Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Aug 18.
Published in final edited form as: Genesis. 2012 Oct 16;51(2):75–82. doi: 10.1002/dvg.22349

Determining how defects in Connexin43 cause skeletal disease

Quynh V Ton 1, M Kathryn Iovine 1,*
PMCID: PMC4540067  NIHMSID: NIHMS410660  PMID: 23019186

Abstract

Gap junction channels mediate direct cell-cell communication via the exchange of second messengers, ions, and metabolites from one cell to another. Mutations in several human connexin (cx) genes, the subunits of gap junction channels, disturb the development and function of multiple tissues/organs. In particular, appropriate function of Cx43 is required for skeletal development in all vertebrate model organisms. Importantly, it remains largely unclear how disruption of gap junctional intercellular communication causes developmental defects. Two groups have taken distinct approaches towards defining the tangible molecular changes occurring downstream of Cx43-based gap junctional communication. Here, these strategies for determining how Cx43 modulates downstream events relevant to skeletal morphogenesis are reviewed.

Keywords: gap junction, Cx43, bone growth, skeletal development

Introduction

Gap junctions are proteinaceous channels that lie in the plasma membranes between adjacent cells, permitting the exchange of small molecules including ions and second messengers (Goodenough et al., 1996; Kumar and Gilula 1996) and playing critical roles in development and homeostasis. Connexins (cx), the subunits of gap junctions, consist of four transmembrane-spanning domains, two extracellular loops, one intracellular loop, and intracellular amino and carboxy ends. Six connexins comprise a connexon, or hemichannel, and a functional gap junction channel is composed of two connexons (one from each cell, see Figure 1). Connexons may be homomeric (same connexin isotype) or heteromeric (two or more connexin isotypes), increasing the potential for specificity differences in the types of molecules shared between cells. For example, mouse Cx43 and Cx45 can assemble into heteromeric gap junctions that exhibit coupling properties distinct from either homomeric type (Martinez at al., 2002). Several studies show that Cx43 is important for the growth and development of the vertebrate skeleton. However, little is known mechanistically regarding how Cx43 regulates events such as cell proliferation and cell differentiation. Thus, the challenge is to identify the tangible events regulated by gap junctional intercellular communication (GJIC). In this review, we will first summarize the important roles of Cx43 in skeletal development. Second, we will discuss two independent experimental strategies aimed at revealing how Cx43 mediates downstream molecular signaling events, in turn altering cell function.

Figure 1.

Figure 1

Open in a new tab

Gap junction channels connect the cytoplasm of adjacent cells. Each connexin contains four transmembrane-spanning domains, with both the amino and carboxy ends located in the cytoplasm. Six connexins comprise a connexon, or hemichannel. Two connexons, one from each cell, dock together at the plasma membrane to make a single gap junction channel. IL, intracellular loop; EL, extracellular loop; TM, transmembrane-spanning domain.

