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.
Highlights
Increasing evidence suggests that the viability of osteocytes embedded within the bone mineral matrix is essential for maintaining skeletal homeostasis, as supported by the increase in apoptotic osteocytes and prevalence of empty lacunae observed in conditions of elevated bone fragility and with old age (Plotkin & Bellido, 2016)
Previous work from our group has demonstrated that the gap junction protein connexin43 (Cx43) is a critical component of the signaling pathway controlling osteocyte survival, as evidenced by the increased osteocyte apoptosis in mice lacking Cx43 in osteoblasts and osteocytes or only in osteocytes (Plotkin et al, 2008; Bivi et al, 2012)
While it has been shown that Cx43 levels in osteocytes are decreased in old mice (Kar et al, 2013), the signaling pathway activated by Cx43 deficiency involved in osteocyte apoptosis with aging is not known
Summary
Increasing evidence suggests that the viability of osteocytes embedded within the bone mineral matrix is essential for maintaining skeletal homeostasis, as supported by the increase in apoptotic osteocytes and prevalence of empty lacunae observed in conditions of elevated bone fragility and with old age (Plotkin & Bellido, 2016). Osteocyte interactions with other cells occur through the release of cytokines to the lacunar–canalicular system that surrounds osteocytes, as well as through cell-to-cell communication via gap junction channels (Plotkin & Bellido, 2013). Previous work from our group has demonstrated that the gap junction protein connexin (Cx43) is a critical component of the signaling pathway controlling osteocyte survival, as evidenced by the increased osteocyte apoptosis in mice lacking Cx43 in osteoblasts and osteocytes or only in osteocytes (Plotkin et al, 2008; Bivi et al, 2012). In addition to increased osteocyte apoptosis, mice lacking osteocytic Cx43 (Cx43DOt) exhibit enhanced endocortical resorption, partially mimicking the phenotype of old mice (Almeida et al, 2007b) and raising the possibility that decreased Cx43 contributes at least in part to the skeletal phenotype of aging. While it has been shown that Cx43 levels in osteocytes are decreased in old mice (Kar et al, 2013), the signaling pathway activated by Cx43 deficiency involved in osteocyte apoptosis with aging is not known
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