Bone remodelling: Connexin 43Shgoofa Walizada, m1502934Gap junction intercellular communication (GJIC) between bone cells like osteoblasts and osteoclast play a vital role in bone remodelling. Connexin 43 (Cx43) gap junctions are abundant in bone cells and aid the exchange of signals to coordinate osteoclast and osteoblastic activity. Research done by Moorer et al1 has shown that Cx43 gap junctions also play an active role in recruiting signalling proteins to the C terminal (CT) domain of Cx43 and not just help movement of signalling molecules to aid bone remodelling. Gap junctions are groups of intermembrane channels that aid diffusion of molecules, usually less than 1.2kDa in size, and transmission of signals across neighbouring bone cells like osteoclasts or osteoblasts. The channels are formed from 6 connexin’s that bind together to form an intracellular hexamer structure in the membrane known as a connexon (hemichannel). These move to the cell surface where it can bind to the connexon in the membrane of another cell to form a channel and gap junction plaques, that lead to gap junction intercellular communication (GJIC). Cx43, Cx45 and Cx46 are all expressed in bone tissue, Zhang et al2 showed that deletion of Cx43 from bone cells lead to a statistically significant decrease in GJIC, so this confirms that Cx43 is the most pre dominant connexin in the bone.Each connexin is made up of 4 transmembrane domains that consist of a cytoplasmic N- and C- termini (NT and CT) and 2 extracellular loops which aids cell to cell recognition. The structure also includes 4 transmembrane domains3. These connexin’s can form different types of connexon structures which can then bind to the hemi channel in another cell membrane. These structures include Homeric channels that involve only one type of connexin protein subunits in the connexon. However, if there is more than 2 different connexin subunits in the connexon it displays a heteromeric arrangement.Cx43 is also known as gap junction alpha 1 (Gja1) protein is encoded by the Gja1 gene. Bone homeostasis occurs to maintain bone strength and mass and is regulated by a balance between the activity of osteoblasts and osteoclasts, known as bone remodelling. Osteoblasts differentiate to form new bone cells, known as osteocytes, whilst osteoclasts counteract this by resorbing bone. For bone remodelling to be effective the bone cells use gap junctions to communicate, to regulate and coordinate the absorption and resorption mechanisms because if it is imbalanced it could lead to bone disorders like osteoporosis or oculo dento-digital dysplasia.To better understand Cx43 role in cell to cell signalling and its effect on skeletal phenotype Moorer et al concentrates in on the Cx43 CT as it is an important part of the Cx43 for maintaining gap junction function1. Truncated Cx43 CT can form gap junctions however the plaques are fewer but larger in size, hence showing Cx43 CT is not enough for optimal gap junction functioning but can none the less provide few channels for signal exchange, so cell-cell communication will not be optimal in these bone cells and may effect bone homeostasis but it will also not be eradicated.Without the CT domain of Cx43 there will be abnormal osteoblastic activity because of an interruption in the communication between the bone cells, required for optimal bone remodelling. Therefore, CT-domain is shown to be required for Cx43 dependant activation of the osteoblast gene. The CT acts as the docking platform for signalling complexes that would regulate the opening and closure of the gap junction so that GJIC can occur effectively. Osteoblasts require the Cx43 CT domain as findings include that with a truncated CT-domain, osteoblastic proteins will not be expressed. Bone formation is a process that involves release of proteins from osteoblasts such as osteocalcin, type 1 collagen and Osteoprotogerin (OPG). OPG binds to Receptor activator of nuclear factor kappa-? ligand (RANKL) to prevent it from binding to the RANK on osteoclast precursors and stimulating its differentiation to osteoclast, therefore OPG can inhibit bone resorption by preventing osteoclast activation. Moorer et el found that when truncating the Cx43 CT (amino acid 258-382) of 6 week old male mice (Gja1-/K258Stop) in vitro lead to a decrease in OPG expression compared to the control (Gja1-/+) showed similar results to previous research done by Plotkin et al4. Meaning more osteoclast cells are expected to be activated and differentiate and therefore more bone resorption would occur in the truncated CT mice, this was confirmed by an increase in osteoclasts on the bone surface. Deletion of Cx43 in cultured osteocytic cells lead to an increase in RANKL, this leads to a higher RANKL to OPG ratio1,4, signifying that there is less OPG available to bind to the RANKL this allows more RANKL to bind to RANK and lead to osteoclast activation