Cx26 affects the in vitro reconstruction of human epidermis

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.     

Cx26 Affects the in Vitro Reconstruction of Human Epidermis

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.     

Gap junctional communication of primary human keratinocytes: characterization by dual voltage clamp and dye transfer.

We have compared dye coupling in pairs of small (less than 10 microns in diameter) and large (greater than 20 microns in diameter) keratinocytes isolated from normal human epidermis, using Lucifer yellow microinjection. Under control conditions, dye coupling was found in only 1 out of the 25 small pairs tested, whereas it was evident in 75% of the large pairs (n = 52). After a 30-min incubation of the latter pairs in the presence of 10(-6) and 10(-4) M all-transretinoic acid (RA), the percentage of coupling was 53% (n = 15; NS) and 7% (n = 14; P less than 0.001), respectively. The almost complete uncoupling observed after 10(-4) M RA was not reversible even 30 min after return to control medium (n = 8). Dual whole-cell patch-clamp recordings from large keratinocyte pairs showed a macroscopic junctional conductance (gj) of 9 +/- 2 nS (n = 43), which was abolished by heptanol (3.5 mM) in a fully reversible way. Compared to heptanol, 10(-4) M RA abolished keratinocyte gj more slowly and irreversibly (n = 10). By contrast, 10(-6) M RA had no significant effect on gj (n = 8). Single-gap junctional channels were also identified between large keratinocytes. Events histograms of 152 transitions from three experiments revealed three main unitary conductances (gamma j) of 45 +/- 4, 78 +/- 4, and 106 +/- 7 pS. The dye coupling results indicate that junctional communication is markedly different in pairs of small and large cells, which showed the phenotype and keratin markers of basal and suprabasal keratinocytes, respectively. In the latter cell type, coupling is ensured by channels of three sizes and is blocked irreversibly by pharmacologic concentrations of RA.     

Human papillomavirus type 16 E5 protein affects cell-cell communication in an epithelial cell line.

The human papillomavirus type 16 (HPV16) E5 protein is considered to have weak oncogenic properties, and its function in infected human keratinocytes is unknown. HPV16 E5 protein has been found to localize to the Golgi apparatus and the plasma membrane. To analyze the effect of E5 on plasma membrane properties, cells from the human keratinocyte cell line HaCaT were transfected with the HPV16 E5 open reading frame under the control of an inducible promoter. The gap junction-mediated cell-cell communication of E5- and vector-transfected cells was analyzed by microinjection of Lucifer yellow to measure dye coupling of the cells. A strong impairment of dye transfer in E5-transfected cells but not in vector-transfected cells was observed, with more than 80% dye transfer inhibition 40 min after injection. This impairment correlated with dephosphorylation of connexin 43, the major gap junctional protein in HaCaT cells. Furthermore, the dye coupling inhibition was not the result of differentiation of the E5-expressing cells, since no overexpression of cytokeratin 1 or filaggrin, markers of HaCaT cell differentiation, could be observed. These results therefore strongly suggest a correlation between expression of the HPV16 E5 open reading frame, impairment of gap junction-mediated dye coupling, and dephosphorylation of connexin 43.     

Decreases in heterologous metabolic and dye coupling, but not in electrical coupling, accompany meiotic resumption in hamster oocyte-cumulus complexes

The temporal relationship between resumption of meiosis and reduction in either heterologous intercellular coupling, or magnitude of oocyte or cumulus cell resting potential in hamster oocyte-cumulus complexes was investigated. Coupling was assessed qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites or electrical current after culture of complexes in various systems previously characterized either to maintain meiotic arrest or to permit meiotic resumption. In each of the three systems which permitted meiotic resumption, cumulus to oocyte metabolic and dye coupling and oocyte to cumulus dye coupling decreased progressively with time after release from meiotic arrest. In contrast, no similar temporal changes in metabolic or dye coupling were observed in any complex after culture in either of the two systems which maintained meiotic arrest. Analysis of the extent of heterologous ionic coupling revealed that in neither direction was a decrease in ionic uncoupling consistently associated with reinitiation of meiosis. Furthermore, while the resting potential of both the oocyte and cumulus cell underwent changes characteristic of each system employed, the level of neither cell membrane potential was specific to meiotic status. These results support the hypothesis that meiotic maturation in hamster oocytes is accompanied by disruption of the integrity of intercellular, non-ionic coupling between the oocyte and its adherent cumulus cells. The data show, however, that no specific alteration either in the extent of ionic coupling or in the oocyte or cumulus cell resting potential is prerequisite for meiotic resumption in this species.     

