Regulation of connexin 43 hemichannels by mechanical loading, integrins and activated kinases in bone osteocytes

Osteocytes are the known mechanosensory cells in bone and are characterized by long dendritic processes. Mechanical signals travel from one osteocyte to another through gap junctions formed at the tips of the dendritic processes. Connexin (Cx) 43 is the major gap junction and hemichannel forming protein in primary osteocytes and osteocyte-like MLO-Y4 cells. Previous studies by our laboratory have shown that the Cx43 hemichannels are sensitive and adaptive towards mechanical stimulation in the form of fluid flow shear stress (FFSS). Upon mechanical stimulation, Cx43 hemichannels play an important role in release of PGE2, an important modulator of bone metabolism. The regulation of Cx43 hemichannels by mechanical stimulation and how these channels are mechanosensitive is not yet known. It is important to understand if the adaptive response of Cx43 hemichannels towards mechanical stimulation is due to altered open state or due to changes in surface expression of Cx43. In this study, I report that both oligomerization and trafficking of Cx43 increases with FFSS. Also, the trafficking of Cx43 was observed to be adaptive as continuous FFSS for longer periods resulted in Cx43 surface levels similar to that of the non-stimulated controls.;In order to understand how Cx43 hemichannels are mechanosensitive, I examined if Cx43 associates with integrins, the known mechanosensors expressed in many cell types. Co-immunoprecipitation experiments suggested that Cx43 associates with integrin alpha5beta1. Dye uptake by Cx43 hemichannels was reduced by siRNA knockdown of integrin alpha5 expression. Direct mechanical stimulation using alpha5 integrin antibody-coated magnetic beads and activation of integrin beta1 using activation antibody (TS2/16) resulted in a significant increase in hemichannel dye uptake. Results from these experiments indicate that integrin alpha5beta1 modulates Cx43 hemichannel opening, thereby molding them to function as mechanosensory channels. 14-3-3theta is observed to mediate this interaction in a PI3-K/AKT dependent manner as inhibitors against PI3-K/AKT pathway disrupted Cx43/integrin alpha5beta1 complex and also inhibited hemichannel dye uptake. Importantly, the activation of AKT is maximal after 0.5hrs of FFSS and follows similar trend as Cx43 hemichannel dye uptake in response to continuous FFSS. In contrast to pAKT, both pERK levels and MAPK-dependent phosphorylation of Cx43 at S279/S282, increased after 24 hrs of continuous FFSS. Accumulation of PGE2 that is released by Cx43 hemichannels during continuous FFSS in the condition media (CM) was observed to be responsible for Cx43 phosphorylation by p44/42 ERK as PGE 2 depleted CM did not have any effect on Cx43 phosphorylation. CM treatment also resulted in reduced hemichannel dye uptake and this inhibition was reversed when the cells were preincubated with a MAPK pathway inhibitor. Therefore, Cx43 hemichannel opening and its interaction with integrin alpha5beta1 is regulated by PI3-K/AKT pathway. On the contrary, p44/42 MAPK pathway causes a decrease in hemichannel dye uptake, probably by regulating the trafficking or opening of Cx43 hemichannels.;The above mentioned results illuminate the biological response of osteocytes to mechanical stimulation, but the precise mechanosensory system of osteocyte that is responsible for sensing the mechanical stimulation is not yet known. Therefore, it is important to understand how osteocytes sense mechanical stimulation and in particular which part (s) of the osteocyte is involved in mechanosensing. In this study, a transwell filter system was used to differentially stimulate dendrites and cell bodies of the osteocytes. It was identified that osteocyte dendrites and glycocalyx surrounding the dendrites is involved in mechanotransduction.;The majority of the studies identifying the role of Cx43 have been performed in osteocyte-like MLO-Y4 cells. To understand the role of Cx43 gap junctions and hemichannels in vivo, transgenic mice overexpressing osteocyte specific Cx43 and Cx43 mutants were generated. In this study, osteocyte specific Cx43 construct with deletion from 130-136 was developed, which is a dominant negative mutant for both gap junctions and hemichannels. Another mutant, Cx43 (R76W) was developed that is dominant negative for gap junctions but forms functional hemichannels. Preliminary studies show that Cx43 (R76W) overexpressing mice display defective bone architecture as observed through micro-CT and one of these mice was reduced in size, indicating abnormal skeletal development. Cx43 and Cx43 (Delta130-136) are yet to be tested for phenotype. These mice will provide better understanding of role of Cx43 in osteocyte function in vivo.;Together, these results suggest that Cx43, integrin alpha5beta1 and 14-3-3theta forms a mechanosensory complex which is involved in transduction of mechanical signals in osteocytes. Cx43 hemichannel function is intricately regulated by both AKT and MAPK signaling pathways which could be crucial in the regulated release of the extracellular anabolic/catabolic signaling factors, such as prostaglandins and other small molecules in response to mechanical loading. Results from this study, sheds light on the importance of Cx43 in osteocyte response to mechanical stimulation and also provide valuable insights into normal osteocyte biology.