Cx43 exhibits conserved functions in the vertebrate skeleton

Defective Cx43 function causes skeletal defects in human, mouse, chick, and zebrafish. Missense mutations in human CX43 result in an autosomal dominant disorder called oculodentodigital dysplasia (ODDD, Paznekas et al., 2003). Patients with ODDD exhibit major skeletal malformations and craniofacial abnormalities such as cranial hyperostosis and broad tubular bones. This disease has additional pleiotropic phenotypes including eye abnormalities leading to vision loss and dental anomalies (reviewed in Pfenniger et al., 2010). The range of phenotypes affecting multiple tissues is likely due to the fact that CX43 is expressed in most cell types. The Gja1jrt/+ mouse carries a mutation coding for a dominant missense mutation in the CX43 gene, and exhibits skeletal and pleiotropic phenotypes similar to those observed in human ODDD (Flenniken et al., 2005). Indeed, recently generated knock-in alleles, where human Cx43-missense mutations replace the endogenous wild-type Cx43 allele in the mouse, also mimic human ODDD (Dobrowolski et al., 2008; Watkins et al., 2011). In contrast, the CX43 knockout mouse (CX43−/−) dies perinatally due to defects in the heart outflow tract and reduced blood flow to tissues (Reaume et al., 1995). Continued investigation revealed that these mice exhibit delayed ossification of both the intramembranous and endochondral skeletons (Lecanda et al., 2000). To further overcome the lethality of CX43−/−, conditional CX43 knockout mouse lines have been generated. While mice lacking Cx43 activity in osteoblasts are viable, they exhibit reduced bone mineral density throughout their skeletons, consistent with phenotypes observed in the complete knockout (Chung et al., 2006; Watkins et al., 2011; Zhang et al., 2012; Bivi et al., 2012). Targeted gene knockdown of CX43 in adult chicks and in chick embryos results in facial defects and limb malformation, respectively (Makarenkova et al., 1999; McGonnell et al., 2003). Finally, in the zebrafish, homozygous mutations in the cx43 gene cause the short fin (sof b123) phenotype, which primarily affects the fin skeleton. The sof b123 mutant exhibits short bony fin rays due to short bony segments and reduced levels of cell proliferation (Iovine et al., 2005). Targeted gene knockdown of cx43 causes embryonic heart defects, suggesting cx43 is essential (Iovine et al., 2005). Collectively, these data demonstrate that Cx43 function is conserved from fish to man and is required for typical skeletal morphogenesis in vertebrates.

There is some evidence that connexins, including Cx43, may exhibit activities independent of, or in addition to, GJIC. For example, since Cx43 in particular has a long carboxy tail exposed to the cytoplasm, it may serve as a signaling platform that functions independent of GJIC. Indeed the carboxy tail of Cx43 is known to bind to the components of signaling complexes including β-catenin and src (Saez et al., 2003; Giepmans, 2004; Herve et al., 2004). Alternatively, the signaling complexes may be activated by GJIC, such that channel function regulates signaling events. There is also evidence that Cx43 hemichannels play important functions in skeletal growth and development (reviewed in Plotkin, 2011). Connexin hemichannels are unpaired connexons that may permit the exchange of small molecules between the cytoplasm and the extracellular space. The focus of this review is on current strategies used to define the cellular events occurring downstream of Cx43 function, whether as a gap junction channel, a hemichannel, or in a channel-independent manner. Thus, it should be noted that while GJIC is often the presumed function for Cx43 in the described studies, its precise role(s) has not been exclusively determined.

Cx43 regulates gene expression in osteoblast cell lines

It is well established that Cx43 enhances the expression of osteoblast genes. Recently, considerable effort has gone into defining the molecular mechanisms underlying Cx43-based regulation of gene expression. This work has been completed using a reporter assay in osteoblast cell lines (i.e. ROS17/2.8 cells and/or MC3T3 cells). The reporter construct contains the promoter of the osteoblast-specific gene osteocalcin upstream of the luciferase coding sequence (Figure 2). Luciferase activity can be precisely calculated and is directly proportional to the level of gene transcription from the osteocalcin promoter. When Cx43 function is high, luciferase activity is high. When Cx43 function is abrogated either by over-expression of Cx45 (which has been shown to reduce Cx43-dependent GJIC by modifying the size and specificity of the heteromeric gap junction channel, Lecanda et al., 1998) or by the addition of pharmacological inhibitors of GJIC (Stains and Civitelli, 2005), luciferase activity is reduced. Using this system, two distinct Cx43-dependent response elements have been identified in the osteocalcin promoter, and a mechanism for Cx43-dependent transcriptional activation has been suggested (described below and shown in Figure 3).

Figure 2.

Figure 2

Open in a new tab

The in vitro system used to evaluate Cx43-dependent regulation of gene expression. The reporter construct contains the osteocalcin (OCN) promoter followed by the coding sequence for the luciferase gene. The level of luciferase transcription is determined by quantitating the amount of luciferase activity. Osteoblast cell lines transfected with this reporter demonstrate that luciferase activity is dependent on the level of Cx43-based function.

Figure 3.

