Genetic Factors Associated with Absolute and Relative Plasma Concentrations of Calcitriol.

BACKGROUND: Little is known regarding factors associated with calcitriol and a relative measure of calcitriol, the calcitriol-24,25-dihydroxyvitamin D3-calcifediol proportion ratio (C24CPR). METHODS: Using a cross-sectional study design, healthy young adults of African and European descent, matched (1:1) on age (±5 years) provided a blood sample in non-summer months (N = 376). Vitamin D metabolites were measured in plasma with HPLC/MS-MS. West African genetic ancestry proportion (WGA) was estimated using STRUCTURE modeling of genetic ancestry-informative markers. Multivariable regression models were used to estimate the association of WGA and vitamin D-pathway gene variants with calcitriol and C24CPR, controlling for days from summer solstice, age, sex, blood pressure, body mass index, dietary vitamin D intake, oral contraceptive/medroxyprogesterone acetate use, smoking, tanning bed use, and time of day. RESULTS: Calcitriol and C24CPR were not highly correlated (rho = 0.14), although both were significantly, positively, and monotonically associated with WGA (Ptrend 0.025 and <0.001, respectively). In fully adjusted models, genetic factors explained a greater proportion of variability in C24CPR (R2 = 0.121 and 0.310, respectively). Variants in genes with associated with calcitriol (CALB1, CYP27B1, GC, and PPARGC1A) differed from those associated with C24CPR (CYP3A43, FGF23, KL, and VDR). CONCLUSIONS: Both absolute and relative measures of calcitriol were significantly higher among African Americans. Otherwise, these biomarkers appear to be genetically distinct. IMPACT: C24CPR may be better suited to personalized medicine, due to a higher proportion of population variability explained by genetic variation and a less skewed distribution.

Tumor necrosis factor-α treatment of HepG2 cells mobilizes a cytoplasmic pool of ERp57/1,25D3-MARRS to the nucleus.

ERp57/PDIA3/1,25-MARRS has diverse functions and multiple cellular locations in various cell types. While classically described as an endoplasmic reticulum (ER) resident protein, ERp57 has a nuclear location sequence (NLS) and can enter the nucleus from the cytosol to alter transcription of target genes. Dysregulation and variable expression of ERp57 is associated with a variety of cancers including hepatocellular carcinoma (HCC). We investigated the dynamic mobility of ERp57 in an HCC cell line, HepG2, to better understand the movement and function of the non-ER resident pool of ERp57. Subcellular fractionation indicated ERp57 is highly expressed in the ER with a smaller cytoplasmic pool in HepG2 cells. Utilizing an ERp57 green fluorescent protein fusion construct created with and without a secretory signal sequence, we found that cytoplasmic ERp57 translocated to the nucleus within 15 min after tumor necrosis factor-α (TNF-α) treatment. Protein kinase C activators including 1,25-dihydroxyvitamin D(3) and phorbol myristate acetate did not trigger nuclear translocation of ERp57, indicating translocation is PKC independent. To determine if an interaction between the rel homology binding domain in ERp57 and the nuclear factor-κB subunit, p65, occurred after TNF-α treatment and could account for nuclear movement, co-immunoprecipitation was performed under control and conditions that stabilized labile disulfide bonds. No support for a functional interaction between p65 and ERp57 after TNF-α treatment was found in either case. Immunostaining for both ERp57-GFP and p65 after TNF-α treatment indicated that nuclear translocation of these two proteins occurs independently in HepG2 cells.

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

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

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.

Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS) binding protein.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) operates through pharmacologically distinct nuclear receptor-mediated and plasma membrane-initiated mechanisms. The nuclear receptor is well described, but the membrane receptor identity remains unproven. A 66 kDa protein from chick intestinal basolateral membranes was isolated previously and identified as a candidate receptor (now termed 1,25D(3)-MARRS). A chicken cDNA library was screened for clones encoding the N-terminal sequence of 1,25D(3)-MARRS. An exact match was found with an insert containing an open coding region for the full-length candidate 1,25D(3)-MARRS protein. Analysis reveals a 5' untranslated region, a precursor translation product with methionine start site, a signal peptide and a translation product of 505 amino acids prior to translation termination site. Prosite analysis predicts potential sites for phosphorylation by casein kinase II cAMP-dependent kinase, protein kinase C, and tyrosine kinase and an N-myristoylation site with high probability of occurrence. Additionally, two conserved domains capable of interacting with Rel homology domains (RHD) are present. Oligonucleotide primers sets designed to amplify unique regions of the sequence produced amplimers of the predicted size from both chicken and rat intestinal cells. Transcription-translation produced a protein that was recognized in Western blot analysis by Ab099, a polyclonal antibody recognizing the N-terminus of the 66 kDa MARRS protein.