Overexpression Of Cx43 Research Articles

Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

Regulation of connexin43 (Cx43) expression affects cell proliferation, differentiation and apoptosis in a gap junctional intercellular communication (GJIC)-independent manner. However, the underlying mechanisms of Cx43-mediated cell cycle suppression are still poorly understood. To elucidate the molecular mechanism of Cx43-mediated cell cycle suppression, we searched for Cx43 interacting proteins by using a proteomics approach. Here, we have identified a Cx43-interacting protein, heat shock cognate protein 70 (Hsc70). We confirmed that Hsc70 directly binds to the C-terminus of Cx43, whereas Hsc54, a splice variant of Hsc70, does not, that Cx43 competes with cyclin D1 for binding to Hsc70, and that the nuclear accumulation of cyclin D1 is reduced by overexpression of Cx43 in a GJIC-independent manner, which is restored by co-overexpression with Hsc70. As a result, the cell proliferation is regulated by Cx43. Our results suggest that Cx43-Hsc70 interaction probably plays a critical role during G1/S progression.

Role of asymmetric methylarginine and connexin 43 in the regulation of pulmonary endothelial function.

Abstract Circulating levels of asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, are increased in patients with idiopathic pulmonary hypertension (IPAH). We hypothesized that ADMA abrogates gap junctional communication, required for the coordinated regulation of endothelial barrier function and angiogenesis, and so contributes to pulmonary endothelial dysfunction. The effects of ADMA on expression and function of gap junctional proteins were studied in human pulmonary artery endothelial cells; pulmonary endothelial microvascular cells from mice deficient in an enzyme metabolizing ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI); and blood-derived endothelial-like cells from patients with IPAH. Exogenous and endogenous ADMA inhibited protein expression and membrane localization of connexin 43 (Cx43) in a nitric oxide/soluble guanosine monophosphate/c-jun-dependent manner in pulmonary endothelial cells, resulting in the inhibition of gap junctional communication, increased permeability, and decreased angiogenesis. The effects of ADMA were prevented by overexpression of DDAHI or Cx43 and by treatment with rotigaptide. Blood-derived endothelial-like cells from IPAH patients displayed a distinct disease-related phenotype compared to cells from healthy controls, characterized by reduced DDAHI expression, increased ADMA production, and abnormal angiogenesis. In summary, we show that ADMA induces pulmonary endothelial dysfunction via changes in expression and activity of Cx43. Cells from IPAH patients exhibit abnormal DDAHI/Cx43 signaling as well as differences in gap junctional communication, barrier function, and angiogenesis. Strategies that promote DDAHI/Cx43 signaling may have an endothelium-protective effect and be beneficial in pulmonary vascular disease.

Connexin43 mediates NF-κB signalling activation induced by high glucose in GMCs: involvement of c-Src

BackgroundNuclear factor kappa-B (NF-κB) signalling plays an important role in diabetic nephropathy. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. The aim of the current study was to investigate the role of Cx43 in the activation of NF-κB induced by high glucose in glomerular mesangial cells (GMCs) and to determine whether c-Src is involved in this process.ResultsWe found that downregulation of Cx43 expression induced by high glucose activated NF-κB in GMCs. Orverexpression of Cx43 attenuated NF-κB p65 nuclear translocation induced by high glucose. High glucose inhibited the interaction between Cx43 and c-Src, and enhanced the interaction between c-Src and IκB-α. PP2, a c-Src inhibitor, also inhibited the tyrosine phosphorylation of IκB-α and NF-κB p65 nuclear translocation induced by high glucose. Furthermore, overexpression of Cx43 or inhibition of c-Src attenuated the upregulation of intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN) expression induced by high glucose.ConclusionsIn conclusion, downregulation of Cx43 in GMCs induced by high glucose activates c-Src, which in turn promotes interaction between c-Src and IκB-α and contributes to NF-κB activation in GMCs, leading to renal inflammation.

