Differential expression of connexins during stratification of human keratinocytes

To assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes.