Regulation of expression of 1,25D3-MARRS/ERp57/PDIA3 in rat IEC-6 cells by TGFβ and 1,25(OH)2D3

We examined the transcriptional regulation of expression of the redox-sensitive Membrane-Associated-Rapid Response, Steroid-binding (1,25D3-MARRS) protein specific for 1,25(OH)2D3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to 1,25(OH)2D3. 1,25D3-MARRS binds and is activated by 1,25(OH)2D3, but is not itself up-regulated by treatment with 1,25(OH)2D3, nor is there a Vitamin D response element (VDRE) in its proximal promoter. We previously reported that transforming growth factor β (TGFβ) increased steady state levels of 1,25D3-MARRS transcript and protein approximately two-fold [Rohe B, Safford SE, Nemere I, Farach-Carson, MC. Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS)-binding protein in rat IEC-6 cells. Steroids 2005;70:458-63]. To determine if this up-regulation could be attributed to the function of a highly conserved consensus smad 3 binding element present in the proximal promoter of the 1,25D3-MARRS gene, we created a promoter-reporter [SEAP] construct that was responsive to TGFβ (200 pM). Deletion or mutation of the smad 3 element greatly reduced the response of the 1,25D3-MARRS promoter to TGFβ. Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 3. Interestingly, although 1,25(OH)2D3 alone did not increase expression of the 1,25D3-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)2D3 and TGFβ shifted the dose-response curve to a lower effective concentration (100 pM peptide). We conclude that TGFβ is a transcriptional regulator of 1,25D3-MARRS expression via a functional smad 3 element and that cross-talk with non-classical 1,25(OH)2D3-stimulated pathways occurs. The findings have broad implications for redox-sensitive signaling phenomena including those that regulate phosphate transport in the intestine.