Differential induction of connexins 26 and 30 in skin tumors and their adjacent epidermis.

Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of CK6, a marker for hyperproliferation, and CK17, a marker for trauma, we discuss that the induction of these gap junctional proteins exceeds a reflection of reactive hyperproliferative or traumatized epidermis. We further discuss the putative roles of these gap junctional proteins in tumor progression.

In vitro isolation and cell culture of vestibular inner ear melanocytes.

INTRODUCTION: Melanocytes of the membranous labyrinth of the inner ear have been described morphologically in various contexts. Nature and functions of these cells are as yet not completely clear, even though several hypotheses exist regarding the same. The limited knowledge is due in part to a lack of methods regarding in vitro cell culture. The aim of this study was to describe conditions for the successful cell culture of vestibular inner ear melanocytes (VIEM), to compare their growth properties with those of epidermal melanocytes, and to characterize them immunohistochemically. MATERIALS AND METHODS: Membranous labyrinth cells from freshly slaughtered sheep were isolated, and melanocytes and fibroblasts subsequently cultured. In addition, melanocytes from the skin of the same sheep were cultured. Antibodies specific to tyrosinase, tyrosinase-related protein 1 (TRP-1/Mel-5), and melanoma-specific antigen A (Melan A) were used to analyze the cultured cells. RESULTS: The proliferation of VIEM was retarded in comparison to epidermal melanocytes. After 14 days, VIEM began to proliferate for the first time, whereas epidermal melanocytes proliferated already after 7 days. In contrast to epidermal melanocytes, the culturing process of VIEM seemed to be dependent on the presence of fibroblasts, and VIEM often accumulated in the vicinity of fibroblasts forming three-dimensional clusters. Moreover, VIEM showed a higher ratio of highly pigmented cells with a round cell shape and small dendrites in comparison to epidermal melanocytes. Immunohistochemical techniques proved the VIEM to be positive for Melan A, TRP-1 and, in the majority of cases, also for tyrosinase. CONCLUSION: We successfully cultured melanocytes of the inner ear vestibular labyrinth for the first time and demonstrated melanocytic characteristics of these cells. This accomplishment will provide the opportunity to investigate VIEM in more detail in future experiments.

Expression and localization of tight junction-associated proteins in human hair follicles.

Tight junctions (TJ) are barrier-forming intercellular junctions selectively sealing cells and controlling the paracellular pathway. They have been well-characterized in simple epithelia and endothelia but have only recently been described in stratified epithelia such as epidermis, oesophagus and oral mucosa. Various epithelial layers which are partly in morphogenic continuity with the epidermis and develop therefrom during early fetal life, build the human hair follicle. The barrier function of these epithelial layers seems to be important for the universal continuity of the barrier represented by the skin. We show the presence of the TJ proteins ZO-1, occludin, and various claudins in the hair follicle and demonstrate their impressive heterogeneous distribution pattern within a given stratum as well as within its different epithelial layers. Coexpression of the various TJ proteins, arguing for typical TJ structures, can be observed especially in cell layers facing the hair shaft and the stratum corneum, and in addition at the border between the outer and inner root sheaths. Usually they are found in close proximity to desmosomal and adherens junction proteins. The morphological and biological importance of these findings and the possible roles of TJ in hair follicles, e.g. in follicular penetration, are discussed.

Organization and formation of the tight junction system in human epidermis and cultured keratinocytes.

Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are discussed.