Studies of the expression of gap junctional protein Connexin43 in dermis and epidermis of human skin : differential modulation by retinoic acid

Guo, Huiming
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Retinoids are effective modulators of proliferation and differentiation of epidermal keratinocytes in vivo and in vitro. Their action in most cases appears due to an alteration in the program of squamous differentiation in normal or diseased epidermal cells. However the mechanism of action of retinoids on skin is not understood at the molecular level. In mouse 10T1/2 cells, the ability of retinoids to suppress neoplastic transformation and enhance growth control is highly correlated with their ability to increase gap junctional intercellular communication and expression of connexin 43. In order to determine if these results have relevance to retinoid action in the human, the interaction of retinoic acid with connexin 43 gene expression in human skin and the potential role of gap junctional communication in the control of differentiation in human skin cells were investigated in the present study. In intact human skin, after two weeks treatment with topical all-trans retinoic acid (RA), levels of Cx43 mRNA and protein increased significantly in epidermis. Immunofluorescence microscopy using an anti Cx43 antibody demonstrated that expression occurred predominantly in the suprabasal layer of epidermis was associated with the thickening of the epidermal layer and the disappearance of the keratinized layer. Furthermore, enhanced Cx43 expression has also been observed in keratinocytes grown on a collagen matrix. Expression was located in suprabasal cells as in intact skin. However, conventional monolayer cultured skin cells are unable to enter a program of normal differentiation and exhibited a differential response to retinoic acid treatment. In keratinocytes increased expression of Cx43 occurred at low (10^-11M ) concentrations whereas inhibition occurred at high (10^-7M ) concentrations, but junctional communication, measured by dye transfer, was not altered over this concentration range. Dermal fibroblasts, in contrast, exhibited a dose-dependent increased expression of Cx43 at concentrations up to 10-7M retinoic acid and' proportionately increased their junctional communication over this dose range. From these observations, it was suggested that regulation of Cx43 gene expression and junctional communication by retinoids in human skin cells is complex. In basal epidermal cells (i.e. those grown on monolayer) Cx43 may not be the major protein involved in intercellular communication. Biochemical changes associated with the differentiation of keratinocytes in the epidermis involve alterations in the pattern of keratin gene expression. Thus keratin gene expression can be used as a marker for determining the process of keratinocyte differentiation. The effect of retinoic acid on keratin 14 expression was studied in 3-dimensional organotypic cultures (raft) where differentiation is allowed. In raft cultures, retinoic acid increased K14 expression. This change of K14 expression by retinoic acid could result from an increase in the number of cells expressing K14. However, it was not shown that the effect of retinoic acid on K14 expression is correlated with Cx43 expression. These data indicate that Cx43 may not be directly involved in the modulation of K14 expression by retinoids. Nevertheless, it may not be excluded that Cx43 plays a role in other aspects of keratinocyte differentiation processes.
Thesis (Ph. D.)--University of Hawaii at Manoa, 1992.
Includes bibliographical references (leaves 164-198).
xv, 198 leaves, bound ill. 29 cm
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Biomedical Sciences (Biophysics); no. 2813
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