RESUMO
Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.
Assuntos
Conexina 43 , Proteína 1 Homóloga a Discs-Large , Queratinócitos , Humanos , Comunicação Celular , Membrana Celular/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Guanilato Quinases/metabolismo , Queratinócitos/metabolismo , Proteína 1 Homóloga a Discs-Large/genética , Proteína 1 Homóloga a Discs-Large/metabolismoRESUMO
Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape-wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape-wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with 'diabetic' conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn-over around diabetic wounds.
Assuntos
Conexinas/metabolismo , Ferimentos e Lesões/metabolismo , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células HaCaT , Células HeLa , Humanos , Insulina/metabolismoRESUMO
Pulmonary hypertension is a serious clinical condition characterised by increased pulmonary arterial pressure. This can lead to right ventricular failure which can be fatal. Connexins are gap junction-forming membrane proteins which serve to exchange small molecules of less than 1 kD between cells. Connexins can also form hemi-channels connecting the intracellular and extracellular environments. Hemi-channels can mediate adenosine triphosphate release and are involved in autocrine and paracrine signalling. Recently, our group and others have identified evidence that connexin-mediated signalling may be involved in the pathogenesis of pulmonary hypertension. In this review, we discuss the evidence that dysregulated connexin-mediated signalling is associated with pulmonary hypertension.
Assuntos
Conexinas/metabolismo , Hipertensão Pulmonar/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Humanos , Hipertensão Pulmonar/patologiaRESUMO
BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.
Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Psoríase/genética , Adulto , Idoso , Biópsia , Conexinas/metabolismo , Conexinas/farmacologia , Epiderme/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Mediadores da Inflamação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Modelos Biológicos , Oligopeptídeos/farmacologia , Peptidoglicano/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Psoríase/patologia , Staphylococcus aureus/fisiologia , Adulto JovemRESUMO
Obesity-associated conditions represent major global health and financial burdens and understanding processes regulating adipogenesis could lead to novel intervention strategies. This study shows that adhesion G-protein coupled receptor 56 (GPR56) gene transcripts are reduced in abdominal visceral white adipose tissue derived from obese Zucker rats versus lean controls. Immunostaining in 3T3-L1 preadipocytes reveals both mitotic cell restricted surface and low level general expression patterns of Gpr56. Gpr56 transcripts are differentially expressed in 3T3-L1 cells during adipogenesis. Transient knockdown (KD) of Gpr56 in 3T3-L1 cells dramatically inhibits differentiation through reducing the accumulation of both neutral cellular lipids (56%) and production of established adipogenesis Pparγ 2 (60-80%), C/ebpα (40-78%) mediator, and Ap2 (56-80%) marker proteins. Furthermore, genome editing of Gpr56 in 3T3-L1 cells created CW2.2.4 and RM4.2.5.5 clones (Gpr56 -/- cells) with compound heterozygous deletion frameshift mutations which abolish adipogenesis. Genome edited cells have sustained levels of the adipogenesis inhibitor ß-catenin, reduced proliferation, reduced adhesion, altered profiles, and or abundance of extracellular matrix component gene transcripts for fibronectin, types I, III, and IV collagens and loss of actin stress fibers. ß-catenin KD alone is insufficient to restore adipogenesis in Gpr56 -/- cells. Together these data show that Gpr56 is required for adipogenesis in 3T3-L1 cells. This report is the first demonstration that Gpr56 participates in regulation of the adipogenesis developmental program. Modulation of the levels of this protein and/or its biological activity may represent a novel target for development of therapeutic agents for the treatment of obesity.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Animais , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Obesidade/metabolismo , Ratos , Ratos ZuckerRESUMO
Mutations in GJB2 encoding Connexin 26 (CX26) are associated with hearing loss and hyperproliferative skin disorders of differing severity including keratitis-ichthyosis-deafness (KID) and Vohwinkel syndrome. A 6-year-old Caucasian girl who presented with recurrent skin rashes and sensorineural hearing loss harboured a heterozygous point mutation in GJB2 (c.424T > C; p.F142L). To characterize the impact of CX26F142L on cellular events. Plasmids CX26WT, CX26F142L, CX26G12R (KID) or CX26D66H (Vohwinkel) were transfected into HeLa cells expressing Cx26 or Cx43 or into HaCaT cells, a model keratinocyte cell line. Confocal microscopy determined protein localization. MTT assays assessed cell viability in the presence or absence of carbenoxolone, a connexin-channel blocker. Co-immunoprecipitation/Western blot analysis determined Cx43:Cx26 interactions. Quantitative real-time polymerase chain reaction assessed changes in gene expression of ER stress markers. Dye uptake assays determined Connexin-channel functionality. F142L and G12R were restricted to perinuclear areas. Collapse of the microtubule network, rescued by co-treatment with paclitaxel, occurred. ER stress was not involved. Cell viability was reduced in cells expressing F142L and G12R but not D66H. Unlike G12R that forms "leaky" hemichannels, F142L had restricted permeability. Cell viability of F142L and G12R transfected cells was greater in HeLa cells expressing Cx43 than in native Cx-free HeLa cells. Co-immunoprecipitation suggested a possible interaction between Cx43 and the three mutations. Expression of CX26F142L and G12R results in microtubule collapse, rescued by interaction with Cx43. The GJB2 mutations interacted with Cx43 suggesting that unique Cx43:Cx26 channels are central to the diverse phenotype of CX26 skin-related channelopathies.