Figure 3

Open in a new tab

Model showing how gap junctions may amplify signals originating from growth-factor mediated cascades. A primary signal originates from a growth factor-receptor interaction that can initiate a response/change in gene expression in cell one (not drawn). At the same time, second messengers resulting from the primary signal transverse gap junction channels to initiate a similar change in gene expression in cell two. This is the secondary signal. Described Cx43-dependent changes include Ras/ERK (triangle) mediated phosphorylation of Sp1 (Stains and Civitelli, 2005) and PKCδ (diamond) mediated phosphorylation of Runx2 (Lima et al., 2009). PKCδ may also independently activate ERK (Niger et al., 2012). Phosphorylated Sp1 and Runx2 favor transcription of certain osteoblast genes.

Using the system described above, a minimal Cx43 response element (CxRE) was identified in the osteocalcin promoter (Stains et al., 2003). It was determined that Cx43 function regulated the level of phosphorylation of the transcription factor Sp1, which in turn bound to the CxRE. Thus, increased phosphorylation of Sp1 favored its recruitment to the CxRE and caused increased gene transcription. In contrast, reduced phosphorylation of Sp1 favored the recruitment of an alternate transcription factor, Sp3, and lead to reduced gene transcription. Continued studies revealed that the phosphorylation of Sp1 occurred through Cx43-dependent activation of the ERK signal transduction cascade (Stains and Civitelli, 2005). Moreover, this Cx43-dependent recruitment of Sp1 was also correlated with recruitment of the osteoblast transcription factor Osx/Sp7 to the osteocalcin promoter (Niger et al., 2011). A second pathway for Cx43-dependent gene transcription occurs via activation of the transcription factor Runx2. Phosphorylated Runx2 interacts with the osteoblast-specific element OSE2, also found in the osteocalcin promoter. Cx43 function leads to activation of PKCδ, which is an intermediate in the FGF2 signaling pathway. Activation of PKCδ leads to the phosphorylation of Runx2 and increased gene transcription (Lima et al., 2009). Alternatively, FGF2 signaling can activate ERK independent of Cx43, also leading to Runx2-phosphorylation (Niger et al., 2012). These data provide evidence that FGF2 and Cx43 synergize to influence Runx2-phosphorylation and therefore increased gene expression of at least a subset of osteoblast-specific genes.

How are the ERK and FGF2 growth factor dependent signaling cascades related to Cx43 function? The Stains group suggests that both primary and secondary responses to growth factor-mediated signal transduction pathways are responsible for coordinated regulation of gene expression among a population of osteoblasts (Figure 3). The primary response occurs in cells expressing appropriate receptors for externally provided cues, such as growth factors. Growth factor binding to its receptor leads to the intracellular production of second messengers, which in turn can be shared with adjacent cells via gap junctions, thereby promoting the same response in neighboring cells (i.e. even when those cells lack the appropriate receptor). The identification of signal transduction cascades working synergistically with Cx43-based gap junctions suggests that one role of gap junctions may be to amplify typical growth-factor induced responses. Moreover, since the carboxy-tail of connexins may act as a signaling center by binding to components of various signaling complexes (Bivi et al., 2011), the mediators of the second messengers may be closely associated with gap junctions. For example, Ras, a mediator of the ERK pathway, is known to associate on the inner leaflet of the plasma membrane and could associate with gap junctions (Stains and Civitelli, 2005). Moreover, PKCδ, a known mediator of FGF2 signaling, was recently shown to physically associate with the carboxy-tail of Cx43 (Niger et al., 2010).

These studies suggested that the Cx43 gap junctions reinforce typical signal transduction cascades by propagating second messengers to neighboring cells that may not have received the primary signal. Such a mechanism allows a signal that is initiated in a small number of cells to be coordinated within a tissue. Furthermore, these studies provide opportunities to begin to identify the relevant second messengers that may permeate Cx43-based gap junction channels (Niger et al., 2012).

