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Molecular Biology and Genetics |
and Retinoid X Receptor 
in Human Keratinocytes1
Department of Dermatology, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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(RAR) and retinoid X receptor
(RXR). We found that exposure of cultured primary human
keratinocytes to UV irradiation (30 mJ/cm2) substantially
reduced (50–90%) RAR and RXR mRNA and protein within 8 h.
The rates of disappearance of RAR and RXR proteins after UV
exposure or treatment with the protein synthesis inhibitor
cycloheximide were similar. UV irradiation did not increase the rate of
breakdown of RAR or RXR but rather reduced their rate of
synthesis. The addition of proteasome inhibitors MG132 and LLvL, but
not the lysosomal inhibitor E64, prevented loss of RAR and RXR
proteins after exposure of keratinocytes to either UV radiation or
cycloheximide. Soluble extracts from nonirradiated or UV-irradiated
keratinocytes possessed similar levels of proteasome activity that
degraded RAR and RXR proteins in vitro.
Furthermore, RAR and RXR were polyubiquitinated in intact cells.
RXR was found to contain two proline, glutamate/aspartate, serine,
and threonine (PEST) motifs, which confer rapid turnover of many
short-lived regulatory proteins that are degraded by the
ubiquitin/proteasome pathway. However, the PEST motifs in RXR did
not function to regulate its stability, because deletion of the PEST
motifs individually or together did not alter ubiquitination or
proteasome-mediated degradation of RXR. These results demonstrate
that loss of RAR and RXR proteins after UV irradiation results
from degradation via the ubiquitin/proteasome pathway. Taken together,
the data here indicate that ubiquitin/proteasome-mediated breakdown is
an important mechanism regulating the levels of nuclear retinoid
receptors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, ß, and
(4)
. In both cultured keratinocytes and human skin
in vivo, RAR and RXR are the predominant retinoid
receptor isoforms (5)
. Upon ligand binding, these
receptors up-regulate transcription of genes containing retinoic acid
response elements (RAREs). In addition, once liganded, both RARs and
RXRs can inhibit expression of certain genes by antagonizing the
transcriptional activity of the activator protein-1 complex
(c-jun/c-fos; Ref. 4
).
We have shown previously that exposure of human skin in vivo
to relatively low levels of UV irradiation causes substantial reduction
of RAR and RXR mRNA and protein (6)
. Loss of
retinoid receptors after UV irradiation was associated with loss of
retinoid-responsive gene expression in human skin. In essence, UV
caused a functional retinoid deficiency.
In the current study, we have investigated the mechanisms of UV
irradiation-induced loss of RAR and RXR in cultured human
keratinocytes. We find that UV irradiation inhibits synthesis of RAR
and RXR proteins. RAR and RXR are degraded with a half-life of
4 h in both UV-irradiated and nonirradiated cells. Reduction of
RAR and RXR after UV irradiation is blocked by inhibitors of
proteasome activity. Furthermore, we demonstrate that RAR and RXR
are substrates for ubiquitination and proteasome-mediated breakdown.
These data reveal a novel mechanism for regulation of retinoid
receptor-dependent signal transduction through the ubiquitin/proteasome
pathway.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, mouse RXR, His-tagged RAR,
and 36B4 cDNA were generously provided by Professor P. Chambon
(Institut de Genetique et de Biologie Moleculaire et Cellulaire,
Strasbourg, France). His-tagged c-Jun expression vector was kindly
provided by Professor D. Bohmann (European Molecular Biology
Laboratory, Heidelberg, Germany). The pCMV-Flag-ubiquitin
expression vector was constructed by inserting the ubiquitin cDNA
excised from a pCMV-His-myc-Ub plasmid (7)
into a
pCMV-Flag expression vector. The pSG5-His-tagged RXR expression
vector was generated by replacing the RAR cDNA in pSG5-His-RAR
with RXR cDNA excised from pSG5-RXR. pSG5-His-RXR was used as
a template in the PCR reaction to generate four pSG5-His-RXR
deletion mutants: His-RXR 
80–115, His-RXR 215–235,
His-RXR 80–115/220–235, and His-RXR 235–467.
