Kanglaite sensitizes colorectal cancer cells to Taxol via NF-κΒ inhibition and connexin 43 upregulation.

Taxol, a first-line anti-tumour drug, has low effectiveness against colorectal cancer. Combination with other agents is an effective strategy to enhance Taxol cytotoxicity. Kanglaite injection is an extract from Coix lacryma-jobi seed and is usually combined with other agents to treat cancer. The aim of this study was to investigate the treatment effect of Taxol combined with Kanglaite on colorectal cancer cell lines. Kanglaite pretreatment followed by Taxol treatment was found to show the best synergism among all combination strategies. This combination also resulted in the smallest tumour volume in a Balb/c mice model. Kanglaite inhibited the expression of nuclear factor (NF)-κΒ and upregulated that of connexin 43, both of which sensitized cancer cells to Taxol. Moreover, Kanglaite increased many cellular variations caused by Taxol, including tubulin polymerization, caspase-3 cleavage, and upregulated expression of survivin and cyclin B1. These results suggest that Kanglaite pretreatment may increase the effect of Taxol on colorectal cancer.

Effects of Electroacupuncture on Interstitial Cells of Cajal (ICC) Ultrastructure and Connexin 43 Protein Expression in the Gastrointestinal Tract of Functional Dyspepsia (FD) Rats.

BACKGROUND Gastrointestinal motility disorder is the main clinical manifestation in functional dyspepsia (FD) patients. Electroacupuncture is effective in improving gastrointestinal motility disorder in FD; however, the underlying mechanism remains unclear. It has been demonstrated that interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract, and the pacemaker potential is transmitted to nearby cells through gap junctions between ICC or ICC and the smooth muscle. Therefore, this study aimed to assess the effects of electroacupuncture on ICC ultrastructure and expression of the gap junction protein connexin 43 (Cx43) in FD rats. MATERIAL AND METHODS The animals were randomized into 3 groups: control, model, and electroacupuncture. Electroacupuncture was applied at Zusanli (ST36) in the electroacupuncture group daily for 10 days, while no electroacupuncture was applied to model group animals. RESULTS Ultrastructure of ICC recovered normally in gastric antrum and small intestine specimens was improved, with Cx43 expression levels in these tissues significantly increased in the electroacupuncture group compared with the model group. CONCLUSIONS These findings indicated that electroacupuncture is effective in alleviating ICC damage and reduces Cx43 levels in FD rats, and suggest that ICC and Cx43 are involved in electroacupuncture treatment in rats with FD to improve gastrointestinal motility disorders.

Tiaogan Qingxin Granule treatment increases myocardial connexin 43 expression in a rat model of arrhythmia.

Myocardial ischemia-induced arrhythmia, especially ventricular arrhythmia, is the main reason for sudden cardiac death. Therefore, ischemic ventricular arrhythmia-targeted treatments are urgently needed. The mechanism of Tiaogan Qingxin Granule in premature ventricular beat (PVB) treatment was explored in arrhythmic rats pretreated with Tiaogan Qingxin Granule. Sprague-Dawley rats (N = 40) were randomly divided into 4 groups: sham-operated, arrhythmia model, Wenxin Granule, and Tiaogan Qingxin Granule. The ischemic arrhythmia model was established by ligating the left anterior descending coronary artery. The Tiaogan Qingxin Granule group was treated intragastrically for 7 days before surgery. Sham-operated rats underwent thoracotomy without coronary artery ligation. Myocardial infarction rate was measured using the triphenyltetrazolium chloride method and Cx43 expression was quantified by western blotting. Compared to the arrhythmia model group, the Tiaogan Qingxin Granule group showed a significant reduction in the myocardial infarct size and myocardial infarction rate (P < 0.01). Cx43 expression in the left ventricular myocardial tissues was significantly lower in the arrhythmia model group than in the sham-operated group (P < 0.01), but significantly higher in the Tiaogan Qingxin Granule group (P < 0.01). Intergroup difference in the relative Cx43 expression between the Tiaogan Qingxin Granule and Wenxin Granule groups was not significant (P > 0.05). Thus, Tiaogan Qingxin Granule reduced the myocardial infarct size, lowered the myocardial infarction rate, and increased Cx43 expression, possibly by increasing blood supply to the cardiac muscles. In conclusion, Tiaogan Qingxin Granule may be useful for treating ischemic PVB.

Effects of aluminum oxide (Al2O3) nanoparticles on ECG, myocardial inflammatory cytokines, redox state, and connexin 43 and lipid profile in rats: possible cardioprotective effect of gallic acid.

