“Connexin 43 is required for bisphosphonate-induced survival of osteoblastic cells but not for bisphosphonate binding”

BOLAND, RICARDO; MORELLI SUSANA; SANTILLÁN GRACIELA; SCODELARO BILBAO, PAOLA GABRIELA; COLICHEO ANDREA; RUSSO DE BOLAND, ANA; VYAS K.; PLOTKIN L.; BELLIDO T.

Philadelphia, Pennsylvania, USA

Congreso; 28TH American Society for Bone and Mineral Research Annual Meeting; 2006

American Society for Bone and Mineral Research

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activation of the extracellular signal regulated kinases ERKs. However, the events elicited by BPs upstream of hemichannel opening remain unknown. BPs only inhibit apoptosis in cells expressing Cx43, and Cx43 confers responsiveness to BPs to unresponsive cells in vitro. In addition, Cx43 is required for the protective effect of BPs against glucocorticoid-induced apoptosis of osteoblastic cells in vivo (shown elsewhere in this meeting). Based on these findings, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. Whole cell binding assays using the BP alendronate labeled with 3H showed the presence of saturable, specific and high affinity binding sites in both ROS 17/2.8 osteoblastic cells and rat calvaria-derived osteoblasts, which express Cx43. 3H-alendronate bound to ROS 17/2.8 cells was displaced by 200-fold excess of unlabeled alendronate, as well as by two other BPs, olpadronate and etidronate, with estimated dissociation constants of 0.65 ± 0.2; 0.6 ± 0.3; and 1 ± 0.15 mM, respectively. Unexpectedly, 3H-alendronate also bound specifically to HeLa cells that do not express Cx43, and to ROS 17/2.8 cells pretreated with a-glycyrrhetinic acid, oleamide or carbenoxolone, agents that disassemble Cx channels. These results indicate that BPs bind to an entity different from Cx43. Because alendronate does not prevent apoptosis of HeLa cells or Cx43-expressing cells pre-treated with Cx channel disassembling agents, these findings also indicate that BP binding is not sufficient to induce survival. Thus, BPs might bind to a protein that, in turn, induces Cx43 hemichannel opening. Because Cx43 interacts with phosphatases and BPs modulate phosphatase activity, we sought for the involvement of phosphatases in BP actions. We found that 3H-alendronate bound to ROS 17/2.8 cells was displaced by the phosphatase substrates p-nitro-phenylphosphate and a-naphthylphosphate. Moreover, p-nitro-phenylphosphate also inhibited BP-induced anti-apoptosis of HeLa cells transfected with Cx43, demonstrating that indeed alendronate binding is required for anti-apoptosis. These findings indicate that, although required for triggering intracellular signaling by BPs, Cx43 is dispensable for BP binding; and they suggest that BPs bind to a Cx43-interacting phosphatase.