Retinoic acid (RA) induces tissue transglutaminase (TGASE) and inhibits terminal differentiation induced either by calcium ion or by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary mouse epidermal cells in culture. The relevance of these effects on cultured cells to the antipromoting action of RA was investigated in female BALB/c and CD-1 mice in vivo. Tissue TGASE was distinguished from epidermal TGASE on the basis of different thermolability at pH 9 or elution from the anion exchanger Mono Q. After topical application of 3 to 5 micrograms (10 to 17 nmol) of RA to the shaved back skin, the specific activity of tissue TGASE increased up to 30-fold primarily in the basal cell fraction of Percoll-separated epidermal cells. Enzyme activity returned to basal levels by 7 days. Treatment with TPA (10 micrograms or 17 nmol/mouse) induced an increase in epidermal TGASE which reached a maximum at 12 h after application, primarily in suprabasal cells. RA applied 1 h before TPA caused no reduction of TPA-induced epidermal TGASE, but the increase in tissue TGASE due to RA was markedly inhibited by TPA. The effects of TPA and RA on TGASE activities in primary epidermal cells in culture were similar to those in vivo except that RA reduced the induction of epidermal TGASE by TPA. In culture the induction of epidermal TGASE by TPA was independent of Ca2+ concentration in the medium above 0.03 mM, but cornified envelope formation was markedly enhanced by Ca2+ above the level required for maintaining a basal cell population (0.03 to 0.05 mM). The TPA-induced formation of cornified envelope in the presence of elevated Ca2+ was completely inhibited by RA if cells were pretreated with RA for 24 h. Our results are consistent with RA causing a reprogramming of epidermal cells that alters their response to differentiation stimuli.
Selenium is an essential dietary micronutrient having numerous health benefits. It is incorporated into proteins (selenoproteins) as selenocysteine (Sec). Selenoprotein synthesis depends on Sec tRNA, a unique eukaryotic tRNA that governs the expression of only a single class of proteins, the selenoproteins. Manipulating Sec tRNA structure and expression can selectively alter selenoprotein synthesis. To understand the importance of selenoprotein expression in skin development and function, we targeted the removal of the Sec tRNA gene ( trsp ) in mouse epidermis, using mammalian K14 promoter based loxP‐Cre technology. Our results show that the knockout mice (Δtrsp) have stunted growth and a shorter life span as compared to control mice. They have flaky and wrinkled skin and histological analysis revealed moderate epidermal hyperplasia along with coagulative necrosis of the epidermis. The knockout mice have sparse hair and histological examination suggested that hair morphogenesis might be affected. Real‐time PCR analysis of skin of knockout and control mice shows a decrease in the expression of selenoprotein mRNAs in knockout mice, with the decrease being most significant in epidermis. These studies report a novel and previously unidentified role of selenoproteins in skin function and development. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.
Abstract— Germicidal ultraviolet light (UVC. 8–10 J/m 2 ) induces ornithine decarboxylase (ODC) in mouse epidermal cells in vitro in a biphasic manner with maxima of 2–3 fold induction at 4–6 h and of 10–20 fold induction at 15–18 h after irradiation. At this dose of UVC overall protein synthesis is inhibited by 10–30% and RNA synthesis by 40–50%. Induction of both ODC peaks is prevented by actinomycin D or cycloheximide. Similar culture factors appear to influence the extent of ODC induction by UVC and by the tumor promoter, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), since the ratio of peak activities is approximately constant at 2, whereas absolute values vary considerably between experiments. If cells are irradiated with UVC and then exposed to TPA, the effects are additive at 10 J/m 2 , less than additive at higher and enhanced at lower doses of UVC.
The human papillomavirus (HPV) transforming genes E6 and E7 are retained and expressed in the majority of cervical cancers implying an important role for these proteins in maintenance of the malignant phenotype. Leukoregulin (LR) and recombinant gamma-interferon (r-IFN-gamma), lymphokines secreted by immune cells present in regressing HPV infections, inhibited transcription of E6/E7 RNAs in several human cervical epithelial cell lines immortalized by recombinant HPV-16, -18, and -33 DNAs. r-IFN alpha was not effective. Reduction in E6/E7 RNA expression was accompanied by inhibition of cell proliferation coincident with an increase in epidermal transglutaminase activity, a marker of squamous differentiation. LR and r-IFN gamma enhanced transcription of class 1 cell surface histocompatibility antigens (HLA) and r-IFN gamma additionally induced HLA class 2 expression. HPV-immortalized cells developed partial resistance to the growth inhibitory effects of lymphokines after malignant transformation or extended propagation in culture. This is the first demonstration that LR and r-IFN gamma selectively inhibit transcription of HPV-transforming genes and suggests a molecular mechanism by which these lymphokines participate in regression of premalignant cells.
Abstract Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.02–0.09 mM. Terminal differentiation and slouching of mature kcratinocytes occur when the calcium concentration is increased to 1.2–1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium‐induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12‐0‐tetradccanoylphorbol‐13‐acetate (TPA) in basal cells is enhanced 20‐fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results arc consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells arc sloughed from the dish, the remaining basal cells proliferate and become resitant to induced differentiation by 1.2 m M calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cell, would be expected to increase in number during remodeling. Clonal expansion of the intitiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2‐stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.
