RESUMO
The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.
Assuntos
Caseína Quinase I , Conexina 43 , Miócitos Cardíacos , Adulto , Animais , Humanos , Camundongos , Caseína Quinase I/genética , Conexina 43/genética , Conexinas , Junções Comunicantes , Camundongos Transgênicos , MutaçãoRESUMO
Small cell lung cancer (SCLC) elicits the generation of autoantibodies that result in unique paraneoplastic neurological syndromes. The mechanistic basis for the formation of such autoantibodies is largely unknown but is key to understanding their etiology. We developed a high-dimensional technique that enables detection of autoantibodies in complex with native antigens directly from patient plasma. Here, we used our platform to screen 1009 human plasma samples for 3600 autoantibody-antigen complexes, finding that plasma from patients with SCLC harbors, on average, fourfold higher disease-specific autoantibody signals compared with plasma from patients with other cancers. Across three independent SCLC cohorts, we identified a set of common but previously unknown autoantibodies that are produced in response to both intracellular and extracellular tumor antigens. We further characterized several disease-specific posttranslational modifications within extracellular proteins targeted by these autoantibodies, including citrullination, isoaspartylation, and cancer-specific glycosylation. Because most patients with SCLC have metastatic disease at diagnosis, we queried whether these autoantibodies could be used for SCLC early detection. We created a risk prediction model using five autoantibodies with an average area under the curve of 0.84 for the three cohorts that improved to 0.96 by incorporating cigarette smoke consumption in pack years. Together, our findings provide an innovative approach to identify circulating autoantibodies in SCLC with mechanistic insight into disease-specific immunogenicity and clinical utility.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Neoplasias Pulmonares/patologia , Autoanticorpos , Processamento de Proteína Pós-TraducionalRESUMO
Small cell lung cancer (SCLC) is a deadly neuroendocrine tumor for which there are no screening methods sensitive enough to facilitate early, effective intervention. We propose targeting the neuroendocrine tumor neoantigen CD133 via antibody-based early detection and PET (immunoPET) to facilitate earlier and more accurate detection of SCLC. Methods: RNA sequencing datasets, immunohistochemistry, flow cytometry, and Western blots were used to quantify CD133 expression in healthy and SCLC patients. CD133 was imaged in vivo using near-infrared fluorescence (NIRF) immunoimaging, and 89Zr immunoPET. Anti(α)-CD133 autoantibody levels were measured in SCLC patient plasma using antibody microarrays. Results: Across 6 publicly available datasets, CD133 messenger RNA was found to be higher in SCLC tumors than in other tissues, including healthy or normal adjacent lung and non-SCLC samples. Critically, the upregulation of CD133 messenger RNA in SCLC was associated with a significant increase (hazard ratio, 2.62) in death. CD133 protein was expressed in primary human SCLC, in SCLC patient-derived xenografts, and in both SCLC cell lines tested (H82 and H69). Using an H82 xenograft mouse model, we first imaged CD133 expression with NIRF. Both in vivo and ex vivo NIRF clearly showed that a fluorophore-tagged αCD133 homed to lung tumors. Next, we validated the noninvasive visualization of subcutaneous and orthotopic H82 xenografts via immunoPET. An αCD133 antibody labeled with the positron-emitting radiometal 89Zr demonstrated significant accumulation in tumor tissue while producing minimal uptake in healthy organs. Finally, plasma αCD133 autoantibodies were found in subjects from cohort studies up to 1 year before SCLC diagnosis. Conclusion: In light of these findings, we conclude that the presence of αCD133 autoantibodies in a blood sample followed by CD133-targeted 89Zr-immunoPET could be an effective early detection screening strategy for SCLC.