Connexin 43 is involved in the generation of human-induced pluripotent stem cells

Although somatic cells can be successfully programmed to create pluripotent stem cells by ectopically expressing defined transcriptional factors, reprogramming efficiency is low and the reprogramming mechanism remains unclear. Previous reports have shown that almost all human connexin (CX) isoforms are expressed by human embryonic stem (hES) cells and that gap junctional intercellular communication (GJIC) is important for ES cell survival and differentiation. However, the CX expression profiles in human induced pluripotent stem (iPS) cells and the role of CXs in the process of reprogramming back to iPS cells remains unknown. Here, we determined the expression levels of most forms of CX in human embryonic fibroblasts (hEFs) and in the hEF-derived iPS cells. A scrape loading/dye transfer assay showed that human iPS cells contained functional gap junctions (GJs) that could be affected by pharmacological inhibitors of GJ function. We found that CX43 was the most dramatically upregulated CX following reprogramming. Most importantly, the ectopic expression of CX43 significantly enhanced the reprogramming efficiency, whereas shRNA-mediated knockdown of endogenous CX43 expression greatly reduced the efficiency. In addition, we found that CX43 overexpression or knockdown affected the expression of E-CADHERIN, a marker of the mesenchymal-to-epithelial transition (MET), during reprogramming. In conclusion, our data indicate that CX43 expression is important for reprogramming and may mediate the MET that is associated with the acquisition of pluripotency.

The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade

ABSTRACT Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP4) and InsP5, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

Loss of connexin43 expression in Ewing's sarcoma cells favors the development of the primary tumor and the associated bone osteolysis

Ewing's sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS–FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC).Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS–FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption.Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.

Abstract 194: Increasing Expression of Connexin43 in Bone Marrow Stem Cells Enhances Their Protective Effects on the Infarcted Heart

Introduction: Coupling of stem cells (SCs) injected into the infarcted heart to endogenous cells that survived injury may enhance SC survival and engraftment and improve their protective effects. Connexin43 (Cx43) gap junctions are some of the first proteins expressed by differentiating SCs transplanted into the heart after myocardial infarction (MI), suggesting that cell coupling is critical for SC survival and differentiation. Hypothesis: Overexpression of Cx43 in SCs will enhance their ability to engraft, differentiate, and improve cardiac function and survival after transplant into the infarcted heart. Methods: Adult mouse SCs isolated from compact bone were sorted for c-kit surface marker using magnetic beads. SCs were transduced with PLL3.7 lentivirus encoding the Cx43 gene (GJA1) at MOI=300 to produce overexpression, which was confirmed by Western analysis on SC lysates. MI was induced in 12-week-old male C57BL/6 mice by ligating the left anterior descending coronary artery, and 40,000 SCs in 20 uL saline were administered immediately after MI via intramyocardial injection. A total of 69 animals were used: 18 received Cx43 SCs, 30 received non-transduced SCs (NTSC), 11 received only saline, and 10 underwent sham surgery. Echocardiography was performed 1 and 2 weeks post-MI. Results: Animals receiving Cx43 SCs had improved survival (90.0%) versus NTSC (76.7%) and saline controls (70.0%). The Cx43 SC group displayed improved ejection fraction versus NTSC and saline controls at 1 week (48.7% v. 43.3%, p=0.04; 30.1%, p<0.0001) and 2 weeks post-MI (45.7% v. 40.4%, p=0.08; 26.8%, p=0.0004). Similar improvements in fractional shortening, stroke volume, and cardiac output were observed. Administration of Cx43 SC and NTSC attenuated remodeling with no difference between the two groups. Both decreased end-diastolic and systolic volumes, increased thickness of the infarct-related anterior wall, and decreased heart weight (HW), HW/body weight and HW/tibia length ratios at 1 and 2 weeks post-MI. Conclusion: All SCs improved survival and cardiac function, and attenuated remodeling. Transplantation of Cx43 SCs improved survival and cardiac function even more than NTSCs, suggesting that Cx43 may play a crucial role in engraftment and differentiation.

Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

S262A mutation abolishes protective effects of connexin 43 against hypothermic preservation–induced injury in cardiomyocytes

Alleviation of cold preservation-induced injury is a critical part of the heart transplantation process. In this study we investigate the protective effect of connexin 43 (Cx43) overexpression against hypothermic preservation-induced injury in cardiomyocytes. Total RNA was prepared from H9c2 cells using TRIzol reagent to construct a recombinant vector pEGFP-c1-Cx43, which was then transformed into Escherichia coli DH5α competent cells. The S262A Cx43 containing a mutant site was obtained by RT-PCR. The protein expression of total Cx43 and p-S262 Cx43 were assessed by Western blot. Cell viability and LDH release in the culture medium was measured. Restrictive enzyme reaction assay and DNA sequencing confirmed that the recombinant vector pEGFP-c1-Cx43 and pEGFP-c1-S262A-Cx43 were constructed correctly. After H9c2 cells were hypothermically preserved in Celsior solution for 12 to 48 hours, the cell viability decreased and LDH release increased. Compared with empty vector cells, overexpression of Cx43 prevented the hypothermic preservation-induced decrease in cell viability and increase in LDH release, which was independent of the absence of gap junctions. S262A mutation prevented S262 phosphorylation of Cx43 and also abolished protection of Cx43 overexpression against cold preservation-induced cardiomyocyte injury. In H9c2 cells hypothermically preserved for up to 48 hours, overexpression of Cx43 could protect against cold preservation-induced injury. Phosphorylation of Cx43 at S262 may play an essential role in the preservation of donor hearts.

Prevention, Treatment, and Recovery Supports for Those with Substance Use Problems: Opportunities for Enhanced Access and Quality of Care

Substance use disorder is one of our nation's most significant public health problems. If unaddressed, an individual's addiction will negatively impact the family, community and the health care system. Despite major strides in the field of addiction treatment, major barriers still exist preventing the problem to be addressed. We propose some recommendations to help improve access to care.

ERK acts in parallel to PKCδ to mediate the connexin43-dependent potentiation of Runx2 activity by FGF2 in MC3T3 osteoblasts

The gap junction protein, connexin43 (Cx43), plays an important role in skeletal biology. Previously, we have shown that Cx43 can enhance the signaling and transcriptional response to fibroblast growth factor 2 (FGF2) in osteoblasts by increasing protein kinase C-δ (PKCδ) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKCδ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKCδ-independent activation of ERK by FGF2 was confirmed by Western blotting, as was the Cx43-dependent enhancement of ERK activation. Consistent with our prior observations for PKCδ, flow cytometry analyses show that Cx43 overexpression enhances the percentage of phospho-ERK-positive cells in response to FGF2, supporting the notion that shared signals among gap junction-coupled cells result in the enhanced response to FGF2. Western blots and luciferase reporter assays performed on osteoblasts cultured under low-density and high-density conditions revealed that cell-cell contacts are required for Cx43 to amplify ERK activation and gene transcription. Similarly, inhibition of gap junctional communication with the channel blocker 18β-glycyrrhetinic acid attenuates the Cx43-dependent enhancement of Runx2-transcriptional activity. In total, these data underscore the importance of cell-cell communication and activation of the ERK and PKCδ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts.

Abstract P016: Human Mesenchymal Bone Marrow--Derived Stem Cells Require Connexin 43 to Form Beating 3-Dimensional Tubes When Cocultured with Neonatal Rat Cardiomyocytes

Mesenchymal stem cells (MSCs) have begun to manifest themselves as a safe and beneficial therapy for restoring cardiac function in failing hearts. However, the mechanism underlying this function is unclear. The present study was initiated to investigate the role of Connexin 43 (Cx43) in hMSC integration, migration and differentiation in co-culture with neonatal rat cardiomyocyte (NRVMs) in vitro. Cx43 is a gap junction connexin which is required for proper heart development and heart electrophysiology. We generated lentiviral constructs for Cx43 knockdown and Cx43 overexpression and used them to alter the level of Cx43 in hMSCs. The effectiveness of these vectors was confirmed by assessing Cx43 levels in hMSCs by Western Blot analysis and by Real Time-PCR. These hMSCs were co-cultured with NRVMs for up to 1 month on poly-lysine and collagen I coated glass cover slips. Co-cultures containing MSCs overexpressing Cx43 exhibited coordinated beating and three-dimensional tube formation in 10 days, whereas 14 days were required for control cells. The hMSCs were integrated into these beating three dimensional tubes. No tubes were observed in co-cultures with Cx43 knockdown hMSCs, rather we observed the formation of small, unconnected beating spheres after 30 days in co-culture. Human MSC-Cx43 knockdown cells were integrated into these spheres and immunofluorescence staining demonstrated that Cx43 expression in hMSC-Cx43 knockdown remained reduced when compared to the control group. Real Time-PCR analysis using human specific primers showed significant upregulation of KDR, smooth muscle actin, Pecam1, CD34, CDH2, and CaCNA1C. Several genes, not initially seen in hMSCs, including Gata4, CDH5, SCN5A, SLC8A1, and KCNQ1, appeared in co-cultures with hMSCs with normal and elevated expression levels of Cx43. However, in co-culture of cardiomyocytes with Cx43 knockdown MSCs we observed downregulation of KDR, CHD2, SCN5A, SLC8A1, KCNQ1 compare to control. These results suggest a strong correlation between the presence of Cx43 and the ability of hMSCs to differentiate into cardiac and endothelial lineages.

