herbimycin and arginyl-glycyl-aspartic-acid

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Topics: Animals; Antibodies; Benzoquinones; Cell Division; Cell Line; Cell Movement; CHO Cells; Cricetinae; Enzyme Inhibitors; Flavonoids; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Insulin-Like Growth Factor Binding Protein 1; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Oligopeptides; Phosphorylation; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Quinones; Receptors, Fibronectin; Rifabutin; Signal Transduction; Time Factors; Tissue Distribution; Trophoblasts

Extracellular matrix facilitates anchorage-dependent cell survival via interaction of its arginine-glycine-aspartate (RGD) motif with integrins. In this report, we describe an unexpected, apoptosis-promoting the effect of immobilized RGD (iRGD) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Mesangial cells cultured on RGD-coated plates showed enhanced susceptibility to TNF-alpha-induced apoptosis. iRGD alone did not affect cell survival. In contrast, iRGD did not facilitate but inhibited apoptosis induced by H(2)O(2). Mitogen-activated protein (MAP) kinases and tyrosine kinases are important mediators for the RGD-integrin signaling. Pretreatment with MAP kinase kinase inhibitor PD098059, c-Jun N-terminal kinase (JNK)-c-Jun/AP-1 inhibitor curcumin or p38 MAP kinase inhibitor SB203580 did not attenuate the apoptosis-promoting effect of iRGD. Consistently, transfection with dominant-negative mutants of extracellular signal-regulated kinases, JNK or p38 MAP kinase did not inhibit the effect of iRGD. In contrast, protein tyrosine kinase inhibitors, genistein, and herbimycin A, abrogated the apoptosis-promoting effect of iRGD. Of note, TNF-alpha-induced apoptosis on uncoated plates was not attenuated by tyrosine kinase inhibitors. These data provide the first evidence that iRGD accelerates certain apoptosis. We identified that the effect was mediated by the tyrosine kinase-dependent, MAP kinase-independent mechanism.

Topics: Animals; Apoptosis; Benzoquinones; Cells, Cultured; Curcumin; Enzyme Inhibitors; Flavonoids; Genes, Dominant; Genistein; Glomerular Mesangium; Hydrogen Peroxide; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; MAP Kinase Signaling System; Microscopy, Phase-Contrast; Mitogen-Activated Protein Kinases; Mutation; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein-Tyrosine Kinases; Pyridines; Quinones; Rats; Rifabutin; Time Factors; Transcription Factor AP-1; Transfection; Tumor Necrosis Factor-alpha

The effects of alpha IIb beta 3 antagonists on the blockade of fibrinogen binding to platelet alpha IIb beta 3 are well documented, however, little is known about their effects on platelet secretion. We compare here the effect of two potent alpha IIb beta 3 antagonists, c7E3 and DMP728, on platelet secretion. Using human platelet-rich plasma, P-selectin expression was measured by flow cytometry and type 1 plasminogen activator inhibitor (PAI-1) secretion as well as beta-thromboglobulin (beta-TG) were determined by ELISA. At various concentrations of the antagonists that inhibited 80-95% of platelet aggregation, neither had any effect on P-selectin expression. In contrast, thrombin-stimulated PAI-1 secretion is only inhibited by c7E3, 49.6% at 3.5 mumol/L (p < 0.05), but not at any other maximally effective anti-aggregatory concentrations of c7E3 or DMP728. Furthermore, a lack of any significant effects on platelet granular secretion of beta-TG induced by either thrombin or ADP was demonstrated with DMP728, c7E3 or LM609. Two protein kinase inhibitors, staurosporine and herbimycin, blocked both ADP and thrombin-induced P-selectin expression at 10 mumol/L, but not PAI-1 secretion. Taken together this suggests that: (1) the mechanism of platelet granular secretion is independent of the integrin alpha IIb beta 3 and (2) the subcellular locations of PAI-1, beta-TG and P-selectin or the signaling mechanisms that regulate their secretion might be different. Although there is no direct effect of platelet alpha IIb beta 3 antagonists on platelet secretion of PAI-1, beta-TG and P-selectin, the present data demonstrates that reduction of platelet number by alpha IIb beta 3 antagonists, via the reduction in thrombus size, might be an alternate mechanism for reduced platelet secretion. In conclusion, a discoupling between the anti-aggregatory and the anti-secretory effects of alpha IIb beta 3 antagonists has been demonstrated.

Topics: Abciximab; Antibodies, Monoclonal; Benzoquinones; beta-Thromboglobulin; Enzyme Inhibitors; Humans; Immunoglobulin Fab Fragments; Lactams, Macrocyclic; Mesylates; Oligopeptides; P-Selectin; Peptides, Cyclic; Plasminogen Activator Inhibitor 1; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Quinones; Rifabutin; Staurosporine

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Chickens; Female; Genistein; Isoflavones; Isoquinolines; Lactams, Macrocyclic; Molecular Sequence Data; Oligopeptides; Osteoclasts; Peptide Fragments; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Stem Cells; Vanadates