Effect of three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 on 25-OH-D3-1 alpha-hydroxylase in isolated mitochondria and on 25-hydroxyvitamin D3 metabolism in cultured kidney cells

Three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)--2-nor-1,3-seco-1,25(OH)2D3 (2-nor analog), 2-oxa-3-deoxy-25-OH-D3 (2-oxa analog), and A-homo-3-deoxy-3,3-dimethyl-2,4-dioxa-25-OH-D3 (A-homo analog)--were tested for their ability to inhibit 25-OH-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) in isolated mitochondria and to alter 25-OH-D3 metabolism in cultured chick kidney cells. The 2-nor and 2-oxa analogs were relatively potent (Kis of 60 and 30 nM, respectively, compared with 170 nM for 1,25(OH)2D3), whereas the A-homo analog was completely ineffective in inhibiting 1 alpha-hydroxylase activity. In contrast, all three analogs were able to repress 1 alpha-hydroxylase and induce 24-hydroxylase activity in cultured chick kidney cells, suggesting that this process is not one of direct action in the mitochondria, but is more likely to be a receptor-mediated one.     

24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs

A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D(3) toxicosis.     

Maternal-perinatal interrelationships of vitamin D metabolism in rats.

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite that ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1.Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet.Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 5/25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected.Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol.Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

Incorportion of oxygen-18 into the 25-position of cholecalciferol by hepatic cholecalciferol 25-hydroxylase.

The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase ('mixed-function oxidase').     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Altered vitamin D metabolism in the kidney of the spontaneously hypertensive rat.

A decrease in plasma Ca2+ and increases in plasma immunoreactive parathyroid hormone (PTH) have been reported in spontaneously hypertensive (SH) rats as compared with normotensive Wistar-Kyoto (WKy) rats. These changes should lead to a higher plasma 1,25(OH)2D (1,25-dihydroxycholecalciferol/1,25-dihydroxyergocalciferol) concentration in SH rat if the kidney responds appropriately. Plasma 1,25(OH)2D, however, has been reported to be normal in SH rats, suggesting possible impairments of vitamin D metabolism in this animal model of hypertension. To test this possibility, we studied the effect of PTH on renal production of 1,25(OH)2D in SH rats before (4 weeks of age) and after (12 weeks of age) the onset of hypertension. Basal serum levels of 1,25(OH)2D were normal in SH rats at both ages. At 4 weeks of age, the rise in serum 1,25(OH)2D after PTH injection (50 units subcutaneously every 2 h; four times) was also normal in SH rats. By contrast, at 12 weeks of age, the rise in serum 1,25(OH)2D was approximately one-half of that in WKy rats, despite the similar rises in serum Ca2+ levels in both groups by PTH injection. The attenuated rise in serum 1,25(OH)2D in SH rats was consistent with the impaired response of renal 1-hydroxylase (25-hydroxycholecalciferol 1 alpha-hydroxylase) activity to PTH. Basal 1,25(OH)2D production by the kidney in SH rat was higher than that in WKy rats both at 4 and 12 weeks of age. These data suggest that, in SH rats: serum 1,25(OH)2D is inappropriately low in relation to the elevated PTH and this may be due, at least in part, to the impaired responsiveness to PTH of renal 1-hydroxylase and to the enhanced metabolism of 1,25(OH)2D, and elevated PTH or other agents may stimulate the 1-hydroxylase in the kidney even before the onset of hypertension.     

Antigenic and catalytic disparity in the distribution of cytochrome P-450-dependent 25-hydroxyvitamin D3-1 alpha- and 24-hydroxylases

Chick 25-hydroxyvitamin D3-1 alpha-hydroxylase, a cytochrome P-450 monooxygenase with a molecular weight of 57 kDa, can be isolated as described by Mandel et al. (1990 b). Under normal physiological circumstances, it occurs exclusively in kidney mitochondria. An isozyme of the 1 alpha-hydroxylase, known as the 24-hydroxylase, which uses the same substrate to yield an isomeric product, is also a cytochrome P-450 monooxygenase, has a molecular weight of 55 kDa, and like-wise occurs in kidney mitochondria. The amino-terminal sequences of the first 10 residues of the two isozymes are 100% homologous. Monoclonal antibodies of the IgM class raised against the 1 alpha-hydroxylase, which quantitatively discriminate against other P-450 cytochromes of mitochondrial or microsomal origin, recognize and interact with the 24-hydroxylase as an antigen. In the present study we show that the intestine, which is the only non-renal tissue with demonstrable 24-hydroxylase activity, gives a positive peroxidase-antiperoxidase immunohistochemical reaction using the monoclonal antibodies against the 1 alpha-hydroxylase. The reactions revealed that the antigen in the kidney is restricted to the cortical proximal tubular cells while in the intestine, the antigen is localized in the enterocytes of the villi. In kidney medullary or intestinal crypt cells, or in liver, heart and lung tissues where 1 alpha-hydroxylase or 24-hydroxylase activity could not be detected using cell or tissue homogenates, the immunohistochemical reactions were also negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Submitochondrial localization of kidney 25-hydroxycholecalciferol 1 alpha-hydroxylase in vitamin D repleted weanling guinea pigs

We have studied the submitochondrial localization of guinea-pig kidney 25-hydroxycholecalciferol 1 alpha-hydroxylase. Treatment of the mitochondrial-enriched fraction with recrystallized digitonin produced mitoplasts bordered by a single membrane and with intact matrix. They contained nearly 90% of the 25-hydroxycholecalciferol 1 alpha-hydroxylase activity and nearly 100% of the cytochrome-c:oxygen oxidoreductase. Amine:oxygen oxidoreductase activity remained mainly in the outer membrane fraction. These data show that 25-hydroxycholecalciferol 1 alpha-hydroxylase has a distribution similar to that of steroid hydroxylases.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.     

A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates

A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.     

1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone

A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin. A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA. Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell. In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3. In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined. However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity. Incubation of viable cell suspensions with 25-hydroxy[26,27-3H]vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2[3H]D3 formation. Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively. The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ. In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity. Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity. Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.     

UVB-induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo-2 cells and in THP-1 macrophages pretreated with a sterol Delta7-reductase inhibitor

Epidermal keratinocytes are able to produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and induce vitamin D activity upon UVB irradiation. To find out whether this property is keratinocyte specific, we investigated this characteristic in two other cell types, namely intestinal CaCo-2 cells and the macrophage-like differentiated THP-1 cells. THP-1 macrophages and preconfluent CaCo-2 cells contain the vitamin D receptor (VDR), possess 25-hydroxylase (CYP2R1 and CYP27A1) and 1alpha-hydroxylase (CYP27B1) activity, and survive the low UVB doses essential for vitamin D3 photoproduction. Upon irradiation, 24-hydroxylase (CYP24) mRNA is induced in both cell types pretreated with the sterol Delta7-reductase inhibitor BM15766 whereby the 7-dehydrocholesterol (7-DHC) content was increased. Transfection studies in CaCo-2 cells with a vitamin D response element-containing construct revealed the involvement of the VDR in this UVB-dependent CYP24 induction. The CYP24 inducing activity in BM15766-pretreated UVB-irradiated CaCo-2 cells and THP-1 macrophages was identified as 1,25(OH)2D3 by combined high-performance liquid chromatography radioimmunoassay. Addition of vitamin D binding protein to the CaCo-2 cells attenuated UVB-induced CYP24 induction suggesting the possibility of a paracrine or autocrine role for the photoproduced 1,25(OH)2D3. In conclusion, preconfluent CaCo-2 cells and THP-1 macrophages are able to induce vitamin D activity upon UVB irradiation and hence combine all parts of the vitamin D photoendocrine system, a characteristic which is therefore not keratinocyte specific.     

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.