Cx43 regulates gene expression in vivo

A second strategy to explore how Cx43 controls gene regulation in a mechanistic pathway is to evaluate Cx43 effects in vivo. Our lab evaluates the fin phenotypes of the sof mutant (Figure 4) to identify Cx43-dependent functions in the skeleton. The fin is composed of multiple bony fin rays and each fin ray consists of multiple bony segments separated by joints. Fin growth occurs at the distal end of the fin ray and can be monitored during typical ontogenetic growth or during regenerative growth following amputation (reviewed in Poss et al., 2003; Akimenko et al., 2003). The fin ray includes several tissues. The bone matrix of opposed hemirays surrounds the central mesenchymal compartment, while the hemirays are surrounded by a multi-layered epithelium. The population of dividing cells contributing to new fin growth is located in the distal mesenchyme, and is referred to as the blastema in the regenerating fin. Skeletal precursor cells (the pre-osteoblasts and pre-joint forming cells) are located laterally in the mesenchymal compartment, adjacent to the blastema. During regeneration, the cx43 mRNA is up-regulated in blastemal cells. We have suggested that communication between the blastema and the skeletal precursor cells could be mediated by Cx43 function (Brown et al., 2009).

Figure 4.

Figure 4

Open in a new tab

Fin length mutants exhibit defects in skeletal morphogenesis. Top: wild-type zebrafish. Middle: sof b123 mutant. Bottom: alf dty86 mutant.

The Iovine lab has taken a molecular genetics approach to identify cx43-dependent genes contributing to skeletal morphogenesis. The original allele, sof b123, exhibits reduced levels of cx43 mRNA, short fins, short bony fin ray segments, and reduced levels of cell proliferation (Iovine et al., 2005). In addition to the sof b123 allele, three alleles coding missense mutations in the cx43 gene were isolated using a non-complementation screen. All four sof alleles are hypomorphic and exhibit reduced fin length, segment length and cell proliferation. Further, morpholino-mediated knockdown of cx43 recapitulates all of the sof phenotypes (Hoptak-Solga et al., 2008). In contrast to the sof mutants, the another long fin (alf dty86, van Eeden et al., 1996, Figure 4) mutant exhibits increased levels of cx43 mRNA, long fins, and stochastic joint failure (i.e. long segments on average). Remarkably, the alf dty86 phenotypes are rescued by cx43 knockdown (Sims et al., 2009). Since the alf dty86 mutant exhibits phenotypes opposite to that of sof, and since cx43 knockdown rescues those phenotypes, we consider alf dty86 to be a mimic of cx43 over-expression. Based on these and other data we further hypothesize that the role of Cx43 in the regenerating fin is to coordinate fin growth/cell proliferation with skeletal patterning/joint formation. In other words, Cx43 mediates two activities: promoting cell proliferation and suppressing joint formation.

In order to identify genes whose expression is regulated by Cx43, we completed a microarray analysis. We generated a list of genes that that are both down-regulated in sof b123 (when cx43 is down-regulated) and up-regulated in alf dty86 (when cx43 is up-regulated). Notably, we found the cx43 gene among the top 50 candidates, suggesting that our approach was successful. From this list, we decided to focus first on the semaphorin3d (sema3d) gene (Ton and Iovine, 2012). Class 3 semaphorins, such as Sema3d, are secreted signaling molecules known to be involved in activating signaling pathways that influence diverse cellular functions (reviewed in Yazdani and Terman, 2006). Expression of sema3d is located in the lateral skeletal precursor cells. Since sema3d is not expressed in the cx43-positive cells, it appears not to be a direct target for Cx43 function. However, since sema3d knockdown recapitulates all of the cx43 knockdown phenotypes (including rescue of the alf dty86 phenotypes), sema3d is a likely candidate for mediating Cx43-dependent functions (Figure 5a).

Figure 5.

