Heterozygous deletion of both sclerostin (Sost) and connexin43 (Gja1) genes in mice is not sufficient to impair cortical bone modeling.

Connexin43 (Cx43) is the main gap junction protein expressed in bone forming cells, where it modulates peak bone mass acquisition and cortical modeling. Genetic ablation of the Cx43 gene (Gja1) results in cortical expansion with accentuated periosteal bone formation associated with decreased expression of the Wnt inhibitor sclerostin. To determine whether sclerostin (Sost) down-regulation might contribute to periosteal expansion in Gja1 deficient bones, we took a gene interaction approach and crossed mice harboring germline null alleles for Gja1 or Sost to generate single Gja1+/-and Sost+/-and double Gja1+/-;Sost+/-heterozygous mice. In vivo µCT analysis of cortical bone at age 1 and 3 months confirmed increased thickness in Sost-/-mice, but revealed no cortical abnormalities in single Gja1+/-or Sost+/-mice. Double heterozygous Gja1+/-Sost+/-also showed no differences in mineral density, cortical thickness, width or geometry relative to wild type control mice. Likewise, 3-point bending measurement of bone strength revealed no significant differences between double Gja1+/-;Sost+/-or single heterozygous and wild type mice. Although these data do not exclude a contribution of reduced sclerostin in the cortical expansion seen in Gja1 deficient bones, they are not consistent with a strong genetic interaction between Sost and Gja1 dictating cortical modeling.

N-cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage-Specific.

N-cadherin inhibits osteogenic cell differentiation and canonical Wnt/ß-catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N-cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N-cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin-insensitive Lrp5A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2-deleted mice, suggesting N-cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and ß-catenin accumulation after administration of an anti-Dkk1 antibody are enhanced in N-cadherin-deficient mice. Thus, although lack of N-cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N-cadherin in osteolineage cells favors bone formation. Hence, N-cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.

Deletion of connexin43 in osteoblasts/osteocytes leads to impaired muscle formation in mice.

It is well-established that muscle forces are necessary for bone development as well as proper bone modeling and remodeling. Recent work has also suggested that bone acts as an endocrine organ that can influence the development of other organs. Connexin43 (Cx43), a gap junction protein that transduces mechanical signals, is an important determinant of cortical bone modeling. Using an osteoblast/osteocyte-specific ablation of the Cx43 gene (Gja1) driven by the 2.3-kb Col1 α1 promoter (cKO) in the mouse, in this study we confirmed reduced cortical bone thickness and density with expanded bone marrow cavity in the cKO humerus. Surprisingly, Gja1 deletion in bone cells also affected skeletal muscle development, resulting in lower fast muscle weight, grip strength, and maximum absolute and specific tetanic forces (60% to 80%, 85%, and 50%, respectively, of WT mice). The normally fast twitch extensor digitorum longus (EDL) muscle exhibited increased slow twitch fibers in cKO mice. These muscle defects were accompanied by a 40% to 60% reduction in mRNA abundance for genes encoding osteocalcin in the humerus, relative to WT mice. Accordingly, both carboxylated and undercarboxylated isoforms of osteocalcin were reduced by over 30% in the circulation of cKO mice. Moreover, the active, undercarboxylated isoform of osteocalcin (glu-OC) promoted myotube formation in C2C12 myoblast cultures, and glu-OC injections to cKO mice rescued EDL muscle cross-sectional area and grip strength in vivo. These findings demonstrate that Cx43 in osteoblasts/osteocytes indirectly modulates skeletal muscle growth and function, potentially via an endocrine effect of glu-OC.

NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms.

