Variable Degrees of 1-{alpha} Hydroxylase Activity—Fine Tuning the Rachitic Rheostat

Thomas O. Carpenter

Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8064

Address all correspondence and requests for reprints to: Thomas O. Carpenter, M.D., Department of Pediatrics, Yale University School of Medicine, P.O. Box 208064, 333 Cedar Street, New Haven, Connecticut 06520-8064. E-mail: . thomas.carpenter{at}yale.edu

What is vitamin D dependency?

Treatment of the endemic vitamin D-deficiency rickets that occurred in the early years of the 20th century in North America revealed an interesting phenomenon. Many patients responded to treatment with pharmacologic vitamin D, and in almost all cases the recurrence of rachitic disease was prevented by providing very small doses of vitamin D, based on a daily nutritional allowance. A small number of patients, however, after having responded favorably to pharmacologic doses of vitamin D, relapsed into the rachitic state when doses were lowered to the standard daily supplementation that prevented rickets in the bulk of the population. This distinct form of rickets was given the name vitamin D-dependent rickets, reflecting the affected children’s unusual dependence on pharmacologic doses of vitamin D required to prevent the rickets. The clinical presentation, similar to severe vitamin D deficiency, also prompted the term pseudo-deficiency rickets. These terms differentiated this group from the more common vitamin D-deficient group (who never recurred when provided the small dietary allowance doses of vitamin D), and the vitamin D-resistant group (who even failed to show a clinical response to pharmacologic doses of vitamin D). A large proportion of vitamin D-resistant cases have been demonstrated to manifest rickets in association with renal phosphate wasting; and, similarly, specific biochemical defects in vitamin D metabolism and action have been identified, which account for a large proportion of cases of vitamin D-dependent rickets.

Vitamin D metabolism and its critical regulation

Intimately tied to these important clinical observations are a lineage of parallel scientific discoveries: the discovery of vitamin D; the detailed description of the vitamin D synthetic pathway; the discovery that a 1{alpha}-hydroxylated metabolite is the important form of the vitamin for systemic calcium homeostasis; and the discovery of the importance of a nuclear receptor of the steroid hormone receptor family in mediating the effects of 1,25 dihydroxyvitamin D (1,25(OH)2D), establishing this metabolite as a classical hormone. The clinical observations have led to speculations regarding the science, and the scientific discoveries have provided the physiological background on which to understand the clinical disorders and provide appropriate therapies.

The past several decades have seen scientific developments that stem from the work of Fraser and Kodicek (1) in 1970, identifying that the bioactive form of vitamin D is a 1{alpha}-hydroxylated metabolite and determining that it is synthesized in the kidney. A number of subsequent studies in the laboratories of Drs. Henry, Norman, Haussler, DeLuca, Rasmussen, and many others focused on the detailed biochemical regulation of the vitamin D 1{alpha}-hydroxylase enzyme (reviewed in Ref. 2).

Vitamin D is either ingested from the diet (thus its legacy as a vitamin) or synthesized in the skin from a metabolite in the cholesterol synthetic pathway, 7-dehydrocholesterol (suggesting a hormonal classification). The conversion of 7-dehydrocholesterol to previtamin D is dependent on the penetration of UV light (290–315 nm) to the stratum spinosum of the skin. These specific wavelengths of UVB light provide the energy to disrupt the covalent bond between carbons 9 and 10 in the B steroid ring of cholesterol. This conversion is followed by rapid, temperature-dependent isomerization of previtamin D to vitamin D, which is found in measurable amounts in the circulation, bound to a specific vitamin D binding protein. Vitamin D is subsequently hydroxylated at the 25 and 1 position carbons to form the active metabolite, 1,25(OH)2 D. The first of these enzymatic hydroxylations is performed by a hepatic P-450 system, vitamin D 25-hydroxylase, producing 25-OHD, a circulating metabolite that is often measured as an index of total body vitamin D stores. 25-OHD has numerous metabolic fates, but of greatest interest is its conversion to 1,25(OH)2D by a specific 25-hydroxyvitamin D 1{alpha}-hydroxylase (1-OHase), which is primarily found in renal tubular mitochondria. It was recognized shortly after the discovery of 1,25(OH)2D that this latter enzymatic reaction is the crucial regulatory control point of 1,25(OH)2D production (2). This feature has led some investigators to regard the 1-OHase as the Holy Grail of vitamin D metabolism (3).

