This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lattuada, D. Articles by Di Blasio, A. M. Search for Related Content PubMed PubMed Citation Articles by Lattuada, D. Articles by Di Blasio, A. M.

Accumulation of Retinoid X Receptor- in Uterine Leiomyomas Is Associated with a Delayed Ligand-Dependent Proteasome-Mediated Degradation and an Alteration of Its Transcriptional Activity

Department of Obstetrics, Gynaecology and Neonatology (D.L., P.V., S.M., S.D.), Fondazione Policlinico-Mangiagalli-Regina Elena Hospital and University of Milan, 20122 Milano, Italy; Department of Obstetrics and Gynecology (M.V.), Clinica Macedonio Melloni and University of Milan, 20129 Milano, Italy; and Molecular Biology Laboratory (J.S., A.M.D), Istituto Auxologico Italiano, 20095 Cusano Milanino, Italy

Address all correspondence and requests for reprints to: Dr. Anna Maria Di Blasio, Molecular Biology Laboratory, Istituto Auxologico Italiano, Via Zucchi 18, 20095 Cusano Milanino, Italy. E-mail: a.diblasio{at}auxologico.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
An alteration of the retinoid pathway can influence the development of uterine leiomyomas in animal models, and retinoids have shown efficacy in inhibiting the growth of this benign tumor both in vitro and in vivo. However, the underlying mechanisms and biological implications are unclear. The present study was based on the demonstration of an accumulation of full-length retinoid X receptor (RXR) in leiomyomas that was not associated with a modification of its gene expression. This accumulation was shown to increase the transcription of the RXR-responsive gene cellular retinoic acid binding protein II (CRABP-II) and to be linked to the cellular redistribution of the receptor and to its retarded degradation via the ubiquitin/proteasome pathway. Accordingly, treatment with a specific proteasome inhibitor but not with protease inhibitors strongly inhibited the degradation of full-length RXR in cells deriving from both myometrium and leiomyoma, but the formation of RXR/ubiquitin conjugates was differentially regulated between the two cell types. Moreover, full-length RXR accumulated in leiomyomas was abnormally phosphorylated at serine/threonine residues relative to myometrial tissue. The ligand to RXR, 9-cis-retinoic acid, induced the receptor breakdown in smooth muscle cells deriving from both normal and tumor tissue, whereas a MAPK-specific inhibitor was able to reduce RXR levels only in leiomyoma cells. These results suggest that switching of the ubiquitin/proteasome-dependent degradation of RXR by phosphorylation in leiomyomas may be responsible for the accumulation of the receptor and the consequent dysregulation of retinoic acid target genes. The ability of retinoids to modify this molecular alteration may be the rationale for their use in the treatment of leiomyomas.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
UTERINE LEIOMYOMATA ARE the most common benign tumors in women of reproductive age, clinically diagnosed in 20–30% of women (1). As the single most common indication for hysterectomy, they are identified in 77% of the patients who have undergone this procedure, over 30% of which are performed for leiomyomata. Notwithstanding the high incidence of this tumor, medical treatments are limited and unable to cure the disease while surgery remains the primary treatment modality. Indeed, the use of GnRH analogs that inhibit ovarian steroid production, induces a remarkable shrinkage of the tumor but it cannot induce its eradication (2). Moreover, prolonged use can lead to severe side effects and cessation of treatment often results in a rapid regrowth of the tumor (3). Therefore, new strategies for the medical treatment of fibroids must be explored (1). Recently, retinoids have shown efficacy in inhibiting the growth of leiomyoma both in vitro and in animal models. A selective ligand of retinoid X receptor (RXR), LGD1069, could limit the progression of microscopic to larger gross tumors in a rat model of the disease (4). Furthermore, the retinoid analog 4-(N-hydroxyphenyl)retinamide (4-HPR) could induce growth inhibition and apoptosis of cells derived from human uterine leiomyomas and this effect was tumor specific because the growth of matched samples of normal myometrium was not inhibited (5).

Retinoids and their synthetic analogs transduce their signals primarily through two classes of nuclear receptors, the retinoic acid receptors (RAR, ß, and ) and the retinoid X receptors (RXR, ß, and ), which act as ligand-depended transcriptional activators and are characterized by a modular domain structure (6). RARs bind to response elements in target gene promoters in response to both all-trans-retinoic acid and 9-cis-retinoic acid (9cRA), whereas RXRs bind and activate transcription in response to only 9cRA. Phosphorylation processes are critical for the transcriptional activity of RAR and RXR because an abnormal phosphorylation might result in the dysregulation of retinoic acid (RA)-target genes (7). Moreover, a link between the phosphorylation status of the receptor and its ubiquitination/degradation has been proposed recently, possibly implicating this mechanism in the carcinogenetic process (8, 9, 10).