Activating-mutations in NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) cause neonatal-onset multisystem inflammatory disease. However, the ontogeny of skeletal anomalies in this disorder is poorly understood. Mice globally expressing the D301N mutation in Nlrp3 (D303N in human) model the human phenotype, including systemic inflammation and skeletal deformities. To gain insights into the skeletal manifestations, we generated mice in which the expression of D301N Nlrp3 (Nlrp3( D301N)) is restricted to myeloid cells. These mice exhibit systemic inflammation and severe osteopenia (∼ 60% lower bone mass) similar to mice globally expressing the knock-in mutation, consistent with the paradigm of innate immune-driven cryopyrinopathies. Because systemic inflammation may indirectly affect bone homeostasis, we engineered mice in which Nlrp3( D301N) is expressed specifically in osteoclasts, the cells that resorb bone. These mice also develop ∼ 50% lower bone mass due to increased osteolysis, but there is no systemic inflammation and no change in osteoclast number. Mechanistically, aside from its role in IL-1ß maturation, Nlrp3( D301N) expression enhances osteoclast bone resorbing ability through reorganization of actin cytoskeleton while promoting the degradation of poly(ADP-ribose) polymerase 1, an inhibitor of osteoclastogenesis. Thus, NLRP3 inflammasome activation is not restricted to the production of proinflammatory mediators but also leads to cytokine-autonomous responses.

Molecular mechanisms of osteoblast/osteocyte regulation by connexin43.

Osteoblasts, osteocytes, and osteoprogenitor cells are interconnected into a functional network by gap junctions formed primarily by connexin43 (Cx43). Over the past two decades, it has become clear that Cx43 is important for the function of osteoblasts and osteocytes. This connexin contributes to the acquisition of peak bone mass and is a major modulator of cortical modeling. We review key data from human and mouse genetics on the skeletal consequences of ablation or mutation of the Cx43 gene (Gja1) and the molecular mechanisms by which Cx43 regulates the differentiation, function, and survival of osteogenic lineage cells. We also discuss putative second messengers that are communicated by Cx43 gap junctions, the role of hemichannels, and the function of Cx43 as a scaffold for signaling molecules. Current knowledge demonstrates that Cx43 is more than a passive channel; rather, it actively participates in the generation and modulation of cellular signals that drive skeletal development and homeostasis.

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype.

To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-ß-BMP pathway, in Osx1(+) cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of ß-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt.

Connexin43 modulates post-natal cortical bone modeling and mechano-responsiveness.

Recent advances have established connexin43 (Cx43) as a key regulator of osteoblast function and of bone response to mechanical stimuli. Work by independent laboratories has consistently demonstrated postnatal development of larger than normal cross-section of long bones after conditional ablation of the Cx43 gene, Gja1, selectively in osteoblasts and/or osteocytes. This phenotype is caused by excessive endocortical bone resorption associated with periosteal expansion and cortical thinning. Review of published data suggests that the earlier in the osteogenic lineage is Gja1 deleted, the more severe is the cortical phenotype, implying functional roles of Cx43 at different stages of the osteoblast differentiation program. Such cortical modeling abnormalities resemble the changes occurring in the cortex upon disuse or aging. Indeed, Cx43 deficiency desensitizes endocortical osteoclasts from activation induced by removal of mechanical load, thus preventing medullary area expansion. The action of Cx43 on cancellous bone is controversial. Furthermore, the absence of Cx43 in osteoblasts and osteocytes results in activation of periosteal bone formation at lower strains than in wild-type bones, suggesting that Cx43 deficiency increased cortical sensitivity to mechanical load. Thus, Cx43 modulates cortical bone modeling in homeostatic conditions and in response to mechanical load by restraining both endocortical bone resorption and periosteal bone formation. Cx43 may represent a novel pharmacologic target for improving cortical bone strength through modulation of mechano-responsiveness.

Enhanced periosteal and endocortical responses to axial tibial compression loading in conditional connexin43 deficient mice.