The effect of connexin43 on the level of vascular endothelial growth factor in human retinal pigment epithelial cells

Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions. Stable cell lines of ARPE-19 were produced in which wild-type Cx43 was either over-expressed, down-regulated by targeted shRNA, or functionally inhibited by co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion were assessed by real-time PCR and ELISA respectively. Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing a dominant-negative mutant of Cx43, and when GJIC was blocked. Over-expression of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells. These studies show that Cx43 protects against oxidative stress-induced VEGF secretion in ARPE-19 cells, and thus has important implications in understanding the pathogenesis of age-related macular degeneration.

Connexin-43 Hemichannels Mediate Cyclic ADP-ribose Generation and Its Ca2+-mobilizing Activity by NAD+/Cyclic ADP-ribose Transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.

Hyperthermia induces upregulation of Connexin43 in the golden hamster neural tube

During early embryonic development, maternal exposure to hyperthermia induces neural tube defects (NTDs). Connexins are essential for the formation of gap junctions and Connexin43 (Cx43) is crucially involved in neural tube development. This study was designed to explore the potential role of Cx43 in NTDs induced by hyperthermia. Using PCR, the Cx43 cDNA was screened from the cDNA library of the neural tube from golden hamsters treated with hyperthermia. By Northern blot, the expression of Cx43 in heat-treated and control groups of the golden hamsters at day 8.5 after mating was detected. Finally, by in situ hybridization and RT-PCR, the expression of Cx43 was examined in the neural tube at different time points after heat treatment. Cx43 was stably expressed in heat-treated and control groups of the golden hamsters, whereas the expression was evidently higher in the heat-treated group. Cx43 expression in the neural tube at different time points after heat treatment was significantly higher than in control groups (p < 0.01). Hyperthermia did not induce any mutations in Cx43 cDNA. Our data provide the first evidence that hyperthermia induces upregulation of Cx43 in the golden hamster neural tube. NTDs caused by hyperthermia may be intimately related with the overexpression of Cx43.

Connexin43 interacts with βarrestin: A pre-requisite for osteoblast survival induced by parathyroid hormone

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43-interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.

Enhanced activation of p21-activated kinase 1 in heart failure contributes to dephosphorylation of connexin 43

We previously showed decreased cellular coupling and dephosphorylation of the gap junctional protein connexin 43 (Cx43) in left ventricular (LV) myocytes from an arrhythmogenic rabbit model of non-ischaemic heart failure (HF) that was associated with a 2.5-fold increase in the amount of protein phosphatase type 2A (PP2A) co-localized with Cx43. Here, we further explore the molecular mechanisms of enhanced dephosphorylation of Cx43 in HF. p21-activated kinase 1 (PAK1) is a serine-threonine protein kinase that has been shown to activate PP2A. We found that total PAK1 and activated PAK1 (PAK1-P(Thr423)) were both increased in HF rabbit LV (vs. controls). PAK1 co-immunoprecipitated (co-IP'd) with Cx43 protein and, with HF, co-IP'd PAK1 and PAK1-P(Thr423) were increased. With failing human LV, PAK1 total protein and PAK1-P(Thr423) were also increased globally and locally (co-IP'd with Cx43). To further explore the role of PAK1 in modulating Cx43 dephosphorylation and intercellular coupling, we overexpressed active PAK1 in isolated LV myocytes from control rabbits and in HEK293 cells with genetically modified overexpression of Cx43 (HEK293-Cx43). PAK1 overexpression in both rabbit myocytes and HEK293-Cx43 cells significantly increased PP2A activity (globally and at the level of Cx43), increased dephosphorylated Cx43, and markedly reduced intercellular dye coupling. These effects were attenuated with PP2A inhibition using okadaic acid (10 nM). PAK1 and PP2A are integral components of a macromolecular complex with cardiac Cx43, and increased activation of associated PAK1 can contribute to enhanced Cx43 dephosphorylation and impaired intercellular coupling that may underlie slow conduction in HF.

