1,25-Dihydroxyvitamin D3 Increases Nuclear Vitamin D3 Receptors by Blocking Ubiquitin/Proteasome-Mediated Degradation in Human Skin

Xiao-Yan Li1, Mohamed Boudjelal1, Jia-Hao Xiao, Zhen-Hui Peng, Agatha Asuru, Sewon Kang, Gary J. Fisher and John J. Voorhees

Department of Dermatology University of Michigan Medical School Ann Arbor, Michigan 48109


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-Dihydroxyvitamin D3 (D3) exerts its effects by binding to and activating nuclear vitamin D3 receptors (VDRs) that regulate transcription of target genes. We have investigated regulation of VDR levels in human skin in vivo and in cultured human keratinocytes. Quantitative ligand-binding analysis revealed that human skin expressed approximately 220 VDRs per cell, which bound D3 with high affinity [(dissociation constant (Kd) = 0.22 nM]. In human skin nuclear extracts, VDR exclusively bound to DNA containing vitamin D3 response elements as heterodimers with retinoid X receptors. Topical application of D3 to human skin elevated VDR protein levels 2-fold, as measured by both ligand-binding and DNA-binding assays. In contrast, the D3 analog calcipotriene had no effect on VDR levels. Topical D3 had no effect on VDR mRNA, indicating that D3 either stimulated synthesis and/or inhibited degradation of VDRs. To investigate this latter possibility, recombinant VDRs were incubated with skin lysates in the presence or absence of D3. The presence of D3 substantially protected VDRs against degradation by human skin lysates. VDR degradation was inhibited by proteasome inhibitors, but not lysosome or serine protease inhibitors. In cultured keratinocytes, D3 or proteasome inhibitors increased VDR protein without affecting VDR mRNA levels. In cells, VDR was ubiquitinated and this ubiquitination was inhibited by D3. Proteasome inhibitors in combination with D3 enhanced VDR-mediated gene expression, as measured by induction of vitamin D3 24-hydroxylase mRNA in cultured keratinocytes. Taken together, our findings indicate that low VDR levels are maintained, in part, through ubiquitin/proteasome-mediated degradation and that low VDR levels limit D3 signaling. D3 exerts dual positive influences on its nuclear receptor, simultaneously stimulating VDR transactivation activity and retarding VDR degradation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear vitamin D3 receptors (VDRs) mediate the effects of 1,25-dihydroxyvitamin D3 (D3) through cis-acting vitamin D3 response elements (VDREs) present in the transcriptional regulatory region of target genes (1, 2). VDR and retinoid X receptors (RXRs) bind as heterodimers (VDR/RXR) to natural VDREs of types DR3 and DR6, which consist of two directly repeated half-sites with a consensus sequence 5'-AGGTCA-3', separated by 3 or 6 bp, respectively. Upon binding D3, the VDR/RXR heterodimers act to stimulate transcription of target genes (3, 4).

Expression of VDR may be positively controlled by D3 (5, 6, 7, 8, 9, 10). Previous studies have shown that D3 up-regulates levels of VDR mRNA in intestinal tissue and osteosarcoma cell lines (8, 9, 11). Alternatively, D3 may increase VDR protein levels posttranslationally by reducing proteolytic degradation of VDR. Although it has been shown that VDR protein is susceptible to proteolysis in vitro (10, 12, 13), the mechanism of VDR degradation in vivo is not well understood.

Skin is a major target tissue for D3 (14). Growth and differentiation of the predominant cell type in skin, the keratinocyte, are regulated by D3 (15, 16, 17). We have previously shown that VDRs are expressed and functionally active in human skin in vivo and cultured human keratinocytes (4, 18). Topical application of D3 to human skin induces expression of the vitamin D3 24-hydroxylase gene, which contains a VDRE within its promoter (3, 19, 20).

In the current study, we have quantified VDR levels in human skin, and investigated regulation of VDR expression and degradation by D3. We find that VDR protein is degraded by the ubiquitin/proteasome pathway, and that D3 binding to VDR inhibits ubiquitination and retards degradation resulting in elevated VDR levels. Inhibition of VDR degradation by D3 or proteasome inhibitors increases VDR-mediated gene expression. Thus, D3 regulates target gene expression through two distinct effects on VDR: stimulation of transactivation activity, and inhibition of proteasome-mediated breakdown.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
D3 Increases Nuclear VDR Protein Levels in Human Skin in Vivo
Direct ligand-binding assays revealed that nuclear extracts from human skin specifically bound D3 with high affinity (Fig. 1aGo). The calculated dissociation constant (Kd) was 0.22 ± 0.04 nM, and the level of VDR was 47.8 ± 3.7 fmol/mg protein (n = 6). Assuming equal expression of VDR among all cells in the skin, the number of VDRs per cell was 221. Treatment of human skin topically for 4 days with 0.05% D3 increased VDR levels by nearly 100% (Fig. 1bGo), compared with vehicle-treated skin.



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Figure 1. D3 Binds with High Affinity to VDR in Human Skin in Vivo

a, Nuclear extracts prepared from human skin cells were incubated with the indicated concentrations of [3H]D3, and specific binding was measured by the charcoal absorption method. The insert shows Scatchard analysis. The calculated dissociation constant and concentration of VDR were 0.22 ± 0.04 nM (n = 6) and 47.8 ± 3.7 fmol/mg protein (n = 6), respectively. b, VDR protein levels in skin treated for 4 days with vehicle (open bar) or 0.05% D3 (hatched bar). VDR levels were measured by ligand-binding, using a single saturating dose (10 nM) of [3H]D3. Data are presented as fold change of D3-treated skin values relative to vehicle-treated skin for each paired sample, and expressed as means ± SEM (n = 11 for paired samples of vehicle-treated and D3-treated skin). *, P < 0.05.