The gap junction protein, connexin43 (Cx43) is involved in mechanotransduction in bone. Recent studies using in vivo models of conditional Cx43 gene (Gja1) deletion in the osteogenic linage have generated inconsistent results, with Gja1 ablation resulting in either attenuated or enhanced response to mechanical load, depending upon the skeletal site examined or the type of load applied. To gain further insights on Cx43 and mechanotransduction, we examined bone formation response at both endocortical and periosteal surfaces in 2-month-old mice with conditional Gja1 ablation driven by the Dermo1 promoter (cKO). Relative to wild type (WT) littermates, it requires a larger amount of compressive force to generate the same periosteal strain in cKO mice. Importantly, cKO mice activate periosteal bone formation at a lower strain level than do WT mice, suggesting an increased sensitivity to mechanical load in Cx43 deficiency. Consistently, trabecular bone mass also increases in mutant mice upon load, while it decreases in WT. On the other hand, bone formation actually decreases on the endocortical surface in WT mice upon application of axial mechanical load, and this response is also accentuated in cKO mice. These changes are associated with increase of Cox-2 in both genotypes and further decrease of Sost mRNA in cKO relative to WT bones. Thus, the response of bone forming cells to mechanical load differs between trabecular and cortical components, and remarkably between endocortical and periosteal envelopes. Cx43 deficiency enhances both the periosteal and endocortical response to mechanical load applied as axial compression in growing mice.

Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice.

The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20 µg/kg) or alendronate (40 µg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface.

Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice.

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1ß. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1ß and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue "spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.

Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis.

We have shown previously that the effect of mechanical loading on bone depends in part on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild-type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks after injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus BtxA-induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone via modulation of osteoclastogenesis on the endocortical surface.

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

N-cadherin and cadherin 11 modulate postnatal bone growth and osteoblast differentiation by distinct mechanisms.

We have previously shown that targeted expression of a dominant-negative truncated form of N-cadherin (Cdh2) delays acquisition of peak bone mass in mice and retards osteoblast differentiation; whereas deletion of cadherin 11 (Cdh11), another osteoblast cadherin, leads to only modest osteopenia. To determine the specific roles of these two cadherins in the adult skeleton, we generated mice with an osteoblast/osteocyte specific Cdh2 ablation (cKO) and double Cdh2(+/-);Cdh11(-/-) germline mutant mice. Age-dependent osteopenia and smaller diaphyses with decreased bone strength characterize cKO bones. By contrast, Cdh2(+/-);Cdh11(-/-) exhibit severely reduced trabecular bone mass, decreased in vivo bone formation rate, smaller diaphyses and impaired bone strength relative to single Cdh11 null mice. The number of bone marrow immature precursors and osteoprogenitor cells is reduced in both cKO and Cdh2(+/-);Cdh11(-/-) mice, suggesting that N-cadherin is involved in maintenance of the stromal cell precursor pool via the osteoblast. Although Cdh11 is dispensable for postnatal skeletal growth, it favors osteogenesis over adipogenesis. Deletion of either cadherin reduces ß-catenin abundance and ß-catenin-dependent gene expression, whereas N-cadherin loss disrupts cell-cell adhesion more severely than loss of cadherin 11. Thus, Cdh2 and Cdh11 are crucial regulators of postnatal skeletal growth and bone mass maintenance, serving overlapping, yet distinct, functions in the osteogenic lineage.

Microfibril-associated glycoprotein-1, an extracellular matrix regulator of bone remodeling.

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Delta) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Delta mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Delta animals and muCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Delta mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Delta mice is likely not due to an osteoblast defect, because MAGP1Delta bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Delta mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Delta bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Delta cells. In vivo, MAGP1Delta mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-kappaB ligand) expression is significantly higher in MAGP1Delta bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Delta mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.

Attenuated response to in vivo mechanical loading in mice with conditional osteoblast ablation of the connexin43 gene (Gja1).

