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Centre National de la Recherche Scientifique (CNRS URA 583), Calcium et Tissu Osseux dans l’organisme en Développement, Hôpital Saint Vincent de Paul, 75014 Paris, France; and Institut National de la Recherche Medicale (INSERM U-349), Biologie Cellulaire et Moléculaire de l’Os et du Cartilage, Hôpital Lariboisière (B.Z.), 75010 Paris, France
Address all correspondence and requests for reprints to: Dr. Rizk-Rabin Marthe, CNRS URA 583/UPR 1524 Hôpital Saint Vincent de Paul, 82 Avenue Denfert Rochereau, 75014 Paris, France.
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Introduction |
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The up-regulation of VDR production by 1,25-(OH)2D3 has been extensively studied in intestine and kidney in vivo (11) and in vitro (12). This regulation is believed to be a transcriptional event, but the issue remains controversial. 1,25-(OH)2D3 undoubtedly increases the concentration of both the VDR protein and its messenger RNA (mRNA) in the intestine and kidneys of vitamin D-depleted rats (13). However, the increase in VDR protein is not always associated with an increase in VDR mRNA (14). Moreover, regulators of VDR production may have different effects depending on the target tissue. Dietary calcium restriction does not increase the intestinal VDR production, but it significantly down-regulates VDR in renal tissue (15). The response of the VDR in both tissues also differs when serum 1,25-(OH)2D3 is altered by changes in dietary phosphorus (16).
The synthesis of VDR is also influenced by the differentiation state of the target cell. Pillai et al. (17) showed that the number and affinity of 1,25-(OH)2D3 VDR in epidermis vary with the differentiation stage of the keratinocytes. This correlation is also observed in vivo (18). However, the influence of nutritional factors on the number of VDR in these target cells is as yet unknown. We previously evaluated the subtle changes in the differentiation of the epidermis caused by vitamin D deficiency and vitamin D treatment or a calcium-enriched diet (19). This study showed that vitamin D and calcium may have distinct functions in the epidermis. A primary function of calcium may be to promote early differentiation, whereas 1,25-(OH)2D3 seems to be involved in promoting later differentiation events.
The present work analyzes the influence of in vivo administration of either vitamin D or calcium on VDR expression in skin keratinocytes of adult rats and compares this influence to that observed in the kidneys and duodena of the same animals. The amounts of VDR mRNA and protein in isolated keratinocytes at different stages of differentiation were also measured to identify the ways in which calcium and vitamin D influenced the differentiation of the epidermis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total serum calcium was measured by standard laboratory techniques on a Hitachi 717 analyzer (Boehringer Mannheim, Indianapolis, IN). Serum 25-hydroxyvitamin D (25OHD) was determined by methanol-chloroform extraction of the sample, chromatography of the extract on an Amprep minicolumn, and a protein binding assay (20). Serum 1,25-(OH)2D was measured using a modification of the receptor binding assay described by Shepard et al. (21). The lower limits of detection were 1 ng/ml for 25OHD and 5 pg/ml for 1,25-(OH)2D.
Preparation of epidermal cells
Keratinocytes were isolated from the skin samples of five rats
per experimental group by a modification of the method of Yuspa and
Harris (22). The skins were stretched and floated on 0.25% trypsin
(Eurobio, Paris, France) in PBS at 4 C for 15 h. The epidermis was
then separated from the dermis with forceps. Care was taken to remove
the lowermost layer of epidermal cells by applying gentle pressure to
the dermis. The epidermal sheet was shaken in Ca- and Mg-free MEM
containing 10% Chelex-treated FCS (Eurobio). Deoxyribonuclease
(0.25%; Sigma Chemical Co., Saint Quentin Fallaver, France) was added
to minimize cell aggregation. The resulting cell suspension was
filtered through three layers of gauze, then through a 200-µm pore
size nylon mesh filter, sedimented through a layer of 10% FCS in MEM,
and resuspended in 0.5% Ficoll (Pharmacia, Orsay, France) in PBS. An
aliquot of this suspension was taken for cell counting. Cell viability
was estimated by trypan blue exclusion. The viability of nucleated
cells was about 85%.