Cell Culture.
Primary human keratinocytes were prepared from skin samples taken from
normal adult volunteers, as described previously (8)
.
Cells were grown as monolayer cultures in serum-free, low-calcium MCDB
153 medium in a humidified incubator with 5% CO2
at 37°C. For UV irradiation, cells were seeded in 10-cm dishes, grown
to 80% confluence, and then exposed to UV while submerged in 6 ml
of Dulbecco’s PBS. Media were then replaced, and plates were returned
to the incubator for the indicated times. Protease inhibitors (see
below) were dissolved in DMSO and added to the cultures immediately
after exposure to UV. HeLa cells were grown in DMEM containing 10%
fetal bovine serum.
UV Source and Irradiation.
Cultured human keratinocytes were irradiated with 30
mJ/cm2 UV using an Ultralite Panelite lamp
containing six FS24T12 UVB-HO bulbs. A Kodacel filter was used to
eliminate wavelengths <290 nm (UVC). The irradiation intensity was
monitored with an I1443 phototherapy radiometer and a SED240/UVB/W
photodetector (International Light, Newbury, MA).
Preparation of Whole-Cell Extracts and Western Blot Analysis.
After treatment, cultured keratinocytes were harvested in PBS by
scraping and pelleted by centrifugation at 500 x g for 5 min at 4°C. Cells were homogenized in 150 µl of
extraction buffer [10 mM Tris (pH 7.4), 300
mM NaCl, 1 mM EDTA, 10
mM MgCl2, 2
mM DTT, 5 mM
phenylmethysulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 0.5% NP40]. The homogenate was centrifuged at
14,000 x g for 15 min, the supernatant was
collected, and protein concentrations were measured using a commercial
Bio-Rad assay.
Equal amounts of whole-cell extract proteins were subjected to 10%
SDS-PAGE, transferred to polyvinylidene difluoride nitrocellulose
membrane, and probed with specific antibodies. Polyclonal antibodies
specific for RAR and RXR and monoclonal antibodies for ß-actin
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Immunoreactive proteins were visualized by enhanced chemiluminescence
detection and quantified by laser densitometry. RAR and RXR that
were overexpressed in HeLa cells were used as standards.
Pulse-Chase Labeling.
Keratinocytes at 80% confluency were preincubated in
cysteine/methionine-free MEM for 2 h and then pulse-labeled with
50 µCi/ml [35S]methionine/cysteine (1175
Ci/mmol) for 1 or 2 h. Cells were washed with PBS twice and then
either exposed or not exposed to UV (30 mJ/cm2).
In some experiments, cells were UV irradiated in PBS before the
addition of [35S]methionine/cysteine. Labeled
cells were placed in serum-free, low-calcium MCDB 153 containing 300
µg/ml methionine/cysteine, harvested 1–24 h after exposure to UV,
and washed with PBS, and whole-cell extracts were prepared, as
described above. Whole-cell extracts (200 µg) were incubated at 4°C
with 5 µl of anti-RAR or anti-RXR antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA). Samples were rotated overnight in 150
µl of immunoprecipitation buffer [10 mM Tris (pH 7.4), 1
mM EDTA, 150 mM NaCl, 10 mM
MgCl2, 2 mM DTT, 5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 0.5% NP40]. Protein G agarose beads (30 µl) were
added, and samples were rotated for 2 h at 4°C. Beads were
washed three times with immunoprecipitation buffer and then subjected
to 12% SDS-PAGE. Gels were dried, visualized, and quantified by STORM
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Proteasome Activity Assay.
Proteasome activity was measured as described by Craiu et
al. (9)
and Dick et al. (10)
.
Whole-cell extracts (50–100 µg) prepared from keratinocytes were
placed in 200 µl of assay buffer [20 mM HEPES,
0.5 mM EDTA (pH 8.0), and 100
nM ATP] containing 50 µM
of one of the following peptide substrates: Suc-Leu-Leu-Tyr-AMC,
Boc-Leu-Arg-Arg-AMC, or FITC-casein (Sigma Chemical Co., St. Louis,
MO). These substrates specifically measure the chymotryptic, tryptic,
and protein hydrolysis activities, respectively, of proteasomes
(9
, 10)
. After incubation for 2 h at 37°C,
reactions were halted by adding 2.5 ml of cold ethanol. Proteasome
activity was monitored by measuring the fluorescence of released AMC at
excitation wavelength 380 nm and emission wavelength 460 nm or FITC at
excitation wavelength 490 nm and emission wavelength 520 nm.
Transient Transfection and Purification of His-tagged Proteins.
HeLa cells were transfected with expression vectors for His-tagged
RAR, RXR, and c-Jun and Flag-tagged ubiquitin using Superfect
(Qiagen, Chatsworth, CA), according to the manufacturer’s
instructions. For purification of expressed His-tagged proteins, the
proteasome inhibitor MG132 was added to the media (final concentration,
50 µM) 36 h after transfection. Eight h 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.
His-RAR, His-RXR, and His-c-Jun were purified using
Ni2+-NTA-agarose (Qiagen, Chatsworth, CA), as
described (11)
. Ubiquitinated His-RAR, His-RXR, and
His-c-Jun were detected by Western analysis using anti-Flag monoclonal
antibody (Sigma Chemical Co., St. Louis, MO). His-RAR, His-RXR,
and His-c-Jun (antibody from Transduction Laboratories, Lexington, KY)
levels were determined by Western analysis.
Northern Blot Analysis of Retinoid Receptors.
Total RNA was isolated from UV-irradiated and nonirradiated cultured
human keratinocytes by guanidine hydrochloride lysis and
ultracentrifugation, as described (12)
. Northern analysis
of total RNA with randomly primed [32P]cDNA
probes for human RAR and RXR and 36B4 (a ribosomal protein used
as an internal control) were performed as described (13)
.
In Vitro Protein Translation and Degradation
Assay.
RAR and RXR proteins were translated in vitro in the
presence of [35S]methionine by TNT T7
reticulocyte lysate as described by the manufacturer Promega Corp.
(Madison, WI), using pSG5-RAR, pSG5-RXR, His-RXR 80–115,
His-RXR 220–235, and His-RXR 80–115/220–235 plasmids
as templates. 35S-labeled RAR or RXR
protein (5 µl) was incubated with keratinocyte whole-cell extract (50
µg), prepared as described above, but in the absence of any protease
inhibitor, in a total volume of 50 µl 20 mM
Tris (pH 7.4), 50 mM NaCl, and 0.2
mM DTT. Reaction mixtures were incubated at
37°C for 2 h and then resolved on 12% SDS-PAGE. Dried gels were
visualized and quantified by STORM PhosphorImager (Molecular Dynamics).
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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and RXR mRNA and Protein in Human
Keratinocytes. and RXR mRNA
(Fig. 1A)
and proteins (Fig. 1B)
. RAR mRNA began to
decline 8 h after UV and was reduced 60% 24 h after
irradiation. RXR mRNA began to decline 4 h after UV irradiation
and was further reduced 80% 24 h after UV. Quantitative analyses
of Western blots (shown in Fig. 1B
) revealed that both
RAR and RXR proteins were reduced 50% within 2 h after
UV exposure and continued to decrease for at least 24 h (Fig. 1B)
.
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and RXR proteins after UV exposure was not
attributable to reduced cell viability or general protein loss. Cell
viability, determined by trypan blue exclusion, was >90% 24 h
after UV treatment (data not shown). ß-actin protein levels, used as
a control, were not affected by UV (Fig. 1
B, inset). In
addition, c-jun and c-fos protein levels were increased within 2 h
after UV irradiation and remained elevated for at least 16 h (data
not shown).
RAR and RXR Turnover Is Rapid in Cultured Keratinocytes and
Not Altered by UV.