INTRODUCTION: In vitro data suggest that gap junctional intercellular communication mediated by connexin43 (Cx43) plays an important role in bone cell response to mechanical stimulation. We tested this hypothesis in vivo in a model of genetic deficiency of the Cx43 gene (Gja1). MATERIALS AND METHODS: Four-month-old female mice with a conditional Gja1 ablation in osteoblasts (ColCre;Gja1(-/flox)), as well as wildtype (Gja1(+/flox)) and heterozygous equivalent (Gja1(-/flox)) littermates (eight per genotype), were subjected to a three-point bending protocol for 5 d/wk for 2 wk. Microstructural parameters and dynamic indices of bone formation were estimated on sections of loaded and control contralateral tibias. RESULTS: ColCre;Gja1(-/flox) mice had significantly thinner cortices, but larger marrow area and total cross-sectional area in the tibial diaphysis, compared with the other groups. The ColCre;Gja1(-/flox) mice needed approximately 40% more force to generate the required endocortical strain. In Gja1(+/flox) mice, the loading regimen produced abundant double calcein labels at the endocortical surface, whereas predominantly single labels were seen in ColCre;Gja1(-/flox) mice. Accordingly, mineral apposition rate and bone formation rate were significantly lower (54.8% and 50.2%, respectively) in ColCre;Gja1(-/flox) relative to Gja1(+/flox) mice. Intermediate values were found in Gja1(-/flox) mice. CONCLUSIONS: Gja deficiency results in thinner but larger tibial diaphyses, resembling changes occurring with aging, and it attenuates the anabolic response to in vivo mechanical loading. Thus, Cx43 plays an instrumental role in this adaptive response to physical stimuli.

Bone loss after temporarily induced muscle paralysis by Botox is not fully recovered after 12 weeks.

To study the effect of unloading followed by reloading on hindlimb bone mineral content (BMC), we used botulinum toxin A (Botox). We studied the timing and degree of recovery upon restoration of muscle function. We also tested to see if reaction to Botox injection occurred as a function of the degree of expression of connexin43 (Cx43). Sixteen mice were divided by gender and genotype; wild-type equivalent (Gja1(+/flox)) and heterozygous (Gja1(+/-)) mice were injected with a single dose of 2U/100 g Botox i.m. in the quadriceps, hamstrings, and posterior calf muscle groups (day 0). Regional BMC was monitored for 12 weeks in the injected and contralateral control limb. Significant bone loss was observed in the injected limb by week 2 in the Gja1(+/flox) mice, and by week 3-4 in the Gja1(+/-) mice. By week 12, BMC in the Botox-treated limb of both male and female wild-type and heterozygous mice was still at least 14% less than that of the noninjected limb. Cortical thickness and BV/TV were lower in the Botox femur compared to the noninjected femur. The heterozygous mice tended to show a slower response to Botox injection as reflected in a slower loss of BMC. Our results show that the rapid and profound bone loss following temporary muscle paralysis is not fully recovered upon restoration of muscle function within 12 weeks. These results underscore the significance of normal muscle function in the maintenance of bone mass.

Role of connexin43 in osteoblast response to physical load.

Gap junctions are hexameric transmembrane channels formed by connexins, and are responsible for direct cell-to-cell communication. The most abundant gap junction protein in bone is connexin43 (Cx43), although connexin45 (Cx45) is also expressed. In the present study, we tested the hypothesis that bone cell responses to mechanical stimulation are dependent on the type of gap junction communication provided by Cx43 in vitro and in an in vivo model of physical load. Application of cyclic stretch to calvaria osteoblasts results in a modest but detectable increase in PGE2 levels, and the amount of PGE2 produced was lower in cells isolated from Cx43 null mice. Mice with an osteoblast-specific deletion of the Cx43 gene were subjected to an in vivo four-point bending protocol on the tibia. This resulted in fast and exuberant formation of woven bone at the region directly below the loading fulcrum in both osteoblast Cx43-deleted and wild-type mice. However, indirect measurement of endosteal bone apposition suggested a less pronounced effect of physical load in Cx43-deficient than in wild-type mice. Taken together, these results indicate that deficiency of Cx43 in osteoblasts attenuates but does not abolish anabolic responses to mechanical strain.