Figure 5

Open in a new tab

Cx43 influences cell proliferation and joint formation by regulating the Sema3d pathway. (a) Expression of the sema3d gene is regulated by cx43. The Sema3d protein is secreted and interacts with its putative receptors, Nrp2a and PlxnA3, to regulate cell proliferation and joint formation respectively. (b) Compartmental expression of the genes in the Cx43 pathway. The cx43 mRNA is up-regulated in the proliferating cells of the blastema (red), while sema3d is up-regulated in the lateral skeletal precursor cells and basal layer of the epidermis (green). The nrp2a gene is expressed in the distal blastema (yellow), where it may regulate cell proliferation in the blastemal cells. The plxna3 gene is expressed in the skeletal precursor cells (blue), where it may regulate joint formation. This figure was originally published in Ton and Iovine, 2012, and is reprinted here with permission.

Semaphorins utilize Neuropilins (Nrps) and Plexins (Plxns) as typical cell-surface receptors. Based on gene expression and gene function of candidate genes encoding the Nrps and Plxns, we found that independent receptors mediate either cell proliferation or joint formation (Ton and Iovine, 2012). For example, the Nrp2a receptor appears to mediate Sema3d-dependent cell proliferation while the PlxnA3 receptor appears to mediate Sema3d-dependent joint formation. Interestingly, knockdown of nrp2a causes an increase in cell proliferation, while knockdown of sema3d causes reduced cell proliferation. We interpret these and other data to suggest that Sema3d inhibits Nrp2a function, thereby promoting cell proliferation by inhibiting a negative signal (Ton and Iovine, 2012). Based on these analyses of a single gene identified by our novel microarray strategy, we established the beginning of a molecular pathway dependent on Cx43 function (Figure 5). Continued insight into Cx43-dependent events may be obtained by placing additional genes identified by the microarray in this pathway. Indeed, of the nine newly validated genes from the microarray, four have established connections to mammalian skeletal diseases (Table 1). We anticipate that continued evaluation of these and other microarray genes will reveal a network of Cx43-dependent events that coordinate tangible cellular functions (such as cell proliferation and joint formation) and thereby control skeletal morphogenesis.

Table 1.

Validated microarray genes with known skeletal relevance

name disease associated with mammalian ortholog OMIM ID
hsp47 osteogenesis imperfecta 600943
hapln1a dwarfism and craniofacial defects (mouse) 115435
twist1b craniosynostosis type I 601622
hoxda13 brachydactyly; syndactyly 142989
Open in a new tab

OMIM: Online Mendelian Inheritance in Man is a database that catalogs disease-causing mutations and phenotypes. Mutations in the mammalian orthologs to the four listed zebrafish genes are known to cause skeletal disease. For complete list of identified genes, please see Ton and Iovine, 2012.

The case for Cx43-dependent GJIC during skeletal morphogenesis

Work from both the Stains lab and the Iovine lab provides evidence that Cx43-based GJIC contributes to skeletal development. For example, recall that Cx43 and FGF2 signals synergize to activate the ERK2-mediated phosphorylation of Runx2 and concomitant increased transcription of the luciferase reporter (Niger et al., 2012). When treated cells were examined using flow cytometry it was possible to demonstrate that the increased luciferase activity was due to an increase in the number of cells responding to the Cx43 and FGF2 signals, and not due to a more robust response from the same number of cells. This finding is consistent with their proposed model that the primary signal from the FGF2 growth factor is amplified via Cx43-based GJIC. Prior work from our lab demonstrated that the three missense alleles of cx43 exhibit reduced levels of GJIC in heterologous assays (these alleles also appear to be capable of trafficking to the plasma membrane, Hoptak-Solga et al., 2007). Moreover, the severity of the segment length and cell proliferation phenotypes is correlated with the reduced level of GJIC (Iovine et al., 2005; Hoptak-Solga et al., 2008), strongly suggesting that reduced Cx43-based GJIC is responsible for the observed skeletal defects.