The precise mechanisms underlying the effects of retinoids on leiomyomas are still to be elucidated. However, the observation of an abnormally high expression of RXR protein in leiomyomata compared with normal human myometrium (2) pointed to a specific role for the retinoid pathway in leiomyoma formation and development. We designed our present analysis of RXR status in leiomyomas relative to the normal tissue to further assess the possible relationship between levels of this receptor and tumor development. These data demonstrate an accumulation of full-length RXR in the benign tumor when compared with the normal tissue, which was not associated with an increase in RXR gene expression. This accumulation was associated with an enhanced phosphorylation of the receptor and a delay in the receptor degradation through the ubiquitin/proteasome pathway with a consequent modulatory effect on transactivating activity. Supporting the rationale for the therapeutic use of retinoids in this context, treatment of leiomyoma smooth muscle cells with the ligand could partly revert this situation by inducing a cellular redistribution and the degradation of the receptor.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Accumulation of Full-Length RXR in Leiomyoma Tissue
We first evaluated RXR expression in leiomyomas and in correspondent myometrial tissues by Western blotting (Fig. 1A). A specific anti-RXR polyclonal antibody (D-20), recognizing the N-terminal segments, detected both full-length relative molecular mass (M_r) 54,000 RXR and two fragmented M_r approximately 45,000 and M_r approximately 42,000 RXRs (8). A significant accumulation of the M_r 54,000 form was detected in leiomyoma samples compared with matched myometrial tissues (Fig. 1A). A less evident accumulation in leiomyoma tissues was also observed for the fragmented M_r approximately 42,000 band, whereas the M_r approximately 45,000 band was barely detectable and only in the tumoral tissue. Figure 1B shows results obtained from the analysis of protein lysates deriving from n = 17 samples of myometrium and n = 17 matched leiomyoma tissues. Densitometric analysis of the M_r 54,000 band relative to the actin band was performed for all samples and showed a statistically significant increase in the amount of the full-length RXR in the benign tumors (Fig. 1B, left panel). Very similar results were obtained using an antibody recognizing a ligand binding sequence in the E-domain (N197), thus reacting with both full-length M_r 54,000 RXR and two similar degraded forms of RXRs (data not shown). In keeping with a previous report (2), overexpression of RXR in leiomyomas was more evident for samples obtained during the proliferative phase of the cycle suggesting an hormonal dependency of this phenomenon (Fig. 1B, right panel). We next assessed whether such an overexpression of RXR in leiomyomas could be dependent on changes in mRNA levels. RXR mRNA was thus quantified in myometrial and leiomyoma tissues deriving from n = 12 patients by real-time-quantitative PCR (RT-qPCR) analysis. There was no significant difference in RXR mRNA levels, normalized by hypoxanthine phosphoribosyltransferase 1 (HPRT-1), between the two tissues (Fig. 1C), thus suggesting that RXR protein accumulation in the benign tumor was determined at post-transcriptional level.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Expression of RXR in Myometrial Tissues and Matched Leiomyomas A, Protein lysates were prepared from myometrial tissues and correspondent leiomyomas and analyzed by Western blotting using the D-20 anti-RXR antibody. Results from representative samples are presented. B, Densitometric analysis was performed for each band of full-length Mr 54,000 RXR deriving from n = 17 matched samples of myometrium and leiomyoma, and the ratio of RXR to actin was calculated. Data were also analyzed as a function of the phase of cycle of the samples; n = 11 samples were in the proliferative, whereas n = 6 were in the secretory phase of the cycle. Data are expressed as mean ± SEM. C, RXR mRNA derived from n = 12 matched tissues has been quantified by RT-qPCR analysis using the HPRT-1 as an endogenous control. Data were analyzed using the comparative Ct method and are expressed as mean ± SEM of RXR relative expression, which corresponds to the 2–Ct. M, Myometrial tissue; L, leiomyoma tissue. *, P < 0.05 vs. correspondent myometrial samples.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Expression of the RA-Inducible Target Gene, CRABP-II, in Myometrial and Matched Leiomyoma Tissues and Cells RT-qPCR analysis of CRABP-II mRNA in smooth muscle cells deriving from myometrium and leiomyoma. Cells were treated with DMSO or 1 µM 9cRA for 24 h. Data were analyzed from n = 3 different experiments with similar results using the comparative Ct method and are represented as mean ± SEM CRABP-II relative expression, which corresponds to the 2–Ct (left panel). RT-qPCR analysis of CRABP-II mRNA in n = 6 matched tissue samples deriving from myometrium and leiomyoma. Data are represented as mean ± SEM CRABP-II relative expression (right panel). MC, Myometrial cells; LC, leiomyoma cells; M, myometrial tissue; L, leiomyoma tissue. *, P < 0.05 vs. corresponding myometrial samples.

Accumulation of Full-Length RXR in Smooth Muscle Cells Deriving from Leiomyomas and RXR Degradation by Its Selective Ligand

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effect of 9cRA Treatment on Levels of RXR Protein and mRNA in Smooth Muscle Cells Deriving from Myometrium and Leiomyoma A, Protein lysates were prepared from smooth muscle cells deriving from myometrium and leiomyoma, treated for 24 h in presence or absence of 1 µM 9cRA, and analyzed by Western blotting using the D-20 anti-RXR antibody. B, Time course of RXR reduction in smooth muscle cells deriving from myometrium and leiomyoma incubated in presence or absence of 1 µM 9cRA for the indicated times. Representative Western blots for RXR and ß-actin (internal control gene) are shown (left panel). Densitometric analysis was performed for each band of full-length Mr 54,000 RXR deriving from n = 8 matched cellular samples treated for 24 h with DMSO or 1 µM 9cRA. The ratio of RXR to ß-actin was calculated. Data are expressed as mean ± SEM (right panel). C, RXR mRNA derived from n = 12 matched cellular samples treated with DMSO or 1 µM 9cRA was quantified by RT-qPCR analysis using the HPRT-1 as an endogenous control. Data were analyzed by using the comparative Ct method and are expressed as mean ± SEM of RXR relative expression, which corresponds to the 2–Ct. MC, Myometrial cells; LC, leiomyoma cells. *, P < 0.05 vs. corresponding untreated samples.

