Claudine Billat

Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402–23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995)Blood 86, 598–606).

Murine erythroleukaemia cells (Friend cells) possess high-affinity binding sites for erythropoietin

Murine erythroleukaemia cells represent erythroid precursors blocked near the CFU‐E or proerythroblast stage. In contrast to their non‐leukaemic equivalents, neither their proliferation nor their differentiation seems to be affected by erythropoietin. However, we show in this paper that both uncommitted and committed, benzidine‐positive, cells bind iodinated erythropoietin. The binding is of high affinity (K d = 490 ± 160 pM) and reversible with a half‐life of the complex of 77 ± 19 min. The number of binding sites is low (300–600 per cell). In contrast the haematopoietic non‐erythroid cell lines HL 60 and L 1210 and the myeloiderythroid human cell line K 562 do not exhibit specific binding. If these binding sites represent true hormone receptors, their presence on a permanent cell line should facilitate erythropoietin receptor purification

Activation of Raf-1 and mitogen-activated protein kinases by erythropoietin and inositolphosphate-glycan in normal erythroid progenitor cells: involvement of protein kinase C.

In this work, we show that erythropoietin and inositolphosphate-glycan activate Raf-1 and the mitogen-activated protein kinases (MAP kinases) in normal erythropoietin-responsive cells. Using a protein kinase C (PKC) activator such as the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate and the PKC inhibitor GF109203X, we investigated a possible involvement of PKC during activation of Raf-1 and MAP kinase by erythropoietin or inositolphosphate-glycan. We found that erythropoietin increased MAP kinase level with a maximum stimulation reached at 5-10 min. Inositolphosphate-glycan and 12-O-tetradecanoyl-phorbol-13-acetate increased MAP kinase activity in the same manner. This activity was inhibited by cell preincubation with GF109203X. Two MAP kinase isoforms were present in erythroid progenitor cells, the 44 and 42 kDa proteins. We report here that erythropoietin, inositolphosphate-glycan, and 12-O-tetradecanoyl-phorbol-13-acetate activated only the p44 form (erk-1) of MAP kinase and the Raf-1 protein. GF109203X was used at a concentration which inhibited by 50% erythroid colonie (CFU-E) proliferation and differentiation induced by erythropoietin or inositolphosphate-glycan. These results support the hypothesis that erythropoietin and inositolphosphate-glycan activate Raf-1 and MAP kinases in normal erythroid progenitor cells and suggest that this activation involves PKC.

Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-γ2 activation in erythropoietin-stimulated cells

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.

Involvement of the Src kinase Lyn in phospholipase C-γ2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.

Involvement of the p38 mitogen‐activated protein kinase pathway in tissue inhibitor of metalloproteinases‐1‐induced erythroid differentiation

We examined the role of the mitogen‐activated protein (MAP) kinase pathway in tissue inhibitor of metalloproteinases‐1 (TIMP‐1)‐mediated cellular effects in a human erythroleukemic cell line UT‐7. We show that TIMP‐1 induced both UT‐7 cell erythroid differentiation and proliferation and tyrosine phosphorylation of many intracellular proteins. Using a panel of phosphospecific antibodies, we also demonstrate that phosphorylation of the p38 and c‐Jun N‐terminal kinases is increased by TIMP‐1 whereas phosphorylation of extracellular signal‐regulated kinase 1/2 is not induced. Moreover, inhibition of the p38 activity by SB203580 significantly reduces erythroid differentiation induced by TIMP‐1, suggesting that the p38 MAP kinase pathway is involved in TIMP‐1‐induced erythroid differentiation.

Erythropoietin induces glycosylphosphatidylinositol hydrolysis. Possible involvement of phospholipase c-gamma(2).

We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-γ2 in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-γ2was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-γ2 hydrolyzed purified [3H]GPI indicating that GPI hydrolysis and PLC-γ2 activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-γ2, whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-γ2activation.

Mode of action of erythropoietin and glucocorticoids on the hepatic erythroid precursor cells: role of prostaglandins.

The hypothesis that prostaglandins, and especially PGE2, are the second messengers of erythropoietin (Ep) and that glucocorticoids inhibit Ep action by inhibiting PG synthesis was tested on the erythroid cell line from fetal rat liver. The optimal (10(-9) M) stimulatory concentration of PGE2 did not reproduce, by far, the maximal effect of Ep on the growth of CFUE erythroid colonies. Ep did not increase PGE2 release in liquid culture media of cell suspensions made of the whole erythroid line or enriched (over 85%) in precursor cells. Ep did not modify the turnover rate of arachidonate. Nevertheless, indomethacin partially inhibited Ep effect on CFUE development, and this inhibition was abolished by PGE2. These results suggest that PGE2 potentiates Ep action but is not its second messenger. Spontaneous PGE2 release in liquid culture media brought about concentrations of the order of 10(-9) M, and 10(-7) M dexamethasone completely inhibited this release. Part of (but not all) the anti-Ep effects of glucocorticoids might thus be mediated this way. Dexamethasone effects required previous protein synthesis.

Effect of the antiglucocorticoid agent RU 38486 on the dexamethasone inhibition of Friend cell differentiation

Glucocorticoid hormones are known to inhibit the erythroid differentiation of Friend cells. The mechanism of action of these hormones has been questioned, and results suggesting an action not involving the nuclear binding of the receptors have been published. We have used the antiglucocorticoid RU 38486 to block the inhibitory effect of dexamethasone on the induced differentiation of Friend cells. Our results strongly suggest a glucocorticoid action involving the binding of classical receptors to the cell nucleus.

In vivo effects of glucocorticoid excess on the contents of the rat fetal liver in erythroid progenitors.

Following a laparotomy of the pregnant rat at 12 days of gestation, erythroid cell suspensions prepared from the fetal livers at 14 days contained an increased proportion of progenitor cells forming colonies after 2 or 7 days of culture. When laparotomy was performed at 14 days and the fetal livers were sampled at 16 days, the opposite effects were observed. Injection of 0.25 mg/kg dexamethasone to the 12 days pregnant rat increased the proportion of erythroid progenitors in suspensions from 14 days fetal livers; injection of 10 mg/kg (a long-acting dose) produced the opposite effects. Both the composition of the erythroid cell line and its environment change between 12 and 14 days, and these modifications might explain the inversion of glucocorticoid effects between these two stages.