High glucose-induced hypertrophy of mesangial cells is reversed by connexin43 overexpression via PTEN/Akt/mTOR signaling

Hypertrophy of glomerular mesangial cells (GMC) is one of the earliest pathological abnormalities in diabetic nephropathy, which correlates with eventual glomerulosclerosis. We have previously proved that this hypertrophy is mediated by downregulation of connexin43 (Cx43) and dysfunction of gap junctional intercellular communication, but the mechanism involved is still unclear. This study aims to investigate whether PTEN/Akt/mammalian target of rapamycin (mTOR) was involved as the downstream molecular signaling of Cx43 in regulating high glucose-induced GMC hypertrophy. GMC were isolated from male Wistar rats at the age of 3 months. Gene transfer technique was used to upregulate Cx43 in GMC, which was validated by western blot and immunofluorescent staining. Forward scatter of flow cytometry and total protein/cell number were examined to testify GMC hypertrophy induced by high glucose (30 mM) and the influence of Cx43 overexpression; western blot was performed to demonstrate the changes of Cx43 and signal protein level and flow cytometry and MTT test were carried out to check cell cycle and proliferation rate, respectively. GMC exposed to a high concentration of glucose presented decreased Cx43, inhibited PTEN, triggered Akt phosphorylation and activated downstream mTOR, leading to stagnancy of cell cycle, decline of proliferation rate and occurrence of hypertrophy. Cx43 overexpression could prevent PTEN inhibition, Akt and mTOR phosphorylation, resulting in restoration of cell cycle and proliferation ability and reversion of GMC hypertrophy. GMC with Cx43 inhibition showed similar PTEN/Akt/mTOR change as stimulated by high glucose. PTEN/Akt/mTOR signaling stimulated by high concentration of glucose is regulated by Cx43 overexpression, which unveils part of the molecular mechanism of Cx43 in regulating hyperglycemia-induced hypertrophy.

Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells.

We previously reported that preconditioning of stem cells with insulin-like growth factor-1 (IGF-1) translocated connexin-43 (Cx-43) into mitochondria, causing cytoprotection. We posit that these preconditioning effects could be simulated by mitochondria-specific overexpression of Cx-43. During IGF-1-induced preconditioning of C57black/6 mouse bone marrow stem cell antigen-1(+) (Sca-1(+)) cells, Cx-43 was mainly translocated onto the mitochondrial inner membrane, which was abrogated by an extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker PD98059. To investigate the role of mitochondrial Cx-43, we successfully designed a vector coding for full-length mouse Cx-43 with a mitochondria-targeting sequence (mito-Cx-43) and cloned into a shuttle vector (pShuttle-IRES-hrGFP-1) for mitochondria-specific overexpression of Cx-43 (mito-Cx-43). Sca-1(+) cells with mito-Cx-43 reduced cytosolic accumulation of cytochrome c, lowered caspase-3 activity, and improved survival during index oxygen-glucose deprivation as determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling and lactate dehydrogenase assays. Computational analysis revealed a B-cell lymphoma-2 (Bcl-2) homology domain-3 (BH3) motif in Cx-43 with a conserved pattern of amino acids consistent with the Bcl-2 family that regulated cytochrome c release. Moreover, computational secondary structure prediction indicated an extended α-helix in this region, a known condition for BH3-driven protein-protein interactions. Cx-43 translocation into mitochondria during preconditioning was ERK1/2-dependent. Expression of mito-Cx-43 simulated the cytoprotective effects of preconditioning in stem cells. Structural features of Cx-43 were shared with the Bcl-2 family as determined by computational analysis.

489 New animal model in colorectal cancer

489 New animal model in colorectal cancer