The objectives of present study were to examine the effects of aluminum oxide (Al2O3) nanoparticles on myocardial functions, electrical activities, morphology, inflammation, redox state, and myocardial expression of connexin 43 (Cx43) and the effect of gallic acid (GA) on these effects in a rat animal model. Forty male albino rats were divided into 4 equal groups: the control (normal) group; the Al2O3 group, rats received Al2O3 (30 mg·kg(-1), i.p.) daily for 14 days; the nano-alumina group, rats received nano-alumina (30 mg·kg(-1), i.p.) daily for 14 days; and the nano-alumina + GA group, rats received GA (100 mg·kg(-1) orally once daily) for 14 days before nano-alumina administration. The results showed disturbed ECG variables and significant increases in serum levels of LDH, creatine phosphokinase (CPK), CK-MB, triglycerides (TGs), cholesterol and LDL, nitric oxide (NO), and TNF-α and myocardial concentrations of NO, TNF-α, and malondialdehyde (MDA), with significant decreases in serum HDL and myocardial GSH, SOD, catalase (CAT), and Cx43 expression in the nano-alumina group. Pretreatment with GA improved significantly all parameters except serum and myocardial NO. We concluded that chronic administration of Al2O3 NPs caused myocardial dysfunctions, and pretreatment with GA ameliorates myocardial injury induced by nano-alumina, probably through its hypolipidaemic, anti-inflammatory, and antioxidant effects and upregulation of Cx43 in heart.

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects.

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200 µL/day) for 5 weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.

Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Connexin 43 (Cx43), a major gap junction protein, is necessary for alveologenesis and plays an important role in the differentiation of type II to type I alveolar epithelial cells. Knockout mice of Cx43 display severe pulmonary hypoplasia (PH). Prenatal administration of retinoic acid (RA) is known to stimulate alveologenesis in nitrofen-induced PH. Recent studies revealed that retinoids upregulate Cx43 expression. We hypothesized that gene expression of Cx43 is downregulated during alveologenesis and that administration of RA upregulates Cx43 expression in the nitrofen-induced PH. METHODS: Pregnant rats were exposed to olive oil or nitrofen on day 9 (D9) of gestation. Retinoic acid was given intraperitoneally on D18, D19, and D20. Fetal lungs were harvested on D18 and D21 and divided into control, nitrofen, control+RA (D21), and nitrofen+RA (D21). The Cx43 expression levels were determined using reverse transcription polymerase chain reaction and immunohistochemistry. RESULTS: On D18 and D21, Cx43 relative messenger RNA expression levels were significantly downregulated in nitrofen compared with those in the control group. On D21, expression levels of Cx43 were significantly upregulated in nitrofen+RA and control+RA compared with those in nitrofen group. Immunohistochemical studies confirmed these results. CONCLUSION: Downregulation of Cx43 expression may interfere with normal alveologenesis. Upregulation of Cx43 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in nitrofen-induced PH.

Omega-3 fatty acids and atorvastatin affect connexin 43 expression in the aorta of hereditary hypertriglyceridemic rats.

Statins and omega-3 polyunsaturated fatty acids (n-3 PUFA) reduce cardiovascular disease incidence during hypertriglyceridemia (HTG). To elucidate possible cardioprotective mechanisms, we focused on gap junction protein connexin 43 (Cx43). Its expression is disturbed during atherogenesis, but little information is available on its expression during HTG. Experiments were performed on adult male hereditary HTG (hHTG) rats treated with n-3 PUFA (30 mg/day) and atorvastatin (0.5 mg/100 g body weight per day) for 2 months. Cx43 expression and distribution in the aorta were investigated by using Western blotting and immunolabeling, followed by quantitative analysis. Transmission electronmicroscopy was used to study ultrastructure of endothelial contact sites. In contrast to age-matched Wistar, Cx43 expression in aorta of hHTG rats was significantly higher (p < 0.05), and prominent Cx43 immunospots were seen in tunica media and less in endothelium of hHTG rats. Changes in Cx43 expression were accompanied by local qualitative subcellular alterations of interendothelial connections. Treatment of hHTG rats with n-3 PUFA and atorvastatin markedly lowered Cx43 expression in aorta and modified connexin distribution in endothelium and media (p < 0.05 vs. untreated hHTG). The protective effect of treatment of HTG was observed on the structural integrity of the endothelium and was readily visible at the molecular level. Results indicate the involvement of altered Cx43 expression in vascular pathophysiology during HTG and during HTG treatment.

Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos.

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

[Effect of Daming capsule on expression of connexin43 isoforms in hyperlipemic rat's cardiac muscle].

