Connexin43-containing gap junctions potentiate extracellular Ca²âº-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2.

Extracellular Ca(2+) can promote dentin sialophosphoprotein (DSPP) expression and odontoblastic differentiation of dental pulp stem cells (DPSCs). Gap junctions mediated by connexin43 (Cx43) allow diffusion of small molecules (such as Ca(2+)) among cells to regulate cell-to-cell communications. However, it is unclear whether Cx43 is required for the Ca(2+)-induced cell differentiation. Here, we found that the influx of extracellular Ca(2+) through L-type Ca(2+) channels increases intracellular free Ca(2+) levels to promote DSPP expression. Cx43 overexpression potentiated the extracellular Ca(2+)-induced DSPP expression via Erk1/2. Flow cytometry analyses showed that Cx43 increased the percentage of p-Erk1/2 positive cells in response to Ca(2+), indicating that Cx43 in DPSCs possibly acts as a traditional gap junction channel, which permits the sharing of signals among coupled cells to make more DPSCs respond to Ca(2+). Furthermore, inhibition of Cx43 function and gap junction communication decreased Ca(2+)-induced the expression of DSPP, suggesting that cell-to-cell contacts are required for Cx43 to promote the Ca(2+)-induced cell differentiation. Similarly, the study performed on DPSCs cultured at low-density and high-density revealed that intercellular contacts are required to potentiate Erk1/2 activity and DSPP expression. In total, this study indicates that Cx43 increases Ca(2+)-induced DSPP expression and odontoblastic differentiation of DPSCs via Erk1/2 through gap junction-mediated cell-to-cell contacts.

Role of connexin43 hemichannels in mechanical stress-induced ATP release in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Our previous studies showed that mechanical stress could induce ATP release in human periodontal ligament (HPDL) cells. By signaling through P2 purinergic receptors, ATP increased the expression and the synthesis of osteopontin and RANKL. In this study, the mechanism of stress-induced ATP release was investigated. MATERIAL AND METHODS: Continuous compressive forces were applied on cultured HPDL cells. The ATP released was measured using luciferin-luciferase bioluminescence. The expression of gap-junction proteins was examined using RT-PCR and western blot analysis. The opening of hemichannels was demonstrated by cellular uptake of a fluorescent dye, 5(6)-carboxyfluorescein, which is known to penetrate hemichannels. Intracellular signal transduction was investigated using inhibitors and antagonists. RESULTS: Mechanical stress induced the release of ATP into the culture medium, which was attenuated by carbenoxolone, a nonspecific gap-junction inhibitor. Addition of meclofenamic acid sodium salt, a connexin43 inhibitor, inhibited ATP release by mechanical stress. Knockdown of connexin43 expression by small interfering RNA reduced the amount of ATP released by mechanical stress, suggesting the role of connexin43 hemichannels. In addition, intracellular Ca(2+) blockers could also inhibit mechanical stress-induced ATP release and the opening of the gap junction. CONCLUSION: Our study demonstrated the involvement of gap-junction hemichannels, especially connexin43, in the stress-induced ATP-release mechanism. Furthermore, this mechanism may be regulated by the intracellular Ca(2+) signaling pathway. These results suggest an important role of gap-junction hemichannels in the function and behavior of HPDL cells.

Functional characterization of connexin43 mutations found in patients with oculodentodigital dysplasia.

Specific mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause an autosomal dominant disorder called oculodentodigital dysplasia (ODDD). Here, we characterize the effects of 8 of these mutations on Cx43 function. Immunochemical studies have shown that most of the mutant proteins formed gap junction plaques at the sites of cell-cell apposition. However, 2 of the mutations (a codon duplication in the first extracellular loop, F52dup, and a missense mutation in the second extracellular loop, R202H, produced full-length connexins that failed to properly form gap junction plaques. Cx43 proteins containing ODDD mutations found in the N-terminus (Y17S), first transmembrane domain (G21R, A40V), second transmembrane domain (L90V), and cytoplasmic loop (I130T, K134E) do form gap junction plaques but show compromised channel function. L90V, I130T, and K134E demonstrated a significant decrease in junctional conductance relative to Cx43WT. Mutations Y17S, G21R, and A40V demonstrated a complete lack of functional electrical coupling even in the presence of significant plaque formation between paired cells. Heterologous channels formed by coexpression of Cx43WT and mutation R202H resulted in electrically functional gap junctions that were not permeable to Lucifer yellow. Therefore, the mutations found in ODDD not only cause phenotypic variability, but also result in various functional consequences. Overall, our data show an extensive range of molecular phenotypes, consistent with the pleiotropic nature of the clinical syndrome as a whole.

Hydroxy apatite microspheres enhance gap junctional intercellular communication of human osteoblasts composed of connexin 43 and 45.

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.

Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response.

BACKGROUND: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction. METHODS: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis. RESULTS: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle. CONCLUSIONS: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital aspect of this work is that it demonstrates that targeted molecular therapeutics, such as JM2, provide the capacity to regulate inflammation in a clinically relevant system.

The G60S connexin43 mutant regulates hair growth and hair fiber morphology in a mouse model of human oculodentodigital dysplasia.

Patients expressing mutations in the gene encoding the gap junction protein Cx43 suffer from a disease called oculodentodigital dysplasia (ODDD). Patients with ODDD are often reported to develop hair that is dry, dull, sparse, and slow growing. To evaluate the linkage between Cx43 and hair growth, structure, and follicle density we employed a mouse model of ODDD that harbors a Cx43 G60S point mutant. Regionally sparse and overall dull hair were observed in mutant mice compared with their wild-type (WT) littermates. However, histological analysis of overall hair follicle density in mutant and WT mice did not reveal any significant differences. After epilation, mutant mouse hair grew back slower, and hair growth was asynchronous. In addition, ultrastructural scanning electron microscopic imaging of hair fibers taken from mutant mice and two patients harboring the G143S mutation revealed severe cuticle weathering. Nodule formation was also observed in the proximal region of hair fibers taken from mutant mice. These results suggest that the G60S mutant mouse model mimics the hair phenotype found in at least some ODDD patients and suggests an important role for Cx43 in hair regeneration, growth, and cuticle formation.

Oogenesis defects in a mutant mouse model of oculodentodigital dysplasia.

The essential role of connexin43 (Cx43) during oogenesis has been demonstrated by the severe germ cell deficiency and arrested folliculogenesis observed in Cx43 knockout mice. Recently, another mutant mouse strain became available (Gja1(Jrt)/+) that carries the dominant loss-of-function Cx43 mutation, Cx43(G60S). Gja1(Jrt)/+ mice display features of the human disease oculodentodigital dysplasia (ODDD), which is caused by mutations in the GJA1 gene. We used this new mutant strain to study how a disease-linked Cx43 mutant affects oogenesis. We found that female mutant mice are subfertile with significantly reduced mating success and small litters. The phosphorylated species of the Cx43 protein are reduced in the mutant ovaries in association with impaired trafficking and assembly of gap junctions in the membranes of granulosa cells, confirming that the mutant protein acts dominantly on its wild-type counterpart. Correspondingly, although starting with a normal abundance of germ cells, ovaries of the mutant mice contain significantly fewer pre-ovulatory follicles and do not respond to superovulation by gonadotropins, which is at least partially the result of reduced proliferation and increased apoptosis of granulosa cells. We conclude that the Gja1(Jrt) mutation has a dominant negative effect on Cx43 function in the ovary, rendering the females subfertile. Given these findings, closer examination of reproductive function in ODDD human females is warranted.

Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide.

Connexins are gap-junction proteins forming hexameric structures in the plasma membranes of adjacent cells, thereby creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. We have employed antisense oligonucleotides (AS) against rat CX43 to study the role of CX43 in dental pulp cells. Cultured dental pulp cells were treated with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was approximately 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS- and S-treated groups at 48 h (P < 0.01). An alkaline phosphatase assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, whereas S-treated pulp cells showed no marked changes. Western blot analysis revealed that heat-shock protein 25 was highly expressed in S-treated cells but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells highly expressed CX45, whereas S-treated cells exhibited high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization, and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.

Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia.

Connexin-protein interactions are believed to be critical for the regulation of gap junctional intercellular communication and for the function of gap junctions formed by these complexes. We have primarily used immunoprecipitation strategies to investigate whether connexin43 binds to selected signaling and cytoskeletal proteins and whether connexin43-protein binding is altered in cultured astrocytes exposed to chemical ischemia/hypoxia, a treatment that resembles ischemia in vivo. Chemical ischemia/hypoxia induced marked dephosphorylation of connexin43, which was accompanied by increased association of connexin43 with c-Src, ERK1/2, and mitogen-activated protein kinase phosphatase-1 and by decreased association between connexin43 and beta-actin. Moreover, we found that endogenous c-Src in normal astrocytes exists primarily in the Triton X-100-soluble membrane fraction, distinct from the Triton-insoluble fraction, which contains gap junctions. After chemical ischemia/hypoxia, c-Src appeared in the Triton-insoluble fraction and was co-immunoprecipitated with connexin43, suggesting that chemical ischemia/hypoxia induced translocation of c-Src to the Triton-insoluble fraction and association with connexin43. Furthermore, the "dephosphorylated" form of connexin43 was immunoprecipitated by a phosphotyrosine antibody, suggesting tyrosine phosphorylation of connexin43 by c-Src. In addition, the association between connexin43 and c-Src was blocked by inhibition of connexin43 dephosphorylation, suggesting that the interaction between connexin43 and c-Src can be regulated by alterations in the phosphorylation state of connexin43. These results identify new binding partners for connexin43 and demonstrate that interactions between connexin43 and protein kinases and phosphatases are dynamically altered as a consequence of connexin43 phosphorylation.

Doubly mutant mice, deficient in connexin32 and -43, show normal prenatal development of organs where the two gap junction proteins are expressed in the same cells.

The connexins are a family of proteins that form the intercellular membrane channels of gap junctions. Genes encoding 13 different rodent connexins have been cloned and characterized to date. Connexins vary both in their distribution among adult cell types and in the properties of the channels that they form. In order to explore the functional significance of connexin diversity, several mouse connexin-encoding genes have been disrupted by homologous recombination in embryonic stem cells. Although those experiments have illuminated specific physiological roles for individual connexins, the results have also raised the possibility that connexins may functionally compensate for one another in cells where they are coexpressed. In the present study, we have tested this hypothesis by interbreeding mice carrying null mutations in the genes (Gjb1 and Gja1) encoding connexin32 (beta 1 connexin) and connexin43 (alpha 1 connexin), respectively. We found that fetuses lacking both connexins survive to term but, as expected, the pups die soon thereafter from the cardiac abnormality caused by the absence of connexin43. A survey of the major organ systems of the doubly mutant fetuses, including the thyroid gland, developing teeth, and limbs where these two connexins are coexpressed, failed to reveal any morphological abnormalities not already seen in connexin43 deficient fetuses. Furthermore, the production of thyroxine by doubly mutant thyroids was confirmed by immunocytochemistry. We conclude that, at least as far as the prenatal period is concerned, the normal development of those three organs in fetuses lacking connexin43 cannot simply be explained by the additional presence of connexin32 and vice-versa. Either gap junctional coupling is dispensable in embryonic and fetal cells in which these two connexins are coexpressed, or coupling is provided by yet another connexin when both are absent.

Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach.

Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.

Antisense oligonucleotide blockade of connexin expression during embryonic bone formation: evidence of functional compensation within a multigene family.