Assuntos
Neoplasias Pulmonares , Tumores Neuroendócrinos , Carcinoma de Pequenas Células do Pulmão , Animais , Humanos , Camundongos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Camundongos Nus , Detecção Precoce de Câncer , Neoplasias Pulmonares/metabolismo , Modelos Animais de Doenças , Biomarcadores , Autoanticorpos , RNA Mensageiro , Linhagem Celular TumoralRESUMO
Pancreatic ductal adenocarcinoma (PDA) is aggressive, highly metastatic and characterized by a robust desmoplasia. Connexin proteins that form gap junctions have been implicated in tumor suppression for over 30 years. Cx43, the most widely expressed connexin, regulates cell behaviors, including migration and proliferation. Thus, we hypothesized that Cx43 could regulate PDA progression. Phosphorylation of Cx43 by Casein Kinase 1 (CK1) regulates gap junction assembly. We interbred the well-established KrasLSL-G12D/+;p48Cre/+ (KC) mouse model of PDA with homozygous "knock-in" mutant Cx43 mice bearing amino acid substitution at CK1 sites (Cx43CK1A) and found profound and surprising effects on cancer progression. Crossing the Cx43CK1A mouse onto the KC background (termed KC;CxCK1A) led to significant extension of lifespan, from a median of 370 to 486 days (p = 0.03) and a decreased incidence of metastasis (p = 0.045). However, when we examined early stages of disease, we found more rapid onset of tissue remodeling in the KC;CxCK1A mouse followed by divergence to a cystic phenotype. During tumorigenesis, gap junctions are increasingly present in stromal cells of the KC mice but are absent from the KC;Cx43CK1A mice. Tail vein metastasis assays with cells derived from KC or KC;CxCK1A tumors showed that KC;CxCK1A cells could efficiently colonize the lung and downregulate Cx43 expression, arguing that inhibition of metastasis was not occurring at the distal site. Instead, stromal gap junctions, their associated signaling events or other unknown Cx43-dependent events facilitate metastatic capacity in the primary tumor.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Caseína Quinase I/genética , Conexina 43/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/patologia , Animais , Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/genética , Junções Comunicantes/genética , Junções Comunicantes/patologia , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Camundongos , Metástase Neoplásica , Fosforilação/genéticaRESUMO
The gap junction protein Connexin43 (Cx43) is highly regulated by phosphorylation at over a dozen sites by probably at least as many kinases. This Cx43 "kinome" plays an important role in gap junction assembly and turnover. We sought to gain a better understanding of the interrelationship of these phosphorylation events particularly related to src activation and Cx43 turnover. Using state-of-the-art live imaging methods, specific inhibitors and many phosphorylation-status specific antibodies, we found phospho-specific domains in gap junction plaques and show evidence that multiple pathways of disassembly exist and can be regulated at the cellular and subcellular level. We found Src activation promotes formation of connexisomes (internalized gap junctions) in a process involving ERK-mediated phosphorylation of S279/282. Proteasome inhibition dramatically and rapidly restored gap junctions in the presence of Src and led to dramatic changes in the Cx43 phospho-profile including to increased Y247, Y265, S279/282, S365, and S373 phosphorylation. Lysosomal inhibition, on the other hand, nearly eliminated phosphorylation on Y247 and Y265 and reduced S368 and S373 while increasing S279/282 phosphorylation levels. We present a model of gap junction disassembly where multiple modes of disassembly are regulated by phosphorylation and can have differential effects on cellular signaling.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Fosforilação , RatosRESUMO
BACKGROUND: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration. METHODS: We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation. RESULTS: Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint. CONCLUSION: Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.
Assuntos
Células-Tronco Embrionárias Humanas , Miócitos Cardíacos , Animais , Diferenciação Celular , Células-Tronco Embrionárias , Cobaias , MiocárdioRESUMO
The gap junction protein connexin 43 (Cx43) is a key player in wound healing, and inhibitors of Cx43, which speed epidermal wound healing, are currently in clinical trials. Here, we provide direct in vivo evidence that specific phosphorylation events on Cx43 change the physiological response during wound healing. Blocking phosphorylation, through mutation of serine residues in Cx43 at the protein kinase C (PKC) or casein kinase 1 (CK1) sites, significantly slowed the rate of wound closure in vivo and in vitro and resulted in a thicker epidermal layer after reepithelialization. Conversely, preventing Cx43 phosphorylation by mitogen-activated protein kinases (MAPKs) through mutation significantly increased the rate of wound closure in vivo Defects in migration, but not proliferation, in all mutants were partially rescued in vitro by changing serine residues to aspartic or glutamic acid. These data prove that specific Cx43 phosphorylation events play an important role at different stages of wound healing. Thus, a clear physiological understanding of the spatiotemporal regulation of kinase activation and consequent effects on gap junctions could lead to a more targeted approach to modulating Cx43 expression during wound healing.
Assuntos
Caseína Quinase I/metabolismo , Conexina 43/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/fisiologia , Proteína Quinase C/metabolismo , Animais , Caseína Quinase I/genética , Linhagem Celular , Conexina 43/genética , Cães , Células Epidérmicas , Imuno-Histoquímica , Cinética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação/genética , Proteína Quinase C/genéticaRESUMO
Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 µM, <1 h for HCh; ~100 µM, >1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-Hdc) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca2+-wave propagation (IC50 64.8 pM) and HCh-mediated dye uptake (IC50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca2+-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca2+-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca2+-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca2+-wave propagation, dye coupling, and dye uptake were largely unaffected (IC50 12.3 µM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca2+-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Peptídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Conexinas/metabolismo , Cães , Células Madin Darby de Rim Canino , Oligopeptídeos , Isoformas de Proteínas/metabolismoRESUMO
Gap junctions contain intercellular channels that enable intercellular communication of small molecules while also serving as a signaling scaffold. Connexins, the proteins that form gap junctions in vertebrates, are highly regulated and typically have short (<2 h) half-lives. Connexin43 (Cx43), the predominate connexin in the myocardium and epithelial tissues, is phosphorylated on more than a dozen serine residues and interacts with a variety of protein kinases. These interactions regulate Cx43 and gap junction formation and stability. Casein kinase 1 (CK1)-mediated phosphorylation of Cx43 promotes gap junction assembly. Using murine knock-in technology and quantitative PCR, immunoblotting, and immunoprecipitation assays, we show here that mutation of the CK1 phosphorylation sites in Cx43 reduces the levels of total Cx43 in the myocardium and increases Cx43 phosphorylation on sites phosphorylated by extracellular signal-regulated kinase (ERK). In aged myocardium, we found that, compared with WT Cx43, mutant Cx43 expression increases ERK activation, phosphorylation of Akt substrates, and protection from ischemia-induced injury. Our findings also uncovered that Cx43 interacts with the hypoxia-inducible protein N-Myc downstream-regulated gene 1 protein (NDRG1) and that Cx43 phosphorylation status controls this interaction and dramatically affects NDRG1 stability. We propose that, in addition to altering gap junction stability, Cx43 phosphorylation directly and dynamically regulates cellular signaling through ERK and Akt in response to ischemic injury. We conclude that gap junction-dependent NDRG1 regulation might explain some cellular responses to hypoxia.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Conexina 43/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Envelhecimento , Animais , Caseína Quinase I/metabolismo , Conexina 43/genética , Cães , Junções Comunicantes/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Miocárdio/metabolismo , Fosforilação/efeitos dos fármacos , Estabilidade Proteica , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Especificidade por SubstratoRESUMO
Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (<1000Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP3) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
Assuntos
Arritmias Cardíacas/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Insuficiência Cardíaca/metabolismo , Sistemas do Segundo Mensageiro , Animais , Arritmias Cardíacas/patologia , Transporte Biológico Ativo , Junções Comunicantes/patologia , Insuficiência Cardíaca/patologia , Humanos , FosforilaçãoRESUMO
Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.
Assuntos
Ciclo Celular/fisiologia , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Conexinas/genética , Junções Comunicantes/genética , Mutação de Sentido Incorreto , Fosforilação , RatosRESUMO
Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43's half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing.