The preceding data demonstrate that UV irradiation reduces both RAR
and RXR mRNA and protein in a time-dependent manner. The decrease in
RAR and RXR mRNA after UV exposure could be the result of
inhibition of RAR and RXR gene transcription and/or increased RNA
degradation. Loss of RAR and RXR proteins preceded loss of their
transcripts, indicating that initial loss of retinoid receptor proteins
occurred via a posttranscriptional mechanism. The initial loss of
retinoid receptor proteins could result from reduced retinoid receptor
protein synthesis and/or accelerated retinoid receptor degradation. To
investigate these possibilities, we first determined the rate of RAR
and RXR protein turnover in nonirradiated keratinocytes.
Keratinocytes were treated with cycloheximide to prevent new protein
synthesis, and RAR and RXR protein levels were determined by
Western analysis.
Both RAR and RXR protein levels were reduced 40% within 2 h
of addition of cycloheximide, compared with their levels in untreated
control keratinocytes (data not shown). Levels of both retinoid
receptors continued to decline at similar rates, with 90% reduction
after 10 h. The rate of RAR and RXR protein breakdown after
cycloheximide treatment was similar to the rate of RAR and RXR
loss after UV exposure. These data suggest that UV irradiation inhibits
RAR and RXR synthesis rather than accelerates their breakdown.
To investigate this possibility, we performed pulse-chase experiments
with [35S]methionine/cysteine to metabolically
label RAR and RXR proteins in keratinocytes. Cells were UV
irradiated or left untreated, then pulsed for 2 h, and chased for
0–8 h. Synthesis of RAR (Fig. 2A)
and RXR (Fig. 2B)
, as assessed by
incorporation of label during the 2-h pulse, was 50% less in
UV-irradiated keratinocytes, as compared with nonirradiated
keratinocytes. During the chase period, the levels of labeled RAR
and RXR declined at similar rates (Fig. 2)
. These data indicate that
UV irradiation inhibits synthesis of RAR and RXR proteins,
without altering the rate of their breakdown. To further substantiate
this latter conclusion, keratinocytes were pulsed with
[35S]methionine/cysteine for 1 h, then
exposed to UV irradiation or left untreated, and chased for 1–24 h.
The rates of loss of labeled RAR (Fig. 3A)
and RXR (Fig. 3B)
were similar in UV
irradiated and nonirradiated keratinocytes. The half-lives of both
retinoid receptors were between 4 and 8 h, regardless of whether
cells had been UV irradiated (Fig. 3)
. Taken together, the above data
indicate that loss of retinoid receptors in human keratinocytes after
UV irradiation results from inhibition of their protein synthesis
through a posttranscriptional mechanism, coupled with inherent
(i.e., not altered by UV irradiation) breakdown.
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and RXR Proteins Are Degraded by the Proteasome Pathway
in Human Keratinocytes. and RXR in human keratinocytes. Keratinocytes
were UV irradiated or treated with cycloheximide and then cultured for
10 h in the presence of inhibitors specific for lysosomal
proteases [E64,
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)
butane; Ref. 14
], or proteasome activity [MG132
(Z-Leu-Leu-Leu-H) and LLvL (N-CBZ-Leu-Leu-Norvalinal); Ref.
15
], or in the presence of vehicle (DMSO). As expected,
RAR and RXR were reduced substantially (75–90%) in
vehicle-treated keratinocytes exposed to UV irradiation (Fig. 4A)
or incubated with cycloheximide (Fig. 4B)
.
Treatment of cells with MG132 and LLvL, but not E64, largely prevented
this loss of retinoid receptors in both UV-irradiated and
cycloheximide-treated cells. In UV-irradiated keratinocytes, MG132 and
LLvL reduced loss of RAR to <30% and completely prevented loss of
RXR (Fig. 4A)
. In cycloheximide-treated cells, addition
of MG132 and LLvL maintained RAR and RXR levels at or above their
respective levels in untreated control cells (Fig. 4B)
. The
proteasome inhibitor lactacystin also inhibited loss of RAR and
RXR in both UV-irradiated and cycloheximide-treated keratinocytes
(data not shown). MG132 and LLvL also maintained RAR and RXR at
their initial levels for at least 10 h in pulse chase experiments
in both UV-irradiated and nonirradiated keratinocytes (data not shown).
