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We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.
The mode of action of coumarin is well suited to the relief of high-protein oedemas. It binds rapidly to the serum albumins, and the protein binding capacity of a substance provides insight into its interaction with sites of biological activity. Together with proteins, inflowing through the damaged capillary endothelium after thermal injury, it is taken up by the monocytes in the inflamed tissues and by the resident macrophages. Coumarin can stimulate these cells to enhanced proteolysis of the engulfed protein which is digested to debris of molecular weight less than 1000. This is on the other hand readily utilisable protein for the large number of monocytes to mature and differentiate into macrophages. Coumarin may dissociate from protein in the tissue and cause local site activation or even activate all cell sites increasing cell numbers or rejuvenate older phagocytic cells and stimulate the reticulo-endothelial system (RES). A passing histamine-like effect of coumarin and other benzopyrones is an indirect stimulant for the RES. The released injurious amines open additional numbers of endothelial intercellular junctions, allowing extra protein and fluid into the interstitial tissues. A reinforced phagocytosis follows and results in more rapid and complete digestion. The action of the drug in causing the opening of intercellular junction is very beneficial. The extra protein is of little consequence, since its inflow is more than compensated for by the other actions of the drug which results in its lysis. Coumarin has also a number of other important actions on the vascular system. It causes constriction of the precapillary sphincters and a dilatation of the arterio-venous anastomosis. The result is an increased blood pressure and flow, and a better oxygen supply of the area. It is able to restore the deformability of the red blood cell to near normal levels and to protect the thrombocyte by membrane stabilizing. The result is a reduced likelihood of thrombosis and embolism. By increasing the glucose uptake and transport the benzopyrones can improve the chance of survival of those cells in stagnant areas of the circulation.
In keratinocyte culture, the cellular distribution of many adhesion markers and the organization of intercellular junctions are controlled by the calcium ion concentration of the medium. We show in the present study that urokinase plasminogen activator (uPA) localization in the human keratinocyte is similarly dependent upon calcium concentration. At 30 microM calcium, uPA is present throughout the cell, often with a perinuclear concentration. Upon calcium elevation to 1.0 mM, uPA is concentrated along the cell-cell borders, where it colocalizes (at the light microscope level) with E-cadherin. Blocking antibody to E-cadherin delays the calcium-induced redistribution of uPA, in a manner very similar to the previously observed delay in redistribution of several adhesion-related markers, including vinculin, desmoplakin, and beta 1 integrin. These data suggest a link between the redistribution of uPA to the cell-cell borders and the calcium-induced organization of intercellular junctions in the human keratinocyte. The presence of uPA along the intercellular borders suggests that this enzyme may be involved in regulation of epidermal adhesion through proteolysis.
Ultrastructural subtypes of endothelial cells in Kaposi's sarcoma were compared with lymphatics and the normal dermal microcirculation in different stages of lesional development. In the earliest patch stage, lymphatic channels, recognised by their dissecting growth pattern and a lack of a basal lamina and pericytes, were prominent. Venous endothelium was recognised by virtue of its multilaminated basal lamina and often showed markedly irregular luminal and abluminal cytoplasmic projections. As the histological stage progressed toward spindle cells, venous endothelium showed a tetrad of changes: dissolution of the basal lamina; fragmentation and disappearance of the pericyte sheath, decreased and often rudimentary intercellular junctions and markedly reduced numbers of Weibel-Palade bodies. These were also features of spindle cells, which were rarely seen to emerge from narrow vascular channels of indeterminate type. Spindle cells showed sparse intercellular junctions and minimal basal lamina but no Weibel-Palade bodies. These progressive venous alterations thus resulted in a mixed intermediate subtype of endothelium with the morphological traits resembling lymphatics as well as venous blood vessels. The mixed subtype included endothelial tubes surrounded by a complete basal lamina but lacking pericytes, and much more commonly, tubes with pericytes but a scanty basal lamina. Both forms had remarkably few or no Weibel-Palade bodies. In the spindle cell stage, normal vessels were largely replaced by the mixed subtype and an indeterminate type of frequently disrupted endothelial tube which lacked a basal lamina as well as a pericytic investment. Dissecting lymphatic channels could not be confidently distinguished from the latter vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.
Early changes in rabbit limb-bud fine structure subsequent to maternal administration of either hydroxyurea (750 mg HU/kg) or methotrexate (19.2 mg MTX/kg) on gestational day 12 have been analyzed in order to identify the morphologic sites of teratogenic action. Forelimb-buds were examined at 15 minutes to 32 hours posttreatment for ultrastructural alterations. HU caused early dispersion of ribosomes, condensation of nuclei, segregation of the granular portion of the nucleoli, and eventual fragmentation of mesenchymal cells. As a consequence of the extensive mesenchymal cellular death, the intercellular space among the remaining viable cells was increased. Cellular debris from fragmented cells was seen phagocytized within cells and in the intercellular space. In contrast, MTX caused an increase in cytoplasmic lipid, angular contours of nuclei, but relatively little cellular death. The most prominent change was a disruption of the normal mesenchymal architecture characterized by greatly increased intercellular space and a consequent reduction in the number of intercellular junctions. Nearly normal cytoarchitecture was restored by 32 hours. It was concluded that the primary morphologic sites of action are indeed different. Nevertheless, MTX and HU shared a common denominator since both caused disruption of intercellular contacts and possible breakdown of intercellular communication: HU through cytolethality, and MTX via disruption of intercellular junctions. This latter phenomenon may help to explain the similarities in limb defects caused by these two drugs.
