We sought to determine the importance of integrins for recovery after acute tubular injury and to investigate the signal transduction pathways for integrin effects on cell cycle regulation involving proliferation and apoptosis. Primary cultures of rat renal proximal tubule epithelial cells were exposed to a superoxide-generating system to induce injury in the absence of overt necrosis. Integrin function was antagonized by the integrin recognition sequence tetrapeptide Gly-Arg-Gly-Asp (GRGD) or monoclonal antibody to β1-integrin. Injured cells had reduced thymidine uptake compared with normal cells. The presence of GRGD during recovery from injury caused a further 44% reduction in DNA synthesis but did not affect DNA synthesis in normal cells. Injured cells had an increased proportion of apoptosis that was further accentuated by exposed to GRGD during recovery. Integrin antagonism also stimulated apoptosis in uninjured cells. To investigate signal transduction mechanisms for this effect of integrins, inhibitors and activators of protein tyrosine kinase (PTK) and protein kinase C (PKC) were evaluated Activation of PKC stimulated cellular proliferation, whereas inhibitors of PKC and PTK had no significant effect. Genistein, a PTK inhibitor, induced apoptosis in normal cells, mimicking the effect of integrin inhibition. On the other hand, PMA, an activator of PKC, prevented cells from becoming apoptotic when exposed to injury plus GRGD. The phosphorylation status of intracellular proteins was evaluated by immunoblotting with antiphosphotyrosine antibody. A similar pattern of decreased phosphorylation was observed after either integrin inhibition, injury, both, or PTK inhibition. These findings suggest that kinase cascades are involved in the effects of integrins on renal epithelial cell proliferation and apoptosis. After injury, an interaction between cells and the extracellular matrix is required for cells to proliferate and contribute to repair rather than to enter an apoptotic pathway.
Bibliographical noteFunding Information:
This work was supported by an extramural grant from Baxter Health-
Care Corporation and by a grant from the National Kidney Foundation of the Upper Midwest. We are grateful to Dr. Suman Setty for the generous gift of antibodies, to Dr. Mark Rosenberg for assistance with immuno- blotting, and to David Chmielewski and Marsha Patten for technical assistance.
- Epithelial cells
- Protein kinase C
- Tubular injury