There are two possibilities to explain how Cx43-dependent GJIC in the blastemal cells influences sema3d gene expression in the adjacent skeletal precursor cells. First, Cx43-GJIC causes changes in gene expression in the cx43-positive compartment that lead to the secretion of an unidentified growth factor. This growth factor will bind to its receptor on the adjacent cells and cause an increase in sema3d expression in the lateral compartment (Figure 6a). Such a mechanism may be revealed by continued examination of genes identified from the microarray analysis. Alternatively, it remains possible that heterotypic gap junctions (i.e. when each cell contributes a connexon composed of different connexins) exist between cells of the blastema and the skeletal precursor cells, directly influencing gene expression between the two compartments (Figure 6b). Expression of a connexin gene has not been identified in the lateral compartment. However, since the zebrafish genome has at least 37 connexin genes (Eastman et al., 2006), one may still be found. This latter model, if true, may represent an in vivo example of second messengers traveling from one cell population to another in order to coordinate changes in gene expression, as elucidated by work from Stains et al.

Figure 6.

Figure 6

Open in a new tab

Examples of how Cx43-dependent GJIC may influence gene expression in the regenerating fin. (a) Up-regulation of cx43 in the blastema (red) may lead to the secretion of a growth factor that can interact with its receptor located on adjacent skeletal precursor cells (green), leading to increased sema3d expression. (b) Heterotypic gap junctions may exist between cells of the blastema and skeletal precursor cells, permitting the direct exchange of secondary messengers. These second messengers may influence sema3d expression.

Conclusions

The goal in defining the relevant Cx43 functions during skeletal morphogenesis is to reveal the initial Cx43-dependent event that regulates changes in cellular function. How do the different connexin functions lead to tangible changes in cell proliferation or cell differentiation? What are the relevant molecules that mediate these functions? Does the channel activity enhance propagation (as suggested in the work of Stains et al.), or is the channel-function secondary to the carboxy-tail as a scaffold for signaling molecules? How does hemichannel function fit in? The strategies described here have revealed tangible events downstream of Cx43 function and have provided novel insights into the types of cellular changes that are regulated by Connexins. In the future, we and others hope to work from these downstream events towards the initial Cx43-dependent function.

Acknowledgments

The authors wish to thank the members of the Iovine lab for providing constructive comments on the manuscript. Some of the work described in this review was supported by the NIH (HD047737) and the NSF (IOS-1145582) to MKI.

