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1.AbstractBone fragility is the product of defects in bone mass and bone quality, both of which show sex-specific differences. Despite this, the cellular and molecular mechanisms underpinning the sexually dimorphic control of bone quality remain unclear, limiting our ability to effectively prevent fractures, especially in postmenopausal osteoporosis. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFβ signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (TβRIIocy−/−mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Since systemic TGFβ inhibition more robustly increases bone mass in female than male mice, we postulated that sex-specific differences in bone quality could likewise result, in part, from dimorphic regulation of PLR by TGFβ. Moreover, since lactation induces PLR, we examined the effect of TGFβ inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGFβ signaling were protected from TGFβ-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacuno-canalicular network, and the flexural strength of female TβRIIocy−/−bone was intact. With lactation, however, bone loss, and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in TβRIIocy−/−bone, relative to wild-type. Indeed, differential control of PTHR1 expression, by TGFβ and other factors, may contribute to dimorphism in PLR regulation in male and female TβRIIocy−/−mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGFβ signaling as a crucial regulator of lactation-induced PLR.

Identifying new genetic determinants of bone mineral density (BMD) and fracture promises to yield improved diagnostics and therapies for bone fragility. However, prioritizing candidate genes from genome-wide screens can be challenging. To overcome this challenge, we prioritized mouse genes that are differentially expressed in aging mouse bone based on whether their human homolog is associated with human BMD and/or fracture. Unbiased RNA-seq analysis of young and old male C57BL/6 mouse cortical bone identified 1499, 1685, and 5525 differentially expressed genes (DEGs) in 1, 2, and 2.5-year-old bone, relative to 2-month-old bone, respectively. Gene-based scores for heel ultrasound bone mineral density (eBMD) and fracture were estimated using published genome-wide association studies (GWAS) results of these traits in the UK Biobank. Enrichment analysis showed that mouse bone DEG sets for all three age groups, relative to young bone, are significantly enriched for eBMD, but only the oldest two DEG sets are enriched for fracture. Using gene-based scores, this approach prioritizes among thousands of DEGs by a factor of 5- to 100-fold, yielding 10 and 21 genes significantly associated with fracture in the two oldest groups of mouse DEGs. Though these genes were not the most differentially expressed, they included Sost, Lrp5, and others with well-established functions in bone. Several others have, as yet, unknown roles in the skeleton. Therefore, this study accelerates identification of new genetic determinants of bone fragility by prioritizing a clinically relevant and experimentally tractable number of candidate genes for functional analysis. Finally, we provide a website (www.mouse2human.org) to enable other researchers to easily apply our strategy. © 2022 American Society for Bone and Mineral Research (ASBMR).

Organism scale mechanical forces elicit cellular scale changes through coordinated regulation of multiple signaling pathways. The mechanisms by which cells integrate signaling to generate a unified biological response remains a major question in mechanobiology. For example, the mechanosensitive response of bone and other tissues requires coordinated signaling by the transforming growth factor beta (TGFβ) and Wnt pathways through mechanisms that are not well-defined. Here we report a new microRNA-dependent mechanism that mediates mechanosensitive crosstalk between TGFβ and Wnt signaling in osteocytes exposed to fluid shear stress (FSS). From 60 mechanosensitive microRNA (miRs) identified by small-RNAseq, miR100 expression is suppressed by in vivo hindlimb loading in the murine tibia and by cellular scale FSS in OCY454 cells. Though FSS activates both TGFβ and Wnt signaling in osteocytes, only TGFβ represses miR-100 expression. miR-100, in turn, antagonizes Wnt signaling by targeting and inhibiting expression of Frizzled receptors (FZD5/FZD8). Accordingly, miR-100 inhibition blunts FSS- and TGFβ-inducible Wnt signaling. Therefore, our results identify FSS-responsive miRNAs in osteocytes, including one that integrates the mechanosensitive function of two essential signaling pathways in the osteoanabolic response of bone to mechanical load.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFβ) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone‐forming and bone‐resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFβ signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFβ signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFβ target gene expression, even in the absence of added TGFβ. Indeed, relative to treatment with TGFβ, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFβ receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFβ family R‐Smads by stimulating multimerization and concurrently activating several TGFβ and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFβ and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Recent advances have revived interest in the concept of osteocyte perilacunar/canalicular remodeling (PLR) and have motivated efforts to identify the mechanisms regulating this process in bone in the context of normal physiology and pathological conditions. Here, we describe several methods that are evaluating morphological changes associated with PLR function of osteocytes.