RXR Degradation in Smooth Muscle Cells Occurs through the Proteasome Proteolytic Pathway and the Formation of RXR/Ubiquitin Conjugates
To determine the proteolytic pathway responsible for ligand-induced breakdown of RXR, smooth muscle cells were treated with a proteasome-specific or with nonspecific protease inhibitors. As shown in Fig. 4A, MG132 (a potent cell-permeable proteasome inhibitor) almost completely inhibited the degradation of full-length RXR induced by 9cRA both in myometrium and leiomyoma cells. Conversely, ALLM (calpain inhibitor II), Ca074 (cathepsin B inhibitor), and E64 (cysteine protease inhibitor) were unable to inhibit 9cRA-mediated degradation. These data suggested that the 9cRA-induced RXR decay in uterine smooth muscle cells occurred through the proteasome proteolytic pathway. Moreover, because protein degradation via the ubiquitin-proteasome pathway involves tagging of the substrates by covalent attachment of multiple ubiquitin molecules (9), we attempted to detect the RXR/ubiquitin conjugates by immunoprecipitation. Cells were treated with 1 µM 9cRA for different times (every hour for up to 27 h) to study the time course of RXR/ubiquitin conjugation. Cellular extracts were immunoprecipitated with an antiubiquitin antibody and the immunoprecipitates were analyzed by immunoblot with an antibody specific for RXR (N197) (Fig. 4B). In myometrial cells, ubiquitinated RXR was detectable after 3–7 h of 9cRA treatment and then was absent. Conversely, in leiomyomas cells, ubiquitinated RXR appeared long afterward. In fact, at 3–7 h of 9cRA treatment, we did not observe any specific signals and ubiquitinated RXR could not be detected before 20 h after 9cRA treatment. To confirm that the slower migrating band represented a ubiquitin conjugate, we performed the following experiments. First, the proteasome inhibitor MG132 was added to the cultured medium under similar experimental conditions. Figure 4C, left panel, shows accumulation of the conjugates in the presence of MG132 in both myometrial and leiomyoma cells. Second, MG132, which acts by inhibiting the degradation of ubiquitinated RXR, was added or not to the tissue extracts and the addition resulted in a more intense band (Fig. 4C, right panel). Taken together, these data suggest that 9cRA induces the formation of RXR/ubiquitin conjugates, but in leiomyoma cells, this effect takes a longer time to occur.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Effect of 9cRA Treatment on Proteasome-Mediated Degradation of Smooth Muscle Cells Deriving from Myometrium and Leiomyoma A, Effect of a proteasome-specific and nonspecific inhibitors on the RA-mediated decline of RXR. Cells were pretreated with inhibitors for 30 min, and 9cRA or DMSO were added to the culture medium. Cells were cultured for an additional 24 h at 37 C. Twenty micromolar MG132, 25 µM ALLM, 1 µM CA074, and 100 µM E64 were added directly into the culture medium. Protein lysates were analyzed by Western blotting using the D-20 anti-RXR antibody, and representative blots for RXR and ß-actin (internal control gene) are shown. B, Time dependence of the 9cRA-mediated ubiquitination of RXR in smooth muscle cells deriving from myometrium and leiomyoma. Cells were incubated in presence or absence of 1 µM 9cRA for the indicated times. Cellular extracts in which MG132 was added to inhibit the degradation of ubiquitinated RXR were immunoprecipitated with an antiubiquitin antibody, and the immunoprecipitates were blotted and probed with the N197 anti-RXR antibody. C, Effect of a proteasome inhibitor on the formation of RXR-ubiquitin conjugates. Cells were pretreated with and without 20 µM MG132 for 30 min, and 9cRA or DMSO were added to the culture medium for an additional 6 h for myometrial cells and for 22 h for leiomyoma cells. Cellular extracts were immunoprecipitated with an antiubiquitin antibody, and the immunoprecipitates were blotted and probed with the N197 anti-RXR antibody (right panel). Protein extracts from myometrial and leiomyoma tissues were prepared in the presence or absence of MG132 (50 µM) in denaturing but not reducing conditions. Extracts were immunoprecipitated with the N197 anti-RXR antibody, and the immunoprecipitates were blotted and probed with an antiubiquitin antibody (left panel). MC, Myometrial cells; LC, leiomyoma cells; M, myometrial tissue; L, leiomyoma tissue.