References

  1. Akimenko MA, Mari-Beffa M, Becerra J, Geraudie J. Old questions, new tools, and some answers to the mystery of fin regeneration. Dev Dyn. 2003;226:190–201. doi: 10.1002/dvdy.10248. [DOI] [PubMed] [Google Scholar]
  2. Bivi N, Lezcano V, Romanello M, Bellido T, Plotkin LI. Connexin43 interacts with βarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone. J Cell Biochem. 2011;112:2920–2930. doi: 10.1002/jcb.23208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bivi N, Condon KW, Allen MR, Farlow N, Passeri G, Brun LR, Rhee Y, Bellido T, Plotkin LI. Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation. J Bone Miner Res. 2012;27:374–89. doi: 10.1002/jbmr.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown AM, Fisher S, Iovine MK. Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish. Dev Dyn. 2009;238:2922–2928. doi: 10.1002/dvdy.22114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung DJ, Castro CH, Watkins M, Stains JP, Chung MY, Szejnfeld VL, Willecke K, Theis M, Civitelli R. Low peak bone mass and attenuated anabolic response to parathyroid hormone in mice with an osteoblast-specific deletion of connexin43. J Cell Sci. 2006;119:4187–98. doi: 10.1242/jcs.03162. [DOI] [PubMed] [Google Scholar]
  6. Dobrowolski R, Sasse P, Schrickel JW, Watkins M, Kim JS, Rackauskas M, Troatz C, Ghanem A, Tiemann K, Degen J, Bukauskas FF, Civitelli R, Lewalter T, Fleischmann BK, Willecke K. The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans. Hum Mol Genet. 2008;17:539–54. doi: 10.1093/hmg/ddm329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eastman SD, Chen TH, Falk MM, Mendelson TC, Iovine MK. Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes. Genomics. 2006;87:265–274. doi: 10.1016/j.ygeno.2005.10.005. [DOI] [PubMed] [Google Scholar]
  8. Flenniken AM, Osborne LR, Anderson N, Ciliberti N, Fleming C, Gittens JE, Gong XQ, Kelsey LB, Lounsbury C, Moreno L, Nieman BJ, Peterson K, Qu D, Roscoe W, Shao Q, Tong D, Veitch GI, Voronina I, Vukobradovic I, Wood GA, Zhu Y, Zirngibl RA, Aubin JE, Bai D, Bruneau BG, Grynpas M, Henderson JE, Henkelman RM, McKerlie C, Sled JG, Stanford WL, Laird DW, Kidder GM, Adamson SL, Rossant J. A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia. Development. 2005;132:4375–4386. doi: 10.1242/dev.02011. [DOI] [PubMed] [Google Scholar]
  9. Giepmans BN. Gap junctions and connexin-interacting proteins. Cardiovasc Res. 2004;62:233–245. doi: 10.1016/j.cardiores.2003.12.009. [DOI] [PubMed] [Google Scholar]
  10. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem. 1996;65:475–502. doi: 10.1146/annurev.bi.65.070196.002355. [DOI] [PubMed] [Google Scholar]
  11. Herve JC, Bourmeyster N, Sarrouilhe D. Diversity in protein-protein interactions of connexins: emerging roles. Biochim Biophys Acta. 2004;166:22–41. doi: 10.1016/j.bbamem.2003.10.022. [DOI] [PubMed] [Google Scholar]
  12. Hoptak-Solga AD, Klein KA, Derosa AM, White TW, Iovine MK. Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication. FEBS Lett. 2007;581:3297–3302. doi: 10.1016/j.febslet.2007.06.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoptak-Solga AD, Nielsen S, Jain I, Thummel R, Hyde DR, Iovine MK. Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration. Dev Biol. 2008;317:541–548. doi: 10.1016/j.ydbio.2008.02.051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iovine MK, Higgins EP, Hindes A, Coblitz B, Johnson SL. Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins. Dev Biol. 2005;278:208–219. doi: 10.1016/j.ydbio.2004.11.005. [DOI] [PubMed] [Google Scholar]
  15. Kumar NM, Gilula NB. The gap junction communication channel. Cell. 1996;84:381–388. doi: 10.1016/s0092-8674(00)81282-9. [DOI] [PubMed] [Google Scholar]
  16. Lecanda D, Towler DA, Ziambaras K, Cheng SL, Koval M, Steinberg TH, Civitelli R. Gap junctional communication modulates gene expression in osteoblastic cells. Mol Biol Cell. 1998;9:2249–2258. doi: 10.1091/mbc.9.8.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R. Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol. 2000;151:931–944. doi: 10.1083/jcb.151.4.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lima F, Niger C, Hebert C, Stains JP. Connexin43 potentiates osteoblast responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-delta/Runx2-dependent mechanism. Mol Biol Cell. 2009;20:2697–2708. doi: 10.1091/mbc.E08-10-1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makarenkova H, Patel K. Gap junction signaling mediated through connexin-43 is required for chick limb development. Dev Biol. 1999;207:380–392. doi: 10.1006/dbio.1998.9171. [DOI] [PubMed] [Google Scholar]
