Degradation of RXRs Influences Sensitivity of Rat Osteosarcoma Cells to the Antiproliferative Effects of Calcitriol

Kirsten Prüfer, Claudia Schröder, Krisztina Hegyi and Julia Barsony

Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Julia Barsony, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Room 422, Bethesda, Maryland 20892. E-mail: jul{at}helix.nih.gov.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several cell lines, including ROS17/2.8 rat osteosarcoma (ROS) cells, contain functional VDRs and RXRs but are resistant to the antiproliferative effects of calcitriol and retinoids. We explored the role of receptor degradation in this hormone resistance. Results of transactivation assays indicated that ROS cells contain insufficient amounts of RXR to activate a DR-1 reporter, and Western blot analyses of cell extracts showed that the degradation of RXR is accelerated and produces an aberrant 45-kDa RXR. We stably expressed functional fluorescent chimeras of VDR and RXR [green fluorescent protein (GFP)-VDR; yellow fluorescent protein (YFP)-RXR] to evaluate degradation mechanisms and the impact of excess receptor expression on antiproliferative effects. Microscopy showed a diminished expression of YFP-RXR in ROS cells compared with the expression in CV-1 cells. Treatment with inhibitors of proteasomal degradation (lactacystin and MG132) selectively enhanced GFP-VDR and YFP-RXR expression and also increased the endogenous levels of VDR and RXR. Expression of GFP-VDR had no effect on the sensitivity of ROS cells to calcitriol. Increases of RXR levels by YFP-RXR expression, drug treatments, or the combination of the two, however, restored the growth-inhibitory effects of calcitriol and 9-cis-RA and restored p21 induction by calcitriol. These studies revealed that an accelerated and aberrant RXR degradation could cause resistance to the antiproliferative effects of calcitriol and retinoids in ROS cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE IMPORTANCE OF hormonal treatment for cancer is well established. Retinoids are used routinely for the treatment of acute promyelocytic leukemia (1) and lymphoma (2). Moreover, clinical trials are under way applying calcitriol and its analogs for the treatment of breast and colorectal cancer (3) and prostate cancer (4). Experimental data suggest that calcitriol and its analogs have potential for the treatment of other cancer types including glioma, melanoma, sarcoma, pancreas, and renal cell carcinoma (5). The efficacy of hormonal cancer treatment is dependent on the expression of hormone receptors. The RAR mediates the effects of retinoids and the VDR mediates the antiproliferative effect of calcitriol. In many tumors, the loss of hormone responsiveness develops as a result of receptor loss. The cancer may be resistant from the beginning or may become resistant after prolonged treatment.

Treatment with RA or calcitriol is not always effective, even in receptor-positive tumors. Dissociation between VDR expression levels and growth-inhibitory effects of calcitriol have been described in several cancer cell lines (6, 7). Although rat osteosarcoma ROS17/2.8 (ROS) cells possess VDR (8) and respond to calcitriol by increases in osteopontin and osteocalcin mRNA levels (9), they do not respond to calcitriol by growth inhibition (10). Responsiveness of leukemia cells to RA is frequently lost after prolonged treatment, although RAR expression remains normal in most cases (11, 12). Thus, although the expression of VDR and RAR is essential for the effects of calcitriol and retinoids, other factors may also be critical for the antiproliferative effects of these hormones.

The mechanisms of hormone resistance are likely to be related to the mechanisms of receptor functions. Type I nuclear receptors, such as the ER, act as homodimers and the ER content of tumors is frequently a good predictor of the efficacy of antiestrogen treatment. In contrast, the type II nuclear receptors, such as VDR and RAR, require heterodimerization with the RXR for high-affinity binding to DNA (13). Recent studies indicated that of the three known subtypes of RXR, RXR{alpha} is functionally the most important in VDR actions (14, 15). RXR expression can be regulated via either transcriptional or posttranscriptional mechanisms. A posttranscriptional regulatory mechanism was indicated by findings on three different osteosarcoma cell lines containing similar amounts of RXR mRNA but varying amounts of RXR protein (16). The sensitivities of these cells to the growth-inhibitory effects of calcitriol or retinoids have not been evaluated.

It has been demonstrated that both RXR (17) and VDR (18, 19, 20) proteins are degraded by the proteasomes. Most substrates for the proteasome are poly-ubiquitinated, as has been shown for VDR (20) and RXR (17). Increased proteasomal activity and increased expression of proteasomes in cancer cells have been correlated with malignancy (21, 22), and proteasomal inhibitors have been shown to inhibit cancer growth (23). These data suggested to us that RXR degradation can be regulated in cancer cells, and the amount of RXR can be just as important for defining calcitriol and retinoid sensitivity as VDR or RAR expression.

In this study, we explored the role of RXR expression in the responsiveness of ROS cells to the growth-inhibitory effects of calcitriol and retinoids and the cell type-specific differences in RXR degradation. We increased the RXR content of ROS cells by stably expressing a yellow fluorescent protein chimera of RXR (YFP-RXR; RYRXR cells) and by inhibiting proteasomal degradation. For control, VDR expression was similarly increased in a subclone of ROS cells by stable expression of a green fluorescent protein chimera of VDR (GFP-VDR, RGVDR cells) and by inhibition of proteasomal degradation. Degradation of endogenous and recombinant RXR was studied in the presence and absence of proteasome and protein synthesis inhibitors. Our studies demonstrated that degradation of RXR is abnormal and accelerated in ROS cells compared with CV-1 cells. Moreover, both proteasomal inhibitors and the introduction of recombinant RXR restored the responsiveness of ROS cells to growth-inhibitory effects of calcitriol and RA. These findings provide evidence for the role of increased RXR degradation in the development of hormone resistance in ROS and possibly in other cancer cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antiproliferative Effects of Calcitriol Correlate with Transcriptional Activity of RXR
We compared the relationship between the transcriptional activities of VDR and RXR and the antiproliferative effects of calcitriol in ROS and CV-1 cells. Transcriptional activities of the endogenous VDR were evaluated using the 24-hydroxylase luciferase (24-OH/Luc) reporter. Treatment with 10 nM calcitriol induced 5 times more luciferase activity in ROS than in CV-1 cells (Fig. 1AGo). Transcriptional activities of the endogenous RXR were measured similarly, using the DR-1 luciferase (DR-1/Luc) reporter. Treatment with 100 nM 9-cis-RA induced twice the luciferase activity in CV-1 than in ROS cells (Fig. 1AGo). Next, we compared the effect of calcitriol on cell growth in ROS and CV-1 cells using [3H]thymidine assays. Increasing doses of calcitriol (10–1,000 nM) induced a small but significant inhibition of [3H]thymidine incorporation in CV-1 cells (P < 0.05; Fig. 1BGo). In contrast, calcitriol (10–1,000 nM) moderately increased [3H]thymidine incorporation (P < 0.001) in ROS cells (Fig. 1BGo). These data indicate discrepancies between the antiproliferative effect and the transcriptional activities of calcitriol and a correlation between the transcriptional activities of RXR and the antiproliferative effects of calcitriol. This prompted us to compare VDR and RXR expression in ROS and CV-1 cells.



