Abstracts
1997 Digestive Disease Week

Basement membrane proteins alter focal adhesion kinase phosphorylation in enterocytes.

ST Summers, BL Bass. Department of Surgery, VA Medical Center and University of Maryland School of Medicine, Baltimore MD.

Epithelial - basement membrane interactions are critical to enterocyte growth and differentiation. The mechanism by which extracellular matrix proteins regulate enterocytes is poorly understood but likely involves integrins, a family of transmembrane proteins which bind specific matrix proteins. Focal adhesion kinase (FAK) is a protein tyrosine kinase (PTK) which is activated by integrins. FAK, like many other PTKs, has been implicated in the regulation of cell proliferation. The objective of our study was to determine if the basement membrane protein laminin (LAM) alters cell growth and FAK phosphorylation. Methods: The human colon cancer derived cell line, HT 29, was used as an enterocyte model. Cells were plated on dishes coated with LAM, collagen I (COL), or nothing. Cell growth was assessed by cell count after 72 hrs. FAK phosphorylation was investigated by SDS-PAGE and western blot analysis of cell lysates prepared from cells in culture for 1 - 4 days using anti-phosphotyrosine or anti-FAK monoclonal antibodies. Blots were analyzed by computer densitometry. Data are expressed as mean ± SEM, with p value determined by t-test. Results: HT 29 proliferation was significantly decreased in cells cultured on LAM, 165 ± 20 x 10³ cells, vs COL 308 ± 19 x 10³ cells (p < .001, n = 5). The content of phosphorylated FAK increased slightly from 1 to 4 days of culture on COL while decreasing significantly in cells cultured on LAM. At 4 days, there was significantly less phosphorylated FAK in cells grown on LAM than COL with no change in the total amount of FAK protein.