Deletion of the auxiliary α2δ1 voltage sensitive calcium channel subunit in osteocytes and late-stage osteoblasts impairs femur strength and load-induced bone formation in male mice.

Osteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel's α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol anchor and regulates the channel's calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10 kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (P < .05) by DXA, reduced relative osteoid surface (P < .05), and altered osteoblast and osteocyte regulatory gene expression (P < .01). Cacna2d1f/f, Cre + male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo micro-computed tomography measurements at the proximal tibia (P < .01) compared to controls, whereas Cacna2d1f/f, Cre + female mice showed impaired 20-wk cancellous and cortical bone ex vivo micro-computed tomography measurements (P < .05) vs controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.

The ability of bone to sense and respond to forces generated during daily physical activities is essential to skeletal health. Although several bone cell types contribute to the maintenance of bone health, osteocytes are thought to be the primary mechanosensitive cells; however, the mechanisms through which these cells perceive mechanical stimuli remains unclear. Previous work has shown that voltage sensitive calcium channels are necessary for bone to sense mechanical force; yet the means by which those channels translate the physical signal into a biochemical signal is unclear. Data within this manuscript demonstrate that the extracellular α2δ1 subunit of voltage sensitive calcium channels is necessary for load-induced bone formation as well as to enable calcium influx within osteocytes. As this subunit enables physical interactions of the channel pore with the extracellular matrix, our data demonstrate the need for the α2δ1 subunit for mechanically induced bone adaptation, thus serving as a physical conduit through which mechanical signals from the bone matrix are transduced into biochemical signals by enabling calcium influx into osteocytes.

Loss of the auxiliary α2δ1 voltage-sensitive calcium channel subunit impairs bone formation and anabolic responses to mechanical loading.

Voltage-sensitive calcium channels (VSCCs) influence bone structure and function, including anabolic responses to mechanical loading. While the pore-forming (α1) subunit of VSCCs allows Ca2+ influx, auxiliary subunits regulate the biophysical properties of the pore. The α2δ1 subunit influences gating kinetics of the α1 pore and enables mechanically induced signaling in osteocytes; however, the skeletal function of α2δ1 in vivo remains unknown. In this work, we examined the skeletal consequences of deleting Cacna2d1, the gene encoding α2δ1. Dual-energy X-ray absorptiometry and microcomputed tomography imaging demonstrated that deletion of α2δ1 diminished bone mineral content and density in both male and female C57BL/6 mice. Structural differences manifested in both trabecular and cortical bone for males, while the absence of α2δ1 affected only cortical bone in female mice. Deletion of α2δ1 impaired skeletal mechanical properties in both sexes, as measured by three-point bending to failure. While no changes in osteoblast number or activity were found for either sex, male mice displayed a significant increase in osteoclast number, accompanied by increased eroded bone surface and upregulation of genes that regulate osteoclast differentiation. Deletion of α2δ1 also rendered the skeleton insensitive to exogenous mechanical loading in males. While previous work demonstrates that VSCCs are essential for anabolic responses to mechanical loading, the mechanism by which these channels sense and respond to force remained unclear. Our data demonstrate that the α2δ1 auxiliary VSCC subunit functions to maintain baseline bone mass and strength through regulation of osteoclast activity and also provides skeletal mechanotransduction in male mice. These data reveal a molecular player in our understanding of the mechanisms by which VSCCs influence skeletal adaptation.

Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice.

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.

miR21 deletion in osteocytes has direct and indirect effects on skeletal muscle in a sex-dimorphic manner in mice.

