Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS)-binding protein in rat IEC-6 cells.
We report the presence of a mammalian equivalent of the avian Membrane-Associated Rapid Response, Steroid (1,25D3-MARRS)-binding protein specific for 1,25(OH)2D3 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 1,25D3-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 beta1 (TGFbeta1) increased steady-state levels of 1,25D3-MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH)2D3 failed to significantly change 1,25D3-MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D3-MARRS protein after 1,25(OH)2D3 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)2D3 sensitive response systems in intestinal cells, 1,25D3-MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca2+ absorption in a variety of avian and mammalian species.
Rohe
Benjamin
B
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
Safford
Susan E
SE
Nemere
Ilka
I
Farach-Carson
Mary C
MC
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007535:Isomerases / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases; D016337:RNA, Catalytic / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D014807:Vitamin D / Q000378:metabolism
D017931:DNA Primers; D006360:Heat-Shock Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014807:Vitamin D; D007535:Isomerases; C513196:PDIA3 protein, rat; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human; D002118:Calcium