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and
RXR. and RXR in human
keratinocytes. To further support this conclusion, we measured
proteasome activity in whole-cell extracts prepared from irradiated and
nonirradiated human keratinocytes using synthetic fluorescent peptides,
specific for chymotryptic and tryptic hydrolyzing activities of
proteasomes (16)
. Keratinocyte extracts exhibited both
chymotrypic and tryptic proteasomal activities. Chymotryptic and
tryptic activities were similar in whole-cell extracts from irradiated
and nonirradiated keratinocytes and were inhibited by the proteasome
inhibitor MG132 but not by the calpain inhibitors I and II (data not
shown). Because the primary function of the proteasome is to hydrolyze proteins into oligopeptides (17) , we also used FITC-conjugated casein as a substrate. This protein substrate is degraded in an ATP-dependent reaction by the 26S complex, without ubiquitination (9) . Whole-cell extracts from both UV-irradiated and nonirradiated keratinocytes cleaved FITC-casein to similar extents. This activity was blocked by MG132 but not by the calpain I and II inhibitors (data not shown).
We next used in vitro-translated
35S-labeled RAR and
35S-labeled RXR as substrates for proteasome
activity in whole-cell extracts from either UV-irradiated or
nonirradiated keratinocytes. Whole-cell extracts from nonirradiated
keratinocytes degraded 48% and 63% of added RAR and RXR,
respectively (Fig. 5)
. Degradation of RAR and RXR was reduced to 2 and 24%,
respectively, by addition of the proteasome inhibitor MG132. Calpain I
and II inhibitors did not prevent degradation of either retinoid
receptor. Similar results were obtained using whole-cell extracts from
UV-irradiated keratinocytes and untreated or UV-irradiated human skin
(data not shown). Taken together, the above data demonstrate that
keratinocytes possess functional proteasome activity that degrades
RAR and RXR and that is unaltered by UV irradiation.
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and RXR Are Ubiquitinated. and RXR could be ubiquitinated in intact cells. HeLa cells
were transfected with His-tagged RAR or RXR or c-Jun expression
plasmids together with Flag or Flag-ubiquitin expression vectors. c-Jun
is known to be ubiquitinated (11)
and therefore served as
a positive control. We used HeLa cells for these experiments because of
their high transfection efficiency, compared with keratinocytes, which
is necessary for purification of expressed His-tagged retinoid
receptors. The rate of turnover of RAR and RXR after UV
irradiation or addition of cycloheximide in HeLa cells is similar to
that observed for keratinocytes (data not shown). Additionally, loss of
RAR and RXR proteins in either UV-irradiated or
cycloheximide-treated HeLa cells is prevented by MG132 and LLvL but not
E64 (data not shown). Therefore, proteasome-mediated degradation of
RAR and RXR appears to be similar in keratinocytes and HeLa
cells. After transfections, equivalent amounts of cell lysates were
used to purify His-tagged retinoid receptors or His-tagged c-Jun by
Ni+2-NTA chromatography. Retinoid receptors and
c-jun, in column eluates, that were ubiquitinated were detected by
Western analysis using anti-Flag antibody.
In column eluates from cells transfected with His-RAR and
Flag-ubiquitin, there was a broad ladder of bands that migrated with
apparent molecular weights larger than RAR (Fig. 6
, left), indicating formation of polyubiquitinated RAR.
These higher molecular weight forms of RAR were only present in
cells transfected with RAR and Flag-ubiquitin; they were absent in
cells transfected with His-tagged RAR or Flag-ubiquitin alone.
Similarly, His-RXR was polyubiquitinated in HeLa cells (Fig. 6
,
middle panel). The appearance of the polyubiquitination
ladder that was observed for RAR and RXR was similar to that
observed for the positive control c-Jun (Fig. 6
, right
panel). Western analysis confirmed that His-RAR, His-RXR,
and His-c-Jun had been efficiently expressed and recovered by
Ni+2-NTA chromatography from transfected cells
(data not shown).