Assuntos
Transporte Biológico/genética , Conexina 26/genética , Conexina 26/metabolismo , Exantema/genética , Perda Auditiva Neurossensorial/genética , Microtúbulos/ultraestrutura , Carbenoxolona/farmacologia , Sobrevivência Celular/genética , Criança , Conexina 43/metabolismo , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Células HaCaT , Células HeLa , Heterozigoto , Humanos , Microtúbulos/efeitos dos fármacos , Mutação , Paclitaxel/farmacologia , Transfecção , Moduladores de Tubulina/farmacologiaRESUMO
Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real-time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa ß (NF-κß) was blocked with BAY 11-7082, CX-channel function was determined by adenosine 5'-triphosphate (ATP) release assays. Enzyme-linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL-6, IL-8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-κß. ATP and IL-6 release were stimulated following 15 min and 1-24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX-channel blockers, CX26-, CX43- and TLR2-KD. The IL-6 response was also reduced by purinergic blockers. CX-signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF-κß, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.
RESUMO
Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.
Assuntos
Conexinas/genética , Eritroceratodermia Variável/genética , Mutação de Sentido Incorreto , Morte Celular , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/biossíntese , Conexina 43/genética , Retículo Endoplasmático/ultraestrutura , Epiderme/patologia , Eritroceratodermia Variável/patologia , Genes Dominantes , Estudos de Associação Genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Células HeLa , Humanos , Queratinócitos , Necrose , Transporte ProteicoRESUMO
Impaired wound healing and ulceration caused by diabetes mellitus, is a significant healthcare burden, markedly impairs quality of life for patients, and is the major cause of amputation worldwide. Current experimental approaches used to investigate the complex wound healing process often involve cultures of fibroblasts and/or keratinocytes in vitro, which can be limited in terms of complexity and capacity, or utilisation of rodent models in which the mechanisms of wound repair differ substantively from that in humans. However, advances in tissue engineering, and the discovery of strategies to reprogramme adult somatic cells to pluripotency, has led to the possibility of developing models of human skin on a large scale. Generation of induced pluripotent stem cells (iPSCs) from tissues donated by diabetic patients allows the (epi)genetic background of this disease to be studied, and the ability to differentiate iPSCs to multiple cell types found within skin may facilitate the development of more complex skin models; these advances offer key opportunities for improving modelling of wound healing in diabetes, and the development of effective therapeutics for treatment of chronic wounds.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Pele/fisiopatologia , Cicatrização/fisiologia , Animais , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Modelos Biológicos , Engenharia Tecidual/métodosRESUMO
This Special Issue relates to the 18th biannual International Gap Junction Conference (IGJC2017), held at the Crowne Plaza Hotel, Glasgow, U.K., from the 29 Julyâ»2 August 2017 [...].
Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Congressos como Assunto , Conexinas/análise , Junções Comunicantes/patologia , Humanos , Mapas de Interação de Proteínas , Pesquisa Translacional Biomédica , Reino Unido , CicatrizaçãoRESUMO
Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.
Assuntos
Conexinas/metabolismo , Animais , Epitélio/metabolismo , Epitélio/patologia , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Cicatrização/fisiologia , Peixe-ZebraRESUMO
Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using Cx43 heterozygous (Cx43+/−) mice and 37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male Cx43+/− mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female Cx43+/−mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female Cx43+/− mice or by pre-incubation of IPAs with 37,43Gap27. Nω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.
Assuntos
Conexina 43/metabolismo , Artéria Pulmonar/metabolismo , Animais , Conexina 43/genética , Endotelina-1/metabolismo , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Genótipo , Hipertensão Pulmonar/metabolismo , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Serotonina/metabolismoRESUMO
In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-ß1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100-100 µM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM-100 µM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-ß1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.
Assuntos
Conexina 43/metabolismo , Técnicas de Silenciamento de Genes , Modelos Biológicos , Peptídeos/farmacologia , Pele/patologia , Cicatrização/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The integumentary system comprises the skin and its appendages, which includes hair, nails, feathers, sebaceous and eccrine glands. In this review, we focus on the expression profile of connexins and pannexins throughout the integumentary system in mammals, birds and fish. We provide a picture of the complexity of the connexin/pannexin network illustrating functional importance of these proteins in maintaining the integrity of the epidermal barrier. The differential regulation and expression of connexins and pannexins during skin renewal, together with a number of epidermal, hair and nail abnormalities associated with mutations in connexins, emphasize that the correct balance of connexin and pannexin expression is critical for maintenance of the skin and its appendages with both channel and non-channel functions playing profound roles. Changes in connexin expression during both hair and feather regeneration provide suggestions of specialized communication compartments. Finally, we discuss the potential use of zebrafish as a model for connexin skin biology, where evidence mounts that differential connexin expression is involved in skin patterning and pigmentation.