BACKGROUND: Osteocytic microRNA21 (miR21) removal alters cytokine production and bone mass by modulating osteoclast and osteoblast differentiation and activity. Removing osteocytic miR21 increases osteoclast/osteoblast numbers and bone mass in male mice, whereas it decreases osteoclasts/osteoblasts without affecting bone mass in female mice. On the other hand, it leads to sex-independent increases in bone mechanical properties. Because changes in bone remodeling and strength affect skeletal muscle through bone-muscle crosstalk, we investigated whether osteocytic miR21 deletion influences skeletal muscle. METHODS: miR21fl/fl mice and 8kbDMP1-Cre mice were mated to obtain miR21-deficient mice primarily in the osteocyte (OtmiR21Δ) and littermate controls (miR21fl/fl). Four-month-old male and female mice were analyzed. Body composition was examined by DXA/Piximus and gene expression was assessed by qPCR. Ex vivo cultures of long bones devoid of bone-marrow cells from male and female 4-month-old were maintained for 48 h. Conditioned media were collected and used for the C2C12 assays. Two-way ANOVA analyses were performed to determine the contributions of genotype and sex and their interaction to the effects of miR21 deficiency. RESULTS: Lean body mass was increased only in female OtmiR21Δ mice, although miR21 levels in soleus muscle were similar in miR21fl/fl (0.05 ± 0.02) and OtmiR21Δ (0.09 ± 0.04) mice. Female, but not male, OtmiR21Δ mice exhibited increased soleus (42%) and gastrocnemius (21%) muscle weight compared to miR21fl/fl littermates. However, muscle strength and gastrocnemius muscle fiber cross-sectional area were unaltered for either sex. Kinase phosphorylation (phospho/total protein ratio) in soleus muscle, measured as a surrogate for kinase activity by means of multiplex analysis, was also selectively changed depending on the mouse sex. Thus, female OtmiR21Δ mice had higher T185/Y187-ERK1/2 but lower S473-Akt phosphorylation than miR21fl/fl controls, while male OtmiR21Δ mice had higher S473-Akt phosphorylation, suggesting sex-dimorphic shifts in anabolic vs. catabolic signaling. Consistently, levels of FOXO3 and MuRF-1, known to be regulated by Akt, were only increased in male OtmiR21Δ mice. Atrogin-1 mRNA levels were upregulated in female OtmiR21Δ mice, suggesting a potential shift in protein regulation. Sex-specific effects were also found by exposing myotube cultures to conditioned media from 48-h-cultured marrow-flushed bones. Thus 5-day differentiated C2C12 myotubes treated with conditioned media of female OtmiR21Δ mice exhibit 12% higher average diameter compared to cells exposed to miR21fl/fl bone conditioned media. Yet, conditioned media from male bones had no effect on myotube size. CONCLUSIONS: We present a novel aspect of bone-muscle crosstalk in which osteocyte-derived miR21 influences skeletal muscle size, but not strength, in female but not male mice; whereas, intracellular signaling alterations resulting from loss of miR21 seem to alter protein dynamics in a sex-dimorphic fashion.

MicroRNA-101a enhances trabecular bone accrual in male mice.

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis.

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) R47H Variant Causes Distinct Age- and Sex-Dependent Musculoskeletal Alterations in Mice.

Previous studies proposed the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone, as a link between brain and bone disease. The TREM2 R47H variant is a known risk factor for Alzheimer's disease (AD), the most common form of dementia. To investigate whether altered TREM2 signaling could contribute to bone and skeletal muscle loss, independently of central nervous system defects, we used mice globally hemizygous for the TREM2 R47H variant (TREM2R47H/+ ), which do not exhibit AD pathology, and wild-type (WT) littermate control mice. Dxa/Piximus showed bone loss in female TREM2R47H/+ animals between 4 and 13 months of age and reduced cancellous and cortical bone (measured by micro-computed tomography [µCT]) at 13 months, which stalled out by 20 months of age. In addition, they exhibited decreased femoral biomechanical properties measured by three-point bending at 13 months of age, but not at 4 or 20 months. Male TREM2R47H/+ animals had decreased trabecular bone geometry but increased ultimate strain and failure force at 20 months of age versus WT. Only male TREM2R47H/+ osteoclasts differentiated more ex vivo after 7 days with receptor activator of nuclear factor κB ligand (RANKL)/macrophage colony-stimulating factor (M-CSF) compared to WT littermates. Yet, estrogen receptor alpha expression was higher in female and male TREM2R47H/+ osteoclasts compared to WT mice. However, female TREM2R47H/+ osteoclasts expressed less complement 3 (C3), an estrogen responsive element, and increased protein kinase B (Akt) activity, suggesting altered estrogen signaling in TREM2R47H/+ cells. Despite lower bone volume/strength in TREM2R47H/+ mice, skeletal muscle function measured by plantar flexion and muscle contractility was increased in 13-month-old female mutant mice. Overall, these data demonstrate that an AD-associated TREM2 variant can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of central neuropathology, potentially mediated through changes in osteoclastic intracellular signaling. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice.

