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Signaling and Regulation

Targeted Disruption of the 25-Hydroxyvitamin D₃ 1-Hydroxylase Gene in ras-Transformed Keratinocytes Demonstrates That Locally Produced 1,25-Dihydroxyvitamin D₃ Suppresses Growth and Induces Differentiation in an Autocrine Fashion¹

¹ Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, Quebec, Canada;
² Center for Prostate Disease Research, Uniformed Services, University of the Health Sciences, Bethesda, MD; and
³ USDA, Agriculture Research Service, National Animal Disease Center, Ames, IA

Requests for reprints: Richard Kremer, Room H4.67, Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1 Canada. Phone: (514) 843-1632; Fax: (514) 843-1712.

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D₃ (25-OHD₃) 1-hydroxylase (1-hydroxylase). 1-Hydroxylase catalyzes the conversion of 25-OHD₃ to 1,25-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 1-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1-hydroxylase gene by homologous recombination. Lack of expression and activity of 1-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 HPK1Aras cells in vitro. It was found that 1-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 1-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.

Key Words: cancer • vitamin D • knockout • keratinocytes • 1-hydroxylase

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