9cRA Mediates RXR Translocation in Smooth Muscle Cells Deriving from Leiomyomas
Immunofluorescence analysis in myometrial cells showed that RXR was mainly localized in the nuclei (Fig. 5A). 9cRA treatment provoked a rapid RXR degradation in agreement with the above-described immunoblot analyses (Fig. 5B). Compared with untreated myometrial cells, in leiomyoma cells, RXR protein was much more dispersed in both the nucleus and the cytoplasm (Fig. 5C). 9cRA led to a rapid intracellular movement of RXR, which appeared to exit from the nucleus, becoming mainly localized in the perinuclear region in perinuclear inclusions (Fig. 5D). Staining of these cells with an antiubiquitin antibody revealed that RXR frequently colocalized with ubiquitin. In line with the observation that RXR was degraded via the proteasome/ubiquitin pathway and that it tended to aggregate when overexpressed, simultaneous treatment with 9cRA and MG132 for 24 h resulted in the formation of perinuclear multiubiquitinated aggregates even in myometrial cells (Fig. 5E). In leiomyoma cells, a similar treatment resulted in the accumulation of RXR protein in both the nucleus and the cytoplasm (Fig. 5F).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Cellular Localization of RXR in Smooth Muscle Cells Deriving from Myometrium and Leiomyoma and Translocation in Response to 9cRA Cells were processed for immunofluorescence using the D-20 anti-RXR antibody (green) and the antiubiquitin antibody (red). Nuclei were counterstained (blue) with DAPI. Myometrial cells were treated for 24 h with DMSO (A) or 1 µM 9cRA (B) or were cotreated with 9cRA and 20 µM MG132 (E). Similarly, leiomyoma cells were treated for 24 h with DMSO (C) or 1 µM 9cRA (D) or were cotreated with 9cRA and 20 µM MG132 (F). In E and F merge, yellow spots represents areas of RXR and ubiquitin colocalization that support the observation that RXR is degraded via the proteasome/ubiquitin pathway.

Increased Phosphorylation of RXR and Increased Levels of Activated MAPKs in Leiomyoma Tissues

View larger version (33K): [in this window] [in a new window]   Fig. 6. Phosphorylation of RXR and p42/p44 MAPK Expression in Myometrial Tissues and Matched Leiomyomas A and B, RXR immunopurified from each lysate with D-20 anti-RXR antibody was subjected to Western blot analysis using specific antibodies to phosphothreonine or phosphoserine. Representative experiments are presented (left panel). Densitometric analysis was performed for each band in n = 10 cases, and the relative intensity of phoshorylated RXR in each sample was calculated normalizing for the bands obtained in the same samples, by Western blot analysis of RXR after immunoprecipitation with the D-20 anti-RXR antibody. Data are expressed as mean ± SEM (right panel). C, Cells were treated for 24 h with 50 nM STS or 100 µM PD98059 in presence or absence of 1 µM 9cRA and analyzed by Western blotting using the D-20 anti-RXR antibody. D, Protein lysates were prepared from myometrial tissues and corresponding leiomyomas and analyzed by Western blotting using antibodies against total and phosphorylated p42/p44 MAPKs. Results from a representative experiment are presented. M, Myometrial tissue; L, leiomyoma tissue; MC, myometrial cells; LC, leiomyoma cells. *, P < 0.05 vs. corresponding myometrial samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Phosphorylation of nuclear receptors induces profound changes in their properties, including alterations in DNA binding capacity, ligand binding, dimerization, coactivator recruitment, and transcriptional activation (7). Both RARs and RXR require phosphorylation of specific serine or threonine residues for maximal transcriptional activity of RAR/RXR heterodimers. However, these nuclear receptors are substrates for a variety of kinases activated by various stimuli, such as cellular stress and peptide growth factors. RAR is a substrate for protein kinase A, p38, c-Jun N-terminal kinase (JNK), and cyclin-dependent kinase 7, whereas RXR is a substrate of JNK and ERK (19). Interestingly, whereas phosphorylation of RAR by p38 (10) or of RAR by JNK (19) acts as a permissive signal, paving the way to the receptor degradation through the ubiquitin-proteasomal pathway, RXR phosphorylation by MAPK can induce resistance to ubiquitin/proteasome degradation. This latter phenomenon has been demonstrated in hepatocellular carcinomas in which phosphorylation of RXR at serine 260, a consensus site of MAPK, was linked to its retarded ubiquitin-mediated degradation by proteasome and to alteration in its expression, metabolism, and signaling, possibly leading to the aberrant growth of the tumor (8, 9). One of the novelties of the present study is to have demonstrated that a mechanism of altered phosphorylation/degradation/transactivation of RXR, comparable with that observed in hepatocellular carcinomas, also characterizes the abnormal, although still benign, proliferation of uterine smooth muscle cells. Thus, regulation of these mechanisms is unlikely to be cell type specific or mutation dependent but is probably largely associated with the phosphorylation status of the receptors.

The observation that overexpression of RXR in leiomyomas was more evident in samples obtained in the proliferative phase of the cycle suggests that, in these cells, estrogens might be involved in the activation of the MAPK pathway. This possibility is not surprising because estradiol-dependent activation of ERK components of the MAPK family appears to be critical in smooth muscle cell proliferation (20, 21).

Controversial results have been reported regarding the biological consequences of RXR phosphorylation. Very recently, Bruck et al. (7) have shown that stress-induced phosphorylation of RXR modulates transcription of several RA target genes in a promoter context-dependent manner and based on the RAR partner. Whereas the phosphorylation of the AF2 domain at serine 265 plays an inhibitory role on the transcription of some RA target genes, phosphorylation of the N-terminal domain controls the transcriptional activity of RAR/RXR heterodimers but not that of RAR/RXR heterodimers. Moreover, it has been underlined that positive effects on activation of transcription can reflect the concomitant phosphorylation of other transcription activators or coregulators that cooperate with RAR/RXR heterodimers, independently from RXR phosphorylation. Expression of Hoxa1, involved in the promotion of cell survival and oncogenic transformation of human epithelial cells have been shown to be activated by phosphorylation of RXR; activation of other genes such as Cyp26 was abrogated by RXR phosphorylation, whereas expression of other genes was unaffected. In leiomyoma cells, activation of the RA target gene CRABP-II by the ligand was enhanced when compared with the normal cells, thus supporting the idea that modifications of retinoid signaling has potent consequences at transcriptional level. We are certainly not able to conclude that the constitutive CRABP-II expression, which is strongly increased in the benign tumor, is merely dependent upon RXR phosphorylation and/or overexpression; other authors have proposed stimulation of the same gene expression in leiomyomas by estrogens (15), but on the other hand, direct activation of CRABP-II gene by steroid hormones has never been demonstrated in humans (22), whereas block of proteasome-mediated degradation has been shown to induce CRABP-II gene expression also in keratinocyte HaCaT cells (17). Interestingly, other RA target genes have been previously demonstrated to be transcriptionally modified in leiomyomas by microarray analysis (15).

To the best of our knowledge, this is also the first study that has demonstrated that 9cRA decreases the levels of its nuclear receptor in uterine smooth muscle cells via proteasome-mediated degradation and a concomitant translocation from the nucleus to the cytoplasm. Given the accumulation of the receptor in the benign tumor, this action was more effective in leiomyoma cells than in normal cells. The RXR breakdown through ubiquitination induced by 9cRA is indeed a common event among different types of normal cells (16, 19). Ubiquitination of nuclear receptors requires ligand binding. Receptor conformational changes induced by ligand are recognized by the ubiquitination enzymes and the subsequent transfer of the ubiquitin polypeptide to the target proteins results ultimately in the proteolysis through the proteasomal complex (19). In leiomyoma, 9cRA and an inhibitor of phosphorylation are additive in their ability to induce the degradation. In contrast, in human hepatocellular carcinoma cells, the ligand affects degradation of RXR only in presence of a MAPK kinase inhibitor (8).

Mechanisms underlying the potential efficacy of RXR-selective ligands for the treatment of leiomyomas are unclear. Reduction in size of the tumors observed in LDG1069-treated animals appeared to be due to an increase in apoptosis (4). In line with this, one of the most promising retinoids for chemioprevention, 4-HPR, was shown to induce apoptosis of leiomyoma cells but not of matched normal myometrial cells (5). It is completely unknown whether, in leiomyomas, these effects are dependent or not upon the binding to nuclear retinoid receptors because other mechanisms cannot be excluded. However, as already suggested (8), the accumulation of retinoid receptor content that confers high sensitivity to the ligand and the consequent induction of transcriptionally regulated genes involved in cell apoptosis may partly explain these therapeutic effects. This is true for the retinoid signaling pathways in general. According to Nagai et al. (23), the neuroblatoma cell line SK-N-DZ expresses high levels of RAR, which confers high sensitivity to all-trans-RA-induced cell death. Therefore, based on reported observations and on the results provided herein, further studies are required to verify the possibility to use retinoids as a potential approach for treating leiomyomas.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Reagents
9cRA, proteasome inhibitor MG132, cysteine protease inhibitor E64, cathepsin B inhibitor CA074, calpain inhibitor II ALLM, and monoclonal antibodies against phosphoserine (clone PSR-45) and phosphothreonine (clone PTR-8) and to ß-actin were obtained from Sigma-Aldrich (St. Louis, MO). Polyclonal anti-RXR antibodies (N197 and D-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) as well as polyclonal antibodies against p42/p44 MAPKs. Antibodies anti-phospho-p42/p44 MAPKs (Thr202/Tyr204) were from New England Biolabs (Beverly, MA). Secondary antibodies as well as Hybond ECL nitrocellulose membranes for Western blotting and protein G-Sepharose were from Amersham Biosciences (Cologno Monzese, Mi, Italy). Assay to detect proteasome activity was purchased from Chemicon International (Temecula, CA). Cell culture medium contained DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), 100 µg/ml streptomycin (Sigma-Aldrich), 2.5 µg/ml fungizone (Sigma-Aldrich), and 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich). Collagenase A was purchased from Roche Molecular Biochemicals (Milan, Italy). TRIzol was from Invitrogen Life Technologies (Carlsbad, CA). All reagents for RT-qPCR were from Applied Biosystems (Foster City, CA).

