We studied the effects of vitamin D deficiency and its correction by vitamin D or calcium-lactose supplementation on vitamin D receptor (VDR) expression in skin keratinocytes, kidney, and duodenum of adult rats. VDR messenger RNA (mRNA) was assayed by Northern blot, and VDR protein was determined immunocytochemically. In addition, four subpopulations of keratinocytes were isolated, characterized for their stages of differentiation, and analyzed for VDR expression. Vitamin D deficiency decreased VDR mRNA in all three tissues. Treatment with vitamin D or calcium-lactose reestablished the VDR mRNA content of the epidermis, but not that of the kidneys, and only the calcium-lactose diet increased duodenal VDR mRNA. The regulation of VDR mRNA in the epidermis was independent of cell differentiation, whereas VDR protein varied with differentiation. The VDR-positive cells in the control rats were at early and advanced states of differentiation. The expression of VDR was decreased by vitamin D deficiency and returned to control values after vitamin D or calcium supplementation. Vitamin D treatment, but not calcium, induced VDR expression in the normally immature population. Vitamin D and calcium, therefore, have distinct, tissue-specific effects on VDR. In epidermis, the posttranscriptional regulation of VDR expression is linked to cell differentiation. Calcium may be a key factor for VDR transcription, whereas both vitamin D and calcium seem to contribute to its posttranscriptional regulation.
The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecalciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)2D3 synthesis was studied by measuring the conversion of [3H] 25(OH)D3 to [3H] 1,25(OH)2D3. The purified product was tested for its ability to compete with synthetic [3H] 1,25(OH)2D3 for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogenous 1,25(OH)2D3 were evaluated by the increase in 25(OH)D3-24 hydroxylase activity. Furthermore the expression of 1,25(OH)2D3 receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)2D3. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D3-24 hydroxylase response to 1,25(OH)2D3 were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)2D3 and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)2D3 produced locally by neighboring immature ones.
The hormonal form of vitamin D appears to be a physiological regulator of the epidermogenesis. While its differentiation-promoting effect is well accepted, there are conflicting reports of its action on keratinocyte proliferation. This study evaluates the specific changes induced by vitamin D treatment in the epidermis of rats nutritionally deprived of vitamin D by cell size analysis, acridine orange flowcytometry, and the immunohistochemical detection of proteins related to the different stages of differentiation (epidermal calcium binding protein and suprabasal keratins recognized by KL1 antibody) The total keratinocyte and isolated keratinocyte subpopulations were studied. Vitamin D deficiency was associated in the total population with a lower percentage of actively proliferating cells and with a lack of differentiation markers. Study of the isolated cell populations demonstrated, however, that small cells were actively proliferating, whereas they were mainly in the resting stage in the normal epidermis. Treatment with vitamin D dramatically increased cell proliferation and stimulated the appearance of differentiation markers. Some of the observed effects, such as an increase in proliferation and the appearance of epidermal calcium binding protein, were due to the normalisation of the vitamin D deficiency-induced hypocalcemia, whereas the expression of suprabasal keratins was directly dependent on vitamin D. We conclude that the action of vitamin D on the epidermis is associated with increases in both proliferation and differentiation of keratinocytes. Vitamin D itself and its resulting action on calcium homeostasis appear to contribute to the observed effects.
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