and hence more bone resorption.This suggests that the GJIC of Cx43 gap junctions in bone can indirectly affect osteoclast activity by regulating the expression of OPG in osteocytes. This also indicates that the Cx43 gap junctions lead to a communication pathway between osteocytes and osteoclast cells. However, when the gap junction is altered, by truncating its CT, this will disrupt communication between the cells and bone homeostasis and lead to a higher level of cortical thinning, cortical porosity and increase in its cross sectional area. This suggests that without the CT domain of Cx43, a more osteopenic phenotype is to be expected because the interruption in cell to cell communication can lead to an increase in osteoclast activation. This is significant as it shows the reason for a more osteopenic skeletal phenotype may be because of the indirect effect of Cx43 gap junction action on osteoclasts and that the Cx43 could perhaps play a more direct role in regulating OPG transcription. Bone remodelling includes osteoblast differentiation, which lays down osteoblasts to regulate ossification. Ossification is the process of laying down new bone and is regulated by many transcription factors acting on mesenchymal cells (Fig 1), one of which includes Runx2 and then downstream to this is where the Osterix transcription factor also required for osteoblast proliferation, to control this process and maintain amount of mesenchymal cells Twist1 acts to inhibit Runx25. Figure 1: Osteoblast differentiation via Wnt/Runx2 pathway; Mesenchymal cells activated via the Runx2 pathway are described as Preosteoblast cells and are committed to the osteoblast lineage and undergo a series of mechanisms to complete osteoblast differentiation The process begins with a continuance of cell proliferation until the cells can leave the cell cycle and start cell differentiation with Osterix into osteoblasts which will then mature into osteocytes6.Gja1-/K258Stop mice displayed decreased levels of mRNA expression of Twist1 and Osterix1. However, with a decrease in Twist1, Runx2 cannot be inhibited and therefore can act on more mesenchymal cells. This shows that a decrease in intact Cx43 gap junction can stimulate osteoblast differentiation and regulate bone formation by downregulating some of the key transcriptional factors required to regulate osteoblastogenesis and therefore bone proliferation rates should be higher. Which was supported by an increase in Serum type 1 collagen N propeptide (P1NP), a marker for bone formation. No gross skeletal difference was identified between Gja1?/K258Stop mice and the control and no difference in body length/weight. Showing that whilst Cx43 CT plays a vital role in bone cell to cell communication the NT and transmembrane domains of Cx43 are enough to maintain normal skeletal structure and to sustain osteocytes. To delve deeper into this hypothesis Periosteal and endosteal cortical bone of the Gja1-/K258Stop mice were analyzed and much like the investigation by Xu et al7 showed an increase in periosteal bone whilst diminishing the endosteal bone, that lead to an increase marrow cavity area. These are new findings that show Cx43 CT truncation can lead to a skeletal phenotype, strongly suggesting that the CT plays a bigger role in maintaining osteocyte and osteoblast cells than just coupling of the cells so that they can share second messengers.Osteoblastogenesis also uses the signalling proteins ERK1/2, PKC? and ?-catenin (Fig.1) known to interact with Cx438. The Wnt/?-catenin pathway regulates adhesion of cells to each other and gene transcription, in the bone it induces the expression of Runx29. Key findings by Moorer et al showed overexpression of a Cx43 CT construct reduces binding of the intact Cx43 to the signaling proteins. This suggests over expressed Cx43 CT and the intact Cx43 compete for signaling proteins, showing that in the bone the Cx43 CT may work as well as the intact Cx43 and are therefore essential for efficient signaling to take place between bone cells. This also shows that Cx43 regulates bone formation through direct interactions with the signaling proteins and providing feedback. Explaining that Cx43 CT plays apart in docking these signaling proteins for optimal osteoblastogenesis so that there is no imbalance in osteoblastic and osteoclastic activityMany studies over recent years have given a better understanding of the involvement of Cx43 and its domains in bone cell communication to regulate bone remodelling, this could lead to developing a means of helping regulate bone formation in diseases that would benefit from these sorts of treatments.Further research in this topic could be done using other connexin or different connexon complexes that are related to osteoblast lineage cells, such as Cx40, Cx45 or Cx46, as the understanding of how these connexin’s contribute to bone homeostasis is still very limited.