To examine the role of gap-junctional intercellular communication in controlling cell proliferation, we have transfected C6 glioma cells with connexin 43 cDNA. The growth of transfected clones was dramatically reduced compared with nontransfected glioma cells. To further characterize the role of gap junctions in controlling proliferation, we have examined the growth of C6 cells cocultured with transfected cells overexpressing connexin 43. Although C6 cells grew at their normal rate when cocultured with nontransfected C6 cells, when cocultured with connexin 43-overexpressing cells they displayed a dramatic reduction in growth rate. Furthermore, a significant, dose-dependent reduction in cell proliferation was noted when C6 cells were cultured in medium conditioned by transfected cells. This effect correlated with the level of connexin 43 expression. These results suggest that the decreased cell proliferation rate of transfected cells and C6 cells cultured with them is due to the secretion of a growth inhibitory factor(s) and that the secretion of this factor may be linked to the level of gap junctional intercellular communication.
Cardiovascular diseases remain to be the 4th rank of top ten causes of mortality in Taiwan in recent years. Atherosclerosis and coronary artery disease, which often culminating in the occurrence of myocardial infarction and congestive heart failure, are responsible for the majority of these death. One of the prominent features of atherosclerotic lesion is local accumulation of lipids, mainly in the forms of cholesteryl ester and free cholesterol, either within cells or extracellularly in matrix. Repeated endothelial injury and enhanced lipid infiltration are critical events in the development of atherosclerosis. Plasma lipoproteins may enter the arterial wall through endothelium, either transcellularly via vesicular transport or paracellularly via intercellular junction. Our previous studies have demonstrated that most of the arterial endothelial cells in mitosis are associated with the leakage of fluorescently labeled albumin and low density lipoproteins. Subsequently, such transendothelial leakage of macromolecules is also shown to be associated with endothelial cell death as assessed by immunocytochemical staining for IgG. These findings suggested that transiently leaky junctions occurring during endothelial cell turnover may provide potentially important pathways for increasing transport or leakage of macromolecules, including atherogenic LDL, across the vascular endothelium. Electron microscopic study using horseradish peroxidase as a tracer revealed markedly widening of intercellular junctions around endothelial cells in mitosis providing direct evidence in support of "cell turnover-leaky junction" theory for the localization of atherogenesis. Hypertension, smoking, diabetes, and hyperlipidemia are well-known major risk factors for atherosclerosis and coronary heart disease. In a series of investigations, we examined the hypothesis that hypertension smoking, diabetes, and hyperlipidemia increase the arterial endothelial cell turnover and hence transendothelial macromolecular transport, which may have some implications in increasing lipid entry and thus, accelerating atherogenesis. Animal experiments were performed in adult male spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) normotensive rats, and Sprague-Dawley (SD) rats. SHRs were used as hypertensive group with WKY rats as normotensive control. SD rats were given nicotine at a dose of 5 mg/Kg body wt/ day in their drinking water to mimic smoking effect over a period of 6 weeks. Diabetes was induced in SD rats by single intraperitoneal injection of 60 mg/Kg body wt of streptozotocin. The duration of diabetes was 6 weeks. Also, SD rats were fed a diet containing 5% cholesterol for 6 weeks to induce hyperlipidemia. Age-matched rats of comparable number served as control for each experimental group. In en face preparations of thoracic aorta, mitotic endothelial cells were identified by hematoxylin staining, immunoglobulin G-containing dying or dead endothelial cells were detected by an indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin (EBA) complexes (5 minutes after intravenous injection) was visualized and quantified by fluorescence microscopy. The results showed that SHR, chronic oral nicotine-treated rats, diabetic, rats, and hyperlipidemic rats, when compared to control rats, had higher values for the frequency of endothelial cell death and the number density of EBA leaky foci in the aorta. These findings suggested that hypertension, cigarette smoking, diabetes mellitus, and hyperlipidemia become risk factors in atherogenesis by increasing the rate of arterial endothelial cell turnover and the associated endothelial cell turnover and the to the consequent enhanced entry of atherogenic lipoproteins into the arterial wall and accelerated atherogenesis.
The intercellular signaling system mediated by connexin channels is crucial for maintaining tissue homeostasis, growth control, development, and synchronized response of cells to stimuli. This review summarizes the structure, assembly, and properties of the components of the complex and diverse connexin system, and their biological functions in skin. The importance of gap junctional intercellular communication for normal development and differentiation of human epidermis as well as the hearing function of the inner ear is illustrated by the examples of erythrokeratodermia variabilis and palmoplantar keratoderma associated with hearing loss. These autosomal dominant inherited disorders are caused by germline mutations in the connexin genes GJB3 (encoding connexin-31) and GJB2 (encoding connexin-26), respectively. Recent functional studies of individual connexin mutations suggest that they may exert a dominant inhibitory effect on normal connexin channel function and perturb gap junctional intercellular communication, resulting in phenotypic manifestation in patients with these disorders.