 
We have previously demonstrated that VDR in human skin binds to a VDRE (DR-3) DNA probe exclusively as a heterodimer with RXR in electrophoretic mobility shift assays (4). This conclusion is supported by the following observations: 1) with nuclear extracts from human skin, unlabeled DR-3 probe, but not mutant DR-3 probe, competes for formation of specific VDRE (DR-3)-bound complexes (4); 2) Specific retarded complexes are completely supershifted with an anti-RXR antibody; and 3) an anti-VDR antibody that prevents DNA binding abolishes formation of specific retarded complexes and complexes supershifted with anti-RXR antibody (4). Therefore, we used electrophoretic mobility supershift assays to examine the effect of D3 treatment on VDR/RXR levels in human skin. Topical treatment of human skin with D3 increased the level of VDR/RXR heterodimers nearly 100%, compared with vehicle-treated skin (Fig. 2Go). In contrast, treatment of skin with a synthetic D3 analog, calcipotriene, which binds with high affinity to VDR and stimulates VDR-mediated transcription (21), did not elevate VDR levels (Fig. 2Go). Thus, two independent methods of measurement, ligand-binding and DNA-binding assays, demonstrate that D3 causes a 100% increase in VDR levels in human skin in vivo.



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Figure 2. Topical Treatment with D3, but not Calcipotriene, Increases VDR/RXR Heterodimers in Human Skin in Vivo

Human skin was treated for 4 days with vehicle (open bar), 0.002% calcipotriene (hatched bar), or 0.002% D3 (solid bar). VDR/RXR complexes in nuclear extracts were quantified by gel retardation assays, as described in Materials and Methods. Insert shows representative VDR/RXR retarded complexes from two subjects. Data are presented as fold change relative to vehicle-treated skin, and expressed as means ± SEM (n = 7–8). *, P < 0.01.

 
D3 Does Not Up-Regulate VDR mRNA in Human Skin in Vivo
To determine whether increased VDR levels seen after D3 treatment resulted from increased VDR gene expression, we measured VDR mRNA levels in vehicle and D3-treated human skin. VDR mRNA levels in both vehicle-treated and D3-treated skin were below the limit of detection for Northern blot analysis. Therefore, skin RNA samples were reverse-transcribed, and VDR mRNA was measured by semiquantitative PCR. RT-PCR was also performed for retinoic acid receptor-{gamma} (RAR{gamma}) mRNA, which is not affected by topical D3 treatment (X. Y. Li, unpublished observation), as an internal control. As shown in Fig. 3Go, low levels of VDR mRNA were detected in vehicle-treated and D3-treated skin from five human subjects. The level of VDR mRNA was not significantly altered by topical D3 treatment in any of these subjects, indicating that increased levels of VDR protein after D3 treatment resulted from either increased VDR translation, and/or decreased VDR degradation.



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Figure 3. Topical Treatment with D3 Does Not Alter VDR mRNA Levels in Human Skin in Vivo

Human skin was treated for 4 days with vehicle or 0.002% D3. Total RNA was extracted and analyzed by semiquantitative RT-PCR assays using primers specific for VDR and RAR{gamma}. RAR{gamma} was used as an internal control. The top panel shows an autoradiograph of a Southern blot containing RT-PCR products corresponding to VDR mRNA from five subjects. The lower panel shows an ethidium bromide-stained agarose gel containing the control RAR{gamma} product from the same five subjects.

 
D3 and Proteasome Inhibitors Protect against VDR Protein Degradation by Extracts from Human Skin
To examine the effect of D3 on VDR breakdown, we measured the capacity of human skin lysates to degrade [35S]VDR in the presence or absence of D3. In the absence of D3, VDR underwent rapid degradation, with a half-life of approximately 2 h (Fig. 4aGo). Addition of D3 reduced VDR degradation by skin lysates by approximately 70% (Fig. 4bGo). To characterize the proteolytic activity in skin lysates responsible for VDR breakdown, we tested the effect of several protease inhibitors. The proteasome inhibitors MG132 and lactacystin (22) significantly blocked VDR degradation, whereas the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and the lysosome protease inhibitor E64 had no effect (Fig. 4bGo). The combination of MG132 and D3 was not significantly better at protecting against VDR degradation by skin extracts than either compound alone (Fig. 4bGo). These results suggest that human skin extracts contain proteasome activity that degrades VDR, and that D3 protects against this degradation. To further substantiate this conclusion, we directly measured proteasome activity in human skin lysates. As shown in Table 1Go, human skin lysates contained proteolytic activity that hydrolyzed proteasome substrates fluorescein isothiocyanate-casein, Suc-Leu-Leu-Tyr-7-amino-4-methylcoumarin (AMC), and Boc-Leu-Arg-Arg-AMC (23, 24). Hydrolysis of each of these substrates was stimulated by ATP and blocked by the specific proteasome inhibitor MG132, but not by calpain inhibitor (Table 1Go). These data indicate that skin lysates contain characteristic proteasome activity.



