0021-972X/01/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2001 by The Endocrine Society
Vol. 86, No. 2
Printed in U.S.A.
Extrarenal Expression of 25-Hydroxyvitamin
D3-1␣-Hydroxylase*
DANIEL ZEHNDER, ROSEMARY BLAND, MARY C. WILLIAMS,
ROBERT W. MCNINCH, ALEXANDER J. HOWIE, PAUL M. STEWART†,
MARTIN HEWISON
AND
Division of Medical Sciences, University of Birmingham, Birmingham, United Kingdom B15 2TH
ABSTRACT
The mitochondrial enzyme 25-hydroxyvitamin D3-1␣-hydroxylase
(1␣-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 1␣-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1␣-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 1␣-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 1␣-hydroxylase in both normal and
diseased tissues. Specific staining for 1␣-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 1␣-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1␣-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin
from these patients 1␣-hydroxylase expression was observed in cells
positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1␣-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 1␣-hydroxylase as an intracrine modulator of vitamin
D function in peripheral tissues. (J Clin Endocrinol Metab 86: 888 –
894, 2001)
T
HE 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 D3-1␣-hydroxylase (1␣-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 1␣hydroxylase 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 1␣-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 1␣-hydroxylase, addition of exogenous 1,25-(OH)2D3 did not inhibit macrophage 1␣-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 1␣-hydroxylase
in extrarenal tissues were different from those observed with
the kidney enzyme. Unfortunately, the absence of nucleotide
and amino acid sequence data for 1␣-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 1␣-hydroxylase in
various species (20 –24). As well as providing insight into the
molecular basis of disorders associated with abnormal renal
1␣-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 1␣-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 1␣-hydroxylase is expressed in both renal and
extrarenal tissues (22, 29). Based on these observations we
have used immunohistochemical techniques previously de-
Received 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.
888
EXTRARENAL EXPRESSION OF 1␣-HYDROXYLASE
scribed for studies of the kidney (5) to characterize the tissue
distribution of extrarenal 1␣-hydroxylase.
Materials and Methods
Tissue preparation
A tissue specimen database from University of Birmingham was
screened for routine surgical samples that were classified as normal.
Psoriatic skin and granulomata-forming diseases such as sarcoidosis and
tuberculosis were also included. For each tissue five different paraffinembedded samples were used to produce tissue sections (5 ␮m thick) on
charged slides (SuperFrost Plus, BDH, Poole, UK).
Immunohistochemistry
Synthesis of a 1␣-hydroxylase antibody was carried out using an
antigenic region of the reported mouse amino acid sequence, peptide
266 –289, as described previously (5, 30). An IgG fraction was subsequently prepared from the immune serum (The Binding Site, Birmingham, UK). Preliminary studies using a human proximal tubule cell line
(HKC-8) that expresses 1␣-hydroxylase activity confirmed the specificity of the antiserum for a 56-kDa 1␣-hydroxylase protein (30). Western
blot analyses identified a single 1␣-hydroxylase protein species in these
cells, and expression of this was down-regulated in the presence of 10
nmol/L 1,25-(OH)2D3 and up-regulated by forskolin. Paraffin-embedded sections were processed in 0.01 mol/L sodium citrate buffer (pH 6.0)
in a pressure cooker at 103 kPa for 2 min. Slides were incubated with
methanol-hydrogen peroxide (1:100) to block endogenous peroxidase
activity and then washed in Tris-buffered saline, pH 7.6. The slides were
then incubated with 1␣-hydroxylase antiserum (1:150) in 10% normal
swine serum for 45 min at 25 C. After rinsing with Tris-buffered saline
for 15 min, donkey antisheep IgG peroxidase conjugate (1:100) was
889
added to sections for 45 min. Staining was developed using 3,3⬘diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer’s hematoxylin. Control sections included 1) omission of primary
antibody, and 2) use of primary antibody preabsorbed with a 100-fold
excess of immunizing peptide.
Western blot analysis
Further characterization of 1␣-hydroxylase protein expression was
carried out by Western blot analysis using homogenates from selected
tissues. As a positive control, lysates were also prepared from HKC-8
cells as described previously (30). Tissue samples and cell preparations
were washed in PBS containing 0.5 mmol/L phenylmethylsulfonylfluoride (Sigma, Poole, UK) and homogenized using a glass-Teflon homogenizer. Cell membranes were pelleted at 2,900 ⫻ g at 4 C for 5 min, and
the supernatant, containing the 1␣-hydroxylase protein, was stored at
⫺80 C. Aliquots of protein were then subjected to SDS-PAGE (5.5 ␮g/
lane) and electroblotted onto Immobilon P membrane as described previously (30). Filters were blocked and incubated with primary antibody
at a 1:500 dilution and with secondary antibody (horseradish peroxidase
conjugated; Amersham Pharmacia Biotech, Aylesbury, UK) at a 1:75,000
dilution. Protein for 1␣-hydroxylase was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech) after exposure of filters to
x-ray film for 10 –30 s. Control experiments were included where 1)
primary antibody was omitted; or 2) primary antibody was preabsorbed
with a 100-fold excess of immunizing peptide. No protein bands were
detected in these controls (data not shown).
Results
Initial studies shown in Fig. 1 were carried out to highlight
the specificity of immunohistochemical methods for detec-
FIG. 1. Immunolocalization of 1␣ -hydroxylase in positive and negative control tissues. A, Expression of 1␣-hydroxylase protein in normal renal
cortex, showing positive staining (brown) in proximal convoluted tubule (arrow), with stronger expression in distal parts of the nephron (double
arrow; magnification, ⫻250). B, Negative control for 1␣-hydroxylase expression in renal cortex (preabsorption of primary antibody with a 100-fold
excess of immunizing peptide; ⫻200). C and D, Extrarenal negative control tissues for 1␣-hydroxylase: liver (⫻100; C) and heart (⫻100; D).
890
JCE & M • 2001
Vol. 86 • No. 2
ZEHNDER ET AL.
tion of 1␣-hydroxylase. Figure 1A demonstrates the presence
of 1␣-hydroxylase immunoreactivity in proximal convoluted
tubules and more distal areas of the nephron (distal convoluted tubule/cortical collecting ducts and thick ascending
loop of Henle). The specificity of this staining was confirmed
by negative controls in which the antiserum was preabsorbed with a 100-fold excess of immunizing peptide (Fig.
1B). The relatively high expression of 1␣-hydroxylase in the
kidney was emphasized by parallel analysis of liver (Fig. 1C)
and heart tissue (Fig. 1D), which showed no staining for the
enzyme.
Further studies were carried out using other extrarenal
tissues that have previously been shown to have 1␣-hydroxylase activity (Fig. 2). In normal skin, epidermal 1␣-hydroxylase expression was restricted to keratinocytes within the
stratum basalis (Fig. 2A), although the enzyme was also
expressed strongly in hair follicles (Fig. 2D). As with kidney
tissue, staining for 1␣-hydroxylase within the skin was completely eliminated by preincubation of antiserum with immunizing peptide (Fig. 2C). In contrast to normal tissue,
psoriatic skin samples showed widespread expression of
1␣-hydroxylase throughout the dysregulated stratum spinosum (Fig. 2B). In normal lymph nodes, 1␣-hydroxylase was
expressed in germinal centers (Fig. 2E). Parallel staining for
the cell surface antigen CD68 indicated that this corresponded to areas that were rich in macrophage populations
(data not shown). Similar results were also obtained with
sections of human tonsil tissue (Fig. 2F). Parallel analyses
using lymph tissue from patients with sarcoidosis showed
strong expression of 1␣-hydroxylase, specifically in granulomata (Fig. 2G). Interestingly, in skin from patients with
sarcoidosis, 1␣-hydroxylase was readily detected in inflammatory infiltrates (CD68-positive cells), but was normally
expressed in basal keratinocytes of the epidermis (Fig. 2H).
The results shown in Fig. 3 indicate that 1␣-hydroxylase is
expressed in a variety of other extrarenal tissues. Analysis of
the colon showed positive staining in epithelial cells (Fig.
3A), although 1␣-hydroxylase was also expressed strongly in
parasympathetic ganglia of the myenteric plexus (Fig. 3B). In
the adrenal gland 1␣-hydroxylase was clearly localized to the
medulla, with background staining observed in the cortex
(Fig. 3D). In the pancreas, 1␣-hydroxylase was clearly detectable in islet cells (Fig. 3C), and expression of the enzyme
was coincident with insulin secretion (data not shown).
Strong immunoreactivity for 1␣-hydroxylase was observed
in both decidualized stromal cells and cytrophoblastic cells
of the placental bed (Fig. 3E) as well as the syncitiotrophoblastic cells (Fig. 3F). The enzyme was also detectable in the
brain, with strong expression in the Purkinje cells of the
cerebellum (Fig. 3G), and in the cerebral cortex (Fig. 3H).
Western blot analyses were carried out to further characterize extrarenal 1␣-hydroxylase expression. The results in
Fig. 4 show the presence of a 56-kDa protein species corresponding to the predicted size of 1␣-hydroxylase in renal
(kidney cortex and medulla) and extrarenal (placenta) tissues. Tissues such as adrenal cortex, which showed no staining for 1␣-hydroxylase in immunohistochemical analyses,
were negative in Western blots. The 1␣-hydroxylase antiserum also identified additional protein species in renal and
extrarenal tissues. A larger band of approximately 65 kDa
was less readily displaced by immunizing peptide than the
56-kDa species and was not observed in cultured HKC-8 cells
(data not shown). A smaller species of approximately 40 kDa
was detectable in placental tissue and could not be competed
out with immunizing peptide.
Discussion
To date, analysis of the expression of 1␣-hydroxylase in
renal and extrarenal tissues has relied upon enzyme activity
studies using tissue homogenates or cultured cells, together
with radiolabeled 25(OH)D3 as substrate. In recent studies
we have addressed this by developing complementary RNA
probes and polyclonal antisera that have been used to characterize the tissue distribution of 1␣-hydroxylase. Contrary
to previous reports, the first of these studies using normal
human kidneys tissue showed that mRNA and protein for
1␣-hydroxylase is expressed throughout the nephron, with
relatively strong expression in distal convoluted tubules and
collecting ducts (5). Enzyme expression appeared to be due
to a single protein species with a size similar to that predicted
by the amino acid sequence. Here we present immunohistochemical analyses that show for the first time the distribution of 1␣-hydroxylase protein in extrarenal tissues. Data
indicate that expression of 1␣-hydroxylase is more widespread than previously thought, suggesting diverse functions for the local production of 1,25-(OH)2D3 in peripheral
tissues.
The discrete distribution of 1␣-hydroxylase in keratinocytes within the stratum basale of the epidermis is consistent
with previous analysis of 1,25-(OH)2D3 production by keratinocytes. In a series of in vitro studies Bikle and co-workers
showed that vitamin D metabolism was dependent on the
stage of keratinocyte development (31, 32). Proliferating cells
that are characteristic of cells within the stratum basale
showed relatively high levels of 1,25-(OH)2D3 synthesis that
decreased as the keratinocytes differentiated toward cornified envelope precursor cells. This was accompanied by an
increase in 24-hydroxylase activity and decreased VDR expression (31). Furthermore, these changes appear to precede
up-regulation of differentiation markers such as transglutaminase activity, suggesting an important autocrine or intracrine role for local 1,25-(OH)2D3 production in the development of the normal epidermis. As a consequence of this,
vitamin D analogs and 1,25-(OH)2D3 have been used with
varying degrees of success as therapy for psoriasis (33, 34).
Topical application of these agents has been shown to attenuate epidermal hyperproliferation and incomplete terminal differentiation as well as localized inflammation. It was
therefore interesting to note the widespread expression of
1␣-hydroxylase throughout the dysregulated stratum spinosum in psoriatic lesions. The paradox that psoriasis is associated with unregulated local synthesis of antiproliferative
1,25-(OH)2D3 may be explained by previous studies showing
that dermal fibroblasts and epidermal keratinocytes from
psoriatics were relatively insensitive to 1,25-(OH)2D3 (35, 36).
Although other groups have failed to confirm this observation (37), the possibility remains that production of 1,25(OH)2D3 in psoriatic skin lesions is similar to that observed
in macrophages from sarcoid granulomata. The localized
EXTRARENAL EXPRESSION OF 1␣-HYDROXYLASE
891
FIG. 2. Expression of 1␣-hydroxylase protein in normal and pathological tissue from skin and lymph nodes. A and B, Immunohistochemical
analysis of normal human skin shows strong expression of 1␣-hydroxylase protein within the stratum basalis of the epidermis (arrow;
magnification, ⫻150; A) and throughout the dysregulated stratum spinosum (arrow) of the epidermis in psoriatic skin (⫻100; B). C, Negative
control for 1␣-hydroxylase expression in the skin (preabsorption of primary antibody with a 100-fold excess of immunizing peptide; ⫻50). D,
Expression of 1␣-hydroxylase protein in hair follicle (⫻250). E–H, Immunolocalization of 1␣-hydroxylase in cells throughout normal human
lymph node (⫻100; E); normal tonsil, with particularly strong staining in germinal centers (arrow; ⫻200; F); lymph node granulomas (arrow;
⫻100; G), and skin affected by sarcoidosis (⫻150; H).
892
ZEHNDER ET AL.
JCE & M • 2001
Vol. 86 • No. 2
FIG. 3. Immunolocalization of 1␣-hydroxylase in colon with strong staining of the epithelium (arrow; magnification, ⫻100; A), parasympathetic
ganglion of the myenteric plexus in bowel (arrow; ⫻200; B), islets of the pancreas (arrow; ⫻200; C), medulla of the adrenal gland (m; ⫻50; D),
decidualized stromal cells (arrow) and trophoblasts (double arrow) in the placental bed (⫻200; E), placental syncytiotrophoblast (arrow; ⫻200;
F), Purkinje cells in the cerebellum (arrow; ⫻100; G), and neurons in the cerebral cortex (arrow; ⫻200; H).
EXTRARENAL EXPRESSION OF 1␣-HYDROXYLASE
FIG. 4. Western blot analysis of 1␣-hydroxylase expression in extrarenal tissue. A, Western analysis using a 1:500 dilution of 1␣hydroxylase antiserum and protein preparations (3.5 ␮g) from normal
placenta (P), adrenal cortex (C), and positive controls [kidney cortex
(KC) and kidney medulla (KM)]. Protein sizes are shown in kilodaltons, with 1␣-hydroxylase at 56 kDa. B, Control Western blot using
identical tissue to A, but with 1␣-hydroxylase antiserum preabsorbed
with a 100-fold excess of immunizing peptide.
release of inflammatory cytokines such as interferon-␥ and
interleukin-2 by activated T cells is likely to provide a potent
stimulus to 1␣-hydroxylase in immature keratinocytes (38).
However, although normal keratinocytes are exquisitely sensitive to exogenously added 1,25-(OH)2D3, similar feedback
control may be absent in cytokine-activated keratinocytes.
Thus, the question remains as to whether the unregulated
expression of 1␣-hydroxylase in psoriasis and sarcoidosis is
due to a common mechanism and, importantly, whether this
contributes to the pathophysiology of these diseases or is a
secondary consequence of the central antigenic challenge.
The dysregulation of 1␣-hydroxylase in psoriatic lesions
contrasts with the apparently normal distribution of the enzyme in sarcoid epidermal tissue. In this case peripheral
synthesis of 1,25-(OH)2D3 appears to be due entirely to subepidermal inflammatory infiltrates, which is consistent with
the macrophage-mediated responses that are associated with
the granulomatous disease (15–19). Expression of 1␣-hydroxylase colocalized with the cell surface antigen CD68 in both
skin and lymph nodes, confirming that macrophages, rather
than lymphocytes, are the most likely source of 1,25-(OH)2D3
production.
Immunohistochemical detection of 1␣-hydroxylase in tissue from the placental bed confirms previous enzyme activity studies that indicated that 1,25-(OH)2D3 may be produced by both decidual and trophoblastic cells (39 – 43). The
functional relevance of this remains unclear, but the enzyme
may contribute both to the materno-fetal transfer of calcium
that is a key feature of third trimester development as well
as to possible immunomodulatory actions during early
stages of placentation. Although Western analyses suggested
that placental 1␣-hydroxylase expression is due to the same
56-kDa protein species found in renal tissues, a smaller band
was also detected that appeared to be less specific in competition with excess immunizing peptide. Further characterization of this species is required.
The presence of 1␣-hydroxylase in pancreatic islet cells
provides a further link between vitamin D and insulin secretion. Recent studies in vivo and in vitro have shown that
893
treatment with 1,25-(OH)2D3 potently enhances insulin secretion in response to glucose (44, 45). A possible role for local
synthesis of 1,25-(OH)2D3 as a modulator of secretory function is emphasized by the further observation that 1␣hydroxylase is detectable in another key secretory tissue,
namely the adrenal medulla. The strong presence of protein
for 1␣-hydroxylase in hair follicles is interesting in view of
the well documented alopecia which is associated with hereditary vitamin D-resistant rickets (46). VDR are known to
be expressed in hair follicles (47), and abnormal expression
of these receptors is thought to be the cause of the hair loss
associated with hereditary vitamin D-resistant rickets. Expression of 1␣-hydroxylase was also relatively strong in
sweat glands (data not shown). This together with the presence of 1␣-hydroxylase in colonic epithelial cells suggests an
association between the enzyme and sites of sodium transport and regulation. A novel role for local 1,25-(OH)2D3
production in salt homeostasis is also supported by our previously reported observations of 1␣-hydroxylase in the distal
nephron, which is the principal site of renal sodium/water
homeostasis (5).
The precise relationship between extrarenal expression of
the enzyme and peripheral synthesis of 1,25-(OH)2D3 remains to be determined and may be dependent on coexpression of other proteins, such as glycoprotein 330, otherwise
known as megalin. Recent studies using megalin-null mice
have shown that this multifunctional protein is essential for
the uptake of the substrate for 1␣-hydroxylase, 25(OH)D3, by
cells of the proximal convoluted tubule (48). Megalin is involved in the endocytosis of 25(OH)D3 which is bound to its
carrier protein, the vitamin D-binding protein. Although this
facilitates the metabolism of 25(OH)D3 to 1,25-(OH)2D3 in the
kidneys, the presence of megalin at extrarenal sites suggests
that regulation of substrate availability may also modulate
1␣-hydroxylase activity in peripheral tissues (49). Key extrarenal tissues that express megalin include the parathyroid
glands, mammary epithelia, and thyroid follicular cells. In
addition, the defective forebrain development associated
with mice lacking megalin suggests a further role in neuroepithelial cell function (50). It was therefore interesting to
note the presence of 1␣-hydroxylase protein in brain and
neuronal tissues, confirming previous Northern blot analyses that indicated that mRNA for the enzyme is strongly
expressed in the brain (21).
The data presented in this study show for the first time the
extrarenal distribution of 1␣-hydroxylase in human tissues.
Immunohistochemical analyses confirm the discrete expression of this enzyme in immature keratinocytes and lymphoid
granulomata, but also reveal novel sites for 1␣-hydroxylase
expression, such as hair follicles, adrenal medulla, pancreatic
islet cells, and the colon. Of particular interest are data that
highlight the dysregulation of 1␣-hydroxylase in psoriatic
keratinocytes. Further analysis of the expression of 1␣hydroxylase in the skin may provide important new information on the link between vitamin D and the pathophysiology of psoriasis.
Acknowledgments
We thank Dr. A. R. Bradwell and The Binding Site for all their help
with preparation of antisera.
894
ZEHNDER ET AL.
References
1. Walters MR. 1992 Newly identified actions of the vitamin D endocrine system.
Endocr Rev. 13:719 –764.
2. Reichel H, Koeffler HP, Norman AW. 1989 The role of the vitamin D endocrine system in health and disease. N Engl J Med. 320:980 –991.
3. Hewison M, O’ Riordan JLH. 1997 Immunomodulatory and cell differentiation effects of vitamin D. In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin
D. San Diego: Academic Press; 447– 462.
4. Haussler MR, Whitfield GK, Haussler CA, et al. 1998 The nuclear vitamin D
receptor: biological and molecular regulatory properties revealed. J Bone
Miner Res. 13:325–349.
5. Zehnder D, Bland R, Walker EA, Bradwell AR, Howie AJ, Hewison M,
Stewart PM. 1999 Expression of 25-hydroxyvitamin D3-1␣-hydroxylase in the
human kidney. J Am Soc Nephrol. 10:2465–2473.
6. Zehnder D, Hewison M. 1999 The renal function of 25-hydroxyvitamin D31␣-hydroxylase. Mol Cell Endocrinol. 151:213–220.
7. Bland R, Zehnder D, Hewison M. 2000 Expression of 25-hydroxyvitamin
D3-1␣-hydroxylase along the nephron: new insights into renal vitamin D
metabolism. Current Opin Nephrol Hyperten. 9:17–22.
8. Adams JS. 1997 Extra-renal production and action of active vitamin D metabolites in human lymphoproliferative diseases. In: Feldman D, Glorieux FH,
Pike JW, eds. Vitamin D. San Diego: Academic Press; 903–922.
9. Bell NH. 1998. Renal and non-renal 25-hydroxyvitamin D-1␣-hydroxylases
and their clinical significance. J Bone Miner Res. 13:350 –353.
10. Bouillon R, Okamura WH, Norman AW. 1995 Structure-function relationships in the Vitamin D endocrine system. Endocr Rev. 16:200 –257.
11. Bouillon R, Garmyn M, Verstuyft A, Segaert S, Casteels K, Mathieu C. 1995
Paracrine role for calcitriol in the immune system and skin creates new therapeutic possibilities for vitamin D analogs. Eur J Endocrinol. 133:7–16.
12. Bunce CM, Brown G, Hewison M. 1997 Vitamin D and haematopoiesis.
Trends Endocrinol Metab. 8:245–251.
13. Taylor RL, Lynch Jr HJ, Wysor WG Jr. 1963 Seasonal influence of sunlight on
hypercalcaemia of sarcoidosis. Am J Med. 34:221–227.
14. Papapoulos SE, Clemens TL, Fraher LJ, Lewin IG, Sandler LM, O’Riordan
JLH. 1979 1,25-Dihydroxycholecalciferol in the pathogenesis of the hypercalcaemia of sarcoidosis. Lancet. 1:627– 630.
15. Barbour GL, Coburn JW, Slatopolsky E, Norman AW, Horst RL. 1981 Hypercalcemia in an anephric patient with sarcoidosis: evidence for extra-renal
generation of 1,25-dihydroxyvitamin D. N Engl J Med. 305:440 – 443.
16. Adams JS, Sharma OP, Gacad MA, Singer FR. 1983 Metabolism of 25hydroxyvitamin D3 by cultured pulmonary alveolar macrophages. J Clin Invest. 72:1856 –1860.
17. Adams JS, Singer FR, Gacad MA, Sharma OP, Hayes MJ, Vouros P, Holick
MF. 1985 Isolation and structural identification of 1,25-dihydroxyvitamin D3
produced by cultured alveolar macrophages in sarcoidosis. J Clin Endocrinol
Metab. 60:960 –966.
18. Adams JS, Gacad MA. 1985 Characterization of 1-␣-hydroxylation of vitamin
D3 sterols by cultured alveolar macrophages from patients with sarcoidosis. J
Exp Med. 161:755–765.
19. Reichel H, Koeffler HP, Bishop JE, Norman AW. 1987 25-Hydroxyvitamin D3
metabolism by lipopolysaccharide-stimulated normal human macrophages.
J Clin Endocrinol Metab. 64:1–9.
20. Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 1997
25-Hydroxyvitamin D3 1␣-hydroxylase and vitamin D synthesis. Science.
277:1827–1830.
21. Fu KG, Lin D, Zhang MYH, Bikle DD, Shackleton CHL, Miller WL, Portale
AA. 1997 Cloning of human 25-hydroxyvitamin D-1␣-hydroxylase and mutations causing vitamin D-dependent rickets type I. Mol Endocrinol.
11:1961–1970.
22. St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. 1997 The
25-hydroxyvitamin D 1-␣-hydroxylase gene maps to the pseudovitamin D
deficiency rickets (PDDR) disease locus. J Bone Miner Res. 12:1552–1559.
23. Monkawa T, Yoshida T, Wakino S, et al. 1997. Molecular cloning of cDNA
and genomic DNA for human 25-hydroxyvitamin D3 1␣-hydroxylase. Biochem Biophys Res Commun. 239:527–533.
24. Kato S, Yanagisawa J, Murayama A, Kitanaka S, Takeyama K. 1998 The
importance of 25-hydroxyvitamin D3 1␣-hydroxylase gene in vitamin D dependent rickets. Curr Opin Nephrol Hypertens. 7:377–383.
25. Kitanaka S, Takeyama K, Murayama A, et al. 1998 Inactivating mutations in
the 25-hydroxyvitamin D3 1␣-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med. 338:653– 661.
26. Wang JT, Lin CJ, Burridge SM, Fu GK, Labuda M, Portale AA, Miller WL.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
JCE & M • 2001
Vol. 86 • No. 2
1998 Genetics of vitamin D 1␣-hydroxylase deficiency in 17 families. Am J Hum
Gen. 63:1694 –1702.
Yoshida T, Monkawa T, Tenenhouse HS, et al. 1998 Two novel 1␣-hydroxylase mutations in French-Canadians with vitamin D dependency rickets.
Kidney Int. 54:1437–1443.
Bikle DD. 1995 1,25-(OH)2D3-regulated human keratinocyte proliferation and
differentiation: basic studies and their clinical application. J Nut. 125:
s1709 –s1714.
Smith SJ, Rucka AK, Berry JL, et al. 1999. Novel mutations in the 1␣hydroxylase (P450cl) gene in three families with pseudovitamin D-deficiency
rickets resulting in loss of functional enzyme activity in blood-derived macrophages. J Bone Miner Res. 14:730 –739.
Bland R, Walker E, Hughes SV, Stewart PM, Hewison M. 1999 Constitutive
expression of 25-hydroxyvitamin D-1␣-hydroxylase in a transformed human
proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium. Endocrinology. 140:2027–2034.
Pillai S, Bikle DD, Elias PM. 1988 1,25-Dihydroxyvitamin D production and
receptor binding in human keratinocytes varies with differentiation. J Biol
Chem. 263:5390 –5395.
Pillai S, Bikle DD. 1991 Role of intracellular free calcium in the cornified
envelope formation of keratinocytes: differences in the mode of action of
extracellular calcium and 1,25-dihydroxyvitamin D. J Cell Physiol. 146:94 –100.
Vandekerkhof PCM. 1995 Biological activity of vitamin D analogs in the skin,
with special reference to antipsoriatic mechanisms. Br J Dermatol. 132:675– 682.
Perez A, Chen TC, Turner A, Raab R, Bhawan J, Poche P, Holick MF. 1996.
Efficacy and safety of topical calcitriol (1,25-dihydroxyvitamin D3) for the
treatment of psoriasis. Br J Dermatol. 134:238 –246.
Abe J, Kondo S, Nishii Y, Kuroki T. 1989 Resistance to 1,25-dihydroxyvitamin
D3 of cultured psoriatic epidermal keratinocytes from involved and uninvolved skin. J Clin Endocrinol Metab. 68:851– 854.
McLaughlin JA, Gange W, Taylor D. 1985 Cultured psoriatic fibroblasts from
involved and uninvolved sites have partial but not absolute resistance to the
proliferation-inhibition activity of 1,25-dihydroxyvitamin D3. Proc Natl Acad
Sci USA. 82:5409 –5412.
Smith EL, Pincus SH, Donovan L, Holick MF. 1988 A novel approach for the
evaluation and treatment of psoriasis. J Am Acad Dermatol. 10:360 –364.
Bikle DD, Pillai S, Gee E, Hincenbergs M. 1988 Regulation of 1,25-dihydroxyvitamin D production in human keratinocytes by interferon-␥. Endocrinology. 124:655– 660.
Tanaka Y, Halloran B, Schnoes HK, DeLuca HF. 1979 In vitro production of
1,25-dihydroxyvitamin D3 by rat placental tissue. Proc Natl Acad Sci USA.
76:5033–5035.
Gray TK, Lester GE, Lorenc RS. 1979 Evidence for extra-renal 1␣-hydroxylation of 25-hydroxyvitamin D3 in pregnancy. Science. 204:1311–1313.
Weisman Y, Harell A, Edelstein S, David M, Spirer Z, Golander A. 1979
1␣,25-Dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis
by human decidua and placenta. Nature. 281:317–319.
Delvin EE, Arabian A. 1987 Kinetics and regulation of 25-hydroxcholecalciferol 1␣-hydroxylase from cells isolated from human term decidua. Eur J Biochem. 163:659 – 662.
Glorieux FH, Arabian A, Delvin EE. 1995 Pseudo-vitamin D deficiency: absence of 25-hydroxyvitamin D 1␣-hydroxylase activity in human placental
decidual cells. J Clin Endorinol Metab. 80:2255–2258.
Bourlon PM, FaureDussert A, Billaudel B. 1997 Modulatory role of 1,25
dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP
pathway in the rat. Br J Pharmacol. 121:751–758.
Ayesha I, Raghunath M, Sesikeran B, Raghuramulu N. 1998 Effect of oral
administration of vitamin D on glucose tolerance and insulin secretion in type
2 diabetes mellitus. Diabetes Nutr Metab. 11:261–265.
Malloy PJ, Pike JW, Feldman D. 1997 Hereditary vitamin D resistant rickets.
In: Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. San Diego: Academic
Press; 765–787.
Reichrath J, Schilli M, Kerber A, Bahmer FA, Czarnetzki BM, Paus R. 1994
Hair follicle expression of 1,25-dihydroxyvitamin D3 receptors during the
murine hair cycle. Br J Dermatol. 131:477– 482.
Nykjaer A, Dragun D, Walther D, et al. 1999 An endocytic pathway essential
for renal uptake and activation of the steroid 25-(OH)vitamin D3. Cell.
96:507–515.
Lundgren S, Carling T, Hjalm G, et al. 1997 Tissue distribution of human
gp330/megalin, a putative Ca2⫹-sensing protein. J Histochem Cytochem.
45:383–392.
Willnow TE, Hilpert J, Armstrong SA, Rohlmann A, Hammer RE, Burns DK,
Herz J. 1996 Defective forebrain development in mice lacking gp330/megalin.
Proc Natl Acad Sci USA. 93:8460 – 8464.