A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2-SH3 domain-containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics.
Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.
Peri-implant lesions are directly linked to the relationship between the implant and the soft tissue. These lesions are rare in the case of pure Ti smooth implants, but appear frequently around the rough-surfaced implants. A marginal inflammation may result in a rapid peri-implant bone loss, since the peri-implant connective tissue constitutes in a less effective barrier than the same tissue surrounding the tooth. The morphopathological examination of the peri-implant tissues revealed after their removal that the gum's mucous membrane is not inflamed, but degenerative lesions were observed both on the epithelium and chorion. The papillary lamina propria contains few cells, but it is rich in collagen fibers; vasodilatation and edema were also present. Multiple areas of lysis of the intercellular junctions were also observed at the intermediary level of the gum peri-implant epithelium (the epithelial cells undergo a process of involution at the intermediary level of the epithelium). Important changes can be seen both at the level of intercellular junction systems and at the junction between the basal extremity of the epithelial cells and the basal membrane.
The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.
Multiple intercalated discs were frequently observed in muscle cells from patients with cardiac hypertrophy of various causes. Evidence is presented that these structures are the intercellular junctions of lateral processes of muscle cells. The sizes and shapes of these processes showed marked variations, depending in part on the plane of sectioning. It is postulated that lateral processes develop in certain cells which have side-to-side intercellular junctions with adjacent cells, and that localized mechanical tensions induce their growth. These tensions are due to shearing forces exerted at side-to-side junctions when contraction occurs at different rates or magnitudes in adjacent cells. These forces lead to asymmetric and complementary growth of sarcomeres along the two sides of the lateral junctions, to reorientation of these junctions and to the eventual formation of lateral processes.
The activity of Src kinases appears to play a role in both assembly and disassembly of tight junction. However, the role of a specific isoform of Src kinase in regulation of tight junction is not known. In the present study the role of c-Src in regulation of epithelial tight junction was investigated in Caco-2 cell monolayers. Oxidative stress (xanthine oxidase + xanthine) induced an activation and membrane translocation of c-Src. The oxidative stress-induced decrease in transepithelial electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 from the intercellular junctions were prevented by PP2. The rates of oxidative stress-induced activation of c-Src, tyrosine phosphorylation of ZO-1 and beta-catenin, decrease in resistance, increase in permeability to inulin, and redistribution of occludin and ZO-1 were significantly greater in cells transfected with wild type c-Src, whereas it was low in cells transfected with kinase-inactive c-SrcK297R mutant, when compared with those in empty vector-transfected cells. The rates of recovery of resistance, increase in barrier to inulin, and reorganization of occludin and ZO-1 into the intercellular junctions during the calcium-induced reassembly of tight junction were much greater in Caco-2 cells transfected with c-SrcK297R as compared with those in cells transfected with empty vector or wild type c-Src. These results show that the dominant-negative expression of kinase-inactive c-Src delays the oxidative stress-induced disruption of tight junction and accelerates calcium-induced assembly of tight junction in Caco-2 cells and demonstrate that oxidative stress-induced disruption of tight junction is mediated by the activation of c-Src.
Lindane (gamma-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 microM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 microM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, alpha-tocopherol (25-100 microM), diphenyl-1,4-phenylene diamine (10-30 microM), and superoxide dismutase (100-400 U/ml). D-mannitol (100-300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. alpha-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with alpha-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.
Signaling via intercellular junctions plays an important role in the regulation of growth and differentiation of epithelial cells. Loss of cell-cell contacts has been implicated in carcinogenesis, tumor progression, and metastasis. Here, we investigated whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was able to stimulate the assembly of adherens junctions and/or desmosomes in cultured human keratinocytes. After 4-day incubation, 1,25-(OH)2D3 caused assembly of adherens junctions, but not desmosomes. The adherens junctions were identified upon known ultrastructural criteria and evidence of the translocation of specific junctional proteins (E-cadherin, P-cadherin, alpha-catenin, and vinculin) to the cell-cell borders. The presence of alpha-catenin and vinculin at cell-cell borders indicated that the adherens junctions were functional. This was further supported by showing that anti E-cadherin antibody inhibited the 1,25-(OH)2D3-induced keratinocyte stratification. A relation between protein kinase C and adherens junction regulation was noticed. 1,25-(OH)2D3-dependent formation of junctions was blocked by the inhibitors of protein kinase C, bisindolylmaleimide and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and treatment of keratinocytes with 1,25-(OH)2D3 caused a rapid activation of protein kinase C and its translocation to the membranes. Formation of intercellular contacts may be an important mechanism of 1,25-(OH)2D3 action in hyperproliferative and neoplastic diseases.