News and Publications

Regulation of Endothelial Cell Migration by Expression and Phosphorylation of the 27-kD Heat-Shock Protein

R.S. Piotrowicz, E. Hickey,* E.G. Levin

* University of Nevada, Reno, NV

We assessed the migration of bovine arterial endothelial cells that had enhanced expression of (1) the wild-type 27-kD heat-shock protein (HSP27) and (2) a form of HSP27 that cannot be phosphorylated. Migration was 2-fold greater for cells expressing the wild-type protein than for control transfectants and was reduced 40% in cells expressing the mutant protein. Because homologs of HSP27 inhibit polymerization of F-actin in vitro and may alter basolateral F-actin content in vivo, we postulated that HSP27 affects the extension of microfilaments essential for cell motility. Expression of the wild-type protein promoted the generation of long cellular extensions, whereas expression of the dominant negative mutant protein resulted in a marked reduction of lamellipodia and in aberrant microfilament morphology at the wound edge. Immunofluorescence combined with phalloidin staining showed colocalization of the HSP27 gene products with lamellipodial microfilament structures. These data suggest that HSP27 regulates migration by affecting the generation lamellipodial microfilaments.

Dual Activities of the 22-, 22.5-, and 24-kD Isoforms of Basic Fibroblast Growth Factor

R.S. Piotrowicz, P.A. Maher, E.G. Levin

Basic fibroblast growth factor 2 (FGF-2) is synthesized as 4 isoforms with molecular weights of 24, 22.5, 22, and 18 kD. Each of the 3 higher molecular weight isoforms is produced by the initiation of translation at 1 of 3 upstream CUG codons. Previously, we showed that export of these 3 isoforms by bovine arterial endothelial cells depends on 17ß-estradiol. To determine whether 24-, 22.5-, and 22-kD FGF-2 affected cell behavior after release from the cell, we evaluated the effect of recombinant higher molecular weight FGF-2 isoforms on the growth and migration of endothelial cells and mammary carcinoma cells.

Treatment with the recombinant proteins inhibited migration of endothelial cells by 45% and the migration of the carcinoma cells by 70%. Inhibition dependent on higher molecular weight FGF-2 isoforms was observed when migration of endothelial cells was stimulated by 18-kD FGF-2 and when growth of vascular endothelial cells and migration of mammary carcinoma cells were stimulated with insulin-like growth factor. In each case, inclusion of an antibody directed against the 55 amino acid amino-terminal end of 24-kD FGF-2 abrogated the inhibition of migration; antibodies to the 18-kD FGF-2 domain had no effect. When endothelial cells were cultured under conditions that promoted export of higher molecular weight isoforms of FGF-2, cell motility was decreased 40%; the decrease could be reversed by the antibodies to the 24-kD FGF-2.

Thus, both recombinant and endogenously produced higher molecular weight isoforms of FGF-2 can inhibit cell migration. In contrast, these isoforms had no effect on endothelial cell growth but stimulated growth of mammary carcinoma cells as well as the 18-kD isoform did (3-fold). Antibodies to the 18-kD domain of 24-kD FGF-2 blocked the growth-promoting activity of the 3 higher molecular weight isoforms of FGF-2, whereas antibodies to the amino-terminal end were ineffective.

These data suggest that higher molecular weight isoforms of FGF-2 have dual activities: an inhibitory effect on cell migration and a stimulating effect on proliferation. The 2 activities can be localized to different parts of the isoforms of FGF-2: inhibitory activity to the amino-terminal 55 amino acids (which are not present in the 18-kD isoform) and growth-promoting activity to the 18-kD domain. Therefore, the ratio of the higher molecular weight isoforms of FGF-2 to the 18-kD FGF-2 in the extracellular space may provide a mechanism of control for angiogenesis and development of mammary tumors.

PUBLICATIONS

Piotrowicz, R.S., Levin, E.G. Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J., in press.