Identification and characterization of 1,25D 3-membrane-associated rapid response, steroid (1,25D 3-MARRS)-binding protein in rat IEC-6 cells

Abstract

We report the presence of a mammalian equivalent of the avian Membrane- Associated Rapid Response, Steroid (1,25D 3-MARRS)-binding protein specific for 1,25(OH) 2D 3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to the steroid hormone. Identification of transcript and protein was achieved using RT-PCR with several specific primer sets, Western blot analysis with two separate antibodies recognizing distinct regions of the protein, ribozyme knockdown and immunohistochemistry. Promoter analysis of the 1000-bp upstream region of the 1,25D 3-MARRS gene in several species revealed the presence of a conserved smad-3 element in the 5′ proximal promoter region, but no classical vitamin D response element (VDRE). Treatment of IEC-6 cells with transforming growth factor β1 (TGFβ1) increased steady-state levels of 1,25D 3-MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH) 2D 3 failed to significantly change 1,25D 3-MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D 3-MARRS protein after 1,25(OH) 2D 3 treatment, suggesting that the protein is subject to membrane-initiated signal pathway activation. Together these data point to complex interactions between the two important 1,25(OH) 2D 3 sensitive response systems in intestinal cells, 1,25D 3-MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca 2+ absorption in a variety of avian and mammalian species.