Pannexin1 (Panx1) is a hemichannel-forming protein that participates in the communication of cells with the extracellular space. To characterize the role of osteoclastic Panx1 on bone, Panx1fl/fl;TRAP-Cre (Panx1ΔOc) mice were generated, and compared to Panx1fl/fl littermates at 6 weeks of age. Total and femoral BMD was ~20% lower in females and males whereas spinal BMD was lower only in female Panx1ΔOc mice. µCT analyses showed that cortical bone of the femoral mid-diaphysis was not altered in Panx1ΔOc mice. In contrast, cancellous bone in the distal femur and lumbar vertebra was significantly decreased in both female and male Panx1ΔOc mice compared to Panx1fl/fl controls and was associated with higher osteoclast activity in female Panx1ΔOc mice, with no changes in the males. On the other hand, vertebral bone formation was decreased for both sexes, resulting from lower mineral apposition rate in the females and lower mineralizing surface in the males. Consistent with an osteoclastic effect in female Panx1ΔOc mice, osteoclast differentiation with RANKL/M-CSF and osteoclast bone resorbing activity in vitro were higher in female, but not male, Panx1ΔOc mice, compared to Panx1fl/fl littermates. Surprisingly, although Panx1 expression was normal in bone marrow stromal-derived osteoblasts from male and female Panx1ΔOc mice, mineral deposition by male (but not female) Panx1ΔOc osteoblasts was lower than controls, and it was reduced in male Panx1fl/fl osteoblasts when conditioned media prepared from male Panx1ΔOc osteoclast cultures was added to the cell culture media. Thus, deletion of Panx1 in TRAP-expressing cells in female mice leads to low bone mass primarily through a cell autonomous effect in osteoclast activity. In contrast, our evidence suggests that changes in the osteoclast secretome drive reduced osteoblast function in male Panx1ΔOc mice, resulting in low bone mass.

6'-Methoxy Raloxifene-analog enhances mouse bone properties with reduced estrogen receptor binding.

Raloxifene (RAL) is an FDA-approved drug used to treat osteoporosis in postmenopausal women. RAL suppresses bone loss primarily through its role as a selective estrogen receptor modulator (SERM). This hormonal estrogen therapy promotes unintended side effects, such as hot flashes and increased thrombosis risk, and prevents the drug from being used in some patient populations at-risk for fracture, including children with bone disorders. It has recently been demonstrated that RAL can have significant positive effects on overall bone mechanical properties by binding to collagen and increasing bone tissue hydration in a cell-independent manner. A Raloxifene-Analog (RAL-A) was synthesized by replacing the 6-hydroxyl substituent with 6-methoxy in effort to reduce the compound's binding affinity for estrogen receptors (ER) while maintaining its collagen-binding ability. It was hypothesized that RAL-A would improve the mechanical integrity of bone in a manner similar to RAL, but with reduced estrogen receptor binding. Molecular assessment showed that while RAL-A did reduce ER binding, downstream ER signaling was not completely abolished. In-vitro, RAL-A performed similarly to RAL and had an identical concentration threshold on osteocyte cell proliferation, differentiation, and function. To assess treatment effect in-vivo, wildtype (WT) and heterozygous (OIM+/-) female mice from the Osteogenesis Imperfecta (OI) murine model were treated with either RAL or RAL-A from 8 weeks to 16 weeks of age. There was an untreated control group for each genotype as well. Bone microarchitecture was assessed using microCT, and mechanical behavior was assessed using 3-point bending. Results indicate that both compounds produced analogous gains in tibial trabecular and cortical microarchitecture. While WT mechanical properties were not drastically altered with either treatment, OIM+/- mechanical properties were significantly enhanced, most notably, in post-yield properties including bone toughness. This proof-of-concept study shows promising results and warrants the exploration of additional analog iterations to further reduce ER binding and improve fracture resistance.

