Tissue inhibitor of metalloproteinase-1 promotes hematopoietic differentiation via caspase-3 upstream the MEKK1/MEK6/p38alpha pathway.

Besides its matrix metalloproteinases inhibitory activity, TIMP-1 exhibits other biological activities such as cell survival and proliferation. The intracellular signalling pathway elicited by TIMP-1 begins to be elucidated. We have shown previously that the caspase-3 and the p38alpha MAP kinase were activated during TIMP-1-induced UT-7 cells erythroid differentiation. In this study, we demonstrated that TIMP-1 differentiating effect can be extended to the IL-3-dependent myeloid murine 32D cell line and human erythroid progenitors derived from cord blood CD34(+) cells. By performing small interfering RNA transfection and using chemical inhibitors, we evidenced that caspase-3 was involved in TIMP-1 differentiating effect. We then identified the MEKK1 kinase as a caspase-3 substrate and demonstrated that the MEKK1/MEK6/p38alpha pathway was activated downstream the caspase-3 in TIMP-1-induced hematopoietic differentiation.

TGFbeta1 effects on functional activity of porcine thyroid cells cultured in suspension.

Thyrotropin (TSH) and transforming growth factor beta 1 (TGFbeta1) have major roles in the regulation of folliculogenesis and differentiation in thyroid cells. Isolated porcine thyroid cells cultured in the presence of TSH on a plastic surface recover a follicular architecture and exhibit normal functional properties. The addition of TGFbeta1 to the culture medium induces important morphological changes and extracellular matrix remodelling. Similarly, thyroid cells lose their ability to organify iodine and their responsiveness to adenylate cyclase. The aim of this study was to analyse the influence of TGFbeta1 on the functional activity of thyrocytes in suspension culture, independent of follicle disruption. In this system, we demonstrate that TGFbeta1 inhibits expression of thyroperoxidase, NADPH oxidase activity, iodine uptake and, consequently, iodine organification. Moreover, TGFbeta1 decreases basal and TSH-stimulated cAMP production and TSH receptor expression. Taken together, these data converge to demonstrate an essential role of TGFbeta1 in the regulation of the thyroid cell function.

TSH-induced differentiated functions correlate with enhancement of phosphotyrosine phosphatase activity in thyroid cells. Effect of phorbol 12-myristate 13-acetate.

TSH-treated pig thyroid cells reorganize into follicle-like structures and exhibit differentiated functions. TSH also induces a phosphotyrosine phosphatase (PTPase) activity evaluated by phosphorylated substrate hydrolysis. Incubation of thyrocytes with various concentrations of 8-bromo-cyclic AMP or forskolin induces an increase of PTPase activity in a dose-dependent manner. During the culture period, adenylyl cyclase sensitivity, protein binding iodine and PTPase activity progressively increase from the first to the fourth day of the culture. Chronic treatment with phorbol 12-myristate 13-acetate (PMA) significantly inhibits PTPase activity during the first 24 h following PMA addition. GF 109203X, a specific inhibitor of protein kinase C, abolishes the inhibitory effect of PMA. Electrophoresis of membrane extracts allowed us to demonstrate a phosphatase activity at 111 kDa (p111). Vanadate inhibits this activity, indicating that p111 is a PTPase. This p111 is significantly reduced in PMA-treated cells. These data suggest that PTPase activity evidenced at 111 kDa is correlated with a differentiated state of primary cultured pig thyroid cells induced by TSH.

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 induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

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.

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)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was 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-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) 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-gamma(2), 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-gamma(2) activation.

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.

Influence of transforming growth factor beta1 (TGF-beta1) on the behaviour of porcine thyroid epithelial cells in primary culture through thrombospondin-1 synthesis.