Sexual dimorphism in the material properties of bone extracellular matrix (ECM) and geometry, which are parameters of bone quality have been recognized (Alswat, 2017; Nieves et al., 2005). However, the molecular mechanisms responsible for dimorphic bone quality remain unclear, limiting our ability to therapeutically target fragility, which is determined by bone quality. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFβ signaling (TβRIIocy-/-), suppresses perilacunar/canalicular remodeling (PLR) and compromises bone quality (Dole et al., 2017). Since we previously demonstrated that female bone mass is more sensitive to TGFβ (Mohammad et al., 2009), we hypothesized that loss of osteocytic TGFβ in females will lead to more severe deterioration of PLR and bone quality. Analysis of female bones, however, surprisingly revealed sexual dimorphism in the TGFβ regulation of osteocyte function. While the male TβRIIocy-/- mice demonstrated reduced trabecular bone mass and repression in expression of requisite PLR genes, including matrix metalloproteinases (Mmp2, Mmp13, and Mmp14), cathepsin K (Ctsk), and tartrate-resistant acid phosphatase (Acp5/TRAP) (Qing et al., 2012), the female TβRIIocy-/- bones were intact. With absence of osteocytic TGFβ signaling, the canaliculi of male TβRIIocy-/- osteocytes were truncated and a severe deterioration of lacuno-canalicular network (LCN) was observed. Unlike the male TβRIIocy-/- mice that exhibited poor bone quality, female TβRIIocy-/- bones were completely protected from bone quality defects and macromechanical bone fragility. Thus, TGFβ-dependent regulation PLR and bone quality is sexually dimorphic. Since, lactation is a potent PLR agonist, we also evaluated the lactation-induced PLR response of TβRIIocy-/- female mice. Compared to WT, lactation-induced bone loss and upregulation of PLR genes was significantly attenuated in the TβRIIocy-/- female mice. Lactation-mediated increases in osteocyte lacunar volume were suppressed in TβRIIocy-/- bones as well. This mitigation in PLR of lactating TβRIIocy-/- mice was analogous to that observed in mice with osteocyte-specific ablation of parathyroid hormone type I receptor (PTHR1) (Qing et al., 2012). Remarkably, lactation-inducible PTHR1 expression was also diminished in TβRIIocy-/- osteocytes. Therefore, osteocyte-intrinsic TGFβ signaling is critical for the maximal induction of PLR during lactation, and TGFβ is an upstream regulator of PTHR1-dependent activation of osteocytic PLR. Indeed, we also found sexual dimorphism in the regulation of osteocytic PTHR1 expression by TGFβ, such that male, but not female, TβRIIocy-/- mice had reduced PTHR1 compared to WT. In summary, we identify a novel PTH-dependent mechanism that protects female bone against the severe bone fragility that results from suppressed TGFβ signaling in osteocytes.