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Figure 4. VDRs Are Rapidly Degraded by Proteasome Activity in Human Skin

a, In vitro-translated [35S]VDR protein was incubated with skin extracts (50 µg) for the indicated times. VDR protein was detected by SDS-PAGE. Inset shows representative VDR protein bands. The control lane shows in vitro translated VDR that was not incubated with skin extract. Triangle indicates position of full-length, nondegraded VDR protein. Changes in intensity of this band were quantified by PhosphorImager and are expressed in the bar graph. Data are presented as fold change relative to control and are means ± SEM (n = 4). *, P < 0.05. b, D3, proteasome inhibitors MG132 and lactacystin (lact), serine protease inhibitor PMSF, and lysosome protease inhibitor E64 were added to the in vitro VDR degradation assay and incubated for 2 h. Data are presented as percent degradation of the [35S]VDR added to the skin extracts. Inset shows representative VDR protein bands. Triangle indicates position of full-length, nondegraded VDR protein, which was quantified by PhosphorImager, and is expressed in the bar graph. Data are means ± SEM (n = 4–6). *, P < 0.05.

 

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Table 1. Human Skin Lysates That Degrade VDR Contain ATP-Stimulated Proteasome Activity

 
VDRs Are a Substrate for Ubiquitination
The above data indicate that VDRs are degraded by proteasome activity in human skin. Since ubiquitination usually targets protein for proteasome-mediated breakdown, using a cell culture model, we examined whether VDRs are a substrate for ubiquitination. HaCaT keratinocytes were transfected with expression vectors for His-tagged VDR and Flag-tagged ubiquitin, or with empty Flag expression vector. After transfection, MG 132 was added to the cultures to prevent proteasome-mediated VDR degradation. His-VDR expressed in the cells was purified and analyzed for ubiquitination by Western blot analysis using anti-Flag antibody (Fig. 5Go, upper panel) or anti-VDR antibody (Fig. 5Go, lower panel).



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Figure 5. VDR Are Ubiquitinated in Cultured Keratinocytes

Human keratinocyte HaCaT cells were transfected with expression vectors for Flag, or Flag-ubiquitin, alone and with His-VDR. Thirty-six hours after transfection, D3 (1 µM, +D3) or vehicle (0.1% ethanol, -D3) was added. Two hours later, MG132 (50 µM) was added, and cells were harvested 8 h later. His-VDR expressed in the cells was purified by nickel chelate chromatography and analyzed for ubiquitination by Western blot analysis using anti-Flag antibody (upper panel) or anti-VDR antibody (lower panel), as described in Materials and Methods. Ubiquitinated VDR appears as a broad band that migrates on SDS-PAGE with apparent molecular masses between 70 kDa and >200 kDa. Results are representative of three experiments.

 
Western analysis with anti-Flag antibody of cells transfected with empty Flag alone (Fig. 5Go, upper panel, lane 1) or with His-VDR, or Flag-ubiquitin alone (Fig. 5Go, upper panel, lane 2) revealed a light diffuse. Ubiquitination of VDR was readily observable in vehicle-treated cells that had been cotransfected with His-VDR and Flag-ubiquitin. Ubiquitinated VDR was detected as a broad band that migrated on SDS-PAGE with apparent molecular masses between 70 kDa and in excess of 200 kDa (Fig. 5Go, upper panel, lane 4). The level of expression of His-VDR was similar in cells cotransfected with His-VDR and empty Flag, or with Flag-ubiquitin (Fig. 5Go, lower left panel, lanes 3–6). With longer exposures, VDR Western blots revealed additional slower migrating bands representing ubiquitinated His-VDR in cells cotransfected with His-VDR and Flag-ubiquitin (Fig. 5Go, lower right panel, lane 4). Treatment of HaCaT keratinocytes with D3 substantially reduced VDR ubiquitination, as demonstrated by the loss of slower migrating bands on Flag (Fig. 5Go, upper panel, lanes 5 and 6) and VDR (Fig. 5Go, lower panels, lane 6) Western blots (Fig. 5Go).

D3 and Proteasome Inhibitors Increase VDR Protein Levels and Functional Activity in Human Skin Keratinocytes
Treatment of cultured human keratinocytes, D3, or proteasome inhibitors, lactacystin or MG132, elevated VDR protein levels (Fig. 6Go). This finding was observed in the absence of any change in VDR mRNA levels (data not shown). To investigate the functional consequences of elevating VDR levels by blocking proteasome-mediated VDR degradation, we examined the effect of proteasome inhibitors on vitamin D3 24-hydroxylase gene expression. Vitamin D3 24-hydroxylase is induced by D3, and this induction is mediated by VDR (3, 4, 19, 20, 25). Treatment of keratinocytes with D3 alone resulted in a 6-fold increase in vitamin D3 24-hydroxylase mRNA expression, while addition of lactacystin alone had essentially no effect, relative to control (Fig. 7Go). Addition of lactacystin plus D3 enhanced induction of vitamin D3 24-hydroxylase mRNA 10-fold relative to control (Fig. 7Go). These data indicate that raising VDR levels by blocking proteasome-mediated VDR degradation results in increased responsiveness of keratinocytes to D3.



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Figure 6. D3 and Proteasome Inhibitors Increase VDR Levels in Cultured Human Keratinocytes

Keratinocytes were treated with 0.1% DMSO (Control), 0.1 µM D3, 20 µM lactacystin, or 20 µM MG132 overnight. VDR levels were determined by Western blot using an anti-VDR antibody (clone 9A7). Results are representative of three experiments.

