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.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways

“…On the other hand, Cx43 deficiency results in alterations in the mineralization and remodeling phases of fracture healing, and is characterized by delayed expression of osteogenic marker genes, increases in GSK-3beta mRNA and active GSK-3beta, consistent with a pronounced decrease in beta-catenin expression. At the same time, alterations in healing of fractures, in Cx43 deficiency, can be rescued by inhibiting GSK-3beta activity using LiCl [32]. BMSCs play an important role in fracture repair, and BMSC commitment to the chondrogenic or osteogenic lineages improves healing, after homing to the site of fracture, by increasing bone formation [71, 72].…”

“…Loiselle et al reported that deletion of Cx43 in osteoblasts/osteocytes results in delay of bone formation and bone remodeling, and impairs mechanical properties33 [32], and the mechanisms by which healing is impaired are related to altered activity of GSK-3beta, and therefore beta-catenin expression, during fracture healing 34 [31]. Several signaling pathways have been shown to participate in the regulation of osteogenesis [13].…”

Osteoblast versus Adipocyte: Bone Marrow Microenvironment-Guided Epigenetic Control

“…MSCs, pre-osteoblasts, osteoblasts, osteocytes, and bone-lining cells all form gap junctions, creating a continuous network and enabling intercellular communication. Of note, FFSS induces conformational changes of Connexin 43 (Cx43) and opens its hemichannels [74, 75]. Deficiency of Cx43 results in lower response of bone to mechanical signals, indicating that Cx43-mediated communication plays a pivotal role in mechanical signal transduction among bone cells.…”

Connexins and pannexins in the skeleton: gap junctions, hemichannels and more

“…Interestingly, the effect of Cx43 deletion in fracture healing appears to be opposite to that of normal bone, since the RANKL/OPG ratio is decreased, while the levels of the Wnt inhibitor sclerostin (i.e. the product of the Sost gene) are increased and β-catenin is decreased in the fracture [122]. In addition, the effect of Cx43 deletion on fracture healing can be reversed by increasing β-catenin stability [122].…”

“…the product of the Sost gene) are increased and β-catenin is decreased in the fracture [122]. In addition, the effect of Cx43 deletion on fracture healing can be reversed by increasing β-catenin stability [122]. These pieces of evidence raise the possibility that Cx43 has distinct roles in bone acquisition/maintenance versus bone healing.…”