Prior studies in our laboratory demonstrated the presence of gap junction proteins (connexins) throughout intramembranous bone formation [Minkoff et al. (1994) Anat Embryol 190:231-241]. In addition, two members of the connexin family of gap junction proteins, connexin 43 (Cx43; Gj alpha 1) and connexin 45 (Cx45; Gj alpha 6), were found by Civitelli et al. [1993; J Clin Invest 91:1888-1896] to be associated, specifically, with osteogenesis. Recently, however, a null mutation in the gene encoding Gj alpha 1 in mice has been produced by Reaume et al. [1995; Science 267:1831-1834]. Gj alpha 1 null homozygotes survived to term but died at birth of heart abnormalities. Examination of the null homozygous embryos, surprisingly, did not reveal overt histological or anatomical abnormalities in any organ system other than the heart. In view of this, the present investigation was initiated in order to evaluate bone formation under conditions in which the expression of Gj alpha 1 and Gj alpha 6, the connexins specifically associated with osteogenesis, had been perturbed, individually as well as in combination. An in vitro system employing organ cultures of dissociated embryonic chick mandibular mesenchyme was employed. Mesenchyme was cultured in the presence and absence of sense and antisense oligodeoxynucleotides (ODN), ranging in length from 15 to 24 mer and containing sequences that included the initiation codon of Gj alpha 1 and of Gj alpha 6. In cultures of mesenchyme, grown for 6 to 13 days in the presence of the combined antisense ODNs to Gj alpha 1 and Gj alpha 6, bone formation was markedly reduced or absent. By contrast, in cultures grown in medium containing the combination of corresponding sense ODNs to both Gj alpha 1 and Gj alpha 6, bone formation was evident. In addition, when cultures were grown in the presence of antisense or sense ODNs to either Gj alpha 1 or Gj alpha 6, individually, bone formation was seen. Immunohistochemical analysis of connexin expression revealed intense immunoreactive signal to Gj alpha 1 and Gj alpha 6 in bone of the control explants, in which no ODNs were present; in those cultures in which either Gj alpha 1 and Gj alpha 6 antisense ODNs were present, however, the expression of the respective connexin protein was either significantly reduced or absent. Further, in those explants in which Gj alpha 1 expression was blocked, immunoreactive signal to Gj alpha 6 appeared to have been amplified in regions of developing bone. These results suggest that, in avian osteogenic tissue, when Gj alpha 1 protein expression has been impeded another related connexin protein (Gj alpha 6) may subserve the functions of the missing connexin. The findings of this study, therefore, support the hypothesis that, within the connexin gene family, functional compensation can occur.

Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers.

Because cell shape and alignment, cell-matrix adhesion, and cell-cell contact can all affect growth, and because mechanical strains in vivo are multiaxial and anisotropic, we developed an in vitro system for engineering aligned, rod-shaped, neonatal cardiac myocyte cultures. Photolithographic and microfluidic techniques were used to micropattern extracellular matrices in parallel lines on deformable silicone elastomers. Confluent, elongated, aligned myocytes were produced by varying the micropattern line width and collagen density. An elliptical cell stretcher applied 2:1 anisotropic strain statically to the elastic substrate, with the axis of greatest stretch (10%) either parallel or transverse to the myofibrils. After 24 h, the principal strain parallel to myocytes did not significantly alter myofibril accumulation or expression of atrial natriuretic factor (ANF), connexin-43 (Cx-43), or N-cadherin (by indirect immunofluorescent antibody labeling and immunoblotting) compared with unstretched controls. In contrast, 10% transverse principal strain resulted in continuous staining of actin filaments (rhodamine phalloidin); increased immunofluorescent labeling of ANF, Cx-43, and N-cadherin; and upregulation of protein signal intensity by western blotting. By using microfabrication and microfluidics to control cell shape and alignment on an elastic substrate, we found greater effects for transverse than for longitudinal stretch in regulating sarcomere organization, hypertrophy, and cell-to-cell junctions.