 


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Figure 7. Proteasome Inhibition Enhances D3-Induced Vitamin D3 24-Hydroxylase Gene Expression

Cultured keratinocytes were treated with vehicle (0.1% DMSO, first and second bars) or 20 µM lactacystin (LAC, third and fourth bars) overnight, and then treated with 0.1 µM D3 for 4 h (second and fourth bars). Total RNA (20 µg) prepared from treated cells was analyzed by Northern blot for vitamin D3 24-hydroxylase and 36B4 (internal control) mRNA levels. Inset shows a representative Northern blot of vitamin D3 24-hydroxylase mRNA (24-OHase mRNA) and 36B4. Vitamin D3 24-hydroxylase hybridization signals were normalized to those of 36B4. Data are presented as fold change of normalized values relative to vehicle-treated keratinocytes. Results are representative of two independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
D3 has been shown to increase the level of its nuclear receptor in a variety of cultured cell types. In some cell types, this increase in VDR protein is associated with elevated VDR mRNA expression (8, 9, 11), while in other cell types D3 elevates VDR protein without altering VDR mRNA levels (10, 12, 26, 27). In vitro, D3 and synthetic D3 analogs that bind to VDR alter VDR’s susceptibility to degradation by proteases such as trypsin, chymotrypsin, and proteinase K (27, 28, 29). These results have led to the conclusion that ligand binding causes conformational changes in VDR that make it less vulnerable to proteolytic attack. In vivo studies utilizing D3-deficient animal models have also demonstrated up-regulation of VDR levels by D3 (13, 30). The present study demonstrates that, in normal human skin in vivo and in cultured human keratinocytes, D3 increases VDR protein without altering VDR mRNA. We identified in human skin a novel mechanism for controlling VDR protein levels by ubiquitin/proteasome-mediated VDR degradation. Furthermore, we demonstrated that inhibition of proteasome activity in cultured human keratinocytes raises VDR levels, which results in increased D3-dependent, VDR-mediated target gene expression. In human skin, RXR levels are 20 times greater than VDR levels (31). Therefore, changes in VDR levels are rate limiting for VDR/RXR-mediated target gene expression.

Topical treatment with D3 caused a 100% increase in VDR levels in human skin in vivo. Interestingly, treatment of skin with the synthetic D3 analog, calcipotriene, which is used clinically to treat psoriasis, had no effect on VDR levels. Although calcipotriene and D3 bind to VDR with similar affinity (21), their binding sites appear to be qualitatively distinct (32). In vitro at physiological concentrations, calcipotriene was much less effective than D3 in protecting VDRs from limited proteolysis (32). These in vitro data are consistent with our in vivo results demonstrating that calcipotriene does not raise VDR levels in human skin. In transient transfection assays employing reporter gene constructs, D3 and calcipotriene activate VDR-mediated transcription to a similar extent (33). These data suggest that calcipotriene may stimulate VDR transactivation activity more efficiently than D3, and thereby compensate for its inability to raise VDR levels. They also suggest that D3-mediated increases in VDR levels are not simply due to increased binding of VDR to DNA.

The ubiquitin/proteasome pathway is the major route of disposal for many short-lived regulatory proteins (22, 34). Our data demonstrate that human skin contains proteasome activity that degrades VDR, and that blocking proteasome activity in cultured keratinocytes increases VDR levels. Proteasome inhibitors have recently been shown to raise VDR levels in osteosarcoma cells (35). These data indicate that VDR levels are regulated in part through continuous proteasome-mediated breakdown. Proteasome-catalyzed degradation limits the levels of other proteins, in addition to VDR. For example, the active, ligand-independent tyrosine kinase receptor ErbB-4 is maintained at low levels by continuous degradation (36). I{kappa}B, the inhibitor of transcription factor nuclear factor-{kappa}B (NF-{kappa}B), is also maintained at a constitutive level by continuous proteasome-mediated breakdown in certain cell types (37). Control of basal VDR levels by continuous degradation likely acts to limit D3 action in human skin, and thereby maintain normal D3 signaling.

In most cases, proteins destined for proteasome-mediated degradation are polyubiquitinated (38). Ubiquitination serves as a basis for recognition of proteins by the 26S proteasome complex. We found that VDR was a substrate for polyubiquitination in HaCaT keratinocytes. Interestly, D3 reduced VDR polyubquitination. These data raise the possiblity that D3 increases VDR levels in skin, in part, by reducing polyubiquitination and consequent proteasome-mediated breakdown. Ubiquitination involves the sequential action of three families of enzymes, termed E1, E2, and E3 (39, 40). Specificity of ubiquitination appears to be conferred by the large number of E2 and E3 enzymes, each of which acts on a restricted subset of substrates (38). The identity of the enzymes that ubiquitinate VDR must await further study. To our knowledge, our study is the first to demonstrate that VDRs are subject to polyubiquitination. A recent report describes ubiquitination of the progesterone receptor (41). Levels of other members of the nuclear receptor superfamily may also be regulated, in part, through the ubiquitin/proteasome pathway.

A common feature of short-lived regulatory proteins is the presence of regions that are rich in proline (P), glutamate (E), serine (S), and threonine (T) residues (42). These PEST sequences confer enhanced susceptibility to proteolysis. The manner in which this process occurs is not entirely clear, but likely involves recognition of PEST sequences by ubiquitinating enzymes or by a component of the proteasome complex. Recognition of PEST sequences by the proteolytic machinery may be direct or may require phosphorylation (42). Interestingly, analysis of VDR by the PEST-FIND computer program (EMBnet, Vienna, Austria) (42), which identifies PEST motifs in proteins, revealed a strongly positive PEST region in the ligand-binding domain of the VDR. This finding suggests that VDRs are rapidly turned over and raises the possibility that D3 binding prolongs VDR half-life by altering the conformation of the PEST sequence. Such an alteration may affect the phosphorylated state of VDR and/or ubiquitination of VDR, thus providing other possible mechanisms by which D3 stabilizes VDR. Identification of the ubiquitination sites in VDR, and better understanding of the role of PEST sequences in VDR proteolysis, will provide insights into the molecular basis for D3 stabilization of VDR. This information may allow the design of synthetic ligands whose potencies could be tailored by varying their ability to protect VDR against degradation by proteasomes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Procurement of Human Skin
Buttock skin from healthy adult human volunteers was treated with vehicle (70% ethanol, 30% polyethylene glycol, and 0.05% butylated hydroxytoluene), calcipotriene [MC903, 0.002% (wt/vol); gift from Dr. Lise Binderup, Leo Pharmaceuticals, Denmark), or D3 [0.002%, or 0.05% (wt/vol); gift from Dr. M. Uskokovic, Hoffmann-LaRoche, Inc., Nutley, NJ] for 4 days under occlusion. Skin samples were obtained as previously described (43). Tissue was placed immediately in ice-cold HBSS for preparation of nuclear extracts, or snap-frozen in liquid nitrogen for RNA preparation. All procedures involving human subjects were approved by the University of Michigan Institutional Review Board, and all participants provided informed written consent.

Preparation of Skin Cell Suspensions
Skin samples were placed in 0.25% (wt/vol) trypsin, 0.1% (wt/vol) EDTA for 30 min at 37 C. Trypsinization was halted by adding 0.05% FBS. Keratinocytes were released from the tissue by scraping. Cell suspensions were passed through a nylon filter to remove residual tissue and washed twice in serum-free DMEM.

Preparation of Nuclear Extracts and Whole-Cell Extracts
Epidermal cells (~2 x 108) were washed twice in PBS and resuspended in 1 ml of buffer [20 mM Tris (pH 8), 20 mM NaCl, 6 mM MgCl2, 0.2% Triton X-100, 1 mM dithiothreitol (DTT), 200 mM sucrose, 1 mM PMSF, 0.02 mg/ml leupeptin, and 0.02 mg/ml pepstatin]. Nuclear and whole-cell extracts containing receptors were prepared as described previously (31, 44).

VDR Ligand-Binding Assays
Measurement of [3H]D3 binding to VDR in nuclear extracts from human skin was performed using the dextran-coated charcoal adsorption method, as described previously (31). [3H]D3 (NEN Life Science Products, Boston, MA) was employed at a saturating concentration of 10 nM to determine levels of VDR by ligand binding. Nonspecific binding, determined in the presence of a 100-fold excess of unlabeled ligand, was subtracted from total binding to give specific binding. Calculation of receptor levels was based on a stoichiometry of one for ligand binding by receptors. Binding data were analyzed according to the method of Scatchard (45).

Gel Mobility Supershift Assays
Gel mobility supershift assays were performed as previously described (44). Briefly, double-stranded oligonucleotides containing consensus VDRE (DR3) were radiolabeled at their 5'-end using {gamma}-[32P]ATP (6000 Ci/mmol; NEN Life Science Products) and T4 polynucleotide kinase. The sequence of the VDRE used was: 5'-tcgact AGGTCA AGG AGGTCA gaga-3' (consensus hexameric half-sites shown in bold capital letters and flanking nucleotides designated in lowercase letters) (46). Radioactive DNA-protein complexes were resolved on nondenaturing 5% polyacrylamide gels, visualized by autoradiography, and quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

RT-PCR Assay
Total RNA was extracted from skin by guanidine isothiocyanate lysis, followed by ultracentrifugation through a CsCl gradient as previously described (47). RNA was then subjected to RT and subsequent PCR amplification with primers specific to VDR, or a D3-insensitive control, RAR-{gamma}. The sequences of the PCR primers were as follows: 5'-AAAAG CACCT GCCGA CCTCG TCCTC-3' and 5'-GCGGC CGATT CCAAA CTCAA GCATT-3' for VDR (48); and 5'-TGCTC CGTGT GCAAT GACAA GTCCT CTGGC-3' and 5'-CATGC CCACT TCGAA GCACT TCTGT AGCCG-3' for RAR-{gamma} (49). Semiquantitative PCR was performed with 0.5 µg RNA for 25 cycles. The RT-PCR products were resolved on 1.5% agarose gels. The PCR product corresponding to RAR-{gamma} mRNA was visualized by staining the gels with ethidium bromide. To detect low levels of VDR PCR product, gels were subjected to Southern blot analysis using an internal 32P-labeled oligonucleotide probe. The sequence of this probe was 5'-TGATG TAGGG TAAGG TGCCT-3' (48).

In Vitro Translation of VDR Protein and Degradation Assay
VDR protein was translated in vitro using a TNT T7-coupled rabbit reticulocyte lysate system (Promega Corp., Madison, WI) and a pXJ40-hVDR expression vector (a gift from Paul MacDonald, St. Louis University, St. Louis, MO) as a template, in the presence of [35S]methionine. Five microliters of 35S-labeled in vitro-translated VDR protein were incubated with 50 µg of human skin lysate in a 50 µl volume of 20 mM Tris, 50 mM NaCl, and 0.2 mM DTT at 37 C for the indicated times. Where indicated, incubations contained 0.5 µl dimethyl sulfoxide (DMSO), 2 mM PMSF, 25 µM E64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino]-butane, Sigma Chemical Co., St. Louis, MO), 50 µM MG132 (Z-Leu-Leu-Leu-H, BIOMOL Research Laboratories, Inc. Plymouth Meeting, PA), or 1 µM D3. SDS sample buffer was added to stop the reaction, and samples were run on 12% SDS-PAGE. The gel was dried and autoradiography was performed. To prepare skin lysates, frozen tissue (100 mg wet weight) was powdered under liquid nitrogen with a mortar and pestel and then homogenized in a glass homogenizer in 20 mM Tris-HCl, pH 7.4, 300 mM NaCl, and 0.2 mM DTT. The resulting homogenate was centrifuged at 10,000 x g for 30 min and the supernatant collected.