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Are Not Required for Ubiquitination.. The first
was located within the A/B domain (amino acids 80–113 in human and
75–108 in mouse), and the second was located within the hinge D domain
(amino acids 215–233). To further investigate regulation of RXR
ubiquitination, we determined the ability of RXR mutant proteins
lacking one or both PEST motifs to be ubiquitinated and degraded by
proteasomes. His-tagged mutant RXR constructs were cotransfected
with Flag-ubiquitin and analyzed for ubiquitination as described above
for wild-type RXR. Ubiquitination of RXR proteins lacking the A/B
domain PEST motif (80–115; Fig. 7
), the D domain PEST motif (200–235; Fig. 7
), or both PEST motifs
(80–115/220–235; Fig. 7
) was readily detectable. The
COOH-terminal half of RXR (235–467, Fig. 7
), which does not
contain any PEST sequences, was also ubiquitinated. The levels of
expression and ubiquitination of all three mutant proteins were
comparable with those of wild-type RXR (Fig. 7)
. These data indicate
that the PEST motifs in RXR are not required for ubiquitination.
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PEST motifs are required for
proteasome-mediated degradation. Each of the three PEST deletion mutant
RXR proteins was translated in vitro and incubated with
proteasome-containing extracts from human skin. The three mutant RXR
proteins were substantially degraded within 2 h, and this
degradation was blocked by proteasome inhibitors MG132 and LLvL (data
not shown). Taken together, the above data indicate that the PEST
motifs in RXR do not function to regulate degradation through the
ubiqitin/proteasome pathway, as has been described for other proteins
(16
, 21, 22, 23, 24)
.
We also identified PEST motifs located within the A/B domain (amino
acids 56–85) and hinge D domain (amino acids 172–192) of RAR. In
addition, a third PEST motif was localized within the COOH-terminal of
the ligand-binding domain (amino acids 413–427). The functional role
of these PEST motifs in the turnover of RAR remains to be
determined.
The above data describe a novel mechanism for regulation of retinoid signaling through ubiquitin/proteasome-mediated degradation of retinoid receptors. Recent data indicate that the vitamin D receptor is a substrate for degradation by proteasomes (25) . We have found recently that the vitamin D receptor is ubiquitinated and degraded by proteasomes in human keratinocytes (26) . In addition, Nawaz et al. (22) reported that estrogen receptor levels, but not progesterone receptor or thyroid hormone receptor levels, are regulated by proteasome degradation, although estrogen receptor ubiquitination has not been demonstrated. These data raise the possibility that ubiquitin/proteasome-mediated breakdown participates in the regulation of the levels of some, but not all, members of the nuclear receptor superfamily. Future research should be directed toward determining which nuclear receptor members are ubiquitinated and toward identification and characterization of the substrate specificity of the ubiquitinating enzymes.
Ubiquitination of RAR and RXR, and their subsequent degradation
by the proteasome, likely functions to terminate the transcriptional
activity of both receptors. The identity and regulation of the enzymes
that ubiquitinate RAR and RXR remain to be determined. In
addition, it is possible that ubiquitination of RAR and RXR
serves not only to target the receptors for degradation but may also
serve to regulate their activities.
Additionally, the role of retinoic acid in regulating retinoid receptor
ubiquitination and turnover has yet to be determined. We found that
pretreatment of keratinocytes with retinoic acid prior to UV or
cycloheximide exposure did not prevent loss of RAR and RXR (data
not shown). This lack of effect of retinoic acid on degradation of
RAR and RXR is in contrast to the effects of ligands on vitamin D
and estrogen receptor turnover. Vitamin D stabilizes the vitamin D
receptor by inhibiting its ubiquitination and subsequent proteasomal
degradation (26)
. In contrast, estradiol induces
proteasome degradation of the estrogen receptor (22
, 27 , 28)
. The possible role of retinoic acid in the turnover of
RAR and RXR proteins warrants further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Babcock Fund for
Dermatological Research and by a grant from the Johnson & Johnson
Corporation. 
2 To whom requests for reprints should be
addressed, at Department of Dermatology, University of Michigan Medical
School, 1150 West Medical Center Drive, Medical Science I, Room 6447,
Ann Arbor, MI 48109-0609. Phone: (734) 763-1469; Fax: (734) 647-0076;
E-mail: dianemch{at}umich.edu
3 The abbreviations used are: RAR, retinoic acid
receptor; RXR, retinoid X receptor; AP-1, activator protein-1; AMC,
7-amino-4-methylcoumarin; PEST, proline, glutamate/aspartate, serine,
and threonine.
