The miR-29-3p family suppresses inflammatory osteolysis.

Osteoclasts are the cells primarily responsible for inflammation-induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA-29-3p family members (miR-29a-3p, miR-29b-3p, miR-29c-3p) promote the function of RANKL-induced osteoclasts, the role of miR-29-3p during inflammatory TNF-α-induced osteoclastogenesis is unknown. We used bulk RNA-seq, histology, qRT-PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR-29-3p family members was decreased by expression of a miR-29-3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM-cre). We found that RANKL-treated monocytic cells expressing the miR-29-3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR-29-3p suppresses macrophage lineage commitment and may have anti-inflammatory effects. In correlation, when miR-29-3p activity was decreased, TNF-α-induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell-autonomous manner in vitro. Further, miR-29-3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF-α signaling sensitivity observed in the miR-29-3p decoy cells. Whereas our previous studies demonstrated that the miR-29-3p family promotes RANKL-induced bone resorption, the present work shows that miR-29-3p dampens TNF-α-induced osteoclastogenesis, indicating that miR-29-3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR-29-3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR-29-3p family a potential therapeutic target for modulating inflammatory response.

Impaired osteocyte maturation in the pathogenesis of renal osteodystrophy.

Pediatric renal osteodystrophy is characterized by skeletal mineralization defects, but the role of osteoblast and osteocyte maturation in the pathogenesis of these defects is unknown. We evaluated markers of osteocyte maturation and programmed cell death in iliac crest biopsy samples from pediatric dialysis patients and healthy controls. We evaluated the relationship between numbers of fibroblast growth factor 23 (FGF23)-expressing osteocytes and histomorphometric parameters of skeletal mineralization. We confirmed that chronic kidney disease (CKD) causes intrinsic changes in bone cell maturation using an in vitro model of primary osteoblasts from patients with CKD and healthy controls. FGF23 co-localized with the early osteocyte marker E11/gp38, suggesting that FGF23 is a marker of early osteocyte maturation. Increased numbers of early osteocytes and decreased osteocyte apoptosis characterized CKD bone. Numbers of FGF23-expressing osteocytes were highest in patients with preserved skeletal mineralization indices, and packets of matrix surrounding FGF23-expressing osteocytes appeared to have entered secondary mineralization. Primary osteoblasts from patients with CKD retained impaired maturation and mineralization characteristics in vitro. Addition of FGF23 did not affect primary osteoblast mineralization. Thus, CKD is associated with intrinsic changes in osteoblast and osteocyte maturation, and FGF23 appears to mark a relatively early stage in osteocyte maturation. Improved control of renal osteodystrophy and FGF23 excess will require further investigation into the pathogenesis of CKD-mediated osteoblast and osteocyte maturation failure.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

Primary osteoblast-like cells from patients with end-stage kidney disease reflect gene expression, proliferation, and mineralization characteristics ex vivo.

Osteocytes regulate bone turnover and mineralization in chronic kidney disease. As osteocytes are derived from osteoblasts, alterations in osteoblast function may regulate osteoblast maturation, osteocytic transition, bone turnover, and skeletal mineralization. Thus, primary osteoblast-like cells were cultured from bone chips obtained from 24 pediatric ESKD patients. RNA expression in cultured cells was compared with RNA expression in cells from healthy individuals, to RNA expression in the bone core itself, and to parameters of bone histomorphometry. Proliferation and mineralization rates of patient cells were compared with rates in healthy control cells. Associations were observed between bone osteoid accumulation, as assessed by bone histomorphometry, and bone core RNA expression of osterix, matrix gla protein, parathyroid hormone receptor 1, and RANKL. Gene expression of osteoblast markers was increased in cells from ESKD patients and signaling genes including Cyp24A1, Cyp27B1, VDR, and NHERF1 correlated between cells and bone cores. Cells from patients with high turnover renal osteodystrophy proliferated more rapidly and mineralized more slowly than did cells from healthy controls. Thus, primary osteoblasts obtained from patients with ESKD retain changes in gene expression ex vivo that are also observed in bone core specimens. Evaluation of these cells in vitro may provide further insights into the abnormal bone biology that persists, despite current therapies, in patients with ESKD.

miR-29 promotes murine osteoclastogenesis by regulating osteoclast commitment and migration.

