Abstract. FG human pancreatic carcinoma cells adhere to vitronectin using integrin o~vB5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an ot2/31-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo av/35-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to otv/55-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGE These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.
Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GIcNAc) residues in the zona pellucida (ShuT, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GIcNAc-agarose and a-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn+÷-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away.The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GIcNAc substrate in the zona pellucida. Under identical conditions, UDPglucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time-and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites.Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.
Integrin avf35 promotes FG carcinoma cell adhesion to vitronectin yet requires protein kinase C (PKC) activation for migration on this ligand. Here we report that this PKCdependent cell motility event requires NF-KB-dependent transcription. Specifically, a component within nuclear extracts prepared from PKC-stimulated FG cells exhibited a significant increase in binding activity to a synthetic oligonucleotide containing a consensus KB sequence. These nuclear DNA-binding complexes were shown to be comprised of p65 and p50 NF-KB/rel family members and appeared functionally active because they promoted transcription of a reporter construct containing a KB site. The NF-KB activation event was directly linked to the av,35 motility response because the NF-KB-binding oligonucleotide, when introduced into FG cells, inhibited cell migration on vitronectin but not on collagen and had no effect on cell adhesion to either ligand. These results suggest that the detected DNA-binding complexes interact with KB transcriptional elements to regulate gene expression required for avp35-dependent cell motility on vitronectin. INTRODUCTION Regulation of cell migration is a critical factor during tissue remodeling associated with angiogenesis, embryogenesis, inflammation, and wound healing (Albelda and Buck, 1990). Upon neoplastic transformation, cells acquire a migratory phenotype that facilitates their ability to metastasize. Integrins are cell surface receptors that mediate cell-matrix interactions critical for cell motility (Ruoslahti, 1991;Hynes, 1992;Leavesley et al., 1992Leavesley et al., , 1993Klemke et al., 1994). Although integrin-mediated cell adhesion is necessary for migration, it is not always sufficient. For example, recent studies have shown that integrin-dependent cell motility, but not attachment, requires the presence of exogenous growth factors Matthay et al., 1993;Klemke et al., 1994), suggesting that cell motility involves activation events not required for adhesion.t Corresponding author.FG pancreatic carcinoma cells attach to vitronectin utilizing integrin av,85, yet are unable to migrate on this ligand even though they attach and migrate on collagen in an a2f31 integrin-dependent manner (Leavesley et al., 1992). These findings indicate that FG cell migration on collagen is constitutive whereas av135-dependent migration on vitronectin requires agonist activation. However, we recently showed that FG cells can be induced to migrate on vitronectin in the presence of soluble growth factors, such as epidermal growth factor and insulin-like growth factor, which act by stimulating a PKC-dependent signaling pathway (Klemke et al., 1994).PKC is known to activate the adhesive properties of some cells by enhancing the affinity of their cell surface integrins (Shattil and Brass, 1987;Hibbs et al., 1991;Valmu et al., 1991;Wilkins et al., 1991; MartinThouvenin et al., 1992). In addition, PKC potentiates signaling events leading to integrin-dependent cell spreading and motility (Vuori and Ruoslahti, 1993;Klemke et al., 1994...
Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk. Losartan (23 mg. kg(-1). day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.
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