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Figure 1. Resistance to the Growth-Inhibitory Effect of Calcitriol in ROS Cells Correlates with Increased RXR Degradation

A, Transcriptional activities of VDR and RXR in CV-1 and ROS cells. After transfection with 24-OH/Luc, cells were treated with 10 nM calcitriol (upper panel). After transfection with DR-1/Luc, cells were treated with 100 nM 9-cis-RA (lower panel). Luciferase activities were normalized with ß-galactosidase activity, and induction of transcription was calculated from values of hormone-treated cells over values from vehicle-treated cells. Mean ± 1 SD from three independent experiments. B, Effects of calcitriol on [3H]thymidine incorporation in ROS ({diamondsuit}) and CV-1 ({blacksquare}) cells. Cells were treated with graded concentrations of calcitriol or vehicle (ethanol, 0.1%) for 48 h as indicated, and [3H]thymidine was added for the final 16 h. [3H]Thymidine incorporation values are given as percent of control. Mean ± 1 SD from four experiments. C, Western blot analysis of VDR and RXR expression in CV-1 and ROS cells. Extracts from each cell type containing 5 µg of protein were separated by electrophoresis and subjected to Western blot analysis for VDR (upper panel). Extracts containing 10 µg of protein from untreated (lane 0) and cycloheximide (10 µg/ml)-treated cells (24 h) were subjected to Western blot analysis using RXR-D20 antibody (lower panel). Note the lower expression of the 54-kDa RXR{alpha} and the higher expression of the 45-kDa protein in ROS cells compared with CV-1 cells.

 
Enhanced RXR Degradation in ROS Cells
We used Western blot analysis to evaluate VDR and RXR levels in extracts from ROS and CV-1 cells. These studies revealed that VDR is expressed in higher concentrations in ROS cells than in CV-1 cells (Fig. 1CGo, upper panel). Densitometric measurements from multiple blots showed that the intensities of VDR-specific 57-kDa bands in samples from CV-1 cells were 42 ± 8% of the intensities in samples from ROS cells. In contrast, RXR expression was lower in ROS cells than in CV-1 cells (Fig. 1CGo, lower panel, treatment 0). Densitometric measurements showed that the RXR-specific 54-kDa band intensities in samples from CV-1 cells were 173 ± 10% (RXR-D20 antibody) of the intensities in samples from ROS cells. Western blot analysis using the RXR444 antibody showed similar results (169 ± 9%). Immunoreactive proteins were also detected in ROS cells at 45 kDa with the RXR-D20 antibody and at 45 kDa and 34 kDa with the RXR444 antibody. Low concentrations of 41- and 44-kDa immunoreactive proteins were detected in CV-1 cells with the RXR-D20 antibody.

The lower levels of RXR in ROS cells could result from reduced protein synthesis or increased RXR degradation. To compare degradation rates in ROS and CV-1 cells, we first inhibited new protein synthesis with cycloheximide (10 µg/ml media) for 24 h and monitored the loss of RXR over time. As evaluated in [35S]methionine incorporation experiments, this concentration of cycloheximide inhibits protein synthesis by 90 ± 1% in CV-1 cells and by 94 ± 0.6% in ROS cells. As shown in Fig. 1CGo, RXR{alpha} expression (54 kDa) decreased by 77 ± 10% after 24 h in ROS cells (Fig. 1CGo, lower panel, treatment 24), and only by 9 ± 9% in CV-1 cells. This experiment indicated that the RXR{alpha} degradation rate is faster in ROS cells than in CV-1 cells.

These findings raised the possibility that RXR degradation could account for the lost responsiveness to the growth-inhibitory actions of calcitriol in ROS cells. To explore this hypothesis in more detail, we developed subclones of ROS and other cells that stably express recombinant RXR{alpha} or VDR. We used fluorescent protein chimeras of these receptors for the easy monitoring of protein expression by fluorescence microscopy. This system allowed studies on receptor degradation and the impact of VDR and RXR overexpression on the sensitivity of ROS cells to the antiproliferative effects of calcitriol.

Expression of GFP-VDR and YFP-RXR Increases Transcriptional Activities
We established subclones of ROS cells that stably express either GFP-VDR (RGVDR cells) or YFP-RXR (RYRXR cells). We then compared transcriptional activities of GFP-VDR with the 24-OH/Luc reporter in ROS-derived cells with activities in a cell line derived from 293 cells (GL48). We also compared YFP-RXR activities with the DR-1/Luc reporter in ROS-derived cells with activities in a cell line derived from CV-1 cells (CYR). Figure 2Go shows cell line-specific differences between maximal responses to hormones. The maximal effects of calcitriol on 24-OH/Luc activities were similar in ROS and in RYRXR cells (Fig. 2Go, upper panel). However, YFP-RXR expression sensitized the RYRXR cells to the effect of calcitriol on transcriptional activity, as evidenced by a 10-fold decrease in the half-maximal effective concentration of calcitriol in RYRXR cells compared with ROS cells (Fig. 2Go, upper panel). Calcitriol induced higher activities in RGVDR cells than in ROS cells, indicating that the presence of GFP-VDR adds to the maximal transcriptional activity of the endogenous VDR (Fig. 2Go, upper panel). Graded concentrations of calcitriol (10 pM to 10 nM) increased 24-OH/Luc activities in RGVDR cells without influencing the half-maximal effective dose (Fig. 2Go, upper panel). Calcitriol effects were also increased in GL48 cells compared with activities in 293 cells (not shown).



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Figure 2. VDR and RXR Expression Correlates with Transcriptional Activities

24-OH/Luc was expressed in ROS, RGVDR, and RYRXR cells (upper panel). Cells were treated 24 h after transfection with graded concentrations of calcitriol for 24 h as indicated. DR-1/Luc was expressed in ROS, RGVDR, RYRXR, and CYR cells (lower panel). Cells were treated with 100 nM 9-cis-RA. Luciferase activities were normalized with ß-galactosidase activities, and induction of transcription was calculated from values from hormone-treated cells over values from vehicle-treated cells. Mean ± 1 SD from three independent experiments.

 
Figure 2Go (lower panel) shows the impact of recombinant receptor expression on the activities of the DR-1/Luc. The effects of 9-cis-RA (100 nM) on luciferase activities were similar in ROS and RGVDR cells (1.2-fold over vehicle-treated control; Fig. 2Go, lower panel), consistent with the notion that they express the same amounts of endogenous RXR. 9-cis-RA induced higher activities in RYRXR cells than in ROS cells, indicating that the YFP-RXR is transcriptionally active (Fig. 2Go, lower panel). Still, 9-cis-RA effects were substantially higher in CYR cells than in RYRXR cells (Fig. 2Go, lower panel).

Together, these results demonstrate that the stably expressed recombinant receptors are transcriptionally active and indicate cell type-specific differences in receptor functions. Thus, these cell lines provide a functionally relevant model for our studies.

RXR Expression Restores the Antiproliferative Effect of Calcitriol and 9-cis-RA in ROS Cells
We used this model of RGVDR and RYRXR cells to evaluate the impact of increased receptor expression on the growth-inhibitory effects of calcitriol and 9-cis-RA. Primarily, we used [3H]thymidine incorporation assays to assess the effect of calcitriol and 9-cis-RA on the proliferation of ROS, RGVDR, and RYRXR cells. Similarly to the lack of effect in ROS cells, calcitriol had no effect on DNA synthesis in RGVDR cells (Fig. 3AGo). On the other hand, transient transfection of CYR cells with a plasmid encoding VDR increased VDR expression and also increased the effect of calcitriol on DNA synthesis; 100 nM calcitriol induced a 40% inhibition. This demonstrated that VDR expression could be the rate-limiting factor for growth-inhibitory effect of calcitriol in CV-1 cells, which expresses low levels of VDR but high levels of RXR.



