β-arrestin/connexin 43 complex anchors ERKs outside the nucleus: a pre-requisite for bisphosphonate anti-apoptotic effect mediated by CX43/ERK in osteocytes

Abstract

Bisphosphonates (BPs) anti-fracture ef cacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of con- nexin (Cx) 43 hemichannels and phosphoryla- tion of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. In- stead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaf- folding protein β-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red uorescent pro- tein (ERK2-RFP) co-localizes with Cx43 fused to green uorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear ac- cumulation in cells transfected with wild type β-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant nega- tive β-arrestin mutant (dnARR) consisting of the β-arrestin-clathrin binding domain that com- petes with endogenous β-arrestin for binding to clathrin. Alendronate activates ERKs in dnARR- transfected cells as effectively as in cells trans- fected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is inef- fective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, β-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs.