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.
Highlights
Connexin 43 (Cx43) is encoded by the Gja1 gene and is the most highly expressed gap junction protein in bone [1]
We further propose that β-catenin expression is reduced as a result of antagonism by Sclerostin and/ or increased GSK-3β activity, and that the fracture healing phenotype in Cx43 deficient mice could be rescued by restoring β-catenin expression through inhibition of GSK-3-β activity with Lithium Chloride (LiCl) treatment
Cx43 is expressed in osteoblasts and osteocytes during fracture healing
Summary
Connexin 43 (Cx43) is encoded by the Gja gene and is the most highly expressed gap junction protein in bone [1]. In addition to regulating osteoblast maturation in vitro, Cx43 plays an important role in bone homeostasis in vivo as demonstrated by the osteopenic phenotype that is associated with several models of Cx43 deficiency in bone [10,18,19]. The development of phenotypes associated with systemic changes in Cx43 expression necessitates the use of conditional deletion constructs to study the functions of Cx43 in bone in vivo postnatally. Loss of Cx43 in mature osteoblasts and osteocytes, by means of the human Osteocalcin promoter driven Cre, results in impaired fracture healing due to defects in bone formation and remodeling [22]. We 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. We further propose that β-catenin expression is reduced as a result of antagonism by Sclerostin and/ or increased GSK-3β activity, and that the fracture healing phenotype in Cx43 deficient mice could be rescued by restoring β-catenin expression through inhibition of GSK-3-β activity with Lithium Chloride (LiCl) treatment
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