Transforming growth factor beta1 (TGF-beta1) is a secreted polypeptide that is thought to play a major role in the regulation of folliculogenesis and differentiation of thyroid cells. On porcine thyroid follicular cells cultured on plastic substratum, TGF-beta1, in a concentration-dependent way, promoted the disruption of follicles, cell spreading, migration and confluency by a mechanism that did not involve cell proliferation. TGF-beta1 strongly activated the production of thrombospondin-1 and (alpha)vbeta3 integrin in a concentration-dependent manner whereas the expression of thyroglobulin was unaffected. Anisomycin, an inhibitor of protein synthesis, inhibited the effect of TGF-beta1 on cell organization. Thrombospondin-1 reproduced the effect of TGF-beta1. In the presence of thrombospondin-1 cells did not organize in follicle-like structures but, in contrast, spreaded and reached confluency independently of cell proliferation. This effect is suppressed by an RGD-containing peptide. The adhesive properties of thrombospondin-1 for thyroid cells were shown to be mediated by both the amino-terminal heparin-binding domain and the RGD domain of thrombospondin-1. Adhesion was shown to involve (alpha)vbeta3 integrin. The results show that TGF-beta1 exerted an influence upon function and behaviour of follicle cells partly mediated by the synthesis of thrombospondin-1 and of its receptor (alpha)vbeta3 integrin.

Glycosyl phosphatidylinositol (GPI)/inositolphosphate glycan (IPG): an intracellular signalling system involved in the control of thyroid cell proliferation.

In porcine thyrocytes, TSH alone does not induce cell growth. Recently, it has been demonstrated that acute stimulation by TSH of porcine thyrocytes leads to release an inositolphosphate glycan (IPG) described as a putative second messenger for various growth factors in different cell types. IPG isolated from porcine thyrocytes induces proliferation of fibroblasts EGFR T17 and porcine thyrocytes. In porcine thyrocytes we have confirmed that cell growth requires the presence of both TSH and insulin. This effect is reproduced by 8-bromo cyclic AMP suggesting a mediation by intracellular cyclic AMP. Cooperative effects between 8-bromo cyclic AMP and IPG have also been evidenced and are in favour of a crosstalk between distinct signalling pathways.

Evidence for a TSH-controlled ectophosphotyrosine phosphatase in pig thyroid cultured cells.

In porcine thyroid cells in primary culture, thyrotropin (TSH) provokes important morphological changes associated with specific thyroidal function recovery. TSH influences cell-cell interactions and follicular morphogenesis. In the present report, we identify an ectotyrosine phosphatase activity controlled by chronic treatment with TSH. Specific substrates and various inhibitors allowed us to characterize this activity. This tyrosine phosphatase is located at the outer surface of thyroid cells as demonstrated by phosphotyrosylhistone hydrolysis. Control of cell viability and trypsin treatment confirm this extracellular localization. These data show that TSH activates ectotyrosine phosphatase activity, suggesting a role for surface protein phosphorylation in regulating specific functions of porcine thyroid cells in suspension.

Flavonoids extracted from fonio millet (Digitaria exilis) reveal potent antithyroid properties.

Digitaria exilis (fonio) is a tiny variety of millet commonly eaten by inhabitants of semiarid regions. A sample of fonio collected right in the middle of a severely iodine-depleted goitrous endemic was submitted to phytochemical investigations in order to assess the potential contributory roles played by vegetable molecules to the goitrogenic processes. The total content of flavonoids amounts to 500 mg/kg of the edible whole cereal grains. Their extraction and identification fail to detect the C-glycosylflavones described in other millet varieties but point out the presence of apigenin (A = 150 mg/kg) and of luteolin (L1 = 350 mg/kg). Ten percent of A and 80% of L1 are present in free form, whereas the remaining 90% of A and 20% of L1 are bound as O-glycosylflavones. Both A and L1 aglycones manifest strong anti-thyroid peroxidase (TPO) activities, resulting in a significant reduction of the hormonogenic capacity of this enzyme. In addition, L1 significantly depresses the cyclic AMP phosphodiesterase, implying a concomitant overproduction of the thyrotropin-dependent nucleotide. These last unreported data are regarded as counteracting to some extent the TPO-mediated goitrogenic properties of L1. Since fonio is devoid of other molecules likely to interfere with the thyroid function, our results are directly and casually attributed to A and L1 found in the customary diet.

Mechanisms of the platelet aggregation induced by activated neutrophils and inhibitory effect of specific PAF receptor antagonists.