Osteocytic miR21 deficiency improves bone strength independent of sex despite having sex divergent effects on osteocyte viability and bone turnover.

Osteocytes play a critical role in mediating cell-cell communication and regulating bone homeostasis, and osteocyte apoptosis is associated with increased bone resorption. miR21, an oncogenic microRNA, regulates bone metabolism by acting directly on osteoblasts and osteoclasts, but its role in osteocytes is not clear. Here, we show that osteocytic miR21 deletion has sex-divergent effects in bone. In females, miR21 deletion reduces osteocyte viability, but suppresses bone turnover. Conversely, in males, miR21 deletion increases osteocyte viability, but stimulates bone turnover and enhances bone structure. Further, miR21 deletion differentially alters osteocyte cytokine production in the two sexes. Interestingly, despite these changes, miR21 deletion increases bone mechanical properties in both sexes, albeit to a greater extent in males. Collectively, our findings suggest that miR21 exerts both sex-divergent and sex-equivalent roles in osteocytes, regulating osteocyte viability and altering bone metabolism through paracrine actions on osteoblasts and osteoclasts differentially in males vs females, whereas, influencing bone mechanical properties independent of sex.

Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength.

Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1-3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males.

Short-term pharmacologic RAGE inhibition differentially affects bone and skeletal muscle in middle-aged mice.

Loss of bone and muscle mass are two major clinical complications among the growing list of chronic diseases that primarily affect elderly individuals. Persistent low-grade inflammation, one of the major drivers of aging, is also associated with both bone and muscle dysfunction in aging. Particularly, chronic activation of the receptor for advanced glycation end products (RAGE) and elevated levels of its ligands high mobility group box 1 (HMGB1), AGEs, S100 proteins and Aß fibrils have been linked to bone and muscle loss in various pathologies. Further, genetic or pharmacologic RAGE inhibition has been shown to preserve both bone and muscle mass. However, whether short-term pharmacologic RAGE inhibition can prevent early bone and muscle loss in aging is unknown. To address this question, we treated young (4-mo) and middle-aged (15-mo) C57BL/6 female mice with vehicle or Azeliragon, a small-molecule RAGE inhibitor initially developed to treat Alzheimer's disease. Azeliragon did not prevent the aging-induced alterations in bone geometry or mechanics, likely due to its differential effects [direct vs. indirect] on bone cell viability/function. On the other hand, Azeliragon attenuated the aging-related body composition changes [fat and lean mass] and reversed the skeletal muscle alterations induced with aging. Interestingly, while Azeliragon induced similar metabolic changes in bone and skeletal muscle, aging differentially altered the expression of genes associated with glucose uptake/metabolism in these two tissues, highlighting a potential explanation for the differential effects of Azeliragon on bone and skeletal muscle in middle-aged mice. Overall, our findings suggest that while short-term pharmacologic RAGE inhibition did not protect against early aging-induced bone alterations, it prevented against the early effects of aging in skeletal muscle.

A let-7-to-miR-125 MicroRNA Switch Regulates Neuronal Integrity and Lifespan in Drosophila.

Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. Here, we show that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo). We first characterize novel adult phenotypes associated with loss of both let-7 and miR-125, which are derived from a common, polycistronic transcript that also encodes a third miRNA, miR-100. Consistent with the coordinate upregulation of all three miRNAs in aging flies, these phenotypes include brain degeneration and shortened lifespan. However, transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction. In contrast, let-7 is predominantly responsible for regulating chinmo during nervous system formation. These results indicate that let-7 and miR-125 function during two distinct stages, development and adulthood, rather than acting at the same time. These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system. Thus, this work not only establishes a key role for the highly conserved miR-125 in aging. It also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target, raising the possibility that such biphasic control may be a general feature of clustered miRNAs.