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Integrated transcriptome and proteome analyses based on new methods for more accurate comparisons indicate that only 40% to 45% of the transcriptome is actually translated.[1-3] This discordance occurs as a consequence of the abundance of noncoding RNA produced, and because mRNA is subject to multilevel, posttranscriptional regulation. These events are largely mediated by regulatory networks established by RNA-binding proteins and RNA species such as small noncoding microRNAs (miRNAs), that orchestrate the concerted production of complex posttranscriptional gene expression networks within specialized cells such as bone-forming osteoblasts. Single-stranded mature miRNAs are known to regulate the posttranscriptional expression of genes by at least two mechanisms: (1) degradation and gene silencing of target mRNA transcripts, and (2) repression of mRNA translation via (partial) complementary base-pair binding with retainment of the RNA silencing complex (reviewed in Shruti and colleagues). Because a single miRNA may target several genes, this is a very efficient mechanism for influencing biological responses. More than 1700 human miRNAs have been identified so far, targeting approximately 40% to 60% of genes in many cell types, but the functions and target genes for most of these miRNAs are still unknown.
Recent data from animal and cellular models suggest that miRNAs play a critical role in skeletal development, osteoclast function, osteogenic lineage progression, adipose tissue–derived stem cell development, and mesenchymal precursor and osteoblastic differentiation under both normal and disease conditions. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), also plays a pivotal in bone homeostasis. However, whereas there are extensive data concerning transcriptional regulation by 1,25D and its cognate nuclear vitamin D receptor (VDR), relatively little is known about the interaction between 1,25D and miRNAs in bone cells. Studies using various nonskeletal neoplastic cells have reported induction of miRNAs by 1,25D,[9, 10] while also describing effects of miRNAs on VDR expression and 1,25D signaling. In other cells miRNAs have been shown to target enzymes associated with the synthesis and catabolism of 1,25D. With this in mind, the aim of the current study was to identify miRNAs in primary human osteoblasts (HOBs) that are induced or suppressed by 1,25D, and to assess the effects of 1,25D-mediated miRNA induction on osteoblastic phenotype and function.
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Recent reports have identified a range of regulatory actions for miRNAs in the generation and function of osteoblasts.[19-24] However, despite its established effects on osteoblast function, little is known about the contribution of vitamin D to miRNA activity in these cells. Data presented in the current study identify two specific miRNAs, miR-637 and miR-1228, that are closely associated with 1,25D responses in HOBs in vitro. As outlined in Fig. 5, the generation of these miRNAs from host genes in response to 1,25D is dependent on distinct mechanisms involving either: (1) conventional VDRE-mediated transactivation of a host gene (LRP1), and concomitant induction of its associated miR-1228; or (2) intronic VDRE-mediated induction of miR-637 in the absence of conventional transactivation of its host gene (DAPK3). In a similar fashion, the target gene effects of miR-637 and miR-1228 are mediated via different mechanisms, with miR-637 acting to stimulate degradation of COL4A1 mRNAs, whereas miR-1228 inhibits translation of the BMP2K protein. These observations highlight an entirely new repertoire of 1,25D functions in bone and suggest that miRNAs are key players in fine-tuning the effects of 1,25D on osteoblast differentiation and function.
Figure 5. Regulation and target activities of miR-637 and miR-1228 in 1,25D-treated HOBs. Schematic representation of miRNA responses to 1,25D in osteoblasts showing: (1) induction of miR-637 transcription via candidate intronic VDRE; (2) associated negative regulation of host gene DAPK3 mRNA transcription; (3) associated inhibition of target gene COL4A1 expression at the level of mRNA degradation; (4) 1,25D induced transcription of the miR-1228 host gene LRP1, with parallel induction of the mirtron miR-1228; and (5) miR-1228 mediated suppression of BMP2K protein expression by inhibition of translation. The effect of 1,25D on regulation of each miRNA activity is influenced by cellular aging as well as osteogenic stimulation. In this way 1,25D promotes HOB differentiation and function via direct effects on gene expression and by indirect regulation of gene expression by specific miRNAs.
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The miR-1228 donor LRP1 is a multifunctional endocytic clearance receptor from the low density lipoprotein receptor (LDLR) family that activates signaling pathways through multiple cytosolic adaptor and scaffold proteins. Through its extracellular domain, LRP1 mediates endocytosis of more than 40 different ligands, with diverse biological roles. Within skeletal tissues, LRP1 has been detected in osteoblasts and chondrocytes,[28, 29] with a potential function in the delivery of lipoproteins and vitamin K to bone. To date, no skeletal phenotypes in mice or humans have been attributed to LRP1 knockout; homozygous null mice exhibit embryonic lethality. Given the availability of conditional and Cre-specific LRP1 transgenic mouse lines (Mouse Gene Informatics, Jackson Laboratory, Bar Harbor, ME, USA; http://www.informatics.jax.org; MGI: 96828), it is possible that osteoblast-specific LRP1 knockout will better clarify the role of LRP1 in skeletal function in vivo. Other LDLR family members such as LRP5/6 play a key role in mediating Wnt signaling responses in the regulation of skeletal homeostasis, and LRP4 is known to control limb and craniofacial development. LRP1 is also known to mediate the canonical Wnt pathway in fibroblasts, and so it is possible that, independent of any actions of its miR-1228 product, the induction of HOB LRP1 alone will be sufficient to influence osteogenic potential via the Wnt pathway. Previous studies have described miRNA targeting of LRP1 expression, but to the best of our knowledge, our data are the first example of miRNA generation linked to LRP1 expression.
The miR-637 donor gene DAPK3 has been postulated to act as a tumor suppressor serine/threonine kinase, but it also plays a role in regulating cell morphology when overexpressed in mammalian cells. There have been no reported studies of DAPK3 in bone, but it is possible that suppression of DAPK3 may itself act as a regulatory switch to limit cell death during 1,25D-mediated differentiation of osteoblasts. In contrast to LRP1 and miR-1228, the induction of HOB miR-637 by 1,25D was associated with decreased expression of its host gene DAPK3. A minority of miRNA genes are located in the introns of protein-coding genes, preferentially in the same orientation as the mRNA, and notably both DAPK3 and miR-637 are in the same orientation on the reverse strand. Thus miR-637 is effectively its own VDRE-containing gene, which can be transcribed to pre-miRNA and then to mature miR-637 independent of its host DAPK3. This will lead to suppression of COL4A1, which is a predicted target of miR-637, but we hypothesize that miR-637 will also act to suppress expression of its host DAPK3 through transcriptional interference. This phenomenon of transcriptional interference has been observed elsewhere as a result of either tandem or convergent transcription of host and nested genes.
The miR-637 target, COL4A1, is found primarily in the basal lamina of blood vessels in many distinct skeletal locations. Primary HOBs and other osteoblastic cell lines express low levels of COL4A1 in vitro, and suppression of COL4A1 has been reported during early stages of osteoblast differentiation. In support of our data, miR-29b was found to be upregulated during osteoblast differentiation and targets many collagens including col4a2, the fibrillation partner of col4a1. It is therefore possible to hypothesize that 1,25D facilitates this process via induction of miR-637 and mRNA targeting of COL4A1. Recently, osteogenin, an extracellular matrix component of bone, was identified as a differentiation factor that initiates endochondral bone formation. Importantly, both osteogenin and transforming growth factor beta (TGF-β) bind avidly to COL4A1, suggesting a functional role in bone development. It is unclear whether COL4A1 expression during osteoblastic differentiation acts as an inhibitor of matrix mineralization as is seen for other bone matrix proteins. Further studies are required to clarify the importance of COL4A1 during vitamin D–induced osteoblastic differentiation.
BMP2K was identified as a gene whose expression was increased during bone morphogenic protein 2 (BMP2)-induced differentiation of a mouse osteoblastic cell line. Stable expression of BMP2K (initially referred to as BMP2-inducible kinase) in MC3T3-E1 osteoprogenitor cells suppressed mature osteoblast function, suggesting that BMP2K plays an important role in attenuating the program of osteoblast differentiation in mineralized tissue. In our study, we show that 1,25D treatment alone can decrease the levels of BMP2K in a BMP-independent manner. This effect was lost following LNA-knockdown of miR-1228, which also impaired 1,25D-mediated induction of alkaline phosphatase and osteocalcin, suggesting a key role for miR-1228 in osteoblast differentiation. In bone, vitamin D and analogs are known to modulate intracellular signaling molecules and the synthesis of ligands and receptors for both TGF-β and BMP pathways. Regulation of BMP2K protein by miR-1228 therefore provides another level of complexity by which vitamin D is able to interface with the BMP2 pathway.
Recent reports have highlighted a possible role for miRNAs in bone diseases such as primary osteoporosis.[46, 47] In this study, we identified two highly evolved miRNAs that are able to mediate novel regulatory responses to vitamin D in osteoblasts. The first, miR-637, is a primate-specific miRNA that was recently identified in the colorectal miRNAome. Although it was discovered 6 years ago, its biological role still remains elusive. Endogenous miR-637 was shown to be downregulated in four hepatocellular carcinoma (HCC) specimens, and overexpression of miR-637 in vitro blocked cell growth and induced apoptosis of HCC. This antiproliferative, tumor suppressor function of miR-637 is consistent with the cell cycle arrest and proapoptotic effects of 1,25D at supraphysiological concentrations. More recent studies have highlighted a musculoskeletal function for miR-637 in which its expression was increased during adipocyte differentiation in human mesenchymal stem cells. Conversely, miR-637 was decreased during osteoblast linage commitment, with osterix (osx), an early osteoblast-specific transcription factor, being a direct mRNA target. It is also interesting to note reports demonstrating osx-mediated induction of VDR in osteoblasts, suggesting that 1,25D-induced miR-637 may be part of a feedback mechanism controlling osx expression. Overall, these data suggest a potential biphasic effect of miR-637 expression; ie, low during initial osteoblast commitment and higher during vitamin D–induced differentiation to potentially maintain low levels of osx.
The 19-nucleotide mature miR-1228 sequence is homologous to mouse miR-667 (Supporting Fig. S3C). The 78% similarity between miR-1228 and miR-667 includes perfect similarity within the crucial seed region. Interestingly, mouse miR-667 is located on chromosome 12 within an intergenic region. The mouse LRP1 is located on chromosome 10, and harbors no intronic miRNAs. This suggests that although mice express a miRNA similar to miR-1228, host gene regulation of these miRNAs is likely to be different between the species. In particular, it is unclear whether mouse miR-667 is regulated by 1,25D in the same fashion as miR-1228. Importantly, given that the seed region for mouse miR-667 is complementary to human miR-1228, the predicted targets for these miRNAs may be similar. In this way, miR-667 may provide an important future animal model to assess the bone impact of 1,25D-induced miRNA in vivo.
Initial studies identified miR-1228 from a mammalian screen of small RNA libraries from Rhesus macaque and human brain tissues, but no evidence of its function has been documented to date. Rather than being a classical miRNA, miR-1228 was predicted to be a mirtron; a short hairpin intron that uses splicing to bypass Drosha cleavage used for the generation of canonical animal miRNAs as an alternative precursor for miRNA biogenesis. Spliced mirtrons are exported out of the nucleus and then cleaved by Dicer and incorporated into RNA silencing complexes. Our study shows that miR-1228 affects one of its targets, BMP2K, via repression of protein translation, and this mode of miRNA-translation repression is known to exist in a range of cells. In future studies it will be interesting to assess in further detail the miRNA:mRNA interactions that are characteristic of miR-1228 and BMP2K. Notably, there was only one miR-1228 binding site within the BMP2K sequence, suggesting that: (1) the singular occupancy of a miR-1228:BMP2K mRNA multiprotein complex is sufficient to ensure aberrant protein translation, or (2) this major duplex, in combination with several different miRNAs, help facilitate translational repression in an additive manner.
Data presented here provide the first evidence for miRNA involvement in 1,25D-mediated regulatory effects in bone. Further elucidation of the role of miRNAs in osteoblast regulation may provide novel strategies for studying the pathogenesis and treatment of vitamin D–associated bone disorders such as rickets and osteoporosis.