Altered Expression of Apoptosis-Associated miRNAs that Regulate IGF-1 Survival Signaling Underlies the Cell Autonomous Requirement of Cx43 for Osteocyte Survival

PACHECO-COSTA R; BRUN LR; SOUTHERN D; REGINATO RD; BIVI N; BELLIDO T; PLOTKIN L

Minneapolis

Congreso; American Society for Bone and Mineral Research Annual Meeting 2012; 2012

American Society for Bone and Mineral Research (ASBMR)

Mice lacking connexin (Cx) 43 in osteocytes exhibit elevated prevalence of osteocyte apoptosis and MLO-Y4 osteocytic cells in which Cx43 is silenced exhibit spontaneous cell death; however, the basis of the cell autonomous requirement of Cx43 for osteocyte viability is unknown. We questioned whether the absence of Cx43 changes the expression of apoptosis-associated microRNAs (miRs), short RNA molecules that act as negative post-transcriptional regulators of gene expression and have profound effects on cell survival. Cx43 expression was stably silenced in MLO-Y4 osteocytic cells using shRNA. Silenced cells exhibit a decrease of more than 80% in the expression of Cx43 at the mRNA and protein levels compared to scramble shRNA-transfected control cells. miR21 and miR218 expression was altered in Cx43-deficient cells out of 47 apoptosis-associated miRs studied (Apoptosis-Associated miRNA Plate Array, Signosis). Results were confirmed by qPCR and showed that miR21 expression was decreased by 50% and that miR218 expression was increased by 500% in Cx43-deficient cells compared to control cells. A target of miR21 is PTEN, the phosphatase that inactivates the Akt-survival pathway; and a target of miR218 is IKKb kinase, which is required for NFkB activation. Akt and NFkB are known mediators of survival signaling activated in several cell types by insulin like growth factor-1 (IGF-1), which also inhibits apoptosis of osteoblasts and osteocytes. We therefore investigated whether the absence of Cx43 interferes with the anti-apoptotic effect of IGF-1. We found that whereas control cells were protected by IGF-1 from glucocorticoid-induced apoptosis, IGF-1 was ineffective in Cx43-deficient MLO-Y4 osteocytic cells. Moreover, whereas wild type HeLa cells that lack endogenous Cx43 were refractory to IGF-1, IGF-1 inhibited glucocorticoid-induced apoptosis in HeLa cells transfected with Cx43. Taken together, these results suggest that dis-regulation of miR21 and miR218 triggered by the absence of Cx43 causes resistance to the survival effect of IGF-1. Moreover, these findings raise the possibility that accumulation of apoptotic osteocytes in Cx43 deficient mice is due to the inability of osteocytes to respond to endogenous IGF-1.