Emmanuelle Petitfrère

Tissue inhibitor of metalloproteinases-1 signalling pathway leading to erythroid cell survival

Tissue inhibitors of metalloproteinases (TIMP) are specific inhibitors of matrix metalloproteinases (MMPs) and thus participate in maintaining the balance between extracellular matrix deposition and degradation in several physio-pathological processes. Nevertheless, TIMP must be regarded as multifunctional proteins involved in cell growth, angiogenesis and apoptosis. The molecular mechanisms induced by TIMP remain largely unknown. In the present study, we provide evidence that TIMP-1 induces a significant anti-apoptotic effect in the human erythroleukaemic cell line UT-7 and in the murine myeloid cell line 32D. Using specific kinases inhibitors, we show that TIMP-1-mediated cell survival is dependent upon Janus kinase (JAK) 2 and phosphoinositide 3-kinase (PI 3-kinase) activities. By transient transfection of dominant-negative Akt in UT-7 cells, we demonstrate that this kinase is crucial for the TIMP-1 anti-apoptotic effect. Moreover, TIMP-1 enhances specific phosphorylation of both Akt and Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death) in a PI 3-kinase-dependent manner and, besides, controls the level of the anti-apoptotic protein Bcl-X(L). We conclude that TIMP-1 induces haematopoietic cell survival via the JAK2/PI 3-kinase/Akt/Bad pathway.

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.

Partial characterization of a glycosylphosphatidyl-inositol (GPI) in porcine thyroid cultured cells.

The aim of this study was to provide information on the structure of glycosylphosphatidylinositol (GPI) and to characterize this novel phospholipid isolated from pig thyroid. We investigated the incorporation of different radioactive precursors: [3H]glucosamine, [3H]inositol, [3H]oleic acid, [32P]orthophosphate and demonstrated the presence of these moieties in the structure of GPI. After labelling and hydrochloric acid hydrolysis, the major part of radioactivity comigrated with glucosamine used as marker. Cleavage of GPI by nitrous acid deamination indicated the presence of a glycosidic bond between the lipid moiety and glucosamine. The fatty acid composition of diacylglycerol was also studied by gas chromatography. Oleic acid was found preferentially to other fatty acids. In a previous paper we reported that a chronic treatment with 0.1 mU/ml thyrotropin (TSH) of thyroid cultured cells for 3 days produced a large increase in the turnover rate of GPI and concomitantly the release of a water-soluble product of GPI hydrolysis: an inositolphosphateglycan (IPG). These findings confirm that GPI may play a role in membrane signalling systems and thyroid cell regulation.

Purification and analysis of the neutral glycan moiety of glycosyl phosphatidylinositol from porcine thyroid cells

Metabolic labelling of inositolphosphate glycan with radioactive precursors is not sufficient to characterize and assess the involvement of the glycosyl phosphatidylinositol/inositolphosphate glycan (GPI/IPG) system in porcine thyroid cell signal transduction machinery. A protocol is described for the isolation and purification of free GPI using differential polarity of lipids and sequential thin layer chromatography. The purification until homogeneity of GPI constitutes a required step for gas chromatographic analysis. Next, successive chemical treatments allowed us to remove the neutral glycan moiety of thyroidal GPI, and its composition was obtained by gas chromatography. The proposed structure is consistent with data available for GPI anchor, but differs from compositional analysis data reported for insulin-sensitive GPI. Our results support the existence in porcine thyroid cells of the GPI/IPG system, which can take part in TSH-dependent signal transduction processes.