Assuntos
Conexinas/metabolismo , Epiderme/metabolismo , Epiderme/fisiologia , Tegumento Comum/fisiologia , Pele/metabolismo , Animais , Humanos , Fenômenos Fisiológicos da PeleRESUMO
The 8th UK Gap Junction meeting was held in Glasgow Caledonian University (GCU) on 5th December 2014. Emeritus Professor Howard Evans presented an overview of 50 years of gap junction research whereas Dr Brant Isakson, University of Virginia, discussed the intriguing role of recently identified pannexin proteins in endothelial function. Forty-five delegates from across the U.K. and the Europe attended the day with 12 talks from young researchers and five posters. This issue of biochemical transactions provides an overview of the highlights of the work discussed throughout the day.
Assuntos
Junções Comunicantes/genética , Pesquisa , HumanosRESUMO
Chronic wounds are not only debilitating to patients, but also impose a huge financial burden on healthcare providers, as current treatments are not particularly effective. Wound healing is a highly co-ordinated process involving a vast array of signalling molecules and different cell types, therefore a substantial amount of research has been carried out in the quest to develop new therapies. The gap junction (GJ) protein connexin43 (Cx43) is one of the many molecules whose expression has been found to be up-regulated in chronic wounds and as a result targeting it may have therapeutic potential. Two different approaches have been adopted to investigate this: knockdown of Cx43 using antisense oligonucleotides and connexin mimetic peptides (CMPs) which inhibit the function of Cx43 without affecting gene expression. These peptides are targeted to the C-terminal domain or the extracellular loops of Cx43 and thus are likely to function by different means. However, both block channel function and have been shown to enhance cell migration rates. In recent years, non-channel functions have emerged for Cx43, many of which are linked to cytoskeletal dynamics and the extracellular matrix (ECM), showing that Cx43 plays diverse roles in co-ordinating wound closure events. It is clear that both CMPs and antisense oligonucleotides hold therapeutic potential, however maintaining Cx43 expression may be beneficial to the cell by preserving other non-channel functions of Cx43. Recent data in the field will be discussed in this article.
Assuntos
Conexina 43/genética , Junções Comunicantes/genética , Cicatrização/genética , Movimento Celular/genética , Conexina 43/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patologia , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Transdução de SinaisRESUMO
Cell-to-cell communication triggered by connexin channels plays a central role in maintaining epidermal homeostasis. Here, we discuss the role of the beta connexin subgroup, where site-specific mutations in at least 4 of these proteins lead to distinctive non-inflammatory and inflammatory hyperproliferative epidermal disorders. Recent advances in the molecular pathways evoked and correlation with clinical outcome are discussed. The latest data provide increasing evidence that connexins in the epidermis are sensors to environmental stress and that targeting aberrant hemichannel activity holds significant therapeutic potential for inflammatory skin disorders.
Assuntos
Conexinas/metabolismo , Homeostase , Dermatopatias/metabolismo , Pele/metabolismo , Animais , Conexinas/química , Conexinas/genética , Humanos , Modelos Biológicos , Pele/patologia , Dermatopatias/genética , Dermatopatias/patologiaRESUMO
Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance.
Assuntos
Movimento Celular/efeitos dos fármacos , Conexinas/farmacologia , Glucose/farmacologia , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Insulina/farmacologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Técnicas de Cultura de Células , Ensaios de Migração Celular , Movimento Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Modelos Teóricos , Oligopeptídeos , Concentração Osmolar , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
UNLABELLED: The liver architecture plays an important role in maintaining hemodynamic balance, but the mechanisms that underlie this role are not fully understood. Hepsin, a type II transmembrane serine protease, is predominantly expressed in the liver, but has no known physiological functions. Here, we report that hemodynamic balance in the liver is regulated through hepsin. Deletion of hepsin (hepsin(-/-) ) in mice resulted in enlarged hepatocytes and narrowed liver sinusoids. Using fluorescent microbeads and antihepsin treatment, we demonstrated that metastatic cancer cells preferentially colonized the hepsin(-/-) mouse liver as a result of the retention of tumor cells because of narrower sinusoids. The enlarged hepatocytes expressed increased levels of connexin, which resulted from defective prohepatocyte growth factor (pro-HGF) processing and decreased c-Met phosphorylation in the livers of hepsin(-/-) mice. Treatment of hepsin(-/-) mice with recombinant HGF rescued these phenotypes, and treatment of wild-type mice with an HGF antagonist recapitulated the phenotypes observed in hepsin(-/-) mice. CONCLUSION: Our findings show that the maintenance of hepatic structural homeostasis occurs through HGF/c-Met/connexin signaling by hepsin, and hepsin-mediated changes in liver architecture significantly enhance tumor metastasis to the liver.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/patologia , Neoplasias Hepáticas/secundário , Fígado/metabolismo , Fígado/patologia , Metástase Neoplásica/patologia , Serina Endopeptidases/metabolismo , Animais , Conexinas/metabolismo , Hemodinâmica , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/genética , Transdução de SinaisRESUMO
Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.