The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression.

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3' untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.

Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Klf4 Promotes Dentinogenesis and Odontoblastic Differentiation via Modulation of TGF-ß Signaling Pathway and Interaction With Histone Acetylation.

Transcription factors bind to cell-specific cis-regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel-like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1-Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA-seq and ATAC-seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF-ß signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal-specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

Evidence of Osteogenic Regulation in Calcific Porcine Aortic Valves.

BACKGROUND: Chemically cross-linked animal tissues, such as porcine aortic valves (PAVs) have many documented advantages over mechanical valves. However, calcification is the major underlying pathologic process that results in bioprosthetic valve failure. Recently, several reports described the expression of noncollagenous bone matrix proteins in bioprosthetic valves and suggested an actively regulated process of tissue repair. METHODS: Thirty-one explanted PAVs with evidence of calcification were collected and examined for the protein expression implicated in myofibroblast activation, osteoblast differentiation, and bone matrix deposition by using immunohistochemistry. RESULTS: The mean duration that PAVs were implanted was 11.5 ± 5.6 years, ranging from 12 months to 28 years. Pearson correlation analysis showed a significant relationship between the duration and valvular calcification (r = 0.3818, P = .034). The number of vimentin-positive mesenchymal cells in explanted PAVs was significantly lower than that of unused PAVs (P < .01). However, increased expression of α-smooth muscle actin (α-SMA) (P < .01), proliferating cell nuclear antigen (PCNA, P < .01), Cbfa1/Runx2 (P < .01), osterix (P = .0126), bone sialoprotein (BSP, P < .01), osteocalcin (P < .01), and osteopontin (P < .01) was found in explanted PAVs. Immunohistochemical staining of alkaline phosphatase (ALP) and osteocalcin was negative in the unused PAVs. In explanted PAVs, the expression level of these 2 proteins was also significantly increased. CONCLUSIONS: Our results support the view that PAV calcification is an actively regulated process with osteogenic signaling activation.

miR-98-TXLNG1 (FIAT)/Sp7 function loop mediates osteoblast mineralization.

OBJECTIVE: To investigate the role of microRNAs (miRNAs) and its mechanism in osteoblast mineralization. MATERIALS AND METHODS: Real-time polymerase chain reaction (PCR), Northern Blot, and Western Blot were used to identify the expression mode of regulators. Overexpression and down-regulation experiments were carried out to study the role of miR-98 and interactions between regulators. Bioinformatics calculation and luciferase reporter assay were used to prove the target gene. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (CHIP), and promoter luciferase reporter assay confirmed the relationship between the regulator and the promoter of miR-98. RESULTS: MiR-98 was up-regulated during osteoblast mineralization. Overexpression of miR-98 promoted osteoblast mineralization. Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a negative regulator of osteoblast differentiation, was confirmed to be a target of miR-98. As a motivator in osteoblast mineralization, Sp7 transcription factor 7 (Sp7) promoted miR-98 transcription by a combination on the promoter region. CONCLUSIONS: Our study showed that miR-98 was an important regulator in osteoblast mineralization and miR-98 carried out its function through a novel miR-98-FIAT/Sp7 regulatory loop. It provides new insights into the roles of miRNAs in osteoblast mineralization.

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

Upregulated osterix expression elicited by Runx2 and Dlx5 is required for the accelerated osteoblast differentiation in PP2A Cα-knockdown cells.

Serine/threonine protein phosphatase 2A (PP2A) is involved in regulating various physiological processes including cell cycle, growth, apoptosis, and signal transduction. Osteoblast differentiation is controlled by main bone specific transcription factors including Osterix, distal-less homeobox 5 (Dlx5), and Runt-related transcription factor 2 (Runx2). We previously reported that knockdown of PP2A Cα increases the expression of Osterix, leading to the accelerated osteoblast differentiation through the upregulation of bone-related genes. In this study, we examined whether Dlx5 and Runx2 are involved in the upregulated Osterix expression in PP2A Cα-knockdown osteoblasts (shPP2A cells). The expression of Dlx5 as well as Osterix was significantly higher in shPP2A cells in the initial stage of osteoblast differentiation compared with the control cells (shCont). The expression of Runx2 protein was also higher in shPP2A cells compared with shCont cells although its mRNA level was comparable. Reduction of Dlx5 or Runx2 decreased Osterix expression and alkaline phosphatase activity in shPP2A cells. Luciferase assay showed that Osterix promoter activity was drastically elevated in shPP2A cells compared with that in shCont cells. The deletion or mutation of the Dlx5 and Runx2 binding sites significantly suppressed Osterix promoter activity in shPP2A cells. These results indicate that Dlx5 and Runx2 are critical factors for the upregulated Osterix expression in shPP2A cells, which is considered to be important for the accelerated osteoblast differentiation in these cells.