Keratinocytes were separated according to their size into four different keratinocyte subpopulations on Ficoll gradient in a specially designed low velocity sedimentation chamber, as previously described (23). This separation method provides mainly basal and early suprabasal cells. The following cell populations were isolated: population 1 (elution volume, 100–170 ml), population 2 (elution volume, 180–230 ml), population 3 (elution volume, 240–320 ml), and population 4 (elution volume, 400–480 ml). The sizes of the cells in these fractions were measured in a Coulter counter (Coulter Electronics, Margeny, France), and their cell cycle stage was determined by flow cytometry using acridine orange staining. Their state of differentiation was identified by the presence of two protein markers. Epidermal calcium binding protein (ECaBP) and suprabasal keratins (KL1) (19). The ECaBP antigen is an early differentiation marker restricted to the transit amplifying basal cells (24), whereas the KL1 antibody recognizes several keratins mainly present in suprabasal differentiated cells (25).
Immunohistochemical analysis
1,25-(OH)2D3 receptors in total
skin sections were identified by immunostaining, and those in separated
cell smears were identified using a modification of the method of
Clemens et al. (26) that was used in a previous work (18).
Skin samples were frozen and cut on a cryostat (4 µm), and the
sections were fixed with acetone. Cell smears from unfractionated and
fractionated populations were prepared using cytospin (Shandon Eragny,
France) and fixed in acetone. Both sections and smears were rehydrated
with 0.1 M Tris-HCl, pH 7.4, and incubated overnight at 4 C
with affinity-purified monoclonal antibody 9A7 to the chick intestinal
1,25-(OH)2D3 receptor (27) (1:200 dilution; a
gift from Dr. M. Haussler) or nonimmune rat IgG (1:200 dilution). The
samples were rinsed twice with 0.1 M Tris-0.05% BSA
(wt/vol), incubated with biotinylated rabbit antirat IgG (1:200
dilution, for 45 min) and streptavidin biotin horseradish peroxidase
(1:200 dilution, for 60 min) at room temperature. Samples were washed
with 0.1 M Tris-HCl, and the immunopositive cells were
visualized by immersion in 0.1 M Tris containing (0.5%)
3,3'-diaminobenzidine tetrahydrochloride with 0.3%
H2O2. The percentage of VDR-positive cells was
calculated by counting cells under the microscope. A minimum of 400
cells were scored per sample.
Ligand binding assay
The available materials was insufficient to analyze the cytosol
1,25-(OH)2D3-binding capacity of the isolated
keratinocyte populations in the experimental groups of 5 rats. Thus, we
tested the parallelism of the ability of keratinocytes to detect the
VDR antibody (immunocytochemical analysis) and its
1,25-(OH)2D3-binding capacity in another group
of control rats. Keratinocyte subpopulations were isolated from the
skin of 30 normal rat neonates, and the immunostaining study was
performed as described above. The 1,25-(OH)2D3
capacity of their cytosol was assessed using a hydroxylapatite batch
assay (28). Briefly, cells (25 x 106) were washed
twice with PBS, sonicated 3 times for 30 sec each time in high salt
buffer (300 mM KCl, 1.5 mM EDTA, 10
mM sodium molybdate, and 1 mM dithiothreitol).
High speed supernatants were obtained (100,000 x g for
1 h), and 200-µl aliquots (0.5 mg protein/ml) were incubated
with 100 IU aprotinin and 2 nM
[3H]1,25-(OH)2D3 for 1 h at
25 C with or without a 50-fold excess of unlabeled
1,25-(OH)2D3. Receptor-bound
1,25-(OH)2D3 was separated from unbound sterol
by absorption of bound hormone onto hydroxylapatite in 10
mM Tris-HCl, pH 7.5.
Preparation of RNA and Northern blot hybridization
Total RNA was extracted from intestine, kidney, and
keratinocytes pooled from five animals in each experimental group using
the guanidium thiocyanate-phenol-chloroform method of Chomczynski and
Sacchi (29). Samples of total RNA (15 µg) were fractionated on
1.2% formaldehyde agarose gels and transferred to nitrocellulose
membrane by diffusion blotting for Northern blot analysis. The filters
were baked at 80 C for 120 min in a vacuum oven.
