Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts

Abstract

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 post-transcriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOB) identified human miRNAs that were differentially regulated by > 1.5-fold following treatment with 1,25D (10nM, 6 hrs), which included miRNAs 637 and 1228. RT-PCR analyses showed that the host gene for miR-1228, low density lipoprotein receptor-related protein 1 (LRP1), was co-induced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1 – 10nM, 6hrs). 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, siRNA inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated down regulation of BMP2K protein expression. This was associated with suppression of pro-differentiation 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 post-transcriptional mechanisms.

Posttranscriptional Regulation of the Inflammatory Marker C-Reactive Protein by the RNA-Binding Protein HuR and MicroRNA 637

“…miR-637, a primate-specific miRNA, was first identified in colorectal tumor tissue (41). While the biological function of miR-637 is still not fully understood, several targets, such as osterix and collagen type IV alpha 1 (COL4A1), have been reported (42,43). Decreased miR-637 expression levels were reported for four hepatocellular carcinoma cell lines; overexpression of miR-637 inhibited growth and enhanced apoptosis by suppressing the expression of the autocrine leukemia-inhibitory factor (LIF) (44), suggesting a tumor suppressor function for miR-637 in hepatocytes.…”

MicroRNAs involved in bone formation

“…Lisse et al investigated regulation of osteoblastic miRNAs by vitamin D [93]. MiR-637 and miR-1228 were upregulated upon vitamin D treatment and were shown to target Col4a1 and Bmp2k (bone morphogenetic protein 2 kinase).…”

DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

“…In contrast, there was accompanying RNApol2 binding activity at the proximal Ddit4 promoter only after 1,25D 3 treatment, suggesting direct transcriptional activation of Ddit4 after ligand stimulation (Figure 3C). For ChIP validation we utilized the murine osteocalcin gene, Bglap , which has no VDREs in its promoter region, nor is it transcriptionally influenced by addition of 1,25D 3 [37]. We also chose the S1 site within intron 3 and 4 of the murine Vdr gene as a potent positive control for both auto-regulatory VDR and RNApol2 binding, as previously shown in MC3T3-E1 murine osteoblasts [38].…”