ROLE OF PROTEOGLYCANS AND CYTOSKELETON IN THE EFFECTS OF TGF-BETA-1 ON RENAL PROXIMAL TUBULE CELLS

Transforming growth factor-beta (TGF-beta) is a critical cell regulatory protein which influences cell growth, cell differentiation and cell chemotaxis. TGF-beta1 has been previously shown to promote a migratorY and adherent transformation of monolayers of renal proximal tubule cells in primary culture to form solid clusters of cells. To better understand the cellular basis of this TGF-beta1 effect, these studies evaluated the influence of TGF-beta1 on the synthesis of proteoglycans and on cytoskeleton rearrangement in rabbit renal proximal tubule cells in primary culture, and their role in this transformation effect of TGF-beta1. Biosynthetic labeling of proteoglycans with S-35 sulfate and enzyme digestion studies demonstrated that TGF-beta1 promoted the synthesis of heparan sulfate proteoglycans in these cells. The importance of proteoglycan synthesis induced by TGF-beta1 in this migration and aggregation process was demonstrated with the use of two chemically-dissimilar proteoglycan synthesis inhibitors: xyloside and galactosamine. Both compounds inhibited TGF-beta1 stimulation of proteoglycan synthesis and diminished TGF-beta1 promoted transformation of proximal tubule cells as assessed by quantitative morphometry. Further experiments evaluated the influence of TGF-beta1 on actin microfilaments with the use of rhodamine conjugated phalloidin staining and immunofluorescent microscopy, and demonstrated that TGF-beta1 provoked a dramatic rearrangement of actin microfilaments into stress fibers. The use of actin microfilament disrupting agents, cytochalasin B and D, attenuated the stress fiber formation promoted by TGF-beta1 and inhibited the TGF-beta1-induced morphologic transformation of these cells. Further studies evaluated these effects on the rate of DNA synthesis in these cells, as assessed with H-3-thymidine incorporation. Proteoglycan synthesis inhibitors significantly diminished the maximal proliferative response of these epithelial cells to epidermal growth factor (EGF). In contrast, actin microfilament disaggregation with cytochalasin B or D did not change the rate of DNA synthesis in response to EGF but did attenuate the antiproliferative effect of TGF-beta1 on EGF-induced DNA synthesis cells. These studies demonstrate that the TGF-beta1 promoted an increase in the production of proteoglycans and a higher ordered structure of the cytoskeleton. Both effects were instrumental in the adhesive migratory response of proximal tubule cells to TGF-beta1 as well as the DNA synthesis rate response to both EGF and TGF-beta1.