title: Mitogen-activated Protein Kinase (MAPK) Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress.
abstract: Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however, sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at Ser(279)-Ser(282), sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pretreated with the MAPK pathway inhibitor. We found that prostaglandin E2 (PGE2) accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at Ser(279)-Ser(282) sites. Depletion of PGE2 from CM, and pre-treatment with a p44/42 ERK pathway-specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. A similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Riquelme
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Sirisha
authors.initials: S
authors.lastname: Burra
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Rekha
authors.initials: R
authors.lastname: Kar
authors.affiliation: Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Lampe
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900. Electronic address: jiangj@uthscsa.edu.
authors.forename: Jean X
authors.initials: JX
authors.lastname: Jiang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004789:Enzyme Activation; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010011:Osteocytes / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013314:Stress, Mechanical
chemical_list: D018031:Connexin 43; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone
keywords: Hemichannels; Mechanical stimulation; channel activation; connexin; mitogen-activated protein kinase (MAPK); osteocyte; phosphorylation