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Figure 3. Distinct Sensitivities of ROS ({diamondsuit}), RGVDR ({blacksquare}), and RYRXR ({blacktriangleup}) Cells to the Antiproliferative Effects of Calcitriol and 9-cis-RA

A, Cells were seeded into 24-well plates and then treated for 48 h with graded doses of calcitriol (upper panel), or 9-cis-RA (lower panel) as indicated. Cells treated with vehicle (ethanol, 0.1%) were used as control. [3H]Thymidine was added for the final 16 h, and [3H]thymidine incorporation into DNA was measured. Values are given as percent of control, mean ± 1 SD (n = 12). B, Calcitriol has no effect on RXR expression in ROS and RGVDR cells whereas it stabilizes RXR in RYRXR cells. ROS, RGVDR, and RYRXR cells were treated with vehicle (-) or 10 nM calcitriol (+) for 16 h. High-salt extracts were prepared and 5-µg protein samples were separated by electrophoresis and subjected to Western blot analysis using the RXR DN197 antibody. C, Calcitriol has no effect on p21 expression in ROS and RGVDR cells, whereas it induces p21 expression in RYRXR cells. ROS, RGVDR, and RYRXR cells were treated with vehicle (-) or 10 nM calcitriol (+) for 16 h. High-salt extracts were then prepared and 10-µg protein samples were separated by electrophoresis and subjected to Western blot analysis using the anti-p21 antibody.

 
In contrast to RGVDR cells, RYRXR cells became highly sensitive to the growth-inhibitory effects of calcitriol. As Fig. 3AGo shows, treatments with graded concentrations of calcitriol (10 pM to 1 µM) caused a dose-dependent inhibition of DNA synthesis in RYRXR cells. This calcitriol responsiveness suggests that RXR levels are important for the antiproliferative actions. In addition, 9-cis-RA (1 nM to 10 µM) also had a dose-dependent inhibitory effect on [3H]thymidine incorporation in RYRXR cells, whereas it did not inhibit DNA synthesis in either ROS or RGVDR cells (Fig. 3AGo). The growth-inhibitory effects of 9-cis-RA were less pronounced than the effects of calcitriol.

We confirmed the differences of calcitriol and 9-cis-RA responsiveness between RYRXR, RGVDR, and ROS cells with cell counting experiments. Cells were counted before and after a 2-d treatment with vehicle, calcitriol, or 9-cis-RA. RYRXR cell number was decreased by 26 ± 9% after treatment with 1 nM calcitriol (P < 0.05), 35 ± 6% after 100 nM calcitriol (P < 0.01), 14 ± 21% after 1 nM 9-cis-RA (NS; P = 0.17), and 40 ± 23% after 100 nM 9-cis-RA (P < 0.001). ROS and RGVDR cell numbers after treatments with calcitriol (0.01 nM, 1 nM, 100 nM) or 9-cis-RA (1 nM and 100 nM) were equal or above the number in vehicle-treated cells, indicating that neither hormone has significant antiproliferative effects in these cell lines. Results were similar after 3 and 4 d of treatment (data not shown).

These data demonstrate that increased RXR expression sensitizes ROS cells to the growth-inhibitory effects of calcitriol and 9-cis-RA, whereas increased VDR expression is ineffective.

RXR Overexpression Allows for Stabilization of RXR by Calcitriol
Another well known effect of calcitriol is to stabilize VDR expression (24). In ROS cells calcitriol induced a 40% increase in VDR levels (not shown). Because VDR acts as heterodimer with RXR, we tested whether calcitriol stabilizes RXR as well. Western blot analyses showed that calcitriol does not stabilize RXR in ROS and RGVDR cells (Fig. 3BGo). Densitometry showed that RXR levels after calcitriol treatment (10 nM, 16 h) were 103 ± 9% of the levels after vehicle in ROS cells and 100 ± 12% in RGVDR cells. However, calcitriol treatment stabilized RXR in RYRXR cells (Fig. 3BGo). Densitometry showed that RXR levels after calcitriol treatment were 123 ± 30% higher than in vehicle-treated cells, and YFP-RXR levels were 38 ± 2% higher than in vehicle-treated cells. This calcitriol effect on RXR stabilization in RYRXR cells can contribute to the sensitivity of RYRXR cells to the growth-inhibitory effect of calcitriol.

RXR Overexpression Restores the Effectiveness of Calcitriol to Induce p21 Expression in ROS Cells
Previous studies demonstrated that calcitriol inhibits cell growth by regulating the expression of proteins involved in cell cycle control. One of the early mediators of this calcitriol-induced cell cycle arrest is p21, a protein that blocks the activity of the cyclin-dependent kinase 2 complexes (25, 26). In MCF-7 cells, calcitriol induced a 2-fold increase in p21 protein levels (27). We compared the effects of 10 nM calcitriol on the expression levels of p21 in ROS, RGVDR, and RYRXR cells to evaluate the potential effect of RXR overexpression on this calcitriol-dependent target. As shown in Fig. 3CGo, calcitriol induces a 120 ± 10% increase in p21 expression in RYRXR cells, whereas it had no effect on p21 expression in either ROS or RGVDR cells. These findings indicate that the ability of calcitriol to increase p21 levels is restored by the overexpression of RXR in ROS-derived cells. This finding supports the notion that selective calcitriol resistance in ROS cells is caused by a defect in RXR function.

Expressions of YFP-RXR and GFP-VDR Are Down-Regulated in ROS Cells
RGVDR, GL48, RYRXR, CYR, and RGFP (ROS cells stably expressing GFP) cells provided us a suitable system with which to study receptor degradation, because the cytomegalovirus promoter drives the transcription of GFP, GFP-VDR, and YFP-RXR in these cells. Consequently, differences in chimeric receptor expression can be generated only by posttranscriptional mechanisms, such as degradation. In addition, these cell lines allowed us to easily detect protein expression with fluorescence microscopy.

First, we compared the expression levels of YFP-RXR in RYRXR cells with the expression levels of YFP-RXR in CYR cells. Remarkably, only 0.6 ± 0.1% of RYRXR cells contained detectable levels of YFP-RXR fluorescence (Fig. 4AGo, panels a and b), whereas the majority of CYR cells contained detectable levels of YFP-RXR fluorescence (Fig. 4AGo, panels c and d). We confirmed the cell type-specific differences of receptor expression by Western blot analysis. Figure 4AGo (lower panel) shows that the 77-kDa YFP-RXR immunoreactivity is much stronger in CYR cells than in RYRXR cells. Next, we compared the expression level of GFP-VDR in RGVDR cells with the expression level of GFP-VDR in GL48 cells. Only 0.7 ± 0.1% of the RGVDR cells contained detectable levels of GFP-VDR fluorescence, even after three subsequent cycles of subcloning and rigorous selections with 1 mM geneticin (Fig. 4BGo, panels a and b). In contrast, GL48 cells typically contained GFP-VDR fluorescence in the majority of cells (Fig. 4BGo, panels c and d).



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Figure 4. Enhanced Degradation of GFP-VDR and YFP-RXR in RGVDR and RYRXR Cells

A, Microscopy shows that the expression of YFP-RXR is undetectable in the majority of the RYRXR cells (a and b), whereas it is easily detectable in the majority of the CYR cells (c and d). Fluorescence images are shown on the left and DIC images of the corresponding fields on the right. Bar, 25 µm. Western blot analyses confirmed that fluorescence intensities accurately represent receptor expression, as YFP-RXR content is lower in RYRXR cells than in CYR cells. Protein extracts (15 µg) were separated by electrophoresis and subjected to Western blot analysis using a GFP antibody. B, Microscopy shows that the expression of GFP-VDR is undetectable in the majority of the RGVDR cells (a and b), whereas it is easily detectable in the majority of the GL48 cells (c and d). Fluorescence images are shown on the left and DIC images of the corresponding fields are shown on the right. Bar, 25 µm. Western blot analyses confirmed that fluorescence intensities accurately represent receptor expression, as GFP-VDR content in RGVDR cells is lower than in GL48 cells. Protein extracts (15 µg) were separated by electrophoresis and subjected to Western blot analysis using a GFP antibody. C, Microscopy showed that RGFP cells express GFP in the majority of cells.