Assuntos
Junções Comunicantes/metabolismo , Proteínas Quinases/metabolismo , Cicatrização , Animais , Conexina 43/química , Conexina 43/metabolismo , Humanos , Modelos Biológicos , FosforilaçãoRESUMO
Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of <5%. Detection of resectable disease improves survival rates, but access to tissue and other biospecimens that could be used to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing â¼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Anticorpos/química , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Ceruletídeo , Diagnóstico Diferencial , Modelos Animais de Doenças , Progressão da Doença , Diagnóstico Precoce , Perfilação da Expressão Gênica , Humanos , Camundongos , Anotação de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/metabolismo , Análise Serial de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially "unzip" and be internalized/endocytosed into the cell that produced each connexin.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Conexina 43/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
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.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Conexina 43/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Western Blotting , Progressão da Doença , Junções Comunicantes/metabolismo , Regulação Neoplásica da Expressão Gênica , Camundongos , FosforilaçãoRESUMO
Vertebrate gap junctions are composed of proteins from the connexin family. Co-immunoprecipitation, in vitro binding and far western experiments demonstrate that mammalian CASK (also known as LIN2) directly interacts with Cx43. Immunoprecipitation studies indicate that the CASK mainly interacts with the hypophosphorylated form of Cx43. Functional co-regulation of these proteins was found in MDCK cells migrating into a scratch wound, where expression of either protein individually inhibits migration but their coexpression abrogates this inhibitory effect. Immunofluorescence shows colocalization of Cx43 and CASK in mouse brain astrocytes and in response to wounding in human foreskin. During wounding, CASK is mobilized to the plasma membrane where it colocalizes with Cx43 and CADM1 1 hour after skin explant wounding. Together, these studies indicate that CASK interaction with Cx43 occurs relatively early in the connexin life cycle and imply a plasma membrane targeting role for the interaction that apparently affects cellular processes including cellular migration and wound healing.
Assuntos
Conexina 43/metabolismo , Guanilato Quinases/metabolismo , Cicatrização/fisiologia , Animais , Astrócitos/metabolismo , Sequência de Bases , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Conexina 43/genética , Cães , Técnicas de Silenciamento de Genes , Guanilato Quinases/antagonistas & inibidores , Guanilato Quinases/genética , Humanos , Imunoglobulinas/metabolismo , Masculino , Camundongos , Fosforilação , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Pele/lesões , Pele/metabolismo , TransfecçãoRESUMO
Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in essentially all tissues. There are 21 connexin genes in the human genome and different tissues express different connexin genes. Most connexins are known to be phosphoproteins. Phosphorylation can regulate connexin assembly into gap junctions, gap junction turnover and channel gating. Given the importance of gap junctions in development, proliferation and carcinogenesis, regulation of gap junction phosphorylation in response to wounding, hypoxia and other tissue insults is proving to be critical for cellular response and return to homeostasis. Connexin43 (Cx43) is the most widely and highly expressed gap junction protein, both in cell culture models and in humans, thus more research has been done on it and more reagents to it are available. In particular, antibodies that can report Cx43 phosphorylation status have been created allowing temporal examination of specific phosphorylation events in vivo. This review is focused on the use of these antibodies in tissue in situ, predominantly looking at Cx43 phosphorylation in brain, heart, endothelium and epithelium with reference to other connexins where data is available. These data allow us to begin to correlate specific phosphorylation events with changes in cell and tissue function. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Assuntos
Encéfalo/metabolismo , Conexina 43/fisiologia , Endotélio/metabolismo , Epitélio/metabolismo , Junções Comunicantes/fisiologia , Miocárdio/metabolismo , Animais , Conexina 43/metabolismo , Genoma Humano , Homeostase , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Fosforilação , Transdução de Sinais , Distribuição TecidualRESUMO
During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43-GFP-4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno-labeled endogenous Cx43-expressing cells. Photo-oxidation of ReAsH-labeled Cx43-GFP-4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse-chase labeling and single label time-lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.
Assuntos
Conexina 43/metabolismo , Mitose/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Conexina 43/química , Conexina 43/genética , Vesículas Citoplasmáticas/metabolismo , Cães , Fibroblastos/citologia , Imunofluorescência , Espaço Intracelular/metabolismo , Ratos , TelófaseRESUMO
Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, co-ordinated contraction of excitable cells, tissue homoeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin 'life cycle', including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1-5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. The present review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology, with an emphasis on events occurring in heart and skin.