Involvement of pRB Family in TGF -dependent Epithelial Cell Hypertrophy

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor ~1 (TGF/~) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGFB did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF/~ blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGFB-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF~induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF/3-induced hypertrophy was inhibited in E7and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF~-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF/3induced blockade at G~/S, the latter due to maintained activity of pRB or a related protein. ENAL tubular hypertrophy occurs in a number of conditions, including diabetes mellitus, loss of renal mass, protein feeding, chronic metabolic acidosis, and potassium deficiency (13). In many of these conditions the hypertrophy has been postulated to cause progressive loss of renal function (6, 53). Despite its importance, the mechanism by which tubular hypertrophy occurs is largely unknown. One possible mechanism is that hypertrophy represents an aborted cell cycle, with cells entering the G, phase and initiating protein synthesis and growth, but failing to progress into S phase. This hypothesis would predict that events associated with entrance into G, would be the same in hyperplastic and hypertrophic processes, but that events associated with the transition between G~ and S phases would be different, with only hyperplastic cells progressing into S phase. pRB, the product of the retinoblastoma gene, plays a key role in regulating the GdS transition (2, 7, 21). Active (hypophosphorylated) pRB inhibits G,/S progression; hyperphosphorylation inactivates pRB, allowing progression into Address all correspondence to Patricia Preisig, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8856. Tel.: (214)648-8628. Fax: (214)648-9100. S phase. Transforming growth factor B (TGFB) exerts an antiproliferative effect in a number of cells (36), which is mediated in part by inhibition of pRB phosphorylation (maintenance of active pRB) and blockade of the cell cycle at G,/S (32). In addition, TGFB has been implicated in renal tubular and vascular smooth muscle cell hypertrophy (17, 42, 46, 52). In the present study, a model of hypertrophy in renal proximal tubule epithelial cells was developed to test the hypothesis that TGFB-mediated hypertrophy is coupled to cell cycle processes. Primary cultures of rabbit proximal tubule cells developed hyperplasia in response to epidermal growth factor (EGF), hypoplasia in response to TGFBI, and hypertrophy in response to the combination of EGF + TGF/~. The ability of TGF/~ to convert EGF-induced hyperplasia to hypertrophy was related to inhibition to pRB phosphorylation and the maintenance of active pRB or a related protein. Materials and Methods Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), except as indicated below. Dulbecco's modified Eagle's medium and Ham's F12 culture media, fetal bovine serum, and trypsin/EDTA were from GIBCO BRL (Gaithersburg, MD); transferrin was from Miles Pentex (Kankakee, IL); penicillin and streptomycin were from BioWhitaker, Inc., M. A. Bioproducts (Walkersville, MD); type 1 collagenase was from Boehringer Mannheim Corp. (Indianapolis, IN); culture dishes were from Corning Glass© The Rockefeller University Press, 0021-9525/95/04/245/10 $2.00 The Journal of Cell Biology, Volume 129, Number 1, April 1995 245-254 245 on F ebuary 3, 2013 jcb.rress.org D ow nladed fom Published April 1, 1995