Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Abstract

SummarySkeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43def cell death. Cx43def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

“…They can induce changes in multiple cellular processes, including cell proliferation, cell differentiation, and cell senescence (Davis et al., 2017; Jing et al., 2015). However, their instability in the extracellular environment limits their application.…”

Connexins and Pannexins in Bone and Skeletal Muscle

“…Consistent with this notion, Cx43 deletion from osteocytes renders a phenotype that resembles that of old animals [40] with increased osteocyte apoptosis and enhanced endocortical resorption and periosteal apposition [19,21]. Furthermore, deletion of Cx43 from osteocytic MLO-Y4, but not from osteoblastic Ob-6, cells leads to spontaneous cell death in culture [38]. The later study showed that Cx43 restrains osteocyte death by maintaining the levels of the microRNA miR21, with the consequent reduction of the expression of the phosphatase and tensin homolog PTEN, resulting in the preservation of the Akt-survival pathway.…”

“…This study did not find changes in Cx43 mRNA or protein in these osteoblastic cells or in basal gap junction communication, suggesting an intrinsic defect on the function of Cx43 gap junctions in response to parathyroid hormone. However, Cx43 expression in osteocyte-enriched whole bone measured by qPCR and by western blotting [38] and in osteocytes assessed by immunohistochemistry [39] is decreased in old mice. The discrepancy among these studies suggests that the reduction in Cx43 expression with aging likely occurs in osteocytes, and not in osteoblasts.…”

Ex Vivo Organ Cultures as Models to Study Bone Biology

“…Interestingly, the ex vivo organ culture model allows the study of the effects of genetic deletions without the need of crossing mice floxed for a particular gene with mice expressing the Cre recombinase, thus reducing costs and time. For instance, in a recent study, calvarial bones from miR21 fl/fl mice were treated with adenovirus‐Cre to delete the miR21 gene . Addition of adenovirus‐Cre decreased miR21 mRNA levels, an effect that, as seen in vivo, was accompanied by decreases in osteocyte viability .…”