Connexin levels regulate keratinocyte differentiation in the epidermis

To understand the role of connexin43 (Cx43) in epidermal differentiation, we reduced Cx43 levels by RNA-mediated interference knockdown and impaired its functional status by overexpressing loss-of-function Cx43 mutants associated with the human disease oculodentodigital dysplasia (ODDD) in rat epidermal keratinocytes. When Cx43 expression was knocked down by 50-75%, there was a coordinate 55-65% reduction in Cx26 level, gap junction-based dye coupling was reduced by 60%, and transepithelial resistance decreased. Importantly, the overall growth and differentiation of Cx43 knockdown organotypic epidermis was severely impaired as revealed by alterations in the levels of the differentiation markers loricrin and involucrin and by reductions in vital and cornified layer thicknesses. Conversely, although the expression of Cx43 mutants reduced the coupling status of rat epidermal keratinocytes by approximately 80% without altering the levels of endogenous Cx43 or Cx26, their ability to differentiate was not altered. In addition, we used a mouse model of ODDD and found that newborn mice harboring the loss-of-function Cx43(G60S) mutant had slightly reduced Cx43 levels, whereas Cx26 levels, epidermis differentiation, and barrier function remained unaltered. This properly differentiated epidermis was maintained even when Cx43 and Cx26 levels decreased by more than 70% in 3-week-old mutant mice. Our studies indicate that Cx43 and Cx26 collectively co-regulate epidermal differentiation from basal keratinocytes but play a more minimal role in the maintenance of established epidermis. Altogether, these studies provide an explanation as to why the vast majority of ODDD patients, where Cx43 function is highly compromised, do not suffer from skin disease.     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.     

Astroglial injury in an ex vivo model: contributions to its analysis in enriched cell cultures

In vitro cell culture models have been proposed to analyze some of the complex structural and functional characteristics involved in astroglial changes after neural injury in vivo. This report contributes to analyze the proposed hypothesis that an experimentally induced discontinuity of a confluent cellular culture could represent a useful model for the analysis of the processes involved in a neural lesion. For this purpose, it was decided to characterize astroglial proliferation and dye coupling state after a “scratch wound” applied to confluent, astrocyte-enriched cell cultures, obtained from several rat brain regions. Proliferation was assessed in terms of bromodeoxyuridine nuclear incorporation as a function of lesion width, serum deprivation, time after confluence, brain region origin, postlesional culture medium changes and agitation, and after application of a gap-junction uncoupling agent. The proliferative reaction after injury was neither cell type-specific nor brain region specific, nor was significantly affected by neither of the above-mentioned variables. Furthermore, injury failed to significantly affect the astroglial dye coupling state. Results suggest that the proliferative response observed under present conditions would depend on the disruption of contact inhibition rather than on astroglial mitogenic signals released from the wound and operating by either extracellular or cell coupling mechanisms. Present results question the validity of astrocyte-enriched cell cultures as an experimental model of neural tissue injury in vivo, as they do not appear to reproduce fundamental characteristics expressed in situ.     

Induction of intercellular communications in epithelial cell cultures

A popular criterion of cell-cell communication in tissue cultures is dye coupling: the ability of the injected fluorescent dye of low molecular weight to be transferred from one cell to another. We report about a new factor which induces cell-to-cell dye coupling in previously uncoupled epithelial sheets. Paradoxically it is the standard fluorescent microscopy itself (that is, blue light of 320- to 480-nm wavelength) which induces rapid morphological alterations of cell culture followed by the transfer of fluorescent dye from one cell to another. Thus monitoring cell-cell dye coupling by fluorescent microscopy may itself induce the dye coupling in previously uncoupled epithelial cells.     

Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system.

We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co-cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co-cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co-culturing melanocytes with keratinocytes, and processing the co-cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co-culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co-cultures inhibited transfer of fluorochrome by approximately 15-44% as assessed by flow cytometry, and of melanosomes by 67-93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.     

Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro

Heterologous intercellular communication was determined qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites and electrical current in hamster oocyte-cumulus complexes during meiotic maturation in vitro and in vivo. In addition, changes in cell resting potentials during maturation were recorded. Significantly less time was required for germinal vesicle breakdown (GVBD) in oocytes matured in vitro than in oocytes stimulated in vivo (1.81 +/- 0.06 hr, N = 13 vs 2.46 +/- 0.07 hr, N = 18, respectively, P less than 0.001). Resting potentials of the oocyte (RP-o) and cumulus cells (RP-c) significantly increased contemporaneously with GVBD in vitro (RP-o: from -18.9 +/- 3.2 mV to -33.2 +/- 2.9 mV, P less than 0.001; RP-c: from -16.3 +/- 1.9 mV to -27.5 +/- 2.6 mV, P less than 0.001) and in vivo after hCG injection (RP-o: from -16.8 +/- 5.9 mV to -30.1 +/- 3.9 mV, P less than 0.001; RP-c: from -15.5 +/- 3.8 mV to -26.3 +/- 3.2 mV, P less than 0.001). RP-o and RP-c progressively increased with time of culture up to 7 hr (maximum time examined) while the values reached maxima in in vivo matured oocytes 4.5 hr post-hCG and subsequently declined concomitant with the onset of cumulus expansion. Cumulus to oocyte coupling decreased progressively with time after release from meiotic arrest both in vitro and in vivo, as assessed by a progressive reduction in transfer of either uridine marker or lucifer yellow from the cumulus cell to the oocyte. By 4.5 hr after hCG injection, cumulus expansion had begun in 100% of complexes examined. Expansion was extensive by 7 hr post-hCG and spread of lucifer yellow from a cumulus cell was limited to very few adjacent cumulus cells. Oocyte to cumulus cell metabolic coupling also decreased progressively with time in both treatment groups. Examination of the extent of heterologous ionic coupling revealed that ionic coupling exhibited biphasic and, bidirectionally parallel, increases during meiotic maturation. While these temporal changes were observed in both groups, the coupling ratios were much greater in those complexes matured in vitro than in vivo. These results show that dye, metabolic, and electrical coupling exist between the immature hamster oocyte and its surrounding cumulus cells but that during the early stages of meiosis, metabolic and dye coupling decrease, while electrical coupling increases biphasically.     