Osteoclast differentiation is regulated by transcriptional, post-transcriptional, and post-translational mechanisms. MicroRNAs are fundamental post-transcriptional regulators of gene expression. The function of the miR-29 (a/b/c) family in cells of the osteoclast lineage is not well understood. In primary cultures of mouse bone marrow-derived macrophages, inhibition of miR-29a, -29b, or -29c diminished formation of TRAP (tartrate-resistant acid phosphatase-positive) multinucleated osteoclasts, and the osteoclasts were smaller. Quantitative RT-PCR showed that all miR-29 family members increased during osteoclast differentiation, in concert with mRNAs for the osteoclast markers Trap (Acp5) and cathepsin K. Similar regulation was observed in the monocytic cell line RAW264.7. In stably transduced RAW264.7 cell lines expressing an inducible miR-29 competitive inhibitor (sponge construct), miR-29 knockdown impaired osteoclastic commitment and migration of pre-osteoclasts. However, miR-29 knockdown did not affect cell viability, actin ring formation, or apoptosis in mature osteoclasts. To better understand how miR-29 regulates osteoclast function, we validated miR-29 target genes using Luciferase 3'-UTR reporter assays and specific miR-29 inhibitors. We demonstrated that miR-29 negatively regulates RNAs critical for cytoskeletal organization, including Cdc42 (cell division control protein 42) and Srgap2 (SLIT-ROBO Rho GTPase-activating protein 2). Moreover, miR-29 targets RNAs associated with the macrophage lineage: Gpr85 (G protein-coupled receptor 85), Nfia (nuclear factor I/A), and Cd93. In addition, Calcr (calcitonin receptor), which regulates osteoclast survival and resorption, is a novel miR-29 target. Thus, miR-29 is a positive regulator of osteoclast formation and targets RNAs important for cytoskeletal organization, commitment, and osteoclast function. We hypothesize that miR-29 controls the tempo and amplitude of osteoclast differentiation.

miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop.

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.

Inactivation of SPARC enhances high-fat diet-induced obesity in mice.

Secreted protein, acidic and rich in cysteine (SPARC), a matricellular protein, modulates extracellular matrix assembly and turnover in many physiological processes. SPARC-null mice exhibit an increased accumulation of adipose tissue. To distinguish between the functions of SPARC in adipogenesis during development and adulthood, we studied wild-type (WT) and SPARC-null mice maintained on a normal (low-fat) or high-fat (HF) diet. On an HF diet, SPARC-null mice exhibited significantly greater weight gain, in comparison to their WT counterparts, and had an enhanced cortical bone area that was likely due to increased mechanical loading. Diet-induced obesity (DIO) was also associated with an increase in vertebral trabecular bone in WT mice, but a significant change in this parameter was not observed in SPARC-null animals. We show that SPARC inhibits mitotic clonal expansion of preadipocytes at an early stage of adipogenesis. Moreover, there were substantially diminished levels of type I collagen in SPARC-null adipose tissue, as well as a reduction in the number of cross-linked, mature collagen fibers. In the absence of SPARC, mice show enhanced DIO. In adult animals, SPARC functions in the production and remodeling of adipose tissue, as well as in the regulation of preadipocyte differentiation.

MicroRNAs regulating TGFß and BMP signaling in the osteoblast lineage.

