Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.     

Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

Background  

Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour.     

Changes in Connexin43 Expression and Localization During Pancreatic Cancer Progression

Gap junctions and gap junction communication have long been recognized to play roles in tissue organization and remodeling through both cell autonomous and intercellular means. We hypothesized that these processes become dysregulated during pancreas cancer progression. Molecular and histological characterization of the gap junction protein, connexin43, during progression of pancreatic ductal adenocarcinoma could yield insight into how these events may contribute to or be modulated during carcinogenesis. In a mouse model of pancreatic ductal adenocarcinoma generated through targeted endogenous expression of Kras(G12D) in the murine pancreas, we examined the evolving expression and localization of connexin43. Overall, connexin43 expression increased over time, and its localization became more widespread. At early stages, connexin43 is found almost exclusively in association with the basolateral membrane of duct cells found in invasive lesions. Connexin43 became increasingly associated with the surrounding stroma over time. Connexin43 phosphorylation was also altered during tumorigenesis, as assessed by migrational changes of the protein in immunoblots. These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma.     

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.     

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs.

Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process.     

pH-dependent dimerization of the carboxyl terminal domain of Cx43

Previous studies have demonstrated that the carboxyl terminus of the gap junction protein Cx43 (Cx43CT) can act as an independent, regulatory domain that modulates intercellular communication in response to appropriate chemical stimuli. Here, we have used NMR, chemical cross-linking, and analytical ultracentrifugation to further characterize the biochemical and biophysical properties of the Connexin43 carboxyl terminal domain (S255-I382). NMR-diffusion experiments at pH 5.8 suggested that the Connexin43 carboxyl terminus (CX43CT) may have a molecular weight greater than that of a monomer. Sedimentation equilibrium and cross-linking data demonstrated a predominantly dimeric state for the Cx43CT at pH 5.8 and 6.5, with limited dimer formation at a more neutral pH. NMR-filtered nuclear Overhauser effect studies confirmed these observations and identified specific areas of parallel orientation within Cx43CT, likely corresponding to dimerization domains. These regions included a portion of the SH3 binding domain, as well as two fragments previously found to organize in alpha-helical structures. Together, these data show that acidification causes Cx43CT dimer formation in vitro. Whether dimer formation is an important structural component of the regulation of Connexin43 channels remains to be determined. Dimerization may alter the affinity of Cx43CT regions for specific molecular partners, thus modifying the regulation of gap junction channels.     

Characterizing the mode of action of extracellular Connexin43 channel blocking mimetic peptides in an in vitro ischemia injury model

BackgroundNon-selective Connexin43 hemichannels contribute to secondary lesion spread. The hemichannel blocking peptidomimetic Peptide5, derived from the second extracellular loop of the human Connexin43 protein, prevents lesion spread and reduces vascular permeability in preclinical models of central nervous system injury. The molecular mode of action of Peptide5, however, was unknown and is described here.MethodsHuman cerebral microvascular endothelial cells and APRE-19 cells were used. Scrape loading was used to assess gap junction function and hypoxic, acidic ion-shifted Ringer solution induced ATP release used to assess hemichannel function. Peptide modifications, including amino acid substitutions and truncations, and competition assays were used to demonstrate Peptide5 functional specificity and site of action respectively.ResultsPeptide5 inhibits Connexin43 hemichannel-mediated ATP release by acting on extracellular loop two of Connexin43, adjacent to its matching sequence within the protein. Precise sequence specificity is important for hemichannel block, but less so for uncoupling of gap junction channels (seen only at high concentrations). The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels. Both the SRPTEKT motif and Peptide5 reduce gap junction communication, but neither uncoupling below 50%.ConclusionsReduced gap junction coupling at high peptide concentrations appears to be relatively non-specific. However, Peptide5 at low concentrations acts upon extracellular loop two of Connexin43 to block hemichannels in a precise, sequence specific manner.General significanceThe concentration dependent and sequence specific action of Peptide5 supports its development for the treatment of retinal injury and chronic disease, as well as other central nervous system injury and disease conditions.     

Changes in uterine oxytocin receptor concentrations throughout the estrous cycle of the cow

The amount of oxytocin (OT) bound by plasma membrane fractions from the bovine myometrium and endometrium on different days of the estrous cycle was determined. In the myometrium, OT was bound to a single class of independent sites with an apparent KD (+/- SE) of 1.2 +/- 0.16 nM (n = 34). There were no differences in the apparent KD for different days of the cycle. The amount of OT bound per mg of membrane protein, however, was greatest near the time of estrus and the least in the luteal phase. Nearly ten times more OT was bound to myometrial membranes on Day 21 than on Day 7 (p less than 0.05). OT was bound to endometrial plasma membranes with KD values ranging from 1.3 to 25.7 nM. The mean apparent KD (+/- SE) with 11 samples was 14.9 +/- 3.2 nM. No consistent differences in KD values were detected among days of the cycle. Because of the large variation in the apparent KD values, we were unable to determine if there were differences in the amount of OT bound to endometrial membranes at different stages of the cycle. The correspondence between myometrial OT receptor concentrations and sensitivity to OT during the estrous cycle suggests that the myometrial response to OT in vivo is regulated at the receptor level as well as or rather than by circulating OT concentrations.     