Changes in junctional communication associated with cell cycle arrest and differentiation of trochoblasts in embryos of Patella vulgata

In early embryos of molluscs, different clones of successively determined trochoblasts differentiate into prototroch cells and together contribute to the formation of a ciliated ring of cells known as the prototroch. Trochoblasts differentiate after cell cycle arrest, which occurs two cell cycles after the commitment of their stem cell. To study the changes of junctional communication in embryos of Patella vulgata in relation to commitment, cell cycle arrest, and differentiation of the trochoblasts, we have monitored electrical coupling as well as transfer of fluorescent dyes. The appearance of dye coupling in embryos of Patella occurs after the fifth cleavage (at the 32-cell stage), when the cell cycles of all embryonic cells become asynchronous and longer. At the 32- and 64-cell stages all cells are well coupled. However, after the 72-cell stage dye transfer to or from any cell of the four interradial clones of four primary trochoblasts becomes abruptly reduced, whereas electrical coupling between these cells and the rest of the embryo can still be detected. From scanning electron microscopical analysis of the cell pattern we conclude that this change in gap junctional communication coincides with cell cycle arrest and with the development of cilia in all four clones of primary trochoblasts. Similarly, after the 88-cell stage the four radial clones of accessory trochoblasts stop dividing, reduce cell coupling, and become ciliated. By the formation of the prototroch, the embryo becomes subdivided into an anterior (pretrochal) and a posterior (posttrochal) domain which will develop different structures of the adult. At the 88-cell stage, the cells within each of these two domains remain well coupled and form two different communication compartments that are separated from each other by the interposed ring of uncoupled trochoblasts. The relations among control of cell cycle, changes in junctional communication, and differentiation are discussed.     

Inhibition of Melanosome Transfer from Melanocytes to Keratinocytes by Lectins and Neoglycoproteins in an In Vitro Model System

We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co‐cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co‐cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co‐culturing melanocytes with keratinocytes, and processing the co‐cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co‐culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co‐cultures inhibited transfer of fluorochrome by approximately 15–44% as assessed by flow cytometry, and of melanosomes by 67–93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.     

Effects of anthrax lethal toxin on human primary keratinocytes

Aims: To investigate the effects of anthrax lethal toxin (LeTx) on human primary keratinocytes. Methods and Results: We show here that human primary keratinocytes are resistant to LeTx‐triggered cytotoxicity. All but one of the MEKs (mitogen‐activated protein kinase kinases) are cleaved within 3 h, and the cleavage of MEKs in keratinocytes leads to their subsequent proteasome‐mediated degradation at different rates. Moreover, LeTx reduced the concentration of several cytokines except RANTES in culture. Conclusions: Our results indicate that primary keratinocytes are resistant to LeTx cytotoxicity, and MEK cleavage does not correlate with LeTx cytotoxicity. Although LeTx is considered as an anti‐inflammatory agent, it upregulates RANTES. Significance and Impact of the Study: According to a current view, the action of LeTx results in downregulation of the inflammatory response, as evidenced by diminished expression of several inflammatory biomarkers. Paradoxically, LeTx has been reported to attract neutrophils to cutaneous infection sites. This paper, which shows that RANTES, a chemoattractant for immune cells, is upregulated after exposure of keratinocytes to LeTx, although a number of other markers of the inflammatory response are downregulated. Our results might explain why the exposure of keratinocytes to LeTx results in the recruitment of neutrophils to cutaneous infection sites, while the expression of several inflammatory biomarkers is diminished.     

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Enhanced Nonlinear Optical Response of Resonantly Coupled Silver Nanoparticle–Organic Dye Complexes

The influence of metal nanoparticles on linear and nonlinear optical properties of surrounding organic molecules has been widely investigated, whereas much less attention has been paid to the influence of molecules on properties of nanoparticles. Here, we employ transient absorption spectroscopy to address the nonlinear optical responses of the resonantly coupled silver nanoparticle–organic dye systems and demonstrate that silver nanoparticles covered with dye molecules show enhanced and spectrally different nonlinear extinction changes from pristine nanoparticles. We identify changes of the plasmon resonance band of nanoparticles induced by excitation of surrounding dye. We attribute these exciton–plasmon coupling effects to the excitation-induced refractive index modifications of the dye layer surrounding a nanoparticle and to the back-transfer of the oscillator strength borrowed by the dye from the nanoparticle.