 
Microscopy showed that in the few ROS cells that expressed visible levels of fluorescent receptors, their subcellular distribution was similar to the distribution in other stably expressing cell lines. GFP-VDR was evenly distributed between the cytoplasm and the nucleus without hormone and was predominantly nuclear after the addition of calcitriol in RGVDR (Fig. 4BGo, panel a) and GL48 cells (Fig. 4BGo, panel c). We confirmed the cell type-specific differences of receptor expression by Western blot analysis. Figure 4BGo (lower panel) shows that the 80-kDa GFP-VDR immunoreactivity is much stronger in GL48 cells than in RGVDR cells. Calcitriol also induced the formation of intranuclear foci in both cell lines (not shown). YFP-RXR was predominantly nuclear with and without hormone in RYRXR (Fig. 4AGo, panel a) and CYR cells (Fig. 4AGo, panel c) and formed intranuclear foci after 9-cis-RA (not shown). These results indicate that the degradation of YFP-RXR and GFP-VDR is increased in ROS cells.

For control, we stably expressed the GFP vector in ROS cells (RGFP cells). As shown in Fig. 4CGo, GFP is expressed in the majority of cells. This finding supports the notion that GFP-VDR and YFP-RXR expressions are selectively decreased in ROS cells.

Proteasomal Inhibitors Restore the Expression of Functional YFP-RXR and GFP-VDR in RYRXR and RGVDR Cells
To clarify the degradation mechanisms, we tested the effects of protease inhibitors on YFP-RXR and GFP-VDR expression by fluorescence microscopy. YFP-RXR expression was detectable in 0.4 ± 0.04% of vehicle-treated RYRXR cells, and GFP-VDR expression was detectable in 0.5 ± 0.05% of the vehicle-treated RGVDR cells. After treatment with MG132, an inhibitor of the 26S proteasome, YFP-RXR, became detectable in 52 ± 1% of RYRXR cells and GFP-VDR became detectable in 99 ± 0.15% of RGVDR cells. Even the highly specific inhibitor of the ß-catalytic subunit of the 20S proteasome, lactacystin, increased expression of YFP-RXR 10- to 100-fold in RYRXR cells and expression of GFP-VDR 100-fold in RGVDR cells. Z-VAD-fmk (100 µM), a caspase-1 inhibitor, restored GFP-VDR fluorescence in 5.8 ± 0.4% of RGVDR cells, whereas it did not affect YFP-RXR fluorescence in RYRXR cells. Neither the caspase-3 inhibitor II nor the calpain inhibitor I restored YFP-RXR or GFP-VDR fluorescence in RYRXR or RGVDR cells. These results argue against the possibility that the decreased YFP-RXR and GFP-VDR expression in ROS subclones is caused by insufficient selection and support the notion that it is caused by increased proteasomal degradation.

To test whether proteasome inhibitors could restore expression of functional receptors, we monitored the hormone-dependent changes in receptor distribution. MG132 treatment (2 µM) for 16 h increased YFP-RXR content both in the nucleus and in the cytoplasm of RYRXR cells (compare Fig. 5AGo, panels a and b, with Fig. 4AGo, panel a). 9-cis-RA addition induced formation of intranuclear YFP-RXR foci (Fig. 5AGo, panel b), indicating that the receptors are functional. Similar MG132 treatment increased the cytoplasmic fraction of GFP-VDR in RGVDR cells (compare Fig. 5AGo, panel c, with Fig. 4BGo, panel a). Calcitriol addition (10 nM) induced translocation of GFP-VDR from the cytoplasm into the nucleus (Fig. 5AGo, panel d). Hormone-induced changes in YFP-RXR and GFP-VDR distributions were also apparent in lactacystin-treated cells. In addition, prolonged treatments with inhibitors promoted the formation of perinuclear GFP-VDR clusters (Fig. 5AGo, panel c). These experiments demonstrated that treatment of ROS cells with proteasomal inhibitors increase the levels of functional YFP-RXR and GFP-VDR.



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Figure 5. Treatments with Proteasomal Inhibitors Increase Chimeric and Endogenous VDR and RXR Levels

A, Microscopy shows the expression of functional receptors in the majority of RYRXR (a and b) and RGVDR (c and d) cells after treatment with 2 µM MG132 for 16 h. Ligand-dependent receptor reorganization signifies functional receptors. 9-cis-RA treatment induces formation of YFP-RXR foci (b) and calcitriol induces translocation of GFP-VDR from the cytoplasm into the nucleus (d). Bar, 25 µm. Lower panel, The increase in receptor expression after treatments with proteasomal inhibitors was confirmed by Western blot analysis. Extracts were made from RGVDR and RYRXR cells after treatments with either 0.2% DMSO (-) or 2 µM lactacystin (+). Extracts containing 5 µg of protein were separated by electrophoresis and probed with either the VDR antibody (RGVDR) or the RXR444 antibody (RYRXR). YFP-RXR was detected as a 77-kDa band, and GFP-VDR was detected as an 80-kDa band. Receptor expression was increased after lactacystin. B, Lactacystin increased endogenous VDR and RXR expression and inhibited the formation of a 45-kDa RXR fragment in ROS cells. Extracts were prepared after treatment with vehicle (-) or lactacystin (+) for 24 h. Protein extracts (5 µg) were separated by electrophoresis and VDR was detected as a 57-kDa band with the VDR antibody (upper panel). RXR was detected as a 54-kDa band with the DN197 antibody (lower left panel) and the RXR444 antibody (lower right panel). A 45-kDa RXR fragment was detected with antibody against the N terminus of RXR (lower right panel), but this was not detected with antibody against the C terminus of the RXR (lower left panel). Lactacystin treatment inhibited the accumulation of this 45-kDa fragment (lower right panel). C, Western blot analysis of extracts from cycloheximide-treated ROS cells showed a time-dependent increase of the 45-kDa RXR fragment levels (left panel). MG132 treatment caused a time-dependent decrease of the 45-kDa RXR fragment levels (right panel). Protein extracts (5 µg) were separated by electrophoresis and probed with RXR D20 antibody. Graph indicates expression of 45-kDa truncated RXR.

 
Proteasomal Degradation Alters Endogenous RXR and VDR Expression in ROS Cells
We used Western blot analysis to confirm findings with microscopy and to analyze degradation of endogenous receptors. Using a VDR antibody, we found that extracts of lactacystin-treated RGVDR cells contain more GFP-VDR than extracts from vehicle-treated RGVDR cells (Fig. 5AGo, lower panel). Densitometry showed that the intensity of the 80-kDa GFP-VDR band in extracts from lactacystin-treated RGVDR cells is 5.5-fold greater then the intensity in extracts from vehicle-treated cells. Using the RXR-DN197 antibody, we confirmed that lactacystin treatment increases YFP-RXR expression in RYRXR cells (Fig. 5AGo, lower panel). Densitometry showed that the intensities of the 77-kDa YFP-RXR band in extracts from lactacystin-treated RYRXR cells were 4.2-fold greater than the intensities in extracts from vehicle-treated cells. Together with calcitriol, lactacystin increased the YFP-RXR levels by an additional 240 ± 50% in RYRXR cells (not shown). These data confirm that proteasomal degradation is responsible for accelerated GFP-VDR breakdown in RGVDR cells and for accelerated YFP-RXR breakdown in RYRXR cells.

Treatment with lactacystin also increased expression of the endogenous VDR and RXR in ROS cells (Fig. 5BGo). The intensity of the endogenous 57-kDa VDR band was 2.6-fold greater in lactacystin-treated than in vehicle-treated ROS cell extracts (Fig. 5BGo, upper panel). Intensities of 54-kDa RXR band in extracts from lactacystin-treated ROS cells were 2.5-fold greater than the intensities in extracts from vehicle-treated cells in blots using the RXR444 antibody (Fig. 5BGo, lower right panel). The reproducibility of this increase was confirmed with Western blots using two additional antibodies (RXR-D20 and RXR-DN197), showing that the 54-kDa band intensities are 1.7-fold greater in extracts from lactacystin-treated cells than in the control extracts (Fig. 5BGo, lower left panel). Together with calcitriol, lactacystin increased the RXR levels by an additional 30 ± 25%. Calpain inhibitor I did not increase RXR levels. This indicates that the sensitivity of the endogenous RXR to inhibitors was similar to the sensitivity of the YFP-RXR.

In addition to the 54-kDa RXR band, a 45-kDa protein was recognized by antibodies against the N terminus of the RXR (Fig. 5BGo, lower right panel) (RXR-D20 and RXR444 antibodies) but not by the antibody against the C terminus of the RXR (RXR-DN197 antibody) (Fig. 5BGo, lower left panel). Lactacystin treatment decreased the expression of this 45-kDa protein 5.6-fold (Fig. 5BGo, lower right panel). The inverse effect of lactacystin on the 54-kDa and 45-kDa protein levels may indicate that the 45-kDa protein is a truncated RXR, a product of proteasomal degradation. This notion was further supported by experiments with cycloheximide treatment. These experiments demonstrated that the expression of the 45-kDa protein increases over time (Fig. 5CGo, left panel). Conversely, in the presence of both cycloheximide and MG132, the 45-kDa protein expression decreased over time (Fig. 5CGo, right panel). The high expression of this RXR degradation product is unique for the ROS cells. As shown in Fig. 1Go, the 45-kDa band intensities were 13-fold greater in ROS cell extracts than in CV-1 cell extracts.

Combined, these results demonstrate the accelerated degradation of RXR and the generation of an RXR degradation product in high quantities in ROS cells.

Overexpression of the 45-kDa RXR Fragment in CYR Cells Inhibits the Antiproliferative Effect of Calcitriol
Because there are several examples for dominant negative effects of a truncated nuclear receptor (28, 29), we performed proliferation assays to test for the possibility that the 45-kDa RXR degradation product from ROS cells may have a dominant negative effect on the growth-inhibitory actions of calcitriol in CYR cells. We generated an expression plasmid encoding a 45-kDa C-terminal truncated RXR by introducing a stop codon at amino acid 372 (RXR372stop) and expressed this truncated RXR in CYR cells together with VDR. For control, cells were transfected with RXR and VDR or with VDR alone. Figure 6Go shows that expression of VDR and RXR enhances the antiproliferative effect of calcitriol compared with the effect in either the mock-transfected CYR cells or CYR cells with VDR alone. Expression of RXR372stop with VDR prevented the antiproliferative effect of calcitriol. Differences were statistically significant (P < 0.05) both at low and high calcitriol doses. These results indicate that the 45-kDa RXR degradation product has a dominant negative effect on this VDR function.



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Figure 6. Truncated 45-kDa RXR (RXR372stop) Has Dominant Negative Effect on Calcitriol-Induced Growth Inhibition

CYR cells were mock transfected, or transfected with expression plasmids as indicated and then treated with vehicle (control) or graded concentrations of calcitriol for 48 h. ATP content was measured using the ViaLight assay. Data are expressed as percent of control; mean ± SEM (n = 8).

 
Inhibition of Proteasomal Degradation Increases VDR and Decreases RXR Transcriptional Activities
Recent studies showed that inhibition of proteasomal degradation inhibits transcriptional activities of several nuclear receptors, including RXR homodimers (30), ER, PR, TR{alpha}, MR, RAR{alpha}, and AR (31). In contrast, GR{alpha} activity is increased by treatment with proteasomal inhibitors. In keratinocytes, treatment with proteasomal inhibitor increased VDR transcriptional activity (20). We evaluated the effects of lactacystin on transcriptional activities of VDR and RXR. Lactacystin treatment in ROS cells increased the calcitriol-induced activity of stably expressed 24-OH/Luc reporter in ROSAI cells by 3-fold (Fig. 7Go, upper panel). Similar results were obtained with transient expression of the same reporter in ROS cells (not shown). Lactacystin treatment did not change 9-cis-RA-induced transcriptional activities of a transiently expressed DR-1/Luc reporter in ROS cells (Fig. 7Go, lower panel). Lactacystin did not change 9-cis-RA-induced DR-1/Luc reporter activities in RYRXR cells either. However, lactacystin decreased 9-cis-RA-induced activities of the DR-1/Luc reporter in CYR cells (Fig. 7Go, lower panel). These data confirm that proteasomal inhibitor treatment increases VDR transcriptional activity, whereas it does not decrease RXR transcriptional activity in ROS-derived cells.



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Figure 7. Lactacystin Treatment Increases VDR and Decreases RXR Transcriptional Activities

ROSAI cells (a ROS-derived cell line stably expressing a 24-OH/Luc reporter) were plated in triplet wells onto 12-well plates (upper panel). After 24 h, cells were treated with 0.2% DMSO or 2 µM lactacystin and with vehicle (open columns) or 10 nM calcitriol (shaded columns). ROS, RYRXR, and CYR cells were transfected with DR-1 luciferase reporter and ß-galactosidase expression plasmid (lower panel). After 24 h, cells were treated with 0.2% DMSO (open columns) or 2 µM lactacystin (shaded columns) and with 100 nM 9-cis-RA or vehicle. Luciferase activities were normalized with ß-galactosidase activities, and induction of transcription was calculated by dividing the values from hormone-treated cells with values from vehicle-treated cells. Mean ± 1 SD from three independent experiments.

 
Inhibition of Proteasomal Degradation Restores Antiproliferative Effects of Calcitriol in RGFP and RGVDR Cells and Potentiates the Effects in RYRXR Cells
To evaluate the significance of accelerated RXR degradation on growth regulation in ROS cells, we tested the antiproliferative effect of calcitriol and 9-cis-RA with simultaneous lactacystin treatment. Lactacystin treatment (2 µM) alone inhibited proliferation of ROS cells by 19 ± 4%, RGFP cells by 23 ± 7%, RGVDR cells by 64.5 ± 12%, and RYRXR cells by 79 ± 8%. This effect of lactacystin is well known; the inhibition is due to the effect on cell cycle proteins. In addition to this effect, lactacystin also increased the sensitivity of ROS, RGVDR, and RYRXR cells to the growth-inhibitory effect of calcitriol and 9-cis-RA. In the presence of lactacystin, 1 nM calcitriol inhibited [3H]thymidine incorporation by 14 ± 6% (P < 0.05), 100 nM calcitriol by 21 ± 13% (P < 0.05), 1 nM 9-cis-RA 14 ± 5 (P < 0.05) and 100 nM 9-cis-RA by 19 ± 10% (P < 0.05) in ROS cells. Lactacystin restored the antiproliferative effects of calcitriol in RGVDR cells, as well (Fig. 8Go, upper panel). The effects of 9-cis-RA were also similar in RGVDR and ROS cells; 1 nM 9-cis-RA inhibited [3H]thymidine incorporation by 20 ± 4% (P < 0.01) and 100 nM 9-cis-RA by 15 ± 8% (P < 0.05). This effect of lactacystin to sensitize ROS and ROS-derived cells to calcitriol and 9-cis-RA was specific and dose dependent. Expression of YFP-RXR in RYRXR cells already permitted the antiproliferative effect of calcitriol and 9-cis-RA. Inhibition of proteasomal degradation shifted the half-maximal effect one range to the left in RYRXR cells (Fig. 8Go, center panel). The sensitizing effect of lactacystin to the antiproliferative effects of calcitriol in RGFP cells was confirmed using the ViaLight assay (Fig. 8Go, lower panel). These results demonstrate that the accelerated proteasomal degradation of RXR is responsible for the hormone resistance in ROS cells and indicate that the antiproliferative effects of calcitriol are proportional to the expression of RXR.



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Figure 8. Lactacystin Treatment Sensitizes RGVDR, RYRXR, and RGFP Cells to the Antiproliferative Effect of Calcitriol

RGVDR cells (upper panel) or RYRXR cells (center panel) were treated with 0.2% DMSO ({diamondsuit}—–{diamondsuit}) or 2 µM lactacystin ({blacksquare}{blacksquare}) together with increasing concentrations of calcitriol or 0.1% ethanol for 48 h. [3H]Thymidine was added for the last 16 h, and [3H]thymidine incorporation into DNA was measured. ATP content was measured using the ViaLight assay. RGFP cells (lower panel) were plated into 96-well plates and then were treated as described above for 72 h. Values from calcitriol-treated cells are expressed as percent of values from ethanol-treated cells. Mean ± 1 SD were determined from three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We demonstrated here that aberrant and accelerated RXR proteasomal degradation is responsible for the resistance of ROS cells to the growth-inhibitory effects of calcitriol and retinoids. Stable cell lines expressing fluorescent protein chimeras of RXR{alpha} and VDR provided us with a suitable system to demonstrate changes in receptor degradation by microscopy and to determine the impact of different functional RXR contents on the antiproliferative effects of calcitriol and retinoids.

The importance of RXR for VDR and RAR functions is well documented. Several studies indicated that RXR is necessary for the growth-inhibitory effects, as well (15, 32). The fact that more RXR is needed for the antiproliferative effect of calcitriol than for transcriptional activity, however, has not been described. We found that the expression of excess RXR selectively increases the growth-inhibitory effect of calcitriol. The specificity of this effect was supported by our finding that excess RXR also restored the effect of calcitriol to increase expression of the cell cycle regulator p21. Similar increases in VDR levels increased the sensitivity of a 24-hydroxylase reporter to calcitriol without influencing the growth-inhibitory effect or p21 levels. The differential sensitivity of genes involved in mediating the effect of calcitriol on cell proliferation has been described previously (33). Separate signal transduction mechanisms for the antiproliferative effect have also been indicated by the need for 100-fold higher calcitriol concentrations (34), by the distinct calcitriol analog specificity (35, 36), and by the selective retention of growth-inhibitory effect in cells harboring a mutation in the VDR gene (37). Our results suggest that RXR is the rate-limiting factor for the antiproliferative effect of calcitriol.

We found a close correlation between RXR{alpha} expression and hormone sensitivity. Reduced levels of RXR{alpha} in ROS cells were associated with complete insensitivity to growth-inhibitory effects of calcitriol and retinoids. Elevated levels of RXR{alpha}, achieved by either proteasomal inhibitor treatment or expression of recombinant receptor, increased sensitivity, and the combination of both inhibitor treatment and recombinant RXR{alpha} expression further increased sensitivity. This correlation agrees with earlier findings on other cancer cells. Ovarian tumor cells lost sensitivity to retinoids when RXR{alpha} levels were reduced by antisense technology (38). The same loss of retinoid sensitivity was found in human monoblastic leukemia cells (32). Stable expression of sense RXR{alpha} in U937 monoblastic leukemia cells increased sensitivity to RA and calcitriol as shown by growth inhibition, cell-surface expression of specific markers, and cell adhesion during differentiation (32). Previous studies also showed that the RXR{alpha} mRNA levels are poor predictors of receptor content and hormone sensitivity (39). These previous results, together with our findings, suggest that posttranscriptional regulation of RXR level is critical for the hormone sensitivity of growth regulation.

Our experiments demonstrated anomalies in RXR{alpha} degradation in ROS cells. Levels of the endogenous RXR and the stably expressed YFP-RXR were both significantly reduced by proteasomal degradation, and data from experiments with cycloheximide showed their accelerated turnover. Moreover, an aberrant 45-kDa protein degradation product accumulated in ROS cells, in addition to the full-length RXR. Bland et al. (40) have also shown this protein in ROS cell extracts and, based on antibody sensitivities, they suggested that it can be a C-terminal truncation product. In experiments with cycloheximide we further supported that this protein is an RXR degradation product. Based on the size of this fragment, antibody sensitivity, lactacystin sensitivity, and the known cleavage sites in the RXR (41), we suggest that the 45-kDa RXR fragment is generated by a cleavage of the RXR after Arg371 by the trypsin-like activity of the proteasome and a subsequent inhibition of the degradation. This truncated RXR exerted a dominant negative effect on the growth inhibition by calcitriol in CYR cells, as well. This dominant negative effect is not without precedent: a dominant negative effect on RXR transcriptional activity has been described for another truncated RXR that was generated by the deletion of the last 20 amino acids at the C terminus (42). The accumulation of the dominant negative truncated RXR, together with an accelerated degradation of the functional RXR, leads to resistance to the antiproliferative effects of calcitriol in ROS cells.

We analyzed the potential catabolic pathways for RXR using microscopy and various inhibitors. Western blot analyses and functional studies demonstrated that the YFP-RXR expression accurately represents the expression and function of the endogenous RXR. Our results are in agreement with previous studies that reported cell type-specific differences in RXR degradation. RXR degradation by caspase cleavage and by the proteasomal pathway in promyelocytic leukemia cells has been reported (43), and this degradation is increased after the addition of ligands. Studies in human and rodent livers showed that the N terminus of RXR{alpha} is cleaved by calpain; this cleavage generated a 44-kDa fragment (44). Cleavage products of RXR by a lysosomal cathepsin L-type protease have been shown as 45-, 43-, and 31-kDa N-terminal truncated proteins in hepatocellular carcinoma cells (45). In ROS cells, we found that RXR degradation is not influenced by inhibition of caspases, calpain, or cathepsin, but it was highly sensitive to proteasomal inhibitors. The mechanisms that lead to accelerated RXR degradation are not known. A possible mechanism would be an interruption of the association between heat shock proteins and RXR, as association with heat shock proteins promotes proper folding of RXR and prevents receptor aggregation. Abnormal chaperone activity could lead to aberrant folding of the RXR, higher susceptibility to proteasomal degradation, and a possible burial of the majority of proteolytic sites (46). Regardless of the mechanism, the generation of a large fragment by proteasomal degradation is not unique for the RXR. In ROS cells a large 50-kDa fragment of VDR was generated by the overexpression of SUG1 and calcitriol treatment; the appearance of this product was prevented by treatment with proteasome inhibitors (18). When combined, our findings provide evidence for a unique regulation of retinoid and calcitriol effects through cell type-specific regulation of RXR degradation.

