1,25–Dihydroxyvitamin D3 Stimulates Transforming Growth Factor–β1 Synthesis by Mouse Renal Proximal Tubular Cells

1,25–Dihydroxyvitamin D3 [1,25–(OH)2 D3] is a secosteroid hormone with effects on cell growth, differentiation and immunoregulatory functions in a number of tissues not primarily involved in mineral metabolism. We recently demonstrated growth–regulating effects of 1,25–(OH)2 D3 on human mesangial cells and proximal tubular cells. To investigate whether 1,25–(OH)2 D3 might also affect the synthesis of cytokines and growth factors in proximal tubular cells, we assessed in the present study the expression and secretion of transforming growth factor–β1 (TGF–β1) in a mouse proximal tubular cell line (MCT) in vitro. TGF–β1 synthesis was measured by a monospecific ELISA in culture supernatant. The secreted TGF–β1 was proven to be biologically active by means of a bioassay system (CCL–64 mink lung epithelial cell proliferation assay). TGF–β1 gene expression was assessed by RT–PCR. To analyze whether TGF–β1 expression mediates the 1,25–(OH)2 D3–induced antiproliferative actions in MCT, proliferation studies in the absence or presence of a blocking monoclonal anti TGF–β1–3 antibody were performed. 1,25–(OH)2 D3 (10–11 to 10–7 M) specifically increased the TGF–β1 protein secretion in MCT with a maximum at 10–8 M. No detectable effect was found with 25 D3 at 10 times higher concentrations. A synthetic 20–epi analogue, MC 1288, increased TGF–β1 secretion up to similar amounts at equimolar concentrations as the natural hormone 1,25–(OH)2 D3. Steady–state TGF–β1 mRNA concentration in MCT was transiently increased by 1,25–(OH)2 D3 between 12 and 24 h, returning to control values at 48 h. Blocking TGF–β1 did not reduce or abrogate the antiproliferative effect of 1,25–(OH)2 D3. In conclusion, 1,25–(OH)2 D3 stimulates TGF–β1 expression in renal proximal tubular cells, a growth factor with anti–inflammatory and profibrotic actions which plays an important role in the development and progression of nephrosclerosis.