Effects of race, sex, and socioeconomic status upon cardiovascular stress responsivity and recovery in youth

In rat brain, expression of the gap junction protein connexin30 increased during the first 3 weeks after birth and reached its maximum after 4 weeks, as shown by analysis with specific connexin30 antibodies. This contrasts with the prenatal onset of connexin43 expression. On cryosections of rat brain, connexin30 immunoreactivity was found near blood vessels and in ependymal as well as in leptomeningeal cells. Expression in the neuropil was first noticed 3 weeks after birth, showing the same spatial pattern of immunoreactivity as connexin43. This late onset of connexin30 expression in astrocytes was also seen in long-term glial cell cultures, where connexin30 was coexpressed with the astrocytic marker proteins S-100beta and glial fibrillary acid protein. In acute brain slices, connexin30 immunofluorescent signals were detected on processes of functionally identified astrocytes. Thus, our results show that connexin30 is expressed in three different cell types of the rodent brain. The late onset of connexin30 expression in astrocytes suggests that this gap junctional protein fulfills a role in intercellular communication among mature astrocytes.     

Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells

Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.     

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (beta-TPA). Within 10 min, beta-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens.     

Rapid turnover of connexin43 in the adult rat heart

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.     

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

Expression of gap junctions in cultured rat L6 cells during myogenesis

Although gap junctions are absent from adult skeletal muscle, they have been described in embryonic and neonatal rat skeletal muscle and in cultured rat myoblasts. In order to determine the precise developmental expression and molecular composition of gap junctions during myogenesis, RNA was isolated from cultures of rat L6 myoblasts and examined using Northern blot analysis with cDNA probes specific for connexin32 and connexin43. Connexin32 mRNA could not be detected in rat myoblast and myotube samples. However, connexin43 mRNA was expressed at high levels in cycling L6 myoblasts and this expression decreased by approximately 60% in L6 myotubes following fusion. Immunofluorescent localization with an antibody specific for connexin43 confirmed the accumulation of connexin43 protein in membranes shared between adjacent myoblasts at 12 hr of culture. By 24 hr of culture, connexin43 disappeared from most cells, only to reappear at 36 hr at a low level that was maintained through 72 hr in culture. Although most myoblasts in these cultures expressed connexin43, myotubes expressed little or no membrane-associated connexin43. Dye transfer experiments established that, at 12 hr of culture, the majority of myoblasts were dye coupled suggesting that connexin43 protein is assembled into functional gap junctions. At 24 hr, the number of coupled cells decreased slightly, while at 48 hr, most of the myoblasts were not dye coupled. These results demonstrate that the expression of connexin43 is temporally correlated with myoblast fusion and may play a role in this process.     

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Gap junctional intercellular communication contributes to the contraction of rat osteoblast populated collagen lattices

The contraction of native collagen lattices by resident mesenchymal cells mimics the organization of collagen during development and repair. Lattice contraction is cell density dependent, suggesting that cell-to-cell communications may contribute to the process. This possibility was investigated by comparing lattice contraction by four rat osteoblastic cell lines: ROS 17/2.8 cells (ROS); ROS transfected with an antisense cDNA sequence of the gap junctional protein connexin 43 (RCx16); ROS transfected with connexin 45 cDNA, a connexin not normally expressed in ROS cells (ROS/Cx45); and ROS transfected with cDNA encoding carboxy-terminal truncated Cx45 (ROS/Cx45tr). The cell coupling indices, which reflect gap junctional communication, were quantitated by the fluorescent dye scrape loading. ROS cells were well coupled (index 3.0), ROS/Cx45tr were better coupled (index 4.2), ROS/Cx45 were poorly coupled (index 1.7), and RCx16 showed no coupling (index 1.1). As determined by immunoblotting, the level of connexin 43 protein was increased in both ROS/Cx45tr and ROS/Cx45 cell lines compared with ROS cells, while the level in RCx16 cells was reduced. ROS populated collagen lattices (PCLs) contracted significantly more at day 5 (177 mm2 to 67 mm2) than ROS/Cx45tr (84 mm2), ROS/Cx45 (108 mm2), or RCx16 (114 mm2). Myosin ATPase activity, which is required for lattice contraction, was equivalent in all four cell lines, indicating that it was not responsible for inhibiting PCL contraction. ROS cells in collagen appeared elongated compared with the other cell lines which were more rounded. These experiments suggest gap junctional communication contributes to PCL contraction by resident osteoblasts.     

Role of gap junctions in lung neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.     

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.     

Different changes in expression and function of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system

In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA synthesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) with 2% dimethylsulfoxide (DMSO) and 10(-7) mol/L glucagon (a DMSO culture system), as we previously reported. In the present cultures, almost confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10(-7) mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10(-7) mol/L glucagon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO and 10(-7) mol/L glucagon. During stimulation of DNA synthesis, both Cx26 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearance of both Cx26 and Cx32 immunoreactivities and gap junction plaques were observed. Gap junctional intercellular communication (GJIC), as measured by lucifer yellow, which indicated the function of Cx32, decreased markedly from before the onset of the S phase. GJIC measured by propidium iodide, which indicated the function of Cx26, decreased from before the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis, Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment control level. Cx32 immunoreactivity and gap junction plaques also recovered. However, the recovery of GJIC measured by lucifer yellow was later than that of Cx32 expression. These results indicated the different changes of expression and function of Cx26 and Cx32 in the hepatocytes during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap junctions during hepatocyte growth and differentiation in vitro.     

Connexin43 is another gap junction protein in the peripheral nervous system

That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 microM forskolin, whereas that of Po increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although Po expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with Po, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.     

Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia

To examine the possible role of gap junctions in mouse skin tumor progression, we generated a panel of mouse skin tissue samples exhibiting normal, hyperplastic, or neoplastic changes and characterized the expression of the gap-junction genes connexin 43 (Cx43) and connexin 26 (Cx26) by in situ hybridization and immunohistochemical analyses. In normal skin, these two gap junction genes were differentially expressed; Cx43 was found predominantly in the less differentiated lower spinous layers, whereas Cx26 was found in terminally differentiating upper spinous and granular layers. In hyperplastic epidermis exhibiting an expansion of the differentiated upper layer, i.e., epidermis with a thickened granular layer or in which the granular layer was replaced with keratinocytes exhibiting tricholemmal differentiation, expression of Cx43 and Cx26 remained segregated in the lower and upper spinous layers, respectively. However, in papillomas, Cx26 was localized in the lower but not upper spinous layer, an expression pattern identical to that of Cx43. In addition, the overall expression levels of both Cx43 and Cx26 appeared to be greatly elevated in the papillomas. It is interesting that such marked alteration in the pattern of Cx26 expression occurred within the context of hyperplastic changes histologically identical to those seen in the nonpapillomous hyperplasias. Interestingly, in neoplastic skin lesions containing a squamous cell carcinoma, Cx43 and Cx26 expression was extinguished. Moreover, expression of Cx43 was also significantly reduced in adjacent apparently nonneoplastic tissues. Overall, these observations show that perturbations in gap-junction gene expression are associated with skin hyperplasia and neoplasia. Such findings suggest a possible role for gap junctions in the malignant conversion of mouse epidermal cells.