Beyond gap junctions: Connexin43 and bone cell signaling

Abstract

Connexin43 (Cx43) is the most abundant gap junction protein expressed in bone cells and plays a central role in cell-to-cell communication in the skeleton. Findings of the last decade uncovered functions of Cx43 hemichannels expressed on unopposed plasma cell membranes as mediators of the communication between bone cells and their extracellular milieu. Additionally, through its cytoplasmic C-terminus domain, Cx43 serves as a scaffolding protein that associates with structural and signaling molecules leading to regulation of intracellular signaling, independent of channel activity. This perspective discusses the evidence demonstrating that via these diverse mechanisms Cx43 is a key component of the intracellular machinery responsible for signal transduction in bone in response to pharmacologic, hormonal and mechanical stimuli. This advance in the knowledge of the role of connexins increases our understanding of the pathophysiological mechanisms that regulate bone cell function and provides new opportunities to treat bone diseases.

Figure 1. Connexin43, hemichannels and gap junction channels

(A) Schematic representation of a Cx43 molecule, showing the amino (NH₂) and carboxy (COOH) domains facing the cytoplasm and the 4 transmembrane domains. Six molecules of Cx43 associate to form a connexon or hemichannel that might be expressed in unopposed cell membranes; and 2 connexons expressed in neighboring cells align to form a gap junction channel. (B) Cx43 expressed in osteoblasts, osteocytes and osteoclasts, forms gap junction channels that allow inter-cellular communication and hemichannels that establish communication between cells and the extracellular milieu. (C) Representative images of OB-6 osteoblastic cells (confocal microscopy, upper panel) and MLO-Y4 osteocytic cells (fluorescence microcopy, middle panel) transfected with Cx43 tagged with green fluorescent protein; and MLO-Y4 osteocytic cells stained with anti-Cx43 antibody (bright field microscopy, lower panel). Localization of Cx43 in areas of cell-cell contact (yellow arrows), unopposed membranes (white and black arrows) and in the perinuclear area (red arrows) is shown. Bars represent 20 µm. The inset in the lower panel corresponds to an MLO-Y4 osteocytic cell stained with non-immune IgG as a negative control to demonstrate the specificity of the anti-Cx43 antibody.

Figure 2. Scaffolding function of connexin43 as a regulator of osteoblast and osteocyte survival

(A) Bisphosphonates induce the transient opening of non-junctional Cx43 hemichannels, likely indirectly by binding to a phosphatase that interact with Cx43. Hemichannel opening results in Src activation, followed by MEK and ERK phosphorylation, which in turn activates survival signaling in osteocytes and osteoblasts. (B) Opening of Cx43 hemichannels by bisphosphonates results in the retention of activated ERKs in the cytoplasm through Cx43/β-arrestin (βA) interactions. This leads to phosphorylation of the cytoplasmic ERK targets p90^RSK, BAD and C/EBPβ. In contrast, ERKs activated by estrogens translocate to the nucleus and activate transcription factors that promote survival. (C) Cx43 sequesters β-arrestin (βA) away from the PTHR1, facilitating cAMP/PKA-mediated downstream signaling promoting osteoblast survival. (D) Mechanical stimulation induces α5β1integrin engagement, followed by FAK/Src/ERK activation. Association of integrins with Cx43 opens the hemichannels. Release of PGE2 through the hemichannels induces autocrine/paracrine signaling through the EP2/4 prostaglandin receptor, cAMP/PI3K activation and inhibition of apoptosis.

Figure 3. Cell Autonomous requirement of Connexin43 for osteocyte survival and control of osteoblast and osteoclast activity

(A) Transmission electron micrographs showing apoptotic and empty lacunae in bones from Cx43^fl/−;OCN-cre mice lacking Cx43 in osteoblasts and osteocytes. Scale bars indicate 2 µm. (B) Increased osteocyte apoptosis in mice lacking Cx43 from osteocytes regulates periosteal apposition and endocortical resorption in cortical bone, by separate mechanisms. Accumulation of empty lacunae adjacent to periosteal surfaces results in decreased sclerostin expression and enhanced bone formation. Apoptotic osteocytes trigger osteoclast recruitment and decreased expression of osteocytic OPG is a permissive event, leading to enhanced resorption on the endocortical surface.

Figure 4. Connexin43 as a mediator of pharmacologic, hormonal and mechanical stimuli

Cx43 is a key component of intracellular machinery responsible for signal transduction in bone in response to pharmacologic, hormonal, and mechanical stimuli.