OBJECTIVE: To find the molecular mechanism of decreasing blood fat effect of Darning capsule on hyperlipemic rat, we study the expression of connexin43 in the myocardium before and after using the capsule. METHOD: Forty Wistar rats were randomly divided into 5 group: control group, hyperlipemia model group, Daming capsule group of high dose, middle dose and low dose (200, 100, 50 mg kg(-1) d(-1)). Each group had 8 rats. Hyperlipemic rat model was made firstly, the blood was obtained via vena caudalis and the indexes of TC, TG, LDL, HDL and NEFA in the serum were measured. The myocardial total RNA was extracted by Trizol method. To compare the expression of connexin43 in the following groups: hyperlipemia, normal and drug, we used the technique of RT-PCR, immunostaining and microconfoul. RESULT: The concentrations of TC, TG, LDL and NEFA in hyperlipemic serum were increased (P <0. 05), while that of HDL was decreased (P <0. 05). After treating with Daming capsule, the concentration of the preceding four indexes were decreased and the concentrations of HDL was increased up to nearly normal level. No significant difference was found in the ECG of the three groups. As compared with the normal group, the mRNA expressions of connexin43 in hyperlipemia group was weakened (P <0.05), while that of the drug group was enhanced(P <0.05). The same result in immunostaining was observed. CONCLUSION: Hyperlipemic rat model was successfully established and Daming capsule has the effect of lowering blood lipid. Furthermore, the molecular mechanism of Darning capsule is related with the change of Cx43 closely.

Tellimagrandin I enhances gap junctional communication and attenuates the tumor phenotype of human cervical carcinoma HeLa cells in vitro.

Tellimagrandin I and chebulinic acid, two hydrolysable tannins, have been shown to exert anti-tumor properties. Dysfunctional gap junctional communication (GJIC) has been recognized as being involved in carcinogenesis. The human cervical carcinoma HeLa cells have been reported to be deficient in functional GJIC. In present study, we investigated whether tellimagrandin I and chebulinic acid might restore functional GJIC in HeLa cells. Both compounds could inhibit the growth of HeLa cells. Either Lucifer yellow transfer assay or calcein transfer assay demonstrated that tellimagrandin I improved GJIC in HeLa cells while chebulinic acid showed no effect on GJIC. The GJIC enhancement by tellimagrandin I occurred along with an increase of Cx43 gene expression at mRNA and protein levels. Exposure to tellimagrandin I also led to inhibition of proliferation and anchorage-independent growth of HeLa cells. In addition, tellimagrandin I decreased the percentage of cells in the G0/G1 and G2/M phases coinciding with an increase in the percentage of cells in the S phase. The accumulation of cells in S phase was coupled with a decreased expression of cyclin A that was critical to the progression of S phase. These results suggested that restoring GJIC might be one explanation for tellimagrandin I antitumor effects, whereas chebulinic acid exerted antitumor action through other pathways.

Expression of connexin43 in rat epithelial cells and fibroblasts.

To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy. And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EA group, n = 10). EA ( 0.5-1. 5 V, 4-16 Hz , 30 min) was applied to "Zusanli" acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected. The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43 was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43 on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91% and 29.53% respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

Expression of connexin43 in rat epithelial cells and fibroblasts / 华中科技大学学报（医学）（英德文版）

To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy. And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EA group, n = 10). EA ( 0.5-1. 5 V, 4-16 Hz , 30 min) was applied to "Zusanli" acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected. The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43 was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43 on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91% and 29.53% respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis.

The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

The preventive effect of green tea on the gap junction intercellular communication in renal epithelial cells treated with a renal carcinogen.

OBJECTIVE: Clinical studies imply that (-)-epigallocatechin-3-gallate (EGCG), a main ingredient of green tea catechins, has a chemopreventive action against cancers and suppresses the proliferation of cancer cells. However, there is no report about its chemopreventive effect for renal cancer. We previously determined that renal carcinogens suppressed the gap junction intercellular communication (GJIC) of renal epithelial cells. In this study, we investigated the effect of EGCG on the GJIC of renal epithelial cells treated with a renal carcinogen. MATERIALS AND METHODS: Mardin-Darby canine kidney (MDCK) cells were used to determine the protective effects of EGCG on dimethylnitrosamine-induced alteration of GJIC and connexin 43 (Cx 43). The maximum concentration of EGCG was determined by the lactate dehydrogenase assay method. The scrape-loading dye transfer method was used to assess the expression and cellular localization of Cx 43. The phosphorylation status of Cx 43 was determined by Western blot analysis. RESULTS: The optimal noncytotoxic concentration of EGCG was determined to be 10 microg/ml. The levels of GJIC and Cx 43 expression were markedly decreased in MDCK cells exposed to dimethylnitrosamine. A 12-h pretreatment with EGCG greatly ameliorated the GJIC-inhibitory effects of dimethylnitrosamine. CONCLUSION: These results suggest that the preservation of GJIC may indicate the chemopreventive effect of green tea on renal epithelial cells treated with a renal carcinogen in vitro.

Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses.

To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of alpha-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of alpha-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473-478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by approximately 4 h after irradiation. The induction of cx43 was observed by mean alpha-particle doses as low as 0.16 cGy, and also in cells exposed to gamma-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses.

Growth hormone-related effects on apoptosis, mitosis, and expression of connexin 43 in bovine in vitro maturation cumulus-oocyte complexes.

Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. Therefore, the aim of our study was to elucidate the mechanisms of GH action during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). As shown by detection of the nuclear cell proliferation-associated antigen Ki-67, COCs matured in vitro in the presence of GH revealed a significantly (P < 0.05) higher proportion of proliferating cumulus cells (12.6%) compared with the COCs matured in the control medium TCM 199 (9.9%). In contrast, the percentage of proliferating cells was not increased by supplementation of the medium with a combination of GH and insulin-like-growth factor I (IGF-I). Apoptosis as determined by TUNEL (terminal doxynucleotidyl transferase mediated dUTP nick-end labeling) was significantly (P < 0.05) reduced in the cumulus cells by GH treatment. COCs matured with a combination of GH and IGF-I revealed the lowest percentage of apoptotic cells (11%). The localization and quantification of the gap junction protein connexin 43 (Cx 43) demonstrated that GH induced a significant decrease in the synthesis of the Cx 43 protein in the cumulus cells. Our results imply that GH increases cumulus expansion by promotion of cell proliferation and inhibition of apoptosis. Whereas the increase in cell proliferation is a direct effect of GH, the antiapoptotic effects of GH during in vitro maturation are modulated by IGF-I. Stimulatory effects of GH on oocyte maturation are correlated with changes in the synthesis of gap junction proteins.

Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(2+) changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca(2+) signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca(2+) signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.

Inhibiting gap junctional intercellular communication alters expression of differentiation markers in osteoblastic cells.

Gap junctional intercellular communication (GJIC) may contribute to cellular differentiation. To examine this possibility in bone cells we examined markers of cellular differentiation, including alkaline phosphatase, osteocalcin, and osteopontin, in ROS17/2.8 cells (ROS), a rat osteoblastic cell line expressing phenotypic characteristics of fully differentiated osteoblasts. We utilized ROS rendered communication deficient either by stable transfection with antisense cDNA to connexin 43 (Cx43), the predominant gap junction protein in bone (RCx16 cells), or by overexpression of Cx45, a gap junction protein not normally expressed in ROS (ROS/Cx45 cells). Both RCx16 and ROS/Cx45 cells displayed reduced dye coupling and Cx43 protein expression relative to ROS, control transfectants, and ROS/Cx45tr, ROS cells expressing carboxylterminal truncated Cx45. Steady-state mRNA levels for osteocalcin as well as alkaline phosphatase activity, two markers of osteoblastic differentiation, were also reduced in poorly coupled RCx16 and ROS/Cx45 cells. On the other hand, steady-state mRNA levels for osteopontin increased slightly in RCx16 and ROS/Cx45 cells. These results suggest that GJIC at least partly contributes to the regulation of expression of markers of osteoblastic differentiation.

The expression of the gap junctional protein Cx43 is restricted to proliferating and non differentiated normal and transformed keratinocytes.

The passage of specific growth modulating signals through gap junctions may regulate the proliferation and differentiation of human keratinocytes. To investigate this, we correlated the proliferation of normal human keratinocytes and a transformed squamous cell carcinoma cell line, SCC4, with the expression of the gap junctional proteins Cx43, 31 and 31.1, known to be expressed by keratinocytes. Proliferation was confined to preconfluent and confluent cultures of normal keratinocytes, falling to undetectable levels once postconfluency was achieved. Cx43, at both the message and protein levels, paralleled these changes, being elevated predominantly in preconfluent and confluent cultures, and downregulated in postconfluency. Similar results were found for Cx31 and 31.1 at the message level. In contrast, the proliferation of SCC4 cells cultured in media supplemented with 5.0% FCS was maintained at a substantial level from preconfluency through 2 weeks postconfluency. Cx43, 31, and 31.1 RNA and Cx43 protein expression mirrored the levels of proliferation within SCC4 cultures. Cx26 and 32 were not found in normal keratinocytes or SCC4 cells at any stage of differentiation. These data, illustrating a tight correlation between proliferation and Cx43, 31 and 31.1 expression, suggest that these connexins may represent proliferation-specific gap junctions within keratinocytes, and may therefore transmit signals that control keratinocyte division.