OBJECTIVE Transforming growth factor β (TGFβ) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFβ signaling to OA. METHODS To identify the role of osteocytic TGFβ signaling in joint homeostasis, we used 16-week-old male mice (n = 9-11 per group) and female mice (n = 7-11 per group) with an osteocyte-intrinsic ablation of TGFβ receptor type II (TβRIIocy-/- mice) and assessed defects in cartilage degeneration, subchondral bone plate (SBP) thickness, and SBP sclerostin expression. To further investigate these mechanisms in 16-week-old male mice, we perturbed joint homeostasis by subjecting 8-week-old mice to medial meniscal/ligamentous injury (MLI), which preferentially disrupts the mechanical environment of the medial joint to induce OA. RESULTS In all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TβRIIocy-/- mice, but not female TβRIIocy-/- mice, had increased cartilage degeneration, increased SBP thickness, and higher levels of SBP sclerostin compared with control mice (all P < 0.05), demonstrating that the role of osteocytic TGFβ signaling on joint homeostasis is sexually dimorphic. With changes in joint mechanics following injury, control mice had increased SBP thickness, subchondral bone volume, and SBP sclerostin expression (all P < 0.05). TβRIIocy-/- mice, however, were insensitive to subchondral bone changes with injury, suggesting that mechanosensation at the SBP requires osteocytic TGFβ signaling. CONCLUSION Our results provide new evidence that osteocytic TGFβ signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFβ signaling as a novel therapeutic target for OA.

Bone fragility associated with obesity is well recognized and is attributed to a cumulative decline in bone quality despite high BMD. Moreover, novel insights into skeletal physiology have revealed that bone-derived factors are key regulators of systemic energy balance. Despite the revelation that bone is both a target and a driver of energy metabolism, it is not entirely clear how obesity compromises the metabolic function of bone and whether deregulated energetics in bone play a causal role in declining bone quality. Transforming growth factor-beta (TGFβ), a key mediator of bone homeostasis, was recently shown by us to play a positive role in osteocytes, where its ablation (TβRIIocy-/-) compromised bone quality. TGFβ signaling has been previously implicated in etiologies of obesity and type 2 diabetes. Also, our unpublished unbiased transcriptomic analysis in TβRIIocy-/- mouse bones (osteocyte-enriched RNA) has shown that osteocytic ablation of TGFβ signaling causes profound changes in the expression of genes implicated in glucose and fatty metabolism. Together these observations led us to hypothesize that osteocytic TGFβ plays an integral role in regulating obesity impacted bone architecture and function in metabolism. Using hyperglycemia to model the metabolic milieu of obesity in vitro, we found that hyperglycemia upregulated TGFβ signaling in osteocytes. We also found that TGFβ acts similarly to hyperglycemia in reprogramming osteocyte cellular metabolism to promote glycolysis at the expense of oxidative phosphorylation. Thus, TGFβ may be a crucial mediator of hyperglycemia-induced metabolic reprogramming in osteocytes. In TβRIIocy-/- mice, inhibition of osteocyte-intrinsic TGFβ signaling reduced age-dependent weight gain and improved glycemic control relative to littermate controls. When subjected to a high-fat diet (HFD, 18 weeks), TβRIIocy-/- mice continued to be leaner, with lower food intake, and higher levels of activity and fatty acid metabolism in the liver compared to HFD fed littermate controls. This implicates osteocyte-intrinsic TGFβ signaling as a crucial regulator of systemic energy metabolism. TβRIIocy-/- mice on HFD also showed increased trabecular and cortical bone mass, increased bone mineral density, and protection against reduced bone strength compared to littermate controls on a similar diet. Overall, our study identifies a novel role for osteocytic TGFβ signaling in regulating energy metabolism and bone health in obesity.

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.

© 2017 Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-β) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-β controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-β signaling (TβRIIocy−/−), we show that TGF-β regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility. Resistance to fracture requires healthy bone mass and quality. However, the cellular mechanisms regulating bone quality are unclear. Dole et al. show that osteocyte-intrinsic TGF-β signaling maintains bone quality through perilacunar/canalicular remodeling. Thus, osteocytes mediate perilacunar/canalicular remodeling and osteoclast-directed remodeling to cooperatively maintain bone quality and mass and prevent fragility.

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.

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.

Background Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation.