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-beta 1 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-beta 1 effect, these studies evaluated the influence of TGF-beta 1 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-beta 1. Biosynthetic labeling of proteoglycans with 35S sulfate and enzyme digestion studies demonstrated that TGF-beta 1 promoted the synthesis of heparan sulfate proteoglycans in these cells. The importance of proteoglycan synthesis induced by TGF-beta 1 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-beta 1 stimulation of proteoglycan synthesis and diminished TGF-beta 1 promoted transformation of proximal tubule cells as assessed by quantitative morphometry. Further experiments evaluated the influence of TGF-beta 1 on actin microfilaments with the use of rhodamine conjugated phalloidin staining and immunofluorescent microscopy, and demonstrated that TGF-beta 1 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-beta 1 and inhibited the TGF-beta 1-induced morphologic transformation of these cells. Further studies evaluated these effects on the rate of DNA synthesis in these cells, as assessed with 3H-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-beta 1 on EGF-induced DNA synthesis cells. These studies demonstrate that the TGF-beta 1 promoted synthesis cells. These studies demonstrate that the TGF-beta 1 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-beta 1 as well as the DNA synthesis rate response to both EGF and TGF-beta 1.
Humes
H D
HD
Department of Internal Medicine, VA Medical Center, Ann Arbor, Michigan.
Nakamura
T
T
Cieslinski
D A
DA
Miller
D
D
Emmons
R V
RV
Border
W A
WA
D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005353:Fibronectins / Q000096:biosynthesis; D006027:Glycosides / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010641:Phenotype; D011509:Proteoglycans / Q000096:biosynthesis*; D011817:Rabbits; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000199:Actins; D005353:Fibronectins; D006027:Glycosides; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; C010807:xylosides; D003571:Cytochalasin B; D004247:DNA