and c-Fos proteins have been described to be important AP-1 factors involved in Epo-induced cell proliferation (45) . Blockade of c-Fos and c-Jun via blockade of ERK activation by U0126 (46) in cells grown under Epo, led to inhibition of Epo-induced TIMP-1 expression, suggesting a role of AP-1 in TIMP-1 gene regulation by Epo. Moreover, c-Jun was shown to be present in the AP-1 complex 3 h after Epo addition (45) in keeping with our results showing that Epo-induced TIMP-1 expression was observed as early as 4 h after stimulation of cells. These results were also in agreement with previous reports on TIMP-1 gene regulation by OSM (38) , where it was found that OSM stimulates c-Fos to bind a transcriptionally responsive AP-1 element within the TIMP-1 promoter. This response element that controls TIMP-1 gene expression was characterized previously. Logan et al. (35) showed that the TIMP-1 promoter contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. STAT3 and STAT1 activation by Epo was not observed in UT-7 cells (17) nor in CD36+ cells (44) and thereby cannot regulate AP-1 sites of TIMP-1 promoter as has been observed for OSM (36 , 37) .

PI 3-kinase activation is considered as a major step in mitogenic and in antiapoptotic signaling pathways (47 , 48) . Several reports suggest that PI 3-kinase could be a major intracellular signaling pathway in the mechanism of action of Epo (9, 10, 11, 12, 13, 14) . Addition of a selective PI 3-kinase inhibitor LY294002 (49) to cell culture medium led to an inhibition of TIMP-1 expression and secretion induced by Epo. Since this inhibitor has no effect on Epo-induced MAP kinase (ERK 1/2) activation, it suggests that PI 3-kinase signaling pathway plays a role in Epo-induced TIMP-1 expression and secretion in UT-7 and erythroid progenitor cells. This pathway was described to be important for proliferation of cells, and our data suggested that it could also act in Epo-mediated TIMP-1 gene regulation.

Activation of MAP kinase and PI 3-kinase pathways in UT-7 cells by GM-CSF or SCF (50 , 51) also induced TIMP-1 secretion. Furthermore, SCF-induced TIMP-1 secretion was inhibited by MEK or PI 3-kinase inhibitors and strengthened the fact that TIMP-1 secretion and expression was regulated by MAP kinase and PI 3-kinase pathways.

Both latent and active forms of MMP-9 were detected in the absence of Epo in culture medium but disappeared upon Epo stimulation of cells concomitant with TIMP-1 induction. Moreover, MMP-9 production seemed to be down-regulated by Epo-activated MAP kinase and PI 3-kinase pathways in UT-7 and CD36+ cells. Indeed, MMP-9 was found in culture media of cells stimulated with Epo when specific inhibitors of MEK, PD98059 and U0126, and of PI 3-kinase, LY294002, were added. Those findings strongly suggest that Epo could regulate in an inverse manner TIMP-1 and MMP-9 production. Only transforming growth factor-ß1 (52) and retinoids (53) were shown previously to down-regulate MMP-1, whereas TIMP-1 was up-regulated. In that sense, Epo appeared distinct from factors such as phorbol esters, IL 1ß (54) , OSM (36) , tumor necrosis factor{alpha}, and GM-CSF (55) which activated MMP-1 or MMP-9 and TIMP-1 in a coordinated fashion. Recently, activation of MAP kinase and PI 3-kinase was shown to repress fibronectin-induced MMP-2 and MMP-9 in T-lymphocytes (40) , but the authors could not detect any regulation of TIMP-1.

It needs to be emphasized that Epo could induce TIMP-1, but not TIMP-2, by activating the same signaling pathways both in UT-7 cells and in normal CD36+ progenitor cells. TIMP-1 production could further potentiate cell growth by directly acting on intracellular pathways (56) ; it could also possess an influence on programmed erythroid cell death, because TIMP-1 was reported to inhibit apoptosis in different cell lines (30 , 31) . Inverse regulation of TIMP-1 and MMP-9 by Epo also suggested that this hormone could actively modulate its cellular microenvironment, thus contributing to the maintenance of the integrity of the bone marrow matrix.

Materials and Methods

Materials.
{alpha}-MEM, Iscove’s modified Dulbecco’s medium, FCS, Trizol, and a random primers DNA labeling system were purchased from Life Technologies, Inc. Recombinant human Epo (specific activity of 120,000 units/mg) was obtained from Roche Molecular Biochemicals. The MEK-inhibitors PD98059, U0126, and the PI 3-kinase inhibitor LY294002, rh-TIMP-1, rh-proMMP-9, and mouse monoclonal anti-TIMP-1 and anti-proMMP-9 antibodies were obtained from Calbiochem (San Diego, CA). MAP kinase and phospho-MAP kinase antibodies were obtained from New England Biolabs, Inc. Hybond-N+ nylon membrane and enhanced chemiluminescence kit were from Amersham Pharmacia Biotech (Orsay, France). BSA, human holotransferrin, and all other reagents were from Sigma.