In conclusion, our data provide a new explanation for the development of selective hormone resistance in ROS cells and demonstrate that proteasomal degradation of receptors has important regulatory functions. There are several interesting aspects of these studies that warrant further investigation. Stable expression of RXR and VDR fluorescent chimeras allowed us to observe the changes in receptor subcellular distribution and expression. These cell lines will also be valuable for studies on the ligand-dependent regulation of receptor turnover (47) in more detail, on receptor interaction with molecular chaperones (48, 49), interactions with corepressors and coactivators, and on receptor phosphorylation (48). Similar cell lines could also be useful for monitoring RXR expression levels in different calcitriol-resistant tumor cells. Our results suggest that calcitriol and retinoid resistance may be reversed by combination treatment with proteasomal inhibitors, such as PS-341 (23). This treatment should be tested in other calcitriol-resistant malignant cells. Potentially, detailed analysis of RXR degradation pathways could reveal new regulatory checkpoints for the cell type-specific actions of VDR, RXR, and other type II nuclear receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
ROS and CV-1 cells were obtained and grown as described previously (50). Cell cultures were maintained in DMEM (Biofluids, Rockville, MD) with 10% FBS (HyClone Laboratories, Inc., Logan, UT), 2 mM glutamine (Invitrogen, Carlsbad, CA), 0.1 µM insulin, and 0.1 mg/ml gentamicin (Invitrogen). For maintaining stable expression of constructs in cells, gentamicin was replaced by 1 mM geneticin. To obtain stable expressing cell lines, ROS cells were transfected with GFP, GFP-VDR, or YFP-RXR. Design and functionality of these plasmids have been described earlier (51). Cell lines derived from ROS cells that stably express GFP (RGFP cells), GFP-VDR (RGVDR cells), and YFP-RXR (RYRXR cells) were established as described previously (51). For control, a clone stably expressing GFP-VDR derived from HEK-293 cells (GL48), and a clone stably expressing YFP-RXR derived from CV-1 cells (CYR) were used (51). The 293 cells and the CV-1 cells were chosen for the high transfection efficiency and for the low expression levels of endogenous VDR. For microscopy, cells were subcultured into glass coverslip chambers (Nalge Nunc International, Naperville, IL). For proliferation assays, 24-well plates were used, and for transactivation assays, 12-well plates were used (Costar, Cambridge, MA). During experiments, cells were maintained in serum-free MEM (Invitrogen) with 1% insulin, transferrin, selenium additive (Collaborative Biomedical Products, Bedford, MA), and 2 mM L-glutamine.

Transactivation Assays
Transcriptional activity of VDR was evaluated using a 24-OH/Luc reporter (gift from Dr. H. F. DeLuca, University of Wisconsin, Madison, WI) (52). This reporter was either stably expressed in ROSAI cells or transiently expressed in CV-1, ROS, RYRXR, RGVDR, and GL48 cells (0.07 µg/well) using Lipofectamine Plus transfection reagents (Invitrogen). One day after transfection, cells were incubated with either 10 nM calcitriol (Solvay Pharmaceuticals, Inc., Duphar, The Netherlands) or vehicle (0.1% ethanol) in media for 24 h. In a subset of experiments, luciferase reporter activity was evaluated in ROSAI cells after 24 h treatment with calcitriol and either lactacystin (2 µM) or vehicle [dimethylsulfoxide (DMSO) 0.2%].

Transcriptional activity of RXR was evaluated with a retinoid X response element DR-1/Luc reporter (gift from S. Minucci, EIO, Milan, Italy). First, the pGL2 standard luciferase vector (Promega Corp., Madison, WI) was modified by insertion of the adenovirus major late promoter sequence (53). Then the DR-1/Luc reporter was constructed by inserting a DR-1 sequence, 5'-AGGTCACAGGTCA-3', at the BglII site of the adenovirus major late promoter/luciferase vector. This DR-1/Luc was characterized in preliminary experiments. The DR-1/Luc was coexpressed with RXR and RAR in CV-1 cells. Graded concentrations of 9-cis-RA and RA (Sigma, St. Louis, MO) activated transcription of this DR-1/Luc (data not shown). In similar coexpression experiments, VDR, ER, and GR failed to activate this reporter after treatments with calcitriol (10 nM), 17ß-E2 (100 nM), and dexamethasone (100 nM) (Sigma), respectively (data not shown). For the current studies, the DR-1/Luc was transfected into CV-1, ROS, RGVDR, RYRXR, and CYR cells (0.07 µg/well). One day after transfection, cells were incubated with either 100 nM 9-cis-RA or vehicle (ethanol 0.1%) in media for 24 h. In a subset of experiments, luciferase reporter activity was evaluated in transfected ROS, RYRXR, and CYR cells treated simultaneously with lactacystin (2 µM) or vehicle (DMSO, 0.2%) and 9-cis-RA for 24 h.

For normalization of transfection efficiency, the ß-galactosidase standardization plasmid pGL3 (0.7 µg/well; Promega Corp.) was used in every experiment. Luciferase activities of cell lysates were determined with reagents from Promega Corp., and ß-galactosidase activities were determined with reagents from Sigma as described previously (50). Luminescence data were normalized with ß-galactosidase activity data and expressed as fold induction relative to vehicle-treated controls. Experiments were done in triplicate and were repeated at least twice. Data are mean ± 1 SD.

Proliferation Assays
Effects of calcitriol and 9-cis-RA on cell growth were assessed with [3H]thymidine incorporation assays, ATP content measurements, and cell counting. For [3H]thymidine incorporation assays, CV-1, ROS, RGVDR, and RYRXR cells were seeded into 24-well plates at 104 cells per well. Then, cells were treated with graded doses of calcitriol (10 pM to 1 µM ), 9-cis-RA (100 pM to 10 µM), or vehicle (0.1% ethanol) for 48 h. In a subset of experiments, cells were treated with lactacystin (2 µM) or DMSO (0.2%) simultaneously with calcitriol, 9-cis-RA, or vehicle. For the last 16 h, 0.5 µCi of [3H]thymidine was added to each well (Amersham Pharmacia Biotech, Piscataway, NJ). Then, media were removed and acid-soluble materials were extracted with trichloroacetic acid (once with 20%, twice with 10%). The acid-resistant pellet was solubilized with 1 N sodium hydroxide and neutralized with 1 N hydrochloric acid. Samples were collected into scintillation vials and measured in a liquid scintillation counter.

Cell counting experiments were performed after 2, 3, and 4 d of hormone treatment. After trypsinization, cells were counted in a Coulter Counter (Beckman Coulter, Inc., Fullerton, CA).

In preliminary experiments, we attempted to use transiently transfected ROS cells. We were unable to measure reproducible effects of calcitriol and 9-cis-RA in these experiments with either [3H]thymidine incorporation assays or with monitoring of cell number, supporting the notion that stable expression of proteins is advantageous for reproducibility of results.

The ViaLight assay (LumiTech, Nottingham, UK) was used to monitor ATP content of ROS and RGFP cells after exposure to calcitriol with and without lactacystin. Cells were plated in 96-well plates and treated with graded concentrations of calcitriol in the presence of lactacystin (2 µM) or vehicle (0.2% DMSO) for 72 h. Subsequently, ATP was measured according to the manufacturer’s instructions. Data are expressed as percent of control. The mean ± 1 SD was calculated from four parallels, and the experiments were repeated at least three times.