The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed platelets in human. It potentiates platelet aggregation induced by agonists in platelet rich plasma as well as in whole blood. This activation involves the phosphoinositide metabolism. Specific PAF receptor antagonists gingkolides (BN 50726, BN 52021, BN 54068, BN 54062, BN 50730, BN 50749, BN 50744) and benzodiazepine Web2086 antagonize this neutrophil-induced platelet aggregation. BN 50,730, BN 50,749 and Web 2086 can fully inhibit this aggregation at the final concentration of 10(-6) M. Preincubation of platelets with synthetic PAF also inhibits this activation through a desensitization of the receptor. These data suggest the major involvement in our model of PAF acether in the platelet-neutrophil interactions.

IPG (inositolphosphate glycan) as a cellular signal for TGF-beta 1 modulation of chondrocyte cell cycle.

The knowledge of transforming growth factor (TGF)-beta receptors has greatly progressed in the recent years. TGF-beta receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-beta to its signaling receptors. In addition, four other proteins that bind TGF-beta 1 or TGF-beta 2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-beta receptors remain an enigma. TGF-beta family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-beta effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-beta 1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-beta 1. Using [3H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-beta, supporting that a PI-anchored molecule gives rise to IPG by TGF-beta-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-beta action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-beta on RAC growth.

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.

Autocrine biological effects of glycosyl inositol phosphate produced by reconstituted pig thyroid follicles: role of pertussis toxin sensitive G proteins.

We examine the autocrine activity of glycosyl inositol phosphate (InP-gly) on thyroid metabolism. The cAMP accumulation promoted by thyrotropin (TSH) or forskolin was modulated by InP-gly, stimulated by the lowest tested concentration (10(-8) M) and progressively inhibited by higher concentrations. Iodide uptake and iodine organification were decreased in a concentration-dependent manner by InP-gly alone, or in the presence of TSH. The IAP component of pertussis toxin blocked the inhibitory action of InP-gly on cAMP accumulation by reconstituted thyroid follicles (RTF), suggesting the participation of Gi protein. But the same treatment with IAP was without effect on iodine metabolism, suggesting that there is a second target for InP-gly, more distal than Gi protein, or coupled to another G protein which is insensitive to the toxin.

[Alterations in erythrocyte membrane. Effect of neutrophil activation]. / Altération de la membrane érythrocytaire. Conséquence de l'activation des neutrophiles.

White blood cells, especially polymorphonuclear neutrophils (PMN), are known to alter some hemorheological parameters. Most of in vitro results have been obtained with passive PMN. Stimulated PMN also lead to other hemorheological changes. In our study, we have firstly activated PMN with opsonized zymosan and collected a PMN-free supernatant after 30 minutes activation. This supernatant was secondarily incubated with erythrocytes either in whole blood or in suspension. After 10 minutes incubation, hemorheological parameters were evaluated: 1) red blood deformability (RBC) (Ektacytometer* Technicon), 2) RBC filtration (Hemorheometer MK 1), 3) RBC aggregation (Erythroaggregometer* Sefam), 4) Plasmatic and whole blood viscosities (Low Shear 30* Contraves). Our results show that activated PMN-supernatant increases rigidity index (IR) of RBC in suspensions (IR of RBC control = 14.59 +/- 3.30 towards RI of incubated RBC = 22.91 +/- 7.06 p less than 0.001). Other rheological parameters remain unchanged. Activated PMN suspenatant influence on RBC is slight but could suggest of an alteration of RBC membrane. With an in vitro model of washed platelets aggregation, we have previously demonstrated that proaggregant activity of PMN supernatant was inhibited by specific Platelet-Activating Factor (PAF-acether) antagonists: BN 52021, BN 50723, Web 2086. We have so compared both effects of activated PMN-supernatant and synthetic PAF-acether on RBC membrane fluidity. Membrane fluidity was studied by fluorescence polarization of 4 probes embedded at different deep in the membrane of intact RBC. Similar modifications of RBC membrane fluidity are observed with either synthetic PAF-acether or PMN supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)