Targeted Disruption of the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene in ras-Transformed Keratinocytes Demonstrates That Locally Produced 1α,25-Dihydroxyvitamin D3 Suppresses Growth and Induces Differentiation in an Autocrine Fashion11 Canadian Institutes of Health Research Grant MT10839 (to R.K.).

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D3 (25-OHD3) 1α-hydroxylase (1α-hydroxylase). 1α-Hydroxylase catalyzes the conversion of 25-OHD3 to 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 is both antiproliferative ( i.e. , suppresses cell growth) and prodifferentiative ( i.e. , induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)2D3 by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1α-hydroxylase alleles in a ras -transformed keratinocyte cell line, HPK1A ras , which typically produces squamous carcinoma in nude mice. To inactivate 1α-hydroxylase expression by HPK1A ras cells, we disrupted both alleles of the 1α-hydroxylase gene by homologous recombination. Lack of expression and activity of 1α-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD3 to 1,25(OH)2D3. We then examined the effect of substrate 25-OHD3 on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1A ras cells in vitro . It was found that 1α-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD3. These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD3. Our results demonstrate, for the first time, that 1α-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.