ABSTRACT
The vitamin D receptor (VDR) mediates vitamin D actions beyond bone health. While VDR activation by 1,25-dihydroxyvitamin D (1,25D) leads to robust transcriptional regulation, less is known about VDR actions in the absence of 1,25D. We analyzed the transcriptomic response to 1,25D in fibroblasts bearing a severe homozygous hereditary vitamin D resistant rickets-related p.Arg30* VDR mutation (MUT) and in control fibroblasts (CO). Roughly 4.5% of the transcriptome was regulated by 1,25D in CO fibroblasts, while MUT cells without a functional VDR were insensitive to 1,25D. Novel VDR target genes identified in human fibroblasts included bone and cartilage factors CILP, EFNB2, and GALNT12. Vehicle-treated CO and MUT fibroblasts had strikingly different transcriptomes, suggesting basal VDR activity. Indeed, oppositional transcriptional effects in basal conditions versus after 1,25D activation were implied for a subset of target genes mostly involved with cell cycle. Cell proliferation assays corroborated this conjectured oppositional basal VDR activity, indicating that precise 1,25D dosage in target tissues might be essential for modulating vitamin D actions in human health.
Subject(s)
Fibroblasts/metabolism , Receptors, Calcitriol/genetics , Transcription, Genetic/drug effects , Transcriptome/genetics , Vitamin D/analogs & derivatives , Cell Proliferation/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Fibroblasts/cytology , Fibroblasts/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Molecular Sequence Annotation , Mutation/genetics , Receptors, Calcitriol/metabolism , Transcriptome/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics , Vitamin D/pharmacologyABSTRACT
BACKGROUND: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. METHODS: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 microg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. RESULTS: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001). CONCLUSIONS: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.
Subject(s)
Collagen Type V , Disease Models, Animal , Lung/metabolism , RNA, Messenger/metabolism , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/metabolism , Transforming Growth Factor beta/metabolism , Administration, Intranasal , Animals , Down-Regulation/drug effects , Female , Gene Expression/drug effects , Humans , RabbitsABSTRACT
HC11, a spontaneously immortalized murine mammary lineage maintains features of normal cells while HC11 H-ras transformed cells (HC11 ras) are tumorigenic. Ras transformation is associated with a lower Vitamin D receptor (VDR) mRNA content. Our goal was to investigate the mechanism underlying VDR mRNA differences between these cells. Although the VDR transcriptional rate measured by run-on assays did not differ between the cells, our data suggested a pos transcriptional mechanism involving higher VDR mRNA degradation in HC11 ras cells which was not due to mutations in its 3'-UTR region since sequences of mRNA obtained from HC11 and HC11 ras cells were identical. Treatment of HC11 ras cells with a farnesyltransferase inhibitor, which prevents ras activation, causing an enhancement of VDR mRNA levels, indicating an association between the ras signaling pathway and VDR mRNA instability. The present work suggests that the decreased mRNA levels in HC11 ras cells might in part be due to an early loss of stability.