Tissue Collection
Tissues were obtained from premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. The indication for surgery was one or more of the following: menorrhagia, dysmenorrhea, pelvic discomfort, rapid tumor growth. At the time of surgery, patients were not receiving any type of hormonal or drug therapy except for nonsteroidal antiinflammatory drugs. Samples were collected from n = 74 leiomyomas and matched myometrium immediately after removal of the uterus or the tumors. In obtaining biopsies from leiomyomas, pathologists gave particular care in avoiding the necrotic central part of the tumor. The myometrium was removed at a distance of 2 cm from both the endometrium and the leiomyomas. Histological evaluation of the endometrium and the patient’s last menstrual period were used to determine the phase of the menstrual cycle. Specimens were assigned to the proliferative phase in n = 45 patients and to the secretory phase in n = 29 patients. The study protocol was approved by the Institutional Review Board of the Department of Obstetrics, Gynaecology and Neonatology of the University of Milan. Patients were informed in detail regarding the aims and procedures of the study and gave their written consent to sample collection.

Cell Preparation and Culture
To isolate smooth muscle cells, samples of leiomyoma and myometrium were minced into small pieces and incubated at 37 C for 2–3 h with 0.1% type A collagenase as described previously (14). Cell suspensions were then centrifuged at 600 x g for 5 min and washed several times. The cell pellet was resuspended in DMEM containing 10% FCS, plated in plastic dishes, and maintained in the same medium at 37 C in 95% air and 5% CO₂. The medium was changed after 24 h to remove unattached cells and subsequently twice a week for a maximum of 4 wk. Flow cytometric analysis showed that CD31-positive cells were less than 2% in both myometrial and leiomyoma cultures, whereas these cells strongly expressed desmin and -smooth muscle actin genes (data not shown). Treatment with and without the different chemicals was carried out with cells at subconfluence. Cells were treated for the indicated periods with the indicated concentrations of chemicals dissolved in dimethylsulfoxide (DMSO) or ethanol at the final concentration of 0.01% in DMEM supplemented with 2% FCS. For each experimental condition, about 5 x 10⁵ cells were used.

RT-qPCR Analysis
Total RNA from tissues and cells was extracted using TRIzol. One microgram of total RNA was reverse transcribed for 2 h at 37 C using the High-Capacity cDNA archive kit. The ABI Prism 7900 sequence detection system was used for RT-qPCR analysis using HPRT-1 as an endogenous control. RT-qPCR was performed using specific primers and probes for RXR or CRABP-II target genes (Assays-on-Demand Gene Expression Products). Validation experiments were performed using the 1:2 diluted templates. Target and reference genes were amplified in separate wells in duplicate. Reaction conditions included 10 µl of 2X TaqMan Universal PCR Master Mix, 1 µl of primers and probes mixture, 50 ng of template cDNA and nuclease-free water to a 96-well reaction plate. The total reaction volume was 20 µl. The cycling conditions were as follows: 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 sec at 95 C, followed by 1 min at 60 C. The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The cycle threshold (Ct) values from each sample were obtained by subtracting the values for the reference gene from the sample Ct. For each experimental sample the 2^–Ct was calculated and data were graphically indicated as relative expression.

Western Blotting
To obtain whole cellular extracts, frozen myometrial and leiomyoma tissues were homogenized in a lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Cell cultures were washed twice with ice-cold PBS and scraped in the same lysis buffer. MG132 (50 µM) was included during the extraction in most samples. Lysates were incubated at 4 C for 30 min and subsequently centrifuged at 13,000 x g for 30 min, and the supernatants were collected for protein analysis. Protein concentration was determined by the bicinchoninic acid method (BCA Protein Assay kit; Pierce Biotechnology, Rockford, IL). Proteins resolved by SDS-PAGE were transferred to Hybond ECL nitrocellulose membranes. Blots were stained with Ponceau to check for loading. After brief washing in TBS-T [25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1% Tween 20], membranes were blocked with 5% skim milk in TBS-T for 2 h at room temperature. Membranes were incubated overnight at 4 C with primary antibodies diluted in 5% skim milk/TBS-T. After several washings with TBS-T, membranes were incubated with peroxidase-conjugated antirabbit or antimouse IgG for 1 h. Bound antibodies were visualized with chemiluminescence (ECL; Amersham Biosciences). To verify equal protein loading, blots were stripped and reprobed with an anti-ß-actin mouse monoclonal antibody. Densitometric analysis was performed using the Total Lab Control Centre software.

Immunoprecipitation
For detection of ubiquitinated or phosphorylated RXR, 70 µl protein G-Sepharose were preincubated with 2 µg antiubiquitin or anti-RXR antibodies for 2 h at 4 C followed by an overnight incubation with 200 µg of proteins from each lysate. Immunoprecipitates were collected by centrifugation, washed four times with PBS and 0.05% BSA, eluted with cracking buffer [187.5 nM Tris-HCl (pH 6.5), 6% sodium dodecyl sulfate (SDS), 15% ß-mercaptoethanol, 30% glycerol, 0.003% bromophenol blue], and boiled for 5 min. Then, they were subjected to Western blot analysis using anti-RXR (1:1000), antiubiquitin (1:800), antiphosphoserine (1:1000), or antiphosphothreonine (1:300) antibodies.