In another set of experiments, we evaluated the effect of a truncated RXR on the sensitivity of CYR cells to the growth inhibitory effect of calcitriol. Point mutations were introduced into the coding sequence of RXR using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Up- and downstream oligonucleotides were designed according to manufacturer’s instructions. To generate a 45-kDa truncated RXR, similar to the degradation product found in ROS cells, a stop codon was introduced after Arg371 by mutating alanine 372 from GCC to TGA (RXR372stop). The accuracy of this new coding sequence was verified by sequence analysis using the ABI Prism sequencing kit (Perkin-Elmer Corp., Norwalk, CT). CYR cells were subcultured into 96-well plates at 3,000 cells per well. The next day cells were transfected with expression plasmids (20 ng/well) using Lipofectamine 2000 reagents (Invitrogen). Expression plasmids were either herring sperm DNA (mock), VDR alone, VDR with RXR, or VDR with RXR372stop. DNA content was normalized with herring sperm DNA. One day after transfection, cells were treated for 2 d with graded concentrations of calcitriol or vehicle (eight wells per group). After treatment, ATP content was measured with the ViaLight assay. Statistical analysis was performed with unpaired t test.

Preparation of Cell Extracts and Immunoblotting
Extracts were prepared from subconfluent ROS, RGVDR, RYRXR, CV-1, GL48, and CYR cells. In one set of experiments, these cells were treated with calcitriol (10 nM), lactacystin (2 µM), a combination of calcitriol and lactacystin, or vehicle (0.2% DMSO) for 24 h. In another set of experiments, ROS and CV-1 cells were treated with the protein synthesis inhibitor cycloheximide (10 µg/ml) for 4, 8, 16, and 24 h in addition to lactacystin or vehicle. The effect of 10 µg/ml cycloheximide on protein synthesis was evaluated in pulse-chase experiments with [35S]methionine according to the method described by Demasi et al. (54). Briefly, ROS and CV-1 cells were subcultured into 24-well plates and incubated in the presence and absence of cycloheximide (10 µg/ml) for 16 h. Then, cells were pulsed for 3 h with [35S]methionine. Cells were lysed in detergent buffer solution (10 mM Tris-HCl, pH 8.0, containing 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS), aliquots were precipitated and washed with trichloric acid, and [35S]methionine incorporation was measured. Data were normalized for cell number.

After treatments, high-salt extracts were prepared from cells as described earlier (55). Briefly, after cells were harvested and had undergone three washing steps with cold PBS (pH 7.4), the suspensions were sonicated in lysis buffer containing 300 mM potassium chloride, 10 mM Tris-HCl (pH 7.4), 10 mM sodium molybdate, 1.5 mM EDTA, 1 mM dithiothreitol, and one protease inhibitor cocktail tablet per 50 ml lysis buffer (Roche Molecular Biochemicals, Indianapolis, IN). Lysates were then ultracentrifuged at 40,000 x g for 1 h, and the supernatants were collected and concentrated with Centricon concentrators (cut-off Mr 10,000, Amicon, Beverly, MA). Until use, cell extracts were kept at -70 C.

Before electrophoresis, samples were denatured for 5 min at 100 C in Tris-Glycine SDS sample buffer (Invitrogen) with 2.5% mercaptoethanol. Cruz Marker molecular weight standards (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as internal standards. Samples containing 5, 10, and 15 µg protein were separated on either 12% or 8% Tris-glycine polyacrylamide gels (Invitrogen) and electrotransferred to ECL-Hybond nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween-20 (PBS-T) for 1 h at room temperature and incubated overnight at 4 C with either of the following primary antibodies: polyclonal rabbit anti-GFP (CLONTECH Laboratories, Inc., Palo Alto, CA) in 1:1,000 dilution, monoclonal mouse anti-VDR (gift from Dr. H. F. DeLuca) in 1:2,500 dilution, polyclonal rabbit anti-RXR{alpha}, N-terminal specific (RXR-D20, Santa Cruz Biotechnology, Inc.) in 1:5,000 dilution, polyclonal rabbit anti-RXR{alpha}, N-terminal specific (RXR444, gift from P. Chambon, CNRS/INSERM, Strasbourg, France) in 1:1,000 dilution, polyclonal rabbit anti-RXR, C-terminal specific (RXR-DN197, Santa Cruz Biotechnology, Inc.) in 1:5,000 dilution, and rabbit anti-p21 (C-19; Santa Cruz Biotechnology, Inc.) in 1:500 dilution. Secondary antibodies were either horseradish peroxidase-labeled Cruz Marker compatible antirabbit secondary antibody (Santa Cruz Biotechnology, Inc.) in 1:7,500 dilution (30-min incubation at room temperature) or biotinylated antimouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 1:2,500 dilution. After washing, membranes were incubated for 1 h with streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) in 1:10,000 dilution. Primary antibodies were diluted in PBS-T with 5% nonfat milk, secondary antibodies were diluted in PBS-T, and washing steps were done with PBS-T. Blots were developed with enhanced chemiluminescence kit reagents according to the manufacturer’s protocol (Amersham Pharmacia Biotech). Specificity of immunoreactivities was confirmed by using multiple antibodies against the same antigen, immunoabsorption of the primary antibody with the antigen, and by omission of the primary antibody (not shown). Densitometric analysis of gels was performed using the Scion Image software (Scion Corp., Frederick, MD).

Evaluation of Receptor Expression and Subcellular Distribution by Microscopy
GFP, GFP-VDR, and YFP-RXR expression was detected using fluorescence microscopy in the presence and absence of different protease inhibitors. The following inhibitors were added to RGVDR and RYRXR cells: the 26S proteasome inhibitor MG132 (2 µM), the 20S proteasome inhibitor lactacystin (2 µM), the caspase inhibitors Z-VAD-fmk (10 and 100 µM) and Z-DVED-fmk (10 and 100 µM), and the calpain inhibitor (10 µM) (all from Calbiochem, La Jolla, CA) for 4 and 16 h. Controls were treated with equal concentrations of vehicle (DMSO) (Sigma). Receptor expression was monitored by viewing and counting fluorescing cells under an Axiovert 100 fluorescent microscope (Carl Zeiss, Thornwood, NY) with a 40x 1.2NA objective, band-pass 475- to 495-nm excitation and 515- to 565-nm emission filters. The number of fluorescing cells was compared with the number of cells determined by differential interference contrast (DIC) imaging in the same field. Ratios of fluorescing to all cells were calculated from at least 100 fields. Values are given as mean ± 1 SE.

To monitor subcellular distribution of receptors, images were collected with an Axiovert 100 fluorescent microscope equipped with a LSM 410 laser-scanning unit (Carl Zeiss). The 488 line of a krypton-argon laser was used for excitation with a band-pass 510- to 525-nm emission filter. Repeated experiments were done with the same parameters.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. S. Minucci (EIO, Milan, Italy) for the retinoid X response element DR-1-Luc plasmid, Dr. P. Chambon (CNRS/INSERM, Strasbourg, France) for the human RXR{alpha} cDNA and RXR{alpha} antibody, and Dr. H. F. DeLuca (University of Wisconsin, Madison, WI) for the p24OH/Luc23 plasmid and the VDR antibody. We thank Colin Segovis for editorial assistance and Daniel Abravanel for performing the ViaLight assays.


    FOOTNOTES
 
Abbreviations: CYR, CV-1 cells stably expressing YFP-RXR; DIC, differential interference contrast; DMSO, dimethylsulfoxide; DR-1/Luc, DR-1 luciferase reporter; GFP-VDR, chimera of green fluorescent protein with VDR; GL48, 293 cells stably expressing GFP-VDR; 24-OH/Luc, 24-hydroxylase luciferase reporter; PBS-T, PBS containing Tween 20; RGFP, ROS cells stably expressing GFP; RGVDR, ROS cells stably expressing GFP-VDR; ROS, rat osteosarcoma ROS17/2.8 cells; RYRXR, ROS cells stably expressing YFP-RXR; YFP-RXR, chimera of yellow fluorescent protein with RXR{alpha}.

Received for publication July 20, 2001. Accepted for publication January 9, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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