Proteasome Activity Assay
To measure proteasome activity, skin lysates (50 to 100 µg) were placed in 200 µl of assay buffer (20 mM HEPES, 0.5 mM EDTA [pH 8.0], 100 nM ATP) with 50 µM of Suc-Leu-Leu-Tyr-AMC, Boc-Leu-Arg-Arg-AMC, or fluorescein isothiocyanate casein (Sigma Chemical Co.). These substrates specifically measure the chymotryptic, tryptic, and protein hydrolysis activities of the proteasomes, respectively (50). After 2 h of incubation at 37 C, reactions were halted by addition of 2.5 ml cold ethanol. Proteasome activity was monitored by measuring the fluorescence of released AMC at excitation wavelength 380 nm and emission wavelength 460 nm, or of fluorescein isothiocyanate at excitation wavelength 490 nm and emission wavelength 520 nm (23, 24).

Keratinocyte Cultures and Treatments
Primary cultures of human keratinocytes were prepared as previously described (44). For determination of the effect of D3 or proteasome inhibitors on endogenous VDR levels, keratinocytes were incubated with vehicle (0.1% ethanol), D3 (0.1 µM), lactacystin (20 µM; Sigma Chemical Co.), or MG132 (20 µM; BIOMOL Research Laboratories, Inc.) for 48 h and then harvested by scraping. Nuclear extracts were prepared as described above for Western blot analysis of VDR protein levels.

To determine the effect of lactacystin on D3 induction of vitamin D3 24-hydroxylase gene expression, cultured keratinocytes were pretreated for 18 h with DMSO (0.1%) or lactacystin (20 µM). D3 (0.1 µM) or vehicle (0.1% ethanol) was added for 4 h, and total RNA was prepared. Northern blot assays for vitamin D3 24-hydroxylase mRNA levels were performed as previously described (18). The human vitamin D3 24-hydroxylase cDNA used as a probe was a gift from Dr. M. Haussler (University of Arizona, Tucson, AZ).

Western Blot Analysis
Nuclear extract proteins (25 µg) were resolved on 10% SDS-PAGE and transferred to a nitrocellulose membrane. Blots were probed with VDR-specific rat monoclonal antibody (clone 9A7, Affinity BioReagents, Inc. Golden, CO). VDR protein was detected using a biotin-labeled secondary antibody, streptavidin-conjugated horseradish peroxidase, and enhanced chemiluminescent substrates.

VDR Ubiquitination
A NdeI/BglII DNA fragment coding for VDR was generated by PCR using Vent DNA polymerase and pXJ40-hVDR plasmid as a template (4). The VDR fragment was sequenced and then ligated into the NdeI/BglII sites of pSG5-His (gift from Dr. Pierre Chambon, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France) to generate a His-tagged VDR mammalian expression vector. The pCMV-Flag-ubiquitin expression vector was constructed by inserting the ubiquitin cDNA, excised from pCMV-His-myc-Ub (gift from Dr. Ron R. Kopito, Stanford University, Stanford, CA) (51), into a pCMV-Flag expression vector (Eastman Kodak Co., Rochester, NY).

Immortalized human keratinocyte HaCaT cells were transfected with His-VDR, Flag-ubiquitin, or empty-Flag expression vectors, using Superfect (Qiagen, Chatsworth, CA). (1.0 µM) or vehicle (0.1% ethanol) was added to the culture 36 h after transfection, and cells were incubated for 2 h before addition of MG132 (50 µM). Eight hours later, cells were lysed in 3–4 ml of 6 M guanidinium-HCl, 0.1 M Na2HPO4/NaH2PO4 (pH 8.0) containing 5 mM imidazol per 100-mm dish, and His-tagged VDR was purified using Ni2+-NTA-agarose (Qiagen) as previously described (52). Ubiquitination of purified VDR was determined by Western analysis using anti-Flag antibody (Sigma Chemical Co.) or anti-VDR antibody.

Statistics
Comparisons among treatment groups were made with the paired t test. Multiple pairwise comparisons were made with the Tukey Studentized range test. All P values are two tailed and significant when <= 0.05.


    ACKNOWLEDGMENTS
 
We thank Suzan Rehbine and Carolyn Petersen for tissue procurement, Li Qin for technical assistance, Ted Hamilton for statistical analysis, Anne Chapple for editorial assistance, and Laura VanGoor for graphics preparation.

This work was supported in part by a research grant from Johnson & Johnson (New Brunswick, NJ) and in part by career development awards awarded to Sewon Kang and J. H. Xiao by the Dermatology Foundation.


    FOOTNOTES
 
Address requests for reprints to: Gary J. Fisher, Ph.D., Department of Dermatology, University of Michigan, Medical Science I, Room 6447, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0609.