Received 8/11/99. Accepted 2/17/00.
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M. Wormke, M. Stoner, B. Saville, K. Walker, M. Abdelrahim, R. Burghardt, and S. Safe The Aryl Hydrocarbon Receptor Mediates Degradation of Estrogen Receptor {alpha} through Activation of Proteasomes Mol. Cell. Biol., March 15, 2003; 23(6): 1843 - 1855. [Abstract] [Full Text] [PDF]
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T. K. Fung, W. Y. Siu, C. H. Yam, A. Lau, and R. Y. C. Poon Cyclin F Is Degraded during G2-M by Mechanisms Fundamentally Different from Other Cyclins J. Biol. Chem., September 13, 2002; 277(38): 35140 - 35149. [Abstract] [Full Text] [PDF]
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G. Boskovic, D. Desai, and R. M. Niles Regulation of Retinoic Acid Receptor alpha by Protein Kinase C in B16 Mouse Melanoma Cells J. Biol. Chem., July 12, 2002; 277(29): 26113 - 26119. [Abstract] [Full Text] [PDF]
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 K. Prufer, C. Schroder, K. Hegyi, and J. Barsony Degradation of RXRs Influences Sensitivity of Rat Osteosarcoma Cells to the Antiproliferative Effects of Calcitriol Mol. Endocrinol., May 1, 2002; 16(5): 961 - 976. [Abstract] [Full Text] [PDF]
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D. L. Osburn, G. Shao, H. M. Seidel, and I. G. Schulman Ligand-Dependent Degradation of Retinoid X Receptors Does Not Require Transcriptional Activity or Coactivator Interactions Mol. Cell. Biol., August 1, 2001; 21(15): 4909 - 4918. [Abstract] [Full Text] [PDF]
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 D. M. Shin, F. R. Khuri, B. Murphy, A. S. Garden, G. Clayman, M. Francisco, D. Liu, B. S. Glisson, L. Ginsberg, V. Papadimitrakopoulou, et al. Combined Interferon-Alfa, 13-cis-Retinoic Acid, and Alpha-Tocopherol in Locally Advanced Head and Neck Squamous Cell Carcinoma: Novel Bioadjuvant Phase II Trial J. Clin. Oncol., June 15, 2001; 19(12): 3010 - 3017. [Abstract] [Full Text] [PDF]
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 X.-C. Xu, W. Y. L. Wong, L. Goldberg, S. C. Baer, J. E. Wolf, W. M. Ramsdell, D. S. Alberts, S. M. Lippman, and R. Lotan Progressive Decreases in Nuclear Retinoid Receptors during Skin Squamous Carcinogenesis Cancer Res., June 1, 2001; 61(11): 4306 - 4310. [Abstract] [Full Text] [PDF]
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 S. Lehmann, C. Paul, and H. Törmä Retinoid Receptor Expression and Its Correlation to Retinoid Sensitivity in Non-M3 Acute Myeloid Leukemia Blast Cells Clin. Cancer Res., February 1, 2001; 7(2): 367 - 373. [Abstract] [Full Text]
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 G. M. Clayton, S. Y. Peak-Chew, R. M. Evans, and J. W. R. Schwabe The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation PNAS, February 1, 2001; (2001) 41611298. [Abstract] [Full Text]
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G. M. Clayton, S. Y. Peak-Chew, R. M. Evans, and J. W. R. Schwabe The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation PNAS, February 13, 2001; 98(4): 1549 - 1554. [Abstract] [Full Text] [PDF]
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) and RXR
) mRNA in cultured keratinocytes after UV
irradiation (30 mJ/cm2). Inset,
representative Northern blots for RAR
) prior to pulse with
[35S]methionine/cysteine for 2 h. Cells were then
washed and placed in media containing unlabeled methionine/cysteine
(300 µg/ml each) for the indicated times. 35S-labeled
RAR