1,25-(OH)2D3 receptor mRNA levels were measured
by hybridization with a 32P-labeled RNA-VDR probe obtained
by in vitro transcription of the receptor complementary DNA
(cDNA; 1.1 kb) insert cloned in the pGEM3 plasmid (a gift from Dr.
J. W. Pike). Filters were stripped and rehybridized to two control
probes. As a control probe and to detect RNA transfer problems and
possible unequal loading of RNA on the gel, glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) cDNA and 1644-bp 18S ribosomal cDNA were both
labeled by random priming. The autoradiographs of the Northern blots
were assessed by scanning densitometry. The amounts of VDR mRNA were
normalized to G3PDH and S18 to adjust for differences in loading.
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Results |
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Effects of calcium and vitamin D on VDR mRNA
Hybridization of VDR mRNA from control animals showed only the
expected band at 4.6 kb in the three target tissues. When normalized to
S18 (not shown) or G3PDH, the amounts of VDR mRNA in the duodenum and
kidney were comparable, but the isolated keratinocytes of the same
animals contained only half the amount of VDR mRNA (Fig 1
).
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). Although
measurement of mRNA is semiquantitative, when using Northern blot
analysis, this analysis showed a marked decrease in the amount of VDR
mRNA in the duodenum and kidney as well as in the keratinocytes.
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). There
was no change in the amount of VDR mRNA in the duodenum or kidney. In
contrast, the vitamin D treatment restored VDR mRNA to normal in the
keratinocytes (Fig. 1).
The role played by the hypocalcemia caused by the vitamin D deficiency
was assessed by feeding vitamin D-deficient rats a vitamin D-deficient
diet enriched with calcium and supplemented with lactose for 1 week.
This diet normalized both serum calcium and PTH, but did not influence
the level of serum vitamin D metabolites, which remained low (Fig. 2).
Unlike vitamin D, the high calcium diet enriched with lactose
significantly enhanced the amount of VDR mRNA in the duodenum (Fig. 1).
In contrast, its effects in kidney and epidermis were similar to those
induced by the vitamin D treatment. Like vitamin D, it did not
influence VDR mRNA in kidney.
Characterization of isolated subpopulations of keratinocytes
Separation of keratinocytes is based on the small, but
progressive, increase in cell volume that takes place during both
progression through the cell cycle and advances in differentiation.
Four cell populations were eluted according to their progression in
cell size (small cells to intermediate large ones), and the relative
percentage of eluted cells per population was measured. In the control
group, the characteristics of differentiation of these cells according
to cell size, cell cycle phase (acridine orange staining), and
expression of differentiation markers in the control group were similar
to those previously described (Table 1) (19).
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Immunohistochemical analysis of the keratinocytes showed profound
changes in the expression of the two protein markers of
differentiation. All cells in the different subpopulations of
keratinocytes from vitamin D-deficient epidermis lacked the expression
of ECaBP and of suprabasal keratins (Fig. 3). Both vitamin D and a calcium-enriched
diet normalized the expression of ECaBP in population 2. In addition,
vitamin D induced the appearance of ECaBP in cells from population 1.
Calcium had no effect on suprabasal keratin expression. In contrast,
vitamin D normalized the pattern of suprabasal keratins in populations
3 and 4 and induced the appearance of these proteins in populations 1
and 2.
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The amounts of VDR mRNA were similar in all cell populations of
normal keratinocytes independent of their cell size or stage of
differentiation (Fig. 4). In vitamin
D-deficient animals, a decrease in VDR mRNA content was observed in
total keratinocytes as well as in all subpopulations. The VDR mRNA in
all cell populations was restored to normal values by oral
supplementations of both calcium and vitamin D. Vitamin D and calcium
were equally effective for this enhancing effect.