Cell Lines and Cell Cultures.
A subclone of the human leukemic cell line UT-7 (57) able to grow in Epo alone was used. These cells were cultured in {alpha}MEM containing 10% FCS supplemented with 2 units/ml Epo. Before each experiment, cells were serum- and growth factor-deprived by incubation overnight in Iscove’s modified Dulbecco’s medium containing 0.2% deionized BSA and 0.2% of human holotransferrin. Then, UT-7 cells were cultured in the same medium with or without 2 units/ml Epo, with 2.5 ng/ml GM-CSF or 50 ng/ml SCF. Normal human erythroid CD36+ progenitors were obtained after 7 days of culture of CD34+ progenitor cells from umbilical cord blood (58) . CD36+ cells were purified from the other cells by using anti-CD36 antibodies coupled to immunomagnetic beads and selected on Mini-MACS columns as described previously (59) . Then, cells were culture in FCS deprived Iscove’s modified Dulbecco’s medium in the presence of Il-3 (10 ng/ml), Il-6 (10 ng/ml), SCF (25 ng/ml), and Epo (2 units/ml) for 24 h. Before each experiment, the cells were deprived of growth factor by a 4 h incubation in Iscove’s modified Dulbecco’s medium containing 0.2% deionized BSA and 25 µg/ml iron-loaded human holotransferrin. After this period, cells were washed and cultured in the same medium with or without Epo (2 units/ml). In some experiments, kinase-inhibitors were added to the culture medium for 48 h. Cell viability was confirmed by trypan blue exclusion, and DNA fragmentation was used to test apoptosis (60). Cell differentiation was assessed by benzidine test (41) .

Total Cellular Extracts.
Cells were starved of growth factor and FCS overnight as described above. Then, they were washed twice with Iscove’s modified Dulbecco’s medium, 25 mM HEPES, and aliquots of cells were preincubated at 37°C for 20 min in Iscove’s modified Dulbecco’s medium with or without kinase-inhibitors before stimulation with 10 units/ml Epo for 10 min. Incubations were stopped with phosphate-buffered saline containing 1 mM Na2VO4. Then, cellular pellets were lysed with 50 µl of 2x loading buffer containing 1 mM Na2VO4, boiled for 5 min, and submitted to Western blot analysis.

RNA Extraction and Northern Blot Analysis.
Total RNA was isolated from cultured cells using Trizol LS reagent according to the manufacturer’s instructions. Extracted total RNA (10–20 µg) was subjected to electrophoresis in 1% agarose containing 2.2 M formaldehyde and transferred to Hybond-N+ membranes in 40 mM NaOH for 2 h. Membranes were then prehybridized in 50% formamide, 1% SDS, 5x Denhardt’s solution, 0.75 M NaCl, 75 mM C6H5O2Na3, 2H2O, and 50 µg/ml salmon sperm DNA at 42°C for 2 h and hybridized overnight by the addition of DNA probes which were 32P-labeled using the Random Primers DNA labeling system. Northern blot analysis of RNA was performed with a 880-bp human TIMP-1 cDNA probe (generously provided by Dr. Marmer, St. Louis, MO) and a human glyceraldehyde-3-phosphate dehydrogenase cDNA probe. Membranes were then washed four times in two-fold concentrated standard saline citrate (SSC: 150 mM NaCl, 15 mM sodium citrate), 0.1% SDS at 42°C and once in the same buffer at 55°C. Then autoradiography was performed using Kodak X-OMAT films.

Western Blot Analysis.
Relative levels of TIMP-1 in culture media were assessed by Western blot analysis. Samples of culture medium (40–150 µl) were mixed with 2x Laemmli sample buffer (1:1), boiled for 5 minutes, and then resolved in 12% SDS-polyacrylamide gel before being transferred to a nitrocellulose filter. Membranes were blocked with 5% milk powder in Tris-buffered saline. Proteins were detected using a mouse monoclonal anti-TIMP-1 or anti-proMMP-9 primary antibody and with horseradish peroxydase-conjugated secondary antibody before adding enhanced chemiluminescence substrate solution and exposing to Kodak X-OMAT film. Signal was quantified by densitometry (Imaging Densitometer GS-670; Bio-Rad) and expressed as arbitrary units (optical density/mm2) for 106 cells. Total cellular extracts were subjected to SDS-PAGE using 10% polyacrylamide gels, and proteins were electrophoretically transferred to protran nitrocellulose membrane and prepared as described above. Proteins were detected using anti-MAP kinase and anti-phospho-MAP kinase antibodies.

Gelatin Zymography.
Cultured media (150 µl) were concentrated using a speed-vac (Concentrator 5301; Eppendorf) and subjected to SDS-PAGE through 10% polyacrylamide gels copolymerized with 0.2 mg/ml gelatin. Gels were washed twice with 2.5% Triton-X-100 and incubated overnight at 37°C in 50 mM Tris-HCl (pH 7.6), 5mM CaCl2, and 200 mM NaCl. Gels were fixed and stained with 0.1% Coomassie Blue G250. After destaining, gelatinolytic signals were quantified by densitometry as above.

Acknowledgments

The authors thank Marie-Line Sowa for expert technical assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Ligue Nationale contre le Cancer, Comités de la Marne, de l’Aube, de l’Aisne, and by Contract 5429 from the Association pour la Recherche sur le Cancer and by the Association Régionale pour l’Enseignement et la Recherche Scientifique. Back

2 To whom requests for reprints should be addressed, at Laboratoire de Biochimie, UFR Sciences Exactes et Naturelles, BP 1039, Université de Reims Champagne-Ardenne, 51687 Reims Cedex 2, France. Fax: 33-03 26-91-32-79; E-mail: claudine.billat{at}univ-reims.fr Back

3 The abbreviations used are: Epo, erythropoietin; JAK, janus kinase; MAP, mitogen activated protein; PI 3-kinase, phosphatidylinositol 3kinase; STAT, signal transducer and activator of transcription; TIMP, tissue inhibitors of metalloproteinase; MMP, matrix metalloproteinase; AP, activating protein; OSM, oncostatin M; MEK, MAP kinase/ERK kinase; II, interleukin; SCF, stem cell factor; CHX, cycloheximide; GM-CSF, granulocyte macrophage-colony stimulating factor. Back

Received for publication 5/24/00. Revision received 9/13/00. Accepted for publication 9/25/00.

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