Extrarenal Expression of 25-Hydroxyvitamin D3-1a-Hydroxylase*

The mitochondrial enzyme 25-hydroxyvitamin D3-1a-hydroxylase (1a-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3, in the kidney. However, enzyme activity assays indicate that 1a-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1a-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1a-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1a-hydroxylase in both normal and diseased tissues. Specific staining for 1a-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Further studies using psoriatic skin highlighted overexpression of 1a-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1a-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin from these patients 1a-hydroxylase expression was observed in cells positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1a-hydroxylase in several extrarenal tissues, such as skin, placenta, and lymph nodes. The function of this enzyme at novel extrarenal sites, such as adrenal medulla, brain, pancreas, and colon, remains to be determined. However, the discrete patterns of staining in these tissues emphasizes a possible role for 1a-hydroxylase as an intracrine modulator of vitamin D function in peripheral tissues. (J Clin Endocrinol Metab 86: 888– 894, 2001) T ACTIVE FORM of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is a pluripotent seco steroid, which, in addition to its direct calciotropic actions, may play an important role in modulating cell proliferation and differentiation (1–4). Synthesis of 1,25-(OH)2D3 from the major circulating form of vitamin D, 25-hydroxyvitamin D3 [25 (OH)D3] is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D3-1a-hydroxylase (1a-hydroxylase), which is classically expressed in the kidney (5–7). However, enzyme activity studies using a variety of tissues have suggested that synthesis of 1,25-(OH)2D3 occurs at several key peripheral sites, including the immune system and skin (3, 8, 9). In contrast to the kidney, which supports the systemic, endocrine actions of 1,25-(OH)2D3, extrarenal 1ahydroxylase appears to act in an autocrine or paracrine fashion by modulating cell differentiation and/or function at a local level (1–4, 10–12). This has generated considerable interest in the possible application of 1,25-(OH)2D3 and its analogs in the treatment of myeloid leukemias (10, 12), psoriasis, and type 1 diabetes (10, 11). The original description of extrarenal 1a-hydroxylase was based on studies of the granulomatous disease sarcoidosis, which supported a link between ectopic synthesis of 1,25(OH)2D3 and the hypercalcemia that is frequently observed with this disorder (13–15). Detailed analysis of lymph node homogenates and pulmonary alveolar macrophages from patients with sarcoidosis demonstrated significant levels of 25(OH)D3 to 1,25-(OH)2D3 conversion (16–19). Importantly, and in contrast to renal 1a-hydroxylase, addition of exogenous 1,25-(OH)2D3 did not inhibit macrophage 1a-hydroxylase. This provided a potential mechanism for the apparently unregulated synthesis of 1,25-(OH)2D3 that is characteristic of the more severe forms of this disease (14, 15). It also suggested that the expression and regulation of 1a-hydroxylase in extrarenal tissues were different from those observed with the kidney enzyme. Unfortunately, the absence of nucleotide and amino acid sequence data for 1a-hydroxylase has, until recently, prevented further characterization of the extrarenal enzyme. However, in the last 2 yr several groups have reported gene and cDNA sequences for 1a-hydroxylase in various species (20–24). As well as providing insight into the molecular basis of disorders associated with abnormal renal 1a-hydroxylase expression (22, 23, 25–27), these studies have generated new information on the ectopic expression of the enzyme. Most notably, the first normal and mutant human cDNAs for 1a-hydroxylase were cloned from keratinocytes, an important extrarenal source of 1,25-(OH)2D3 (11, 22, 28). Studies indicate that a similar messenger ribonucleic acid (mRNA) for 1a-hydroxylase is expressed in both renal and extrarenal tissues (22, 29). Based on these observations we have used immunohistochemical techniques previously deReceived April 26, 2000. Revision received October 4, 2000. Accepted November 1, 2000. Address all correspondence and requests for reprints to: Dr. Martin Hewison, Division of Medical Sciences, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, Birmingham, United Kingdom B15 2TH. E-mail: [email protected]. * This work was funded in part by a Medical Research Council project grant. † Medical Research Council Senior Clinical Fellow. 0021-972X/01/$03.00/0 Vol. 86, No. 2 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright © 2001 by The Endocrine Society