Acute connexin43 temporal and spatial expression in response to ischemic stroke

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.     

Temporal regulation of connexin phosphorylation in embryonic and adult tissues

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in tissues and are important in development, tissue/cellular homeostasis, and carcinogenesis. Genome databases indicate that there are at least 20 connexins in the mouse and human. Connexin phosphorylation has been implicated in connexin assembly into gap junctions, gap junction turnover, and cell signaling events that occur in response to tumor promoters and oncogenes. Connexin43 (Cx43), the most widely expressed and abundant gap junction protein, can be phosphorylated at several different serine and tyrosine residues. Here, we focus on the dynamic regulation of Cx43 phosphorylation in tissue and how these regulatory events are affected during development, wound healing, and carcinogenesis. The activation of several kinases, including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein kinase, and pp60src kinase, can lead to the phosphorylation of different residues in the C-terminal region of Cx43. The use of antibodies specific for phosphorylation at defined residues has allowed the examination of specific phosphorylation events both in tissue culture and in vivo. These new antibody tools and those under development will allow us to correlate specific phosphorylation events with changes in connexin function.     

Distribution pattern of connexin 43, a gap junctional protein, during the differentiation of mouse heart myocytes

In the cardiac muscle, the electrical coupling of myocytes by means of gap (or communicating) junctions, allows the action potentials to be propagated. Connexin 43 (CX 43) is the major constitutive protein of the gap junctions in the mammalian myocardium. In this organ, the abundance of CX 43 and of its messenger, as well as the spatial expression of this protein, are developmentally regulated. These findings are complemented by the results presented in this article, which deals with the distribution of CX 43 in the ventricular myocytes of mouse heart during differentiation, between the 11 days post coitum embryo stage and adulthood. By immunoelectron microscopy experiments on ultrathin sections of cardiac ventricular tissue of one-week-old mouse, we have provided confirmation that the anti-CX 43 antibodies used here specifically recognized the gap junctions. Double labeling immunofluorescence experiments have been undertaken to localize, within the same cells, either CX 43 and desmin, or CX 43 and Con A or WGA receptor sites. From the earliest stage investigated (11 days post coitum) onwards, expression of CX 43 is always associated with desmin-positive cells, that is, with the myocytes. Up to birth, there is in the ventricular wall a gradient of expression of CX 43 which is superimposable on a gradient of expression of desmin. Immunoreactivity to anti-CX 43 and anti-desmin antibodies is high in the sub-endocardial trabeculae and low (or even undetectable for CX 43, in the early stages) in the sub-epicardial cell layers. In the embryonic stages, the expression sites of CX 43 are visible in the form of small dots, whose abundance increases as development proceeds. During these stages, the immunoreactive sites are distributed in a relatively homogeneous pattern throughout the membrane of the myocytes. One week after birth, the CX 43 expression is restricted to the two ends of the myocytes (where the intercalated discs develop), and the adjacent lateral regions. This polarization of CX 43 is more pronounced at the two and three weeks post natal stages and in the fully differentiated ventricular myocytes (adult stage) CX 43 is only present in the intercalated discs.     

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system – chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) – in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

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. Dev. Genet. 21:187–200, 1997. © 1997 Wiley-Liss, Inc.     

miR-125b Inhibits Connexin43 and Promotes Glioma Growth

MicroRNA is strongly associated with tumor growth and development. This study examined the potential roles of miR-125b in glioma growth. We found that miR-125b promotes glioma cell line growth and clone formation, and protects the glioma cells from apoptosis in vitro. The miR-125b-transfected glioma cells also demonstrated increased growth after in vivo transplantation. We further identified that miR-125b inhibits Connexin43 expression, and the overexpression of Connexin43 antagonizes the effects of miR-125b in cell growth and anti-apoptosis. We conclude that miR-125b regulates glioma growth partly through Connexin43 protein.     

过表达的趋化因子CXCL12通过PI3K/Akt途径促进骨髓基质干细胞Connexins的表达

研究过表达的趋化因子CXCL12是否通过PI3K/Akt信号途径促进了骨髓基质干细胞膜上缝隙连接蛋白Connexin40、Connexin43和Connexin45的表达。采用重组DNA技术使骨髓基质干细胞过表达趋化因子CXCL12,采用Western blot法测定趋化因子CXCL12过表达后骨髓基质干细胞Connexin40、Connexin43、Connexin45、Akt、pAkt表达的变化情况,并用CXCL12受体和Akt途径阻断剂明确CXCL12和PI3K/Akt途径在这一过程中的作用。基因重组后骨髓干细胞过表达了趋化因子CXCL12,CXCL12过表达使骨髓基质干细胞膜上Connexin40、Connexin43和Connexin45表达明显增多,且CXCL12的这一作用是通过PI3K/Akt这一途径实现的。趋化因子CXCL12通过PI3K/Akt途径使骨髓基质干细胞膜上Connexin40、Connexin43和Connexin45的表达增加,可促进移植后干细胞与宿主心肌细胞形成有效的电耦合,从而有利于移植后干细胞的存活并发挥治疗作用。