Much work in the late 1970s and early 1980s characterized the regulation, kinetics, and other biochemical properties of this complex enzyme system (2, 4). The 1-OHase is part of the electron transport machinery of the inner mitochondrial membrane, requiring molecular oxygen and facilitating the delivery of electrons through a redox pathway involving a flavoprotein reductase, a renal ferrodoxin, and a unique cytochrome P450 that renders specificity for the 25-OH vitamin D substrate. The major regulatory features of enzyme activity include stimulation by PTH and hypophosphatemia and inhibition by 1,25(OH)2 D itself. Calcitonin has been shown to stimulate enzyme activity under certain conditions, and evidence has been presented to suggest that extracellular calcium, independent of PTH may also regulate this enzyme (5, 6). Certain intracellular toxins have been shown to interfere with 1-OHase activity, and the growth hormone/IGF-I axis may modulate phosphate regulatory effects.

From speculation to proof: 1-OHase and vitamin D-dependent rickets

Scientific and clinical observations have continued to contribute complementary aspects of the vitamin D story, weaving the pieces together in useful and interesting ways. Very shortly after the discovery of 1,25(OH)2D, the group at Hospital for Sick Children in Toronto reasoned that vitamin D-dependent rickets must be an inborn error of vitamin D metabolism due to a genetic defect of the 1-OHase enzyme complex (7), although assays of vitamin D metabolites were not available at the time. This study described five patients with characteristic clinical and biochemical features of rickets that failed to respond to low-to-moderate doses of vitamin D administered under observation in the hospital for a 2-month period. Subsequent investigations determined that although high, pharmacologic doses of vitamin D or 25 hydroxyvitamin D (25-OHD) were required to correct the rachitic and biochemical abnormalities, minute doses of 1,25(OH)2D were effective in this regard. The response to very high doses of vitamin D was explained by an expected high concentration of 25-OHD resulting from this therapy. Very high levels of this precursor were speculated to have some cross-reactivity with 1,25(OH)2D, or alternatively to be sufficiently high to yield a very limited, but sufficient degree of conversion to 1,25(OH)2D, albeit in the absence of a functional enzyme.

This important work, together with the emerging laboratory investigations of the 1-OHase enzyme complex, led to the very plausible hypothesis that mutations in the gene for the enzyme could result in the clinical disorder described as vitamin D-dependent rickets. The first evidence that this indeed was the case was provided by Glorieux et al. (8). These workers established that placental 1-OHase was defective in patients with vitamin D-dependent rickets. They then mapped vitamin D-dependent rickets in the French Canadian population to chromosome 12q13-q14 (9) and subsequently used a vitamin D 24-hydroxylase probe to identify and clone 1-OHase cDNA (10). These techniques mapped the human 1-OHase gene to 12q13.1-q13.3, the same chromosomal region as vitamin D-dependent rickets, thereby providing strong evidence for this hypothesis.

The discovery of mutations in the gene

Investigators in Tokyo and San Francisco subsequently identified inactivating mutations in the 1-OHase gene in vitamin D-dependent rickets (11, 12). Kitanaka et al. (11), of the Tokyo group, cloned the human 1-OHase cDNA and identified mutations in four unrelated patients, all with unique, homozygous missense mutations. Using a reporter gene assay, no activity was evident from any of the mutant 1-OHase constructs when expressed in vitro. In a similar study, Fu et al. (12), of the San Francisco group, cloned and sequenced the 1-OHase cDNA and demonstrated that primary keratinocyte cultures from an affected patient with vitamin D-dependent rickets had no detectable activity. The patient studied was a compound heterozygote for two mutations, a frame shift, and a deletion. Other mutations have now been identified (13).

