Abstract
Previous studies have shown that collagen gel overlay induced selective proteolysis of focal adhesion complex proteins in Madin-Darby canine kidney (MDCK) cells. In this study, we examined whether morphological and biochemical changes were present in cells cultured on collagen gel. We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas, but not vinculin, were decreased within 1 h when MDCK cells were cultured on collagen gel. Collagen gel-induced selective decrease of focal adhesion proteins was observed in all lines of cells examined, including epithelial, fibroblastic, and cancer cells. Matrigel also induced selective down-regulation of focal adhesion proteins. However, cells cultured on collagen gel- or matrigel-coated dishes did not show any changes of focal adhesion proteins. These data suggest that the physical nature of the gel, i.e. the rigidity, is involved in the expression of focal adhesion proteins. The collagen gel-induced down-regulation of focal adhesion complex proteins was caused by reduction of protein synthesis and activation of proteases such as calpain. Overexpression of a dominant negative mutant of discoidin domain receptor 1 (DDR1) or FAK-related non-kinase (FRNK) did not prevent collagen gel-induced down-regulation of the focal adhesion complex protein, whereas an anti-alpha2beta1 integrin-neutralizing antibody completely blocked it. Taken together, our results indicate that the rigidity of collagen gel controls the expression of focal adhesion complex proteins, which is mediated by alpha2beta1 integrin but not DDR1.
Highlights
Adhesion of cells to the extracellular matrix (ECM)1 is a crucial event in multi-cellular organisms for the modulation of cellular processes such as cell growth, differentiation, and apoptosis [1,2,3,4]
We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas, but not vinculin, were decreased within 1 h when Madin-Darby canine kidney (MDCK) cells were cultured on collagen gel
Collagen Gel Induced Down-regulation of Focal Adhesion Complex Proteins—To understand the mechanism of collagen gel-induced changes in MDCK cells, we first examined the expression of focal adhesion complex proteins in MDCK cells cultured on dish, collagen gel-coated dish, or collagen gel
Summary
The collagen gel-coated dishes were air dried and washed twice by normal culture medium prior to the use. To prepare collagen-coated agarose gel, the agarose dissolved in normal culture medium was heated by microwave and allowed for gelation at room temperature. The PCR protocol was performed with the FAK, talin, or c-Src primers at 94 °C for 2 min, 53 °C for 30 s, and 72 °C for 1 min (35 cycles), followed by 72 °C for 10 min. Measurement of Protein Synthesis Rates—MDCK cells were cultured on dish, collagen gel-coated dish, or collagen gel for 3, 7, 17, or 23 h. Immunofluorescence and Confocal Study—MDCK cells cultured on dish, collagen gel coated dish, or collagen gel for 4 h were washed three times with phosphate-buffered saline (PBS) and fixed using 4% paraformaldehyde prepared in PBS for 20 min at room temperature. The immunofluorescent images were taken by confocal microscopy (Leica, TCS-SP2)
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