Jay A. White

cDNA Cloning of Human Retinoic Acid-metabolizing Enzyme (hP450RAI) Identifies a Novel Family of Cytochromes P450 (CYP26)

Retinoids, including all-trans-retinoic acid (RA) and its stereoisomer 9-cis-RA play important roles in regulating gene expression, through interactions with nuclear receptors, during embryonic development and in the maintenance of adult epithelial tissues (Chambon, P. (1995) Rec. Prog. Horm. Res. 50, 317–32; Mangelsdorf, D. J., and Evans, R. M. (1995)Cell 83, 841–850; Petkovich, M. (1992) Annu. Rev. Nutr. 12, 443–471). Evidence suggests that 4-hydroxylation of RA inside the target cell limits its biological activity and initiates a degradative process of RA leading to its eventual elimination. However, 18-hydroxylation and glucuronidation may also be important steps in this process. In this paper, we describe the cloning and characterization of the first mammalian retinoic acid-inducible retinoic acid-metabolizing cytochrome P450 (hP450RAI), which belongs to a novel class of cytochromes (CYP26). We demonstrate that hP450RAI is responsible for generation of several hydroxylated forms of RA, including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. We also show that hP450RAI mRNA expression is highly induced by RA in certain human tumor cell lines and further show that RA-inducible RA metabolism may correlate with P450RAI expression. We conclude that this enzyme plays a key role in RA metabolism, functioning in a feedback loop where RA levels are controlled in an autoregulatory manner.

Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

Expression and Activity of Vitamin D-Metabolizing Cytochrome P450s (CYP1α and CYP24) in Human Nonsmall Cell Lung Carcinomas1

Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.

Modified-Release Calcifediol Effectively Controls Secondary Hyperparathyroidism Associated with Vitamin D Insufficiency in Chronic Kidney Disease

Background/Aims: Vitamin D insufficiency drives secondary hyperparathyroidism (SHPT) and is associated with increased cardiovascular mortality in patients with chronic kidney disease (CKD). SHPT is poorly addressed by current vitamin D repletion options. The present study evaluated a novel investigational vitamin D repletion therapy: a modified-release (MR) formulation of calcifediol designed to raise serum 25-hydroxyvitamin D in a gradual manner to minimize the induction of CYP24 and, thereby, improve the SHPT control. Methods: This randomized, double-blind, placebo-controlled trial evaluated MR calcifediol in CKD subjects (n = 78) with plasma intact parathyroid hormone (iPTH) >70 pg/ml and serum total 25-hydroxyvitamin D <30 ng/ml. Subjects received daily treatment for six weeks with oral MR calcifediol (30, 60 or 90 µg) or a placebo. Results: More than 90% of subjects treated with MR calcifediol achieved serum 25-hydroxyvitamin D levels ≥30 ng/ml versus 3% of subjects treated with placebo (p < 0.0001). Mean plasma iPTH decreased from baseline (140.3 pg/ml) by 20.9 ± 6.2% (SE), 32.8 ± 5.7 and 39.3 ± 4.3% in the 30, 60 and 90 µg dose groups, respectively, and increased 17.2 ± 7.8% in the pooled placebo group (p < 0.005). No clinically significant safety concerns arose during MR calcifediol treatment. Conclusion: Oral MR calcifediol appears safe and highly effective in treating SHPT associated with vitamin D insufficiency in CKD.

Position Statement from ADA/AACE/EASD/TES in Response to a Recently Published Letter to the Editor in The Lancet and an Editorial Addressing the Israeli-Palestinian Fighting in Gaza

Derek LeRoith, MD, PhD, Editor in Chief, Endocrine Practice R. Mack Harrell , MD, President, American Association of Clinical Endocrinologists George Grunberger, MD, President Elect, American Association of Clinical Endocrinologists Leonard Wartofsky, MD, Editor in Chief, The Journal of Clinical Endocrinology and Metabolism Andrea C. Gore, PhD, Editor in Chief, Endocrinology Margaret Wierman, MD, Acting Editor in Chief, Endocrine Reviews Stephen R. Hammes, MD, PhD, Editor in Chief, Molecular Endocrinology Carol A. Lange, PhD, Editor in Chief, Hormones and Cancer Richard J. Santen, MD, President, Endocrine Society George L. Bakris, MD, Editor in Chief, American Journal of Nephrology