1 These authors contributed equally to this work. Back

Received for publication November 16, 1998. Revision received June 8, 1999. Accepted for publication July 12, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW 1998 The nuclear vitamin D receptor: biological and molecular regulatory properties revealed [Review]. J Bone Miner Res 13:325–349[Medline]
  2. DeLuca HF, Zierold C 1998 Mechanisms and functions of vitamin D [Review]. Nutr Rev 56:S4–S10
  3. Zou A, Elgort MG, Allegretto EA 1997 Retinoid X receptor (RXR) ligands activate the human 25-hydroxyvitamin D3-24-hydroxylase promoter via RXR heterodimer binding to two vitamin D-responsive elements and elicit additive effects with 1,25-dihydroxyvitamin D3. J Biol Chem 272:19027–19034[Abstract/Free Full Text]
  4. Li XY, Xiao JH, Feng X, Qin L, Voorhees JJ 1997 Retinoid X receptor-specific ligands synergistically up-regulate 1,25-dihydroxyvitamin D3-dependent transcription in epidermal keratinocytes in vitro and in vivo. J Invest Dermatol 108:506–512[Abstract]
  5. Mangelsdorf DJ, Pike JW, Haussler MR 1987 Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation. Proc Natl Acad Sci USA 84:354–358[Abstract]
  6. McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, O’Malley BW 1987 Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science 235:1214–1217[Medline]
  7. Goto H, Chen KS, Prahl JM, DeLuca HF 1992 A single receptor identical with that from intestine/T47D cells mediates the action of 1,25-dihydroxyvitamin D3 in HL-60 cells. Biochim Biophys Acta 1132:103–108[Medline]
  8. Mahonen A, Pirskanen A, Keinanen R, Maenpaa PH 1990 Effect of 1,25-(OH)2 D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells. Biochim Biophys Acta 1048:30–37[Medline]
  9. Mahonen A, Pirskanen A, Maenpaa PH 1991 Homologous and heterologous regulation of 1,25-dihydroxyvitamin D3 receptor mRNA levels in human osteosarcoma cells. Biochim Biophys Acta 1088:111–118[Medline]
  10. Arbour NC, Prahl JM, DeLuca HF 1993 Stabilization of the vitamin D receptor in rat osteosarcoma cells through the action of 1,25-dihydroxyvitamin D3. Mol Endocrinol 7:1307–1312[Abstract]
  11. Costa EM, Feldman D 1986 Homologous up-regulation of the 1, 25-(OH)2 vitamin D3 receptor in rats. Biochem Biophys Res Commun 137:742–747[Medline]
  12. Wiese RJ, Uhland-Smith A, Ross TK, Prahl JM, DeLuca HF 1992 Up-regulation of the vitamin D receptor in response to 1,25-dihydroxyvitamin D3 results from ligand-induced stabilization. J Biol Chem 267:20080–20086
  13. Strom M, Sandgren ME, Brown TA, DeLuca HF 1989 1,25-dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo. Proc Natl Acad Sci USA 86:9770–9773[Abstract]
  14. Berger U, Wilson P, McClelland RA, Colston K, Haussler MR, Pike JW, Coombes RC 1988 Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissue. J Clin Endocrinol Metab 67:607–613[Abstract]
  15. Hosomi J, Hosoi J, Abe E, Suda T, Kuroki T 1983 Regulation of terminal differentiation of cultured mouse epidermal cells by 1{alpha},25-dihydroxyvitamin D3. Endocrinology 113:1950–1957[Abstract]
  16. Bikle DD, Gee E, Pillai S 1993 Regulation of keratinocyte growth, differentiation, and vitamin D metabolism by analogs of 1,25-dihydroxyvitamin D3. J Invest Dermatol 101:713–718[Abstract]
  17. Bikle DD, Pillai S 1993 Vitamin D, calcium, and epidermal differentiation [Review]. Endocr Rev 14:3–19[Medline]
  18. Kang S, Li XY, Duell EA, Voorhees JJ 1997 The retinoid X receptor agonist 9-cis retinoic acid and the 24-hydroxylase inhibitor ketoconazole increase activity of 1,25-dihydroxyvitamin D3 in human skin in vivo. J Invest Dermatol 108:513–518[Abstract]
  19. Akeno N, Saikatsu S, Kawane T, Horiuchi N 1997 Mouse vitamin D-24-hydroxylase: molecular cloning, tissue distribution, and transcriptional regulation by 1{alpha},25-dihydroxyvitamin D3. Endocrinology 138:2233–2240[Abstract/Free Full Text]
  20. Armbrecht HJ, Chen ML, Hodam TL, Boltz MA 1997 Induction of 24-hydroxylase cytochrome P450 mRNA by 1,25-dihydroxyvitamin D and phorbol esters in normal rat kidney (NRK-52E) cells. J Endocrinol 153:199–205[Abstract]
  21. Kragballe K 1995 Calcipotriol: a new drug for topical psoriasis treatment [Review]. Pharmacol Toxicol 77:241–246[Medline]
  22. Fenteany G, Schreiber SL 1998 Lactacystin, proteasome function, and cell fate [Review]. J Biol Chem 273:8545–8548[Free Full Text]
  23. Craiu A, Gaczynska M, Akopian T, Gramm CF, Fenteany G, Goldberg AL, Rock KL 1997 Lactacystin and clasto-lactacystin ß-lactone modify multiple proteasome ß-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation. J Biol Chem 272:13437–13445[Abstract/Free Full Text]
  24. Dick R, Cruikshank A, Grenier L, Melandri FD, Nunes SL, Stein RL 1996 Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin ß-lactone. J Biol Chem 271:7273–7376[Abstract/Free Full Text]
  25. Chen ML, Heinrich G, Ohyama YI, Okuda K, Omdahl JL, Chen TC, Holick MF 1994 Expression of 25-hydroxyvitamin D3-24-hydroxylase mRNA in cultured human keratinocytes. Proc Soc Exp Biol Med 207:57–61[Abstract]
  26. Strom M, Krisinger J, DeLuca HF 1991 Isolation of an mRNA that encodes a putative intestinal alkaline phosphatase regulated by 1, 25-dihydroxyvitamin D3. Biochim Biophys Acta 1090:299–304[Medline]
  27. van den Bemd GC, Pols HA, Birkenhager JC, van Leeuwen JP 1996 Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060. Proc Natl Acad Sci USA 93:10685–10690[Abstract/Free Full Text]
  28. Nayeri S, Carlberg C 1997 Functional conformations of the nuclear 1{alpha},25-dihydroxyvitamin D3 receptor. Biochem J 327:561–568[Medline]
  29. Vaisanen S, Juntunen K, Itkonen A, Vihko P, Maenpaa PH 1997 Conformational studies of human vitamin-D receptor by antipeptide antibodies, partial proteolytic digestion and ligand binding. Eur J Biochem 248:156–162[Abstract]
  30. Zineb R, Zhor B, Odile W, Marthe R 1998 Distrinct, tissue-specific regulation of vitamin D receptor in the intestine, kidney, and skin by dietary calcium and vitamin D. Endocrinology 139:1844–1852[Abstract/Free Full Text]
  31. Fisher GJ, Talwar HS, Xiao JH, Datta SC, Reddy AP, Gaub MP, Rochette-Egly C, Chambon P, Voorhees JJ 1994 Immunological identification and functional quantitation of retinoic acid and retinoid X receptor protein in human skin. J Biol Chem 269:20629–20635[Abstract/Free Full Text]
  32. Nayeri S, Kahlen JP, Carlberg C 1996 The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2). Nucleic Acids Res 24:4513–4518[Abstract/Free Full Text]
  33. Carlberg C, Mathiasen IS, Saurat JH, Binderup L 1994 The 1,25-dihydroxyvitamin D3 (VD) analogues MC903, EB1089 and KH1060 activate the VD receptor: homodimers show higher ligand sensitivity than heterodimers with retinoid X receptors. J Steroid Biochem Mol Biol 51:137–142[CrossRef][Medline]
  34. Rolfe M, Chiu MI, Pagano M 1997 The ubiquitin-mediated proteolytic pathway as a therapeutic area. J Mol Med 75:5–17[CrossRef][Medline]
  35. Masuyama H, MacDonald PN 1998 Proteasome-mediated degradation of the vitamin D receptor (VDR) and a putative role for SUG1 interaction with the AF-2 domain of VDR. J Cell Biochem 71:429–440[CrossRef][Medline]
  36. Vecchi M, Carpenter G 1997 Constitutive proteolysis of the ErbB-4 receptor tyrosine kinase by a unique sequential mechanism. J Cell Biol 139:995–1003[Abstract/Free Full Text]
  37. Krappmann D, Wulczyn FG, Scheidereit C 1996 Different mechanisms control signal-induced degradation and basal turnover of the NF-{kappa}B inhibitor I{kappa}B{alpha} in vivo. EMBO J 15:6716–6726[Abstract]
  38. Pickart CM 1997 Targeting of substrates to the 26S proteasome [Review]. FASEB J 11:1055–1066[Abstract/Free Full Text]
  39. Ciechanover A 1994 The ubiquitin-proteasome proteolytic pathway. Cell 79:13–21[Medline]
  40. Hochstrasser M 1996 Ubiquitin-dependent protein degradation. Annu Rev Genet 30:405–439[CrossRef][Medline]
  41. Nawaz Z, Lonard D, Dennis A, Smith C, O’Malley B 1999 Proteosome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci USA 96:1858–1862[Abstract/Free Full Text]
  42. Rechsteiner M, Rogers SW 1996 PEST sequences and regulation by proteolysis [Review]. Trends Biochem Sci 21:267–271[CrossRef][Medline]
  43. Fisher GJ, Esmann J, Griffiths CEM, Talwar HS, Duell EA, Hammerberg C, Elder JT, Finkel LJ, Karabin GD, Nickoloff BJ, Cooper KD, Voorhees JJ 1991 Cellular, immunologic, and biochemical characterization of topical retinoic acid-treated human skin. J Invest Dermatol 96:699–707[Abstract]
  44. Xiao JH, Durand B, Chambon P, Voorhees JJ 1995 Endogenous retinoic acid receptor (RAR)-retinoic X receptor (RXR) heterodimers are the major functional forms regulating retinoids-responsive elements in adult human keratinocytes. Binding of ligands to RAR only is sufficient for RAR-RXR heterodimers to confer ligand-dependent activation of hRAR beta 2/RARE (DR5). J Biol Chem 270:3001–3011[Abstract/Free Full Text]
  45. Scatchard G 1949 The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672
  46. Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65:1255–1266[Medline]
  47. Elder JT, Fisher GJ, Zhang QY, Eisen D, Krust A, Kastner P, Chambon P, Voorhees JJ 1991 Retinoic acid receptor gene expression in human skin. J Invest Dermatol 96:425–433[Abstract]
  48. Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, O’Malley BW 1988 Cloning and expression of full length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 85:3294–3298[Abstract]
  49. Krust A, Kastner P, Petkovich M, Zelent A, Chambon P 1989 A third human retinoic acid receptor, hRAR-{gamma}. Proc Natl Acad Sci USA 86:5310–5314[Abstract]
  50. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL 1994 Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761–771[Medline]
  51. Ward CL, Omura S, Kopito RR 1995 Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83:121–127[Medline]
  52. Treier M, Staszewski LM, Bohmann D 1994 Ubiquitin-dependent c-Jun degradation in vivo is mediated by the {delta} domain. Cell 78:787–798[Medline]