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

ABSTRACT: 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 ?g/kg) or alendronate (40 ?g/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.

SUBMITTER: Watkins MP

PROVIDER: S-EPMC3432742 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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

Watkins Marcus P MP Norris Jin Yi JY Grimston Susan K SK Zhang Xiaowen X Phipps Roger J RJ Ebetino Frank H FH Civitelli Roberto R

Bone 20120629 4

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 tra ...[more]

PMID: 22750450

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Project description:The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild-type or singly hemizygous animals. Cortical bone analysis by micro-computed tomography (μCT) revealed that 8-week-old male, compound Gja1+/- Runx2+/- mice have a marked increase in cross-sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1+/- Runx2+/- mice closely approximate the cortical bone phenotypes seen in osteoblast-specific Gja1-conditional knockout models. Furthermore, μCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2-day-old Gja1+/- Runx2+/- neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2+/- neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research.

Project description:ObjectiveThis study used microcomputed tomography (micro-CT) to evaluate the effects of ovariectomy on the trabecular bone microarchitecture and cortical bone morphology in the femoral neck and mandible of female rats.Materials and methodsTwelve female Wister rats were divided into two groups: the control and ovariectomized groups. The rats in the ovariectomized group received ovariectomy at 8 weeks of age; all the rats were sacrificed at 20 weeks of age, and their mandibles and femurs were removed and scanned using micro-CT. Four microstructural trabecular bone parameters were measured for the region below the first mandibular molar and the femoral neck region: bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular separation (TbSp), and trabecular number (TbN). In addition, four cortical bone parameters were measured for the femoral neck region: total cross-sectional area (TtAr), cortical area (CtAr), cortical bone area fraction (CtAr/TtAr), and cortical thickness (CtTh). The CtTh at the masseteric ridge was used to assess the cortical bone morphology in the mandible. The trabecular bone microarchitecture and cortical bone morphology in the femoral necks and mandibles of the control group were compared with those of the ovariectomized group. Furthermore, Spearman's correlation (rs) was conducted to analyze the correlation between the osteoporosis conditions of the mandible and femoral neck.ResultsRegarding the trabecular bone microarchitectural parameters, the BV/TV of the trabecular bone microarchitecture in the femoral necks of the control group (61.199±11.288%, median ± interquartile range) was significantly greater than that of the ovariectomized group (40.329±5.153%). Similarly, the BV/TV of the trabecular bone microarchitecture in the mandibles of the control group (51.704±6.253%) was significantly greater than that of the ovariectomized group (38.486±9.111%). Furthermore, the TbSp of the femoral necks in the ovariectomized group (0.185±0.066 mm) was significantly greater than that in the control group (0.130±0.026mm). Similarly, the TbSp of the mandibles in the ovariectomized group (0.322±0.047mm) was significantly greater than that in the control group (0.285±0.041mm). However, the TbTh and TbN trends for the mandibles and femoral necks were inconsistent between the control and ovariectomized groups. Regarding the cortical bone morphology parameters, the TtAr of the femoral necks in the ovariectomized group was significantly smaller than that in the control group. There was no significant difference in the TtAr, CtAr, or CtTh of the femoral necks between the control and ovariectomized groups, and no significant difference in the CtTh of the mandibles between the control and ovariectomized groups. Moreover, the BV/TV and TbSp of the mandibles were highly correlated with those of the femurs (rs = 0.874 and rs = 0.755 for BV/TV and TbSp, respectively). Nevertheless, the TbTh, TbN, and CtTh of the mandibles were not correlated with those of the femoral necks.ConclusionAfter the rats were ovariectomized, osteoporosis of the trabecular bone microarchitecture occurred in their femurs and mandibles; however, ovariectomy did not influence the cortical bone morphology. In addition, the parametric values of the trabecular bone microarchitecture in the femoral necks were highly correlated with those of the trabecular bone microarchitecture in the mandibles.

Project description:BackgroundIndividuals with Fragile X syndrome (FXS) and autism spectrum disorder (ASD) exhibit an array of symptoms, including sociability deficits, increased anxiety, hyperactivity, and sensory hyperexcitability. It is unclear how endocannabinoid (eCB) modulation can be targeted to alleviate neurophysiological abnormalities in FXS as behavioral research reveals benefits to inhibiting cannabinoid (CB) receptor activation and increasing endocannabinoid ligand levels. Here, we hypothesize that enhancement of 2-arachidonoyl-sn-glycerol (2-AG) in Fragile X mental retardation 1 gene knock-out (Fmr1 KO) mice may reduce cortical hyperexcitability and behavioral abnormalities observed in FXS.MethodsTo test whether an increase in 2-AG levels normalized cortical responses in a mouse model of FXS, animals were subjected to electroencephalography (EEG) recording and behavioral assessment following treatment with JZL-184, an irreversible inhibitor of monoacylglycerol lipase (MAGL). Assessment of 2-AG was performed using lipidomic analysis in conjunction with various doses and time points post-administration of JZL-184. Baseline electrocortical activity and evoked responses to sound stimuli were measured using a 30-channel multielectrode array (MEA) in adult male mice before, 4 h, and 1 day post-intraperitoneal injection of JZL-184 or vehicle. Behavior assessment was done using the open field and elevated plus maze 4 h post-treatment.ResultsLipidomic analysis showed that 8 mg/kg JZL-184 significantly increased the levels of 2-AG in the auditory cortex of both Fmr1 KO and WT mice 4 h post-treatment compared to vehicle controls. EEG recordings revealed a reduction in the abnormally enhanced baseline gamma-band power in Fmr1 KO mice and significantly improved evoked synchronization to auditory stimuli in the gamma-band range post-JZL-184 treatment. JZL-184 treatment also ameliorated anxiety-like and hyperactivity phenotypes in Fmr1 KO mice.ConclusionsOverall, these results indicate that increasing 2-AG levels may serve as a potential therapeutic approach to normalize cortical responses and improve behavioral outcomes in FXS and possibly other ASDs.

Project description:Inhibition of sclerostin with sclerostin antibody (Scl-Ab) results in stimulation of bone formation on cancellous (Cn), endocortical (Ec), and periosteal (Ps) surfaces in rodents and non-human primates. With long-term dosing of Scl-Ab, the increase in bone formation is not sustained, attenuating first on Cn surfaces and later on Ec and Ps surfaces. In Cn bone, the attenuation in bone formation (self-regulation) is associated with transcriptional changes in the osteocyte (OCy) that would limit mitogenesis and are sustained with continued dosing. The expression changes in Cn OCy occur coincident with a decrease in osteoprogenitor (OP) numbers that may directly or indirectly be a consequence of the transcriptional changes in the OCy to limit OP proliferation. To characterize the Scl-Ab-mediated changes in cortical (Ct) bone and compare these changes to Cn bone, densitometric, histomorphometric, and transcriptional analyses were performed on femur diaphyses from aged ovariectomized rats. Animals were administered 50 mg/kg/wk of Scl-Ab or vehicle for up to 6 months (183 days), followed by a treatment-free period (up to 126 days). Scl-Ab increased Ct mass and area through day 183, which declined slightly when treatment was discontinued. Ps and Ec bone formation was sustained through the dosing on both Ct surfaces, with evidence of a decline in bone formation only at day 183 on the Ec surface. This is in contrast to Cn bone, where reduced bone formation was observed after day 29. TaqMan analysis of 60 genes with functional roles in the bone using mRNA isolated from laser capture micro-dissection samples enriched for Ec osteoblasts and Ct OCy suggest a pattern of gene expression in Ct bone that differed from Cn, especially in the OCy, and that corresponded to observed differences in the timing of phenotypic changes. Notable with Scl-Ab treatment was a "transcriptional switch" in Ct OCy at day 183, coincident with the initial decline in bone formation on the endocortex. A consistent sustained increase of expression for most genes in response to Scl-Ab was observed from day 8 through day 85 at the times of maximal bone formation on both Ct surfaces; however, at day 183, this increase was reversed, with expression of these genes generally returning to control values or decreasing compared to vehicle. Genes exhibiting this pattern included Wnt inhibitors Sost and Dkk1, though both had been up-regulated until the end of dosing in Cn OCy. Changes in cell cycle genes such as Cdkn1a and Ndrg1 in Ct OCy suggested up-regulation of p53 signaling, as observed in Cn OCy; however, unlike in Cn bone, p53 signaling was not associated with decreased bone formation and was absent at day 183, when bone formation began to decline on the Ec surface. These data demonstrate involvement of similar molecular pathways in Ct and Cn bone in response to Scl-Ab but with a different temporal relationship to bone formation and suggest that the specific mechanism underlying self-regulation of Scl-Ab-induced bone formation may be different between Cn and Ct bone.

Dataset Information

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

Publications

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

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