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), the concentrations of VDR protein were different (Figs. 3 and 5). Immunostaining for VDR showed that the proportions of stained
cells as well as the intensity and the subcellular location of the
staining varied in the four populations (Fig. 3). In the control group,
few receptor-positive cells were found in populations 1 and 4. The
staining was weak and mainly peripheral and discontinuous. There were
significantly more receptor-positive cells in populations 2 and 3, with
moderate to strong cytoplasmic staining as well as nuclear staining
(Fig. 5).
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and 5).
The normalization of calcium independent of vitamin D status also
enhanced the production of VDR in keratinocytes. Cells in populations 2
and 3 had VDR immunoreactivities similar to those found in normal rats.
Calcium also increased the VDR response in population 1, but staining
remained weak and discontinuous compared with that of vitamin D-treated
keratinocytes (Figs. 3 and 5).
Immunocytochemical expression of the VDR protein and
1,25-(OH)2D3 binding
affinity in isolated subpopulations of keratinocytes
To determine whether the staining was of VDR, the presence of the
receptor was also studied by ligand binding assay in isolated
keratinocyte populations of newborn rat epidermis. In these
populations, the variation in the proportion of stained cells and that
in the intensity and location of staining were roughly similar to those
in keratinocytes isolated from adult skin (Table 2). The percentage of cells expressing
the VDR antigen varied in parallel with the ligand binding affinity of
the receptor for 1,25-(OH)2D3 in the cell
cytosol (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with our findings for kidney and intestine, several researchers have been unable to demonstrate an induction of VDR mRNA in the intestine or kidney of vitamin D-deficient rats by exogenous 1,25-(OH)2D3 (10, 31). Yet, vitamin D treatment may have increased the VDR protein content of these target tissues, as exogenous 1,25-(OH)2D3 (14) and stimulation of endogenous 1,25-(OH)2D3 (10) have been shown to increase the amounts of VDR protein levels in the intestine and kidney, without any concomitant increase in VDR mRNA. The absence of regulation of VDR mRNA by its ligand is not surprising, as many studies (13, 14, 15, 32) have suggested that the increase in VDR produced by 1,25-(OH)2D3 is not due primarily to new receptor synthesis, but results from posttranslational events.
Calcium is another regulator of VDR. It sensitizes renal cells to stimulation of VDR protein production by 1,25-(OH)2D3 (32, 33). Our results for vitamin D-deficient rats given a high calcium diet for 1 week show that neither the normalization of serum calcium nor the decrease in circulating PTH affects the renal VDR mRNA concentration. However, changes in the regulation of VDR protein cannot be excluded. Surprisingly, the supplementation of vitamin D-deficient rats with calcium and lactose enhanced the VDR mRNA content of the intestine. Lactose is known to promote the intestinal absorption of calcium via a vitamin D-independent mechanism that may involve a prolonged stay of calcium in the duodenum and a subsequent increase in the passive absorption of calcium (34). We, therefore, postulate that the observed regulation of VDR mRNA in the intestine may be due to the local high calcium concentration in the duodenum, rather than to the increased circulating calcium or to other related biological changes, such as the decrease in circulating PTH, for example.
The serum PTH level may contribute to the regulation of VDR. However, regulation of VDR by PTH is still controversial. In in vitro experiments using ROS17/2,8 cells (rat osteosarcoma cells), PTH was found to down-regulate VDR and VDR mRNA and to block homologous up-regulation of VDR in vivo (35). The opposite was seen in osteoblast-like cells (UMR-106), in which PTH and PTH-related peptide up-regulate the VDR protein (36). In our experiments, hyperparathyroidism and low serum levels of calcium were associated with the low levels of vitamin D metabolites in the vitamin D-deficient group. However, the tissue differences in the regulation of VDR expression make it difficult to include PTH as a potential regulator of VDR expression in the present conditions. We can only postulate that the failure of 1,25-(OH)2D3 to up-regulate the receptor in kidney and intestine in the vitamin D-treated group may be related to the remaining high levels of circulating PTH despite normal serum calcium in this group.
The regulation of VDR mRNA in the epidermis is more sensitive to changes in dietary vitamin D and calcium than that in the two other tissues studied. One week of vitamin D treatment was sufficient to enhance VDR mRNA in this tissue, but this effect may be indirect, as normalization of serum calcium alone was sufficient to increase the VDR mRNA, without any change in the vitamin D status. Our findings suggest that the induction of VDR mRNA by vitamin D is mediated by its action on calcium homeostasis and that calcium may act as a transcriptional factor. Similarly, both vitamin D and calcium increased the number of keratinocytes containing the VDR protein.