Immunofluorescence
Myometrial and leiomyoma smooth muscle cells were washed twice with PBS, fixed in 4% paraformaldehyde, permeabilized with 1% Triton X-100, thoroughly washed, and then blocked with 10% normal goat serum (NGS). Cells were incubated overnight with primary antibodies overnight at 4 C. Immunoreaction controls involved the omission of the primary antibody. After several washings with 10% PBS/NGS, cells were incubated with the appropriate secondary antibody for 1 h, washed several times, and mounted in fluorsave solution (Calbiochem, Cambridge, MA). Antimouse Cy3- and antirabbit Cy2-conjugated antibodies were supplied by Jackson ImmunoResearch (West Grove, PA). Nuclei were stained with 4,6-diamidine-2-phenylindole, dihydrochloride (DAPI). Samples were observed under a Leica DMIR2 microscope, and digital images were assembled using Imaging Software LeicaFW4000.

Analysis of Proteasome Activity
Treated and control cell cultures were processed as described under Western blotting to obtain cell lysates, which were used to assay the chemotryptic hydrolysis activity of 20S proteasome by measuring the fluorescence of 7-amido-4-methylecoumarin (AMC) generated from peptide- (AMC) linked substrates. Briefly, samples containing 200 µg of proteins were pretreated with and without the inhibitor lactacystin (0.25 mM final concentration) and incubated for 15 min at room temperature in assay buffer [25 mM HEPES (pH 7.5), 0.5 mM EDTA, 0.05% Nonidet P-40, and 0.001% SDS] before adding the proteasome substrate Suc-Leu-Leu-Tyr-AMC (50 µM). Proteasome activity was monitored by measuring the fluorescence of released AMC at excitation wavelength of 380 nm and emission wavelength of 460 nm.

Statistical Analysis
Data are expressed as mean ± SEM. Difference between groups were compared by unpaired Student’s t test or ANOVA and Fisher’s protected least significant difference test as posttest, as appropriate. A value of P < 0.05 was considered to be statistically significant.

	FOOTNOTES

 
The authors have nothing to disclose.

First Published Online December 14, 2006

Abbreviations: AMC, 7-Amido-4-methylecoumarin; 9cRA, 9-cis-retinoic acid; CRABP-II, cellular retinoic acid binding protein II; DAPI, 4,6-diamidine-2-phenylindole, dihydrochloride; DMSO, dimethylsulfoxide; FCS, fetal calf serum; 4-HPR, 4-(N-hydroxyphenyl)retinamide; HPRT-1, hypoxanthine phosphoribosyltransferase 1; JNK, c-Jun N-terminal kinase; M_r, relative molecular mass; NGS, normal goat serum; RA, retinoic acid; RAR, retinoic acid receptor; RT-qPCR, real-time-quantitative PCR; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate.