In this issue of the Journal of Clinical Endocrinology and Metabolism, Wang et al. (14), of the San Francisco group, have furthered the group’s earlier work regarding the molecular basis of this disease. The current paper describes five novel mutations and reports that two patients with a mild clinical and biochemical phenotype have mutations in 1-OHase that confer partial activity in vitro. The study would suggest that there can be variable degrees of vitamin D dependence, and it raises questions regarding the potential physiological significance of subtle alterations in the function of this enzyme.

Of note, the Tokyo group has demonstrated a mutation in a mildly affected individual, however no residual enzyme activity was detected in vitro (15).

Back to the laboratory: animal models and studies of genetic regulation of the enzyme

Recent studies in mice with targeted ablation of the 1-OHase gene have produced an animal model of vitamin D-dependent rickets. In the study by Dardenne et al. (16), the defect in 1,25(OH)2D production is complete, resulting in nondetectable serum levels of 1,25(OH)2D levels associated with an increase in 25(OH)D levels. The eventual development of osteomalacia and disruptive growth plate architecture validate this as an animal model of the human disease. In the study by Panda et al. (17), similar findings are described. However, uterine hypoplasia and T cell defects are evident in the latter model, an interesting finding in view of similar abnormalities described in the vitamin D receptor null mouse (18), and provide evidence that vitamin D plays important roles in the reproductive and immune systems. Interestingly, the characteristic alopecia seen in both patients and mice with nonfunctional vitamin D receptors is not recapitulated in these animal models, suggesting that the vitamin D receptor is the critical piece for development of alopecia.

A number of more recent studies have further characterized the 1-OHase gene and its regulation. Yamagata et al. (19) have shown that 1-OHase mRNA is detected at 13 d gestation in the mouse, in the interior regions of embryonic kidneys. Both the murine and human genes have nine exons with well conserved exon/intron organization. Immunohistochemistry and in situ hybridization have demonstrated that primary sites of expression are in the distal convoluted tubule, the cortical and medullary parts of the collecting ducts, and the papillary epithelia (20). These data differ from earlier studies that projected that proximal tubules were the major site of enzyme activity. Indeed, as determined in the earlier enzyme studies of previous decades, the genetic regulation of 1-{alpha} hydroxylase gene occurs with PTH and calcitonin; negative regulation with 1,25(OH)2 D is also evident (21). A variety of other tissues express 1-OHase; however, their importance for circulating levels is less important than those contributed by the kidney. Extrarenal sites of 1,25(OH)2 D synthesis probably result in 1,25(OH)2 D production for the local or nearby tissue. It is also likely that regulation of extrarenal production of 1,25(OH)2 D is under somewhat different control than the renal enzyme.

Variable degrees of vitamin D dependency

The current study has identified five new mutations of this gene disorder. Three of the five mutations described are missense mutations, and two of these reveal significant residual enzyme activity when expressed in an in vitro cell culture system. The partial enzyme activity in vitro correlated with a mild biochemical phenotype, confirming the likely expectation that structure-function themes are evident in the 1-OHase system. This is analogous to numerous other genetic disorders and in particular mutations in other cytochrome P450 hydroxylase systems such as those observed in patients with congenital adrenal hyperplasia. The mutation that results in the greatest residual enzyme activity is proposed as resulting in conformational changes that would allow for substrate binding but with decreased affinity for the ferrodoxin component of the electron transport cascade, which would limit enzyme efficiency. Indeed, it will be of interest to see whether further studies of such patients reveal mutations in the ferrodoxin or flavoprotein reductase components of the enzyme complex, as described over 30 yr ago.

The finding of partial enzyme activity may explain the effect of pharmacologic doses of vitamin D, or the vitamin D-dependent nature of the disorder. More importantly, the finding raises the possibility that other amino acid substitutions could result in very modest functional alterations in the enzyme. Should this be the case, one may begin to ask whether polymorphisms in 1-OHase could result in increased susceptibility to diseases such as nutritional rickets and osteoporosis.

Acknowledgments

Footnotes

Abbreviations: 1-OHase, 25-Hydroxyvitamin D 1{alpha}-hydroxylase; 25-OHD, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25 dihydroxyvitamin D.

Received April 9, 2002.

Accepted April 9, 2002.

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