This review showcases miRNAs contributing to the regulation of bone forming osteoblasts through their effects on the TGFß and BMP pathways, with a focus on ligands, receptors and SMAD-mediated signaling. The goal of this work is to provide a basis for broadly understanding the contribution of miRNAs to the modulation of TGFß and BMP signaling in the osteoblast lineage, which may provide a rationale for potential therapeutic strategies. Therefore, the search strategy for this review was restricted to validated miRNA-target interactions within the canonical TGFß and BMP signaling pathways; miRNA-target interactions based only bioinformatics are not presented. Specifically, this review discusses miRNAs targeting each of the TGFß isoforms, as well as BMP2 and BMP7. Further, miRNAs targeting the signaling receptors TGFßR1 and TGFßR2, and those targeting the type 1 BMP receptors and BMPR2 are described. Lastly, miRNAs targeting the receptor SMADs, the common SMAD4 and the inhibitory SMAD7 are considered. Of these miRNAs, the miR-140 family plays a prominent role in inhibiting TGFß signaling, targeting both ligand and receptor. Similarly, the miR-106 isoforms target both BMP2 and SMAD5 to inhibit osteoblastic differentiation. Many of the miRNAs targeting TGFß and BMP signaling components are induced during fracture, mechanical unloading or estrogen deprivation. Localized delivery of miRNA-based therapeutics that modulate the BMP signaling pathway could promote bone formation.

Inhibition of miR-29 Activity in the Myeloid Lineage Increases Response to Calcitonin and Trabecular Bone Volume in Mice.

The miR-29-3p family (miR-29a, miR-29b, miR-29c) of microRNAs is increased during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. In vivo, activation of a miR-29-3p tough decoy inhibitor in Cre recombinase under the control of the lysozyme 2 promoter-expressing cells (myeloid lineage) resulted in mice displaying enhanced trabecular and cortical bone volume because of decreased bone resorption. Calcitonin receptor (Calcr) is a miR-29 target that negatively regulates bone resorption. CALCR was significantly increased in RANKL-treated miR-29-decoy osteoclasts, and these cells were more responsive to the inhibitory effect of calcitonin on osteoclast formation. Further, cathepsin K (Ctsk), which is critical for resorption, was decreased in miR-29-decoy cells. CALCR is a Gs-coupled receptor and its activation raises cAMP levels. In turn, cAMP suppresses cathepsin K, and cAMP levels were increased in miR-29-decoy cells. siRNA-mediated knock-down of Calcr in miR-29 decoy osteoclasts allowed recovery of cathepsin K levels in these cells. Overall, using a novel knockin tough decoy mouse model, we identified a new role for miR-29-3p in bone homeostasis. In RANKL-driven osteoclastogenesis, as seen in normal bone remodeling, miR-29-3p promotes resorption. Consequently, inhibition of miR-29-3p activity in the myeloid lineage leads to increased trabecular and cortical bone. Further, this study documents an interrelationship between CALCR and CTSK in osteoclastic bone resorption, which is modulated by miR-29-3p.

miR-433-3p suppresses bone formation and mRNAs critical for osteoblast function in mice.

MicroRNAs (miRNAs) are key posttranscriptional regulators of osteoblastic commitment and differentiation. miR-433-3p was previously shown to target Runt-related transcription factor 2 (Runx2) and to be repressed by bone morphogenetic protein (BMP) signaling. Here, we show that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Multiple pathways regulate osteogenesis. Using luciferase-3' untranslated region (UTR) reporter assays, five novel miR-433-3p targets involved in parathyroid hormone (PTH), mitogen-activated protein kinase (MAPK), Wnt, and glucocorticoid signaling pathways were validated. We show that Creb1 is a miR-433-3p target, and this transcription factor mediates key signaling downstream of PTH receptor activation. We also show that miR-433-3p targets hydroxysteroid 11-ß dehydrogenase 1 (Hsd11b1), the enzyme that locally converts inactive glucocorticoids to their active form. miR-433-3p dampens glucocorticoid signaling, and targeting of Hsd11b1 could contribute to this phenomenon. Moreover, miR-433-3p targets R-spondin 3 (Rspo3), a leucine-rich repeat-containing G-protein coupled receptor (LGR) ligand that enhances Wnt signaling. Notably, Wnt canonical signaling is also blunted by miR-433-3p activity. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433-3p tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating that miR-433-3p decreases osteoblast activity. Overall, we showed that miR-433-3p is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, Wnt, and endogenous glucocorticoids. Local delivery of miR-433-3p inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Inhibition of miR-29-3p isoforms via tough decoy suppresses osteoblast function in homeostasis but promotes intermittent parathyroid hormone-induced bone anabolism.

miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/ßcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.

Rac1 Inhibition Via Srgap2 Restrains Inflammatory Osteoclastogenesis and Limits the Clastokine, SLIT3.

The Rac1-specific guanosine triphosphatase (GTPase)-activating protein Slit-Robo GAP2 (Srgap2) is dramatically upregulated during RANKL-induced osteoclastogenesis. Srgap2 interacts with the cell membrane to locally inhibit activity of Rac1. In this study, we determined the role of Srgap2 in the myeloid lineage on bone homeostasis and the osteoclastic response to TNFα treatment. The bone phenotype of mice specifically lacking Srgap2 in the myeloid lineage (Srgap2 f/f :LysM-Cre; Srgap2 conditional knockout [cKO]) was investigated using histomorphometric analysis, in vitro cultures and Western blot analysis. Similar methods were used to determine the impact of TNFα challenge on osteoclast formation in Srgap2 cKO mice. Bone parameters in male Srgap2 cKO mice were unaffected. However, female cKO mice displayed higher trabecular bone volume due to increased osteoblast surface and bone formation rate, whereas osteoclastic parameters were unaltered. In vitro, cells from Srgap2 cKO had strongly enhanced Rac1 activation, but RANKL-induced osteoclast formation was unaffected. In contrast, conditioned medium from Srgap2 cKO osteoclasts promoted osteoblast differentiation and had increased levels of the bone anabolic clastokine SLIT3, providing a possible mechanism for increased bone formation in vivo. Rac1 is rapidly activated by the inflammatory cytokine TNFα. Supracalvarial injection of TNFα caused an augmented osteoclastic response in Srgap2 cKO mice. In vitro, cells from Srgap2 cKO mice displayed increased osteoclast formation in response to TNFα. We conclude that Srgap2 plays a prominent role in limiting osteoclastogenesis during inflammation through Rac1, and restricts expression of the paracrine clastokine SLIT3, a positive regulator of bone formation. © 2019 American Society for Bone and Mineral Research.

miR-29 suppression of osteonectin in osteoblasts: regulation during differentiation and by canonical Wnt signaling.

The matricellular protein osteonectin, secreted protein acidic and rich in cysteine (SPARC, BM-40), is the most abundant non-collagenous matrix protein in bone. Matricellular proteins play a fundamental role in the skeleton as regulators of bone remodeling. In the skeleton, osteonectin is essential for the maintenance of bone mass and for balancing bone formation and resorption in response to parathyroid hormone (PTH). It promotes osteoblast differentiation and cell survival. Mechanisms regulating the expression of osteonectin in the skeleton and in other tissues remain poorly understood. We found that the proximal region of the mouse osteonectin 3' untranslated region (UTR) contains a well-conserved, dominant regulatory motif that interacts with microRNAs (miRs)-29a and -29c. Transfection of osteoblastic cells with miR-29a inhibitors increased osteonectin protein levels, whereas transfection of miR-29a precursor RNA decreased osteonectin. miR-29a and -29c were increased during osteoblastic differentiation in vitro. The up-regulation of these miRNAs correlated with decreased osteonectin protein during the matrix maturation and mineralization phases of late differentiation. In contrast, osteonectin transcript levels remained relatively constant during this process, implying repression of translation. Treatment of osteoblasts with LiCl induced miR-29a and -29c expression and decreased osteonectin synthesis. When cells were treated with Dickkopf-1 (Dkk-1), miR-29a and -29c expression was repressed. These data suggest that canonical Wnt signaling, which is increased during osteoblastic differentiation, induces expression of miR-29. Osteonectin and miR-29 are co-expressed in extra-skeletal tissues, and the post-transcriptional mechanisms regulating osteonectin in osteoblasts are likely to be active in other cell systems.

MicroRNAs are Critical Regulators of Osteoclast Differentiation.

PURPOSE OF REVIEW: Our goal is to comprehensively review the most recent reports of microRNA (miRNA) regulation of osteoclastogenesis. We highlight validated miRNA-target interactions and their place in the signaling networks controlling osteoclast differentiation and function. RECENT FINDINGS: Using unbiased approaches to identify miRNAs of interest and reporter-3'UTR assays to validate interactions, recent studies have elucidated the impact of specific miRNA-mRNA interactions during in vitro osteoclastogenesis. There has been a focus on signaling mediators downstream of the RANK and CSF1R signaling, and genes essential for differentiation and function. For example, several miRNAs directly and indirectly target the master osteoclast transcription factor, Nfatc1 (e.g. miR-124 and miR-214) and Rho-GTPases, Cdc42 and Rac1 (e.g. miR-29 family). SUMMARY: Validating miRNA expression patterns, targets, and impact in osteoclasts and other skeletal cells is critical for understanding basic bone biology and for fulfilling the therapeutic potential of miRNA-based strategies in the treatment bone diseases.

Accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in osteonectin-null mice.

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone. In the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 microg/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin-/- mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH.

Increased Notch 1 expression and attenuated stimulatory G protein coupling to adenylyl cyclase in osteonectin-null osteoblasts.

Osteonectin, or secreted protein acidic and rich in cysteine, is one of the most abundant noncollagen matrix components in bone. This matricellular protein regulates extracellular matrix assembly and maturation in addition to modulating cell behavior. Mice lacking osteonectin develop severe low-turnover osteopenia, and in vitro studies of osteonectin-null osteoblastic cells showed that osteonectin supports osteoblast formation, maturation, and survival. The present studies demonstrate that osteonectin-null osteoblastic cells have increased expression of Notch 1, a well-documented regulator of cell fate in multiple systems. Furthermore, osteonectin-null cells are more plastic and less committed to osteoblastic differentiation, able to pursue adipogenic differentiation given the appropriate signals. Notch 1 transcripts are down-regulated by inducers of cAMP in both wild-type and osteonectin-null osteoblasts, suggesting that the mutant osteoblasts may have a defect in generation of cAMP in response to stimuli. Indeed, many bone anabolic agents signal through increased cAMP. Wild-type and osteonectin-null osteoblasts generated comparable amounts of cAMP in response to forskolin, a direct stimulator of adenylyl cyclase. However, the ability of osteonectin-null osteoblasts to generate cAMP in response to cholera toxin, a direct stimulator of Gs, was attenuated. These data imply that osteonectin-null osteoblasts have decreased coupling of Gs to adenylyl cyclase. Because osteonectin promotes G protein coupling to an effector, our studies support the concept that low-turnover osteopenia can result from reducing G protein coupled receptor activity.

MicroRNA variants as genetic determinants of bone mass.

Single nucleotide polymorphisms (SNPs) are the most abundant genetic variants that contribute to the heritability of bone mass. MicroRNAs (miRNAs, miRs) are key post-transcriptional regulators that modulate the differentiation and function of skeletal cells by targeting multiple genes in the same or distinct signaling pathways. SNPs in miRNA genes and miRNA binding sites can alter miRNA abundance and mRNA targeting. This review describes the potential impact of miRNA-related SNPs on skeletal phenotype. Although many associations between SNPs and bone mass have been described, this review is limited to gene variants for which a function has been experimentally validated. SNPs in miRNA genes (miR-SNPs) that impair miRNA processing and alter the abundance of mature miRNA are discussed for miR-146a, miR-125a, miR-196a, miR-149 and miR-27a. SNPs in miRNA targeting sites (miR-TS-SNPs) that alter miRNA binding are described for the bone remodeling genes bone morphogenetic protein receptor 1 (Bmpr1), fibroblast growth factor 2 (Fgf2), osteonectin (Sparc) and histone deacetylase 5 (Hdac5). The review highlights two aspects of miRNA-associated SNPs: the mechanism for altering miRNA mediated gene regulation and the potential of miR-associated SNPs to alter osteoblast, osteoclast or chondrocyte differentiation and function. Given the polygenic nature of skeletal diseases like osteoporosis and osteoarthritis, validating the function of additional miRNA-associated SNPs has the potential to enhance our understanding of the genetic determinants of bone mass and predisposition to selected skeletal diseases.

The microRNA-29 family in cartilage homeostasis and osteoarthritis.

UNLABELLED: MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family was also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFß1 decreased expression of miR-29a, b, and c (3p) in primary chondrocytes, whilst IL-1ß increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB, and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, and CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways. KEY MESSAGES: Expression of the miR-29 family is regulated in cartilage during osteoarthritis. SOX9 represses expression of the miR-29 family in chondrocytes. The miR-29 family is regulated by TGF-ß1 and IL-1 in chondrocytes. The miR-29 family negatively regulates Smad, NFκB, and canonical Wnt signalling. Several Wnt-related genes are direct targets of the miR-29 family.

A single nucleotide polymorphism in osteonectin 3' untranslated region regulates bone volume and is targeted by miR-433.

Osteonectin/SPARC is one of the most abundant noncollagenous extracellular matrix proteins in bone, regulating collagen fiber assembly and promoting osteoblast differentiation. Osteonectin-null and haploinsufficient mice have low-turnover osteopenia, indicating that osteonectin contributes to normal bone formation. In male idiopathic osteoporosis patients, osteonectin 3' untranslated region (UTR) single-nucleotide polymorphism (SNP) haplotypes that differed only at SNP1599 (rs1054204) were previously associated with bone mass. Haplotype A (containing SNP1599G) was more frequent in severely affected patients, whereas haplotype B (containing SNP1599C) was more frequent in less affected patients and healthy controls. We hypothesized that SNP1599 contributes to variability in bone mass by modulating osteonectin levels. Osteonectin 3' UTR reporter constructs demonstrated that haplotype A has a repressive effect on gene expression compared with B. We found that SNP1599G contributed to an miR-433 binding site, and miR-433 inhibitor relieved repression of the haplotype A, but not B, 3' UTR reporter construct. We tested our hypothesis in vivo, using a knock-in approach to replace the mouse osteonectin 3' UTR with human haplotype A or B 3' UTR. Compared with haplotype A mice, bone osteonectin levels were higher in haplotype B mice. B mice displayed higher bone formation rate and gained more trabecular bone with age. When parathyroid hormone was administered intermittently, haplotype B mice gained more cortical bone area than A mice. Cultured marrow stromal cells from B mice deposited more mineralized matrix and had higher osteocalcin mRNA compared with A mice, demonstrating a cell-autonomous effect on differentiation. Altogether, SNP1599 differentially regulates osteonectin expression and contributes to variability in bone mass, by a mechanism that may involve differential targeting by miR-433. This work validates the findings of the previous candidate gene study, and it assigns a physiological function to a common osteonectin allele, providing support for its role in the complex trait of skeletal phenotype. © 2014 American Society for Bone and Mineral Research.

Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls.

Osteonectin function in bone was investigated by infrared analysis of bones from osteonectin-null (KO) and wildtype mice (four each at 11, 17, and 36 weeks). An increase in mineral content and crystallinity in newly formed KO bone and collagen maturity at all sites was found using FTIR microspectroscopy and imaging; consistent with osteonectin's postulated role in regulating bone formation and remodeling. Mineral and matrix properties of tibias of osteonectin-null mice and their age- and background-matched wildtype controls were compared using Fourier-transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) at 10- and 7-mm spatial resolution, respectively. The bones came from animals that were 11, 17, and 36 weeks of age. Individual FTIRM spectra were acquired from 20 x 20 microm areas, whereas 4096 simultaneous FTIRI spectra were acquired from 400 x 400 microm areas. The FTIRM data for mineral-to-matrix, mineral crystallinity, and collagen maturity were highly correlated with the FTIRI data in similar regions. In general, the osteonectin-null mice bones had higher mineral contents and greater crystallinity (crystal size and perfection) than the age-matched wildtype controls. Specifically, the mineral content of the newly forming periosteal bone was increased in the osteonectin-null mice; the crystallinity of the cortical bone was decreased in all but the oldest animals, relative to the wildtype. The most significant finding, however, was increased collagen maturity in both the cortical and trabecular bone of the osteonectin-null mice. These spectroscopic data are consistent with a mechanism of decreased bone formation and remodeling.