Proliferation and Differentiation of Cultured Human Keratinocytes is Modulated by 1,25(OH)₂D₃ and Synthetic Vitamin D₃ Analogues in a Cell Density‐, Calcium‐ and Serum‐Dependent Manner*

Abstract

Abstract:The natural form of vitamin D3, 1,25-dihydroxyvitamin D3 ( 1,25(OH)2D3) decreases proliferation and promotes terminal differentiation of cultured human epidermal keratinocytes. The purpose of this study was to find out to what extent the culture conditions determine the sensitivety of keratinocytes to 1,25(OH)2D3 and synthetic vitamin D analogues.Human keratinocytes were grown in microplates. Cell proliferation (MTTassay) and differentiation (quantity of transglutaminase type I) were measured consecutively in the same monolayer. When vitamin D3 analogues were added to 50-60% confluent keratinocytes grown in serum-free keratinocyte growth medium with 0.09 mM CaZ+, stimulation of the proliferation was either minimal or non-existent, while differentiation was unaffected or slightly inhibited. There was no difference in the sensitivity to 1,25(OH)2D3 and the vitamin D3 analogues. When 1,25(OH)2D3 was added to less confluent keratinocytes (30%) a marked antiproliferative effect was observed. Addition of 3% charcoal stripped foetal calf serum further enhanced the antiproliferative effect of 1,25(OH)2D3, and a difference in the sensitivity of the vitamin D3 analogues was noted. If, finally, the Ca2+ concentration was raised to 0.3 mM, 1,25(OH)2D3 and the vitamin D3 analogues stimulated differentiation. Also, a biphasic effect on proliferation occurred: stimulation at low vitamin D concentrations and inhibition at higher concentrations. Furthermore, keratinocytes became more sensitive to the synthetic vitamin D3 analogues than to 1.25(OH)2D3: KH1060>EB1089>GS1500~EBl2l3>calcipotriol> 1,25(0H)2D3. For all compounds tested differentiation occurred at concentrations 10 to 30 times lower than for proliferation. These results indicate that the sensitivity to vitamin D3 analogues as well as the direction of the response to vitamin D3 analogues is dependent on the keratinocyte density, the availability of serum and Ca2+ concentrations.The active form of vitamin D3, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), decreases the proliferation and increases the terminal differentiation of cultured mouse (Hosomi et al. 1983) and human (Bikle et al. 1993;Smith et al. 1986) epidermal keratinocytes. The action of vitamin D3 is probably mediated through the classical intracellular vitamin D receptor present in the epidermis (Berger et al. 1988). The effect of 1,25(OH)$3 on keratinocyte proliferation may be mediated by an altered expression of transforming growth factor p (Kim et al. 1992) and c-myc (Matsumoto et al. 1990), while differentiation may be induced via increased transglutaminase and involucrin transcription (Su et al. 1994). In addition, vitamin D3 has nongenomic actions involving altered phosphoinositide metabolism (Sorensen et al. 1993, protein kinase C activation (Hannafin et al. 1995, and a rise in intracellular Ca2+ (Jones et al. 1994). The ef- fect of 1,25(OH)2D3 on intracellular Ca2+ may be important for the differentiation-promoting effect of 1,25(OH)2D3. The effect of vitamin D3 on epidermal proliferation and dif...

Effects of Vitamin D₃ on Keratinocyte Proliferation and Differentiation in vitro: Modulation by Ligands for Retinoic Acid and Retinoid X Receptors

“…In contrast, the addition of epi dermal growth factor to the culture medium potentiated the effect of l,25(OH)2D3. The culture conditions in the present study were chosen based on experiments showing that the antiproliferative and differentiation-pro moting effects of l,25(OH)2D3 in human kér atinocyte cultures were optimal under these conditions [23].…”

Molecular mechanism of vitamin D receptor action

“…Calcitriol exhibits a dual effect on keratinocytes in culture where it stimulates proliferation of keratinocytes committed to 463 Vol. 47,1998 Vitamin D receptor function differentiate, while inhibiting the proliferation of undifferentiated cells [200,201]. Topical application of calcitriol and 20-epi vitamin D analogues MC 1288, MC 1301 and KH 1060 induce epidermal cell proliferation of normal mouse skin [202,203].…”

Vitamin D and the Pathophysiology of Inflammatory Skin Diseases

“…At low concentration (10 -9 M or less), 1,25(OH) 2 D 3 was found to enhance keratinocyte proliferation, while at high concentration (greater than 10 -8 M ), it inhibited the proliferation and promoted the differentiation [11,12]. Several other factors like density of cells, calcium concentrations and presence or absence of serum influence the effect of vitamin D on in vitro keratinocyte proliferation [13]. The antiproliferative action of vitamin D on keratinocytes is mediated by the decreased expression of c-myc and cyclin D and by the increased expression of the cell cycle inhibitors p21 cip and p27 kip [1,14].…”