1,25–Dihydroxyvitamin D<sub>3</sub> Stimulates Transforming Growth Factor–β1 Synthesis by Mouse Renal Proximal Tubular Cells

Abstract

1,25–Dihydroxyvitamin D<sub>3</sub> [1,25–(OH)<sub>2</sub> D<sub>3</sub>] 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)<sub>2</sub> D<sub>3</sub> on human mesangial cells and proximal tubular cells. To investigate whether 1,25–(OH)<sub>2</sub> D<sub>3</sub> 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)<sub>2</sub> D<sub>3</sub>–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)<sub>2</sub> D<sub>3</sub> (10<sup>–11</sup> to 10<sup>–7</sup> M) specifically increased the TGF–β1 protein secretion in MCT with a maximum at 10<sup>–8</sup> M. No detectable effect was found with 25 D<sub>3</sub> 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)<sub>2</sub> D<sub>3</sub>. Steady–state TGF–β1 mRNA concentration in MCT was transiently increased by 1,25–(OH)<sub>2</sub> D<sub>3</sub> 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)<sub>2</sub> D<sub>3</sub>. In conclusion, 1,25–(OH)<sub>2</sub> D<sub>3</sub> 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.