However, there are differences in the posttranslational effects of these two regulators when studied at the cellular level. Although the VDR mRNA contents were similar in the four keratinocyte populations, the amounts of VDR protein differed with the differentiation state of the keratinocytes. Vitamin D deficiency decreased the amounts of both VDR protein and its mRNA in the cells. However, vitamin D and calcium treatments had distinct effects on the expression of the VDR protein that were not directly related to their effects on VDR mRNA. The production of VDR in populations 2 and 3 was sensitive to normalization of the extracellular calcium concentration, whether obtained by calcium or vitamin D treatment. In contrast, a new and impressive production of VDRs was observed after vitamin D treatment in cells of population 1, which normally do not contain it. This effect was not found in rats fed the high calcium-lactose diet and, therefore, appears to result from a specific action of vitamin D rather than from the calcium concentration in the close vicinity of the keratinocytes.
The present results suggest an association between the regulation of VDR production and the parameters of differentiation. In the vitamin D-replete group of rats, keratinocytes from populations 2 and 3 produced the VDR protein. Population 2 cells also contained a protein marker of pre-differentiation (ECaBP) of keratinocytes (24), whereas population 3 expressed suprabasal keratin levels (KL1), a marker of advanced differentiation (24, 25). VDR was absent from small immature cells (population 1) and from the more differentiated ones (population 4). In addition, the decrease in the amounts of VDR in vitamin D-deficient animals was associated with a lack of the two protein markers of differentiation, reflecting changes in the cell phenotype and a state of arrest (19). Third, the new synthesis of VDR in population 1 of rats treated with vitamin D was associated with the early onset of both protein markers in the small cells of this population, which are normally immature. This may indicate acceleration of differentiation (19). Finally, the resynthesis of VDR in cells from population 2 in response to calcium or vitamin D treatment was associated with the appearance of ECaBP, a predifferentiation marker (24). In contrast, the association between VDR production and the reinduction of suprabasal keratins in population 3 was observed in the vitamin D-supplemented group, but not in the calcium-treated group.
Thus, VDR is regulated by both calcium and vitamin D in the epidermis, but at distinct levels. Calcium may be a key factor for enhancing the transcription of the VDR. As in kidney (32, 33) or osteoblast-like cells (37), a normal calcium condition may be required for vitamin D to stimulate the production of the epidermal receptor. Calcium also promotes early differentiation of keratinocytes, as indicated by the presence of ECaBP in these cells (19). Further studies are required to determine whether part of this calcium effect is mediated by the increase in the VDR production, as this increase may potentiate the activity of the low 1,25-(OH)2D in vitamin D-deficient animals. Part of the action of vitamin D on skin differentiation and VDR synthesis may be linked to its effects on calcium homeostasis. However, vitamin D has additional effects; it can induce the synthesis of suprabasal keratins and accelerate the differentiation of small immature keratinocytes in population 1 and their synthesis of VDR. It is not known at present whether vitamin D acts directly on the KL1 gene or indirectly, via its general action on cell differentiation, as no vitamin D response element has yet been demonstrated on the promoter of this gene. In any case, sufficient amounts of 1,25-(OH)2D3 and VDR are required for these effects, unlike the situation for the induction of early differentiation.
In conclusion, vitamin D and calcium have distinct and tissue-specific effects on VDR production. Neither serum calcium nor vitamin D levels appear to mediate changes in VDR mRNA in the kidneys of vitamin D-deficient rats, at least during the first week of treatment, but regulation of VDR protein synthesis cannot be excluded. In contrast, the VDR mRNA in the duodenum may respond to the calcium content of the diet. Extracellular calcium appears to be a major regulator of transcription of the VDR gene in the epidermis, whereas both vitamin D and calcium may contribute to the posttranscriptional regulation of VDR in this tissue.
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Acknowledgments |
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Received August 25, 1997.
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