  20. Martinez AD, Hayrapetyan V, Moreno AP, Beyer EC. Connexin43 and connexin45 form heteromeric gap junction channels in which individual components determine permeability and regulation. Circ Res. 2002;90:1100–1107. doi: 10.1161/01.res.0000019580.64013.31. [DOI] [PubMed] [Google Scholar]
  21. McGonnell IM, Green CR, Tickle C, Becker DL. Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face. Dev Dyn. 2001;222:420–438. doi: 10.1002/dvdy.1208. [DOI] [PubMed] [Google Scholar]
  22. Niger C, Hebert C, Stains JP. Interaction of connexin43 and protein kinase C-delta during FGF2 signaling. BMC Biochem. 2010;11:14. doi: 10.1186/1471-2091-11-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Niger C, Lima F, Yoo DJ, Gupta RR, Buo AM, Hebert C, Stains JP. The transcriptional activity of osterix requires the recruitment of Sp1 to the osteocalcin proximal promoter. Bone. 2011;49:683–692. doi: 10.1016/j.bone.2011.07.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Niger C, Buo AM, Hebert C, Duggan BT, Williams MS, Stains JP. ERK acts in parallel to PKCδ to mediate the connexin43-dependent potentiation of Runx2 activity by FGF2 in MC3T3 osteoblasts. Am J Physiol Cell Physiol. 2012;302:C1035–C1044. doi: 10.1152/ajpcell.00262.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paznekas WA, Boyadjiev SA, Shapiro RE, Daniels O, Wollnik B, Keegan CE, Innis JW, Dinulos MB, Christian C, Hannibal MC, Jabs EW. Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet. 2003;72:408–418. doi: 10.1086/346090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfenniger A, Wohlwend A, Kwak BR. Mutations in connexin genes and disease. Eur J Clin Inv. 2010;41:103–116. doi: 10.1111/j.1365-2362.2010.02378.x. [DOI] [PubMed] [Google Scholar]
  27. Plotkin LI. Connexin43 and bone: not just a gap junction protein. Actual osteol. 2011;7:79–90. [PMC free article] [PubMed] [Google Scholar]
  28. Poss KD, Keating MT, Nechiporuk A. Tales of regeneration in zebrafish. Dev Dyn. 2003;226:202–210. doi: 10.1002/dvdy.10220. [DOI] [PubMed] [Google Scholar]
  29. Reaume AG, de Sousa PA, Kulkarni S, Langille BL, Zhu D, Davies TC, Juneja SC, Kidder GM, Rossant J. Cardiac malformation in neonatal mice lacking connexin43. Science. 1995;267:1831–1834. doi: 10.1126/science.7892609. [DOI] [PubMed] [Google Scholar]
  30. Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev. 2003;83:1359–1400. doi: 10.1152/physrev.00007.2003. [DOI] [PubMed] [Google Scholar]
  31. Sims K, Jr, Eble DM, Iovine MK. Connexin43 regulates joint location in zebrafish fins. Dev Biol. 2009;327:410–418. doi: 10.1016/j.ydbio.2008.12.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stains JP, Lecanda F, Screen J, Towler DA, Civitelli R. Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters. J Biol Chem. 2003;278:24377–24387. doi: 10.1074/jbc.M212554200. [DOI] [PubMed] [Google Scholar]
  33. Stains JP, Civitelli R. Gap junctions regulate extracellular signal-regulated kinase signaling to affect gene transcription. Mol Biol Cell. 2005;16:64–72. doi: 10.1091/mbc.E04-04-0339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ton QT, Iovine MK. Semaphorin3d mediates Cx43-dependent phenotypes during fin regeneration. Dev Biol. 2012;366:195–203. doi: 10.1016/j.ydbio.2012.03.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van Eeden FJ, Granato M, Schach U, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh R, Mullins MC, Odenthal J, Warga RM, Nusslein-Volhard C. Genetic analysis of fin formation in the zebrafish, Danio rerio. Development. 1996;123:255–62. doi: 10.1242/dev.123.1.255. [DOI] [PubMed] [Google Scholar]
  36. Watkins M, Grimston SK, Norris JY, Guillotin B, Shaw A, Beniash E, Civitelli R. Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling. Mol Biol Cell. 2011;22:1240–51. doi: 10.1091/mbc.E10-07-0571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yazdani U, Terman JR. The semaphorins. Genome Biol. 2006;7:211. doi: 10.1186/gb-2006-7-3-211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y, Paul EM, Sathyendra V, Davison A, Sharkey N, Bronson S, Srinivasan S, Gross TS, Donahue HJ. Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone. PLoS One. 2011;6:e23516. doi: 10.1371/journal.pone.0023516. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES