Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.
When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms.
Orthopaedic Hospital Research Center, University of California Los Angeles, Los Angeles, CA, USA.
Thomas S
TS
Lisse
Rene F
RF
Chun
Sandra
S
Rieger
John S
JS
Adams
Martin
M
Hewison
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D050071:Bone Density Conservation Agents / Q000494:pharmacology*; D002117:Calcitriol / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D024141:Collagen Type IV / Q000096:biosynthesis / Q000235:genetics; D064550:Death-Associated Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D050071:Bone Density Conservation Agents; C547475:COL4A1 protein, human; D024141:Collagen Type IV; C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; C576906:MIRN1228 microRNA, human; C569538:MIRN637 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D015675:Osteocalcin; C579101:DAPK3 protein, human; D064550:Death-Associated Protein Kinases; D000469:Alkaline Phosphatase; D002117:Calcitriol