Received for publication May 15, 2006. Accepted for publication December 5, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Walker CL, Stewart E 2005 Uterine fibroids: the elephant in the room. Science 308:1589–1592[Abstract/Free Full Text]
2. Tsibris J, Porter K, Jazayeri A, Tzimas G, Nau H, Huang H, Kuparadze K, Porter G, O’Brien W, Spellacy W 1999 Human uterine leiomyomata express higher levels of peroxisome proliferator-activated receptor , retinoid X receptor , and all-trans retinoic acid than myometrium. Cancer Res 59:5737–5744[Abstract/Free Full Text]
3. Sozen I, Arici A 2002 Interactions of cytokines, growth factors, and the extracellular matrix in the cellular biology of uterine leiomyomata. Fertil Steril 78:1–12[CrossRef][Medline]
4. Gamage SD, Bischoff ED, Burroughs KD, Lamph WW, Gottardis MM, Walker CL, Fuchs-Young R 2000 Efficacy of LGD1069 (Targretin), a retinoid X receptor-selective ligand, for treatment of uterine leiomyoma. J Pharmacol Exp Ther 295:677–681[Abstract/Free Full Text]
5. Broaddus R, Xie S, Hsu C, Wang J, Zhang S, Zou C 2004 The chemopreventive agents 4-HPR and DFMO inhibit growth and induce apoptosis in uterine leiomyomas. Am J Obstet Gynecol 190:686–692[CrossRef][Medline]
6. Lefebvre P, Martin PJ, Flajollet S, Dedieu S, Billaut X, Lefebvre B 2005 Transcriptional activities of retinoic acid receptors. Vitam Horm 70:199–264[CrossRef][Medline]
7. Bruck N, Bastien J, Bour G, Tarrade A, Plassat J, Bauer A, Adam-Stitah S, Rochette-Egly C 2005 Phosphorylation of the retinoid X receptor at the omega loop, modulates the expression of retinoic-acid-target genes with a promoter context specificity. Cell Signal 17:1229–1239[CrossRef][Medline]
8. Matsushima-Nishiwaki R, Okuno M, Adachi S, Sano T, Akita K, Moriwaki H, Friedman S, Kojima S 2001 Phosphorylation of retinoid X receptor at serine 260 impairs its metabolism and function in human hepatocellular carcinoma. Cancer Res 61:7675–7682[Abstract/Free Full Text]
9. Adachi S, Okuno M, Matsushima-Nishiwaki R, Takano Y, Kojima S, Friedman S, Moriwaki H, Okano Y 2002 Phosphorylation of retinoid X receptor suppresses its ubiquitination in human hepatocellular carcinoma. Hepatology 35:332–340[CrossRef]
10. Gianni M, Tarrade A, Nigro E, Garattini E, Rochette-Egly C 2003 The AF-1 and AF-2 domains of RAR2 and RXR cooperate for triggering the transactivation and the degradation of RAR2/RXR heterodimers. J Biol Chem 278:34458–34466[Abstract/Free Full Text]
11. Siddiqui NA, Loughney A, Thomas EJ, Dunlop W, Redfern CP 1994 Cellular retinoid binding proteins and nuclear retinoic acid receptors in endometrial epithelial cells. Hum Reprod 9:1410–1416[Abstract/Free Full Text]
12. Van den Bogaerdt A, El Ghalbzouri A, Hensbergen P, Reijnen L, Verkerk M, Kroon-Smits M, Middelkoop E, Ulrich M 2004 Differential expression of CRABP-II in fibroblasts derived from dermis and subcutaneous fat. Biochem Biophys Res Commun 315:428–433[CrossRef][Medline]
13. Durand B, Saunders M, Leroy P, Leid M, Chambon P 1992 All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs. Cell 71:73–85[CrossRef][Medline]
14. Mangioni S, Vigano P, Lattuada D, Abbiati A, Vignali M, Di Blasio AM 2005 Overexpression of the Wnt5b gene in leiomyoma cells: implications for a role of the Wnt signaling pathway in the uterine benign tumor. J Clin Endocrinol Metab 90:5349–5355[Abstract/Free Full Text]
15. Arslan AA, Gold LI, Mittal K, Suen TC, Belitskaya-Levy IT, Tang MS, Toniolo P 2005 Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review. Hum Reprod 20:852–863[Abstract/Free Full Text]
16. Nomura Y, Nagaya T, Hayashi Y, Kambe F, Seo H 1999 9-cis-Retinoic acid decreases the level of its cognate receptor, retinoid X receptor, through acceleration of the turnover. Biochem Biophys Res Commun 14:729–733
17. Boudjelal M, Voorhees J, Fisher G 2002 Retinoid signaling is attenuated by proteasome-mediated degradation of retinoid receptors in human keratinocyte HaCaT cells. Exp Cell Res 10:130–137
18. Chegini M, Kornberg L 2003 Gonadotropin releasing hormone analogue therapy alters signal transduction pathways involving mitogen-activated protein and focal adhesion kinases in leiomyoma. J Soc Gynecol Invest 10:21–26[CrossRef]
19. Srinivas H, Juroske D, Kalyankrishna S, Cody D, Price R, Xu X, Narayanan R, Weigel N, Kurie J 2005 c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor . Mol Cell Biol 25:1054–1069[Abstract/Free Full Text]
20. Barbarisi A, Petillo O, Di Lieto A, Melone MA, Margarucci S, Cannas M, Peluso G 2001 17-ß estradiol elicits an autocrine leiomyoma cell proliferation: evidence or a stimulation of protein kinase-dependent pathway. J Cell Physiol 186:414–424[CrossRef][Medline]
21. Finlay GA, York B, Karas RH, Fanburg BL, Zhang H, Kwiatkowski DJ, Koonan DJ 2004 Estrogen-induced smooth muscle cell growth is regulated by tuberin and associated with altered activation of platelet-derived growth factor receptor-ß and ERK-1/2. J Biol Chem 28:23114–23122
22. Lu M, Mira-y-Lopez R, Nakajo S, Nakaya K, Jing Y 2005 Expression of estrogen receptor , retinoic acid receptor and cellular retinoic acid binding protein II genes is coordinately regulated in human breast cancer cells. Oncogene 24:4362–4369[CrossRef][Medline]
23. Nagai J-I, Yazawa T, Okudela K, Kigasawa H, Kitamura H, Osaka H 2004 Retinoid acid induces neuroblastoma cell death by inhibiting proteasomal degradation of retinoic acid receptor . Cancer Res 64:7910–7917[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   RXRα

Coregulators:   CRABPII

Ligands:   9-cis-Retinoic acid

This article has been cited by other articles:

				M. Zaitseva, B. J. Vollenhoven, and P. A.W. Rogers Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells Hum. Reprod., May 1, 2008; 23(5): 1076 - 1086. [Abstract] [Full Text] [PDF]

				M. Zaitseva, B. J. Vollenhoven, and P. A.W. Rogers Retinoic acid pathway genes show significantly altered expression in uterine fibroids when compared with normal myometrium Mol. Hum. Reprod., August 1, 2007; 13(8): 577 - 585. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lattuada, D. Articles by Di Blasio, A. M. Search for Related Content PubMed PubMed Citation Articles by Lattuada, D. Articles by Di Blasio, A. M.