title: Inhibition of GSK-3β rescues the impairments in bone formation and mechanical properties associated with fracture healing in osteoblast selective connexin 43 deficient mice.
abstract: Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.
authors.affiliation: Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, Pennsylvania, United States of America.
authors.forename: Alayna E
authors.initials: AE
authors.lastname: Loiselle
authors.forename: Shane A J
authors.initials: SA
authors.lastname: Lloyd
authors.forename: Emmanuel M
authors.initials: EM
authors.lastname: Paul
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Lewis
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Donahue
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001696:Biomechanical Phenomena; D002146:Bony Callus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002113:Calcification, Physiologic; D018031:Connexin 43 / Q000172:deficiency / Q000235:genetics; D005260:Female; D005269:Femur / Q000187:drug effects / Q000293:injuries / Q000378:metabolism; D017102:Fracture Healing / Q000187:drug effects* / Q000235:genetics; D050723:Fractures, Bone / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D018021:Lithium Chloride / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010011:Osteocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D053244:Osteoprotegerin / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019415:Torque; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C495267:CTNNB1 protein, mouse; D018031:Connexin 43; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D053244:Osteoprotegerin; D053245:RANK Ligand; C486030:Sost protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D051176:beta Catenin; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride