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Original Studies |
-Hydroxylase1
Division of Medical Sciences, University of Birmingham, Birmingham, United Kingdom B15 2TH
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: m.hewsion{at}bham.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase
(1-hydroxylase), which is classically expressed in the kidney
(5, 6, 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, 2, 3, 4, 10, 11, 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, 14, 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, 17, 18, 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, 21, 22, 23, 24). As well as providing insight into the molecular
basis of disorders associated with abnormal renal 1-hydroxylase
expression (22, 23, 25, 26, 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 described for studies of the
kidney (5) to characterize the tissue distribution of
extrarenal 1-hydroxylase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 paraffin-embedded samples were used to produce tissue sections (5 µm 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 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 x 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
µg/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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were
carried out to highlight the specificity of immunohistochemical methods
for detection 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.
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-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).
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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).
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-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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 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, 16, 17, 18, 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, 40, 41, 42, 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 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.
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Acknowledgments |
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Footnotes |
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2 Medical Research Council Senior Clinical Fellow.
Received April 26, 2000.
Revised October 4, 2000.
Accepted November 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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type I. Mol Endocrinol. 11:1961–1970.-hydroxylase gene maps to
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Bone Miner Res. 12:1552–1559.[CrossRef][Medline]
-hydroxylase. Biochem Biophys Res Commun. 239:527–533.
-hydroxylase gene in vitamin D dependent
rickets. Curr Opin Nephrol Hypertens. 7:377–383.[Medline]
-hydroxylase gene in patients with
pseudovitamin D-deficiency rickets. N Engl J Med. 338:653–661.-hydroxylase
deficiency in 17 families. Am J Hum Gen. 63:1694–1702.[CrossRef][Medline]
-hydroxylase mutations in French-Canadians with vitamin D
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-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.
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evidence for direct regulation of vitamin D metabolism by calcium. Endocrinology. 140:2027–2034.. Endocrinology. 124:655–660.[Abstract]
-hydroxylation of 25-hydroxyvitamin
D3 in pregnancy. Science. 204:1311–1313.,25-Dihydroxyvitamin D3
and 24,25-dihydroxyvitamin D3 in vitro synthesis
by human decidua and placenta. Nature. 281:317–319.[CrossRef][Medline]
-hydroxylase from cells isolated from
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-hydroxylase activity in human placental decidual cells. J
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 W. B. Ogunkolade, B. J. Boucher, S. A. Bustin, J. M. Burrin, K. Noonan, N. Mannan, and G. A. Hitman Vitamin D Metabolism in Peripheral Blood Mononuclear Cells Is Influenced by Chewing "Betel Nut" (Areca catechu) and Vitamin D Status J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2 |
