Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression

SW-1353 human chondrosarcoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were propagated at 37°C with 5% CO in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Inc., Manassas, VA, USA) containing 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 U/mL penicillin, 100 μL/mL streptomycin, and 2 mM glutamine. Cells were washed three times with Hanks' balanced salt solution (HBSS) and passaged 1:10 using 0.25% trypsin (Mediatech, Inc.). Experiments were performed with cells from passages 10 to 30, and subsequent cultures were refreshed from frozen stocks.

The synthetic rexinoid LG268 was kindly provided by Ligand Pharmaceuticals (San Diego, CA, USA). LG268 and the PPARγ ligands rosiglitazone and GW-9662 were solubilized in dimethylsulfoxide, stored in 10 μM aliquots at -20°C, and added to culture media at varying concentrations. Recombinant human IL-1β (Promega Corporation, Madison, WI, USA) was solubilized in sterile H₂O, stored in 10 μg/mL aliquots at -80°C, and added to media at 1 ng/mL. For most experiments, SW-1353 cells were grown to approximately 90% confluence in six-well plates and washed twice with HBSS to remove trace serum and waste metabolites. Two milliliters of serum-free DMEM supplemented with 0.2% lactalbumin hydrosylate (DMEM/LH) and appropriate concentrations of LG268 and/or rosiglitazone were added for 1 to 24 hours. IL-1β was then added to the media for an additional 1 to 24 hours followed by cell harvest.

After experimental treatment, the cells were washed twice with cold 1× phosphate-buffered saline (PBS), scraped off the plate, and homogenized using QIAshredder spin columns (Qiagen Inc., Valencia, CA, USA). Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen Inc.) in accordance with the manufacturer's instructions, including DNA contamination removal by on-column treatment with the RNase-Free DNase Kit (Qiagen Inc.). The reverse transcription (RT) reaction was performed on 4 μg of purified total RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) with oligo(dT) or random hexamer primers (Applied Biosystems, Foster City, CA, USA) for mRNA and heterogeneous nuclear RNA (hnRNA) studies, respectively. The RT reactions were performed in a PTC-100 thermal cycler (MJ Research, now part of Bio-Rad Laboratories, Inc., Hercules, CA, USA). Real-time polymerase chain reaction (PCR) was performed using the SYBR Green PCR Master Mix kit (Applied Biosystems) in accordance with the manufacturer's instructions. PCRs were run with experimental triplicates and machine (on-plate) duplicates or triplicates for each sample. To enable quantitative between-plate comparisons, standard curves were generated with each mRNA assay. Both experimental and standard reactions were run using 125 ng each of the appropriate forward and reverse primers for the MMPs analyzed (sequences described previously in [21]). Target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression and reported as mean copies ± standard deviation of target gene mRNA per copy of GAPDH mRNA. Several real-time RT-PCR experiments in which standard curve plasmids were not available were performed. In these cases, the relative mRNA levels of the experimental gene under different treatment conditions were normalized to GAPDH mRNA levels using the 2^-ΔΔCt statistical method [29].

Trichloracetic acid (TCA) protein precipitation and Western blotting were performed as described previously [21]. Briefly, SW-1353 cells were grown to confluency in six-well plates in DMEM with 10% FBS. The media were aspirated, the cells were washed with HBSS, and 2 mL of DMEM/LH was added to each well. Cells were pretreated for 24 hours with rosiglitazone, LG268, or both, and IL-1β was added for an additional 24 hours. Protein was TCA-precipitated from 1 mL of media from each well and resuspended in 40 mL of Laemmli buffer. Samples were resolved using Tris-HEPES-SDS precast 10% polyacrylamide gels (catalog number 25201; Pierce, Rockford, IL, USA) and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). The membranes were probed for MMP-1 using a polyclonal rabbit anti-human MMP-1 antibody (AB8105; Chemicon International, Temecula, CA, USA) or for MMP-13 with a polyclonal MMP-13 antibody generously provided by Peter Mitchell (Pfizer Inc, New York, NY, USA). Protein bands were visualized by incubation with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Danvers, MA, USA) and enhanced chemiluminescence analysis with the Western Lightning reagent (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA, USA).

This assay was performed as previously described [21, 30]. Briefly, fibrillar collagen preparations were made from Vitrogen 100 bovine type I collagen (Cohesion Technologies, Inc., Palo Alto, CA, USA) in accordance with the manufacturer's instructions. The collagen solution was diluted to 2 mg/mL and the pH was adjusted to 7.3 ± 0.2. Once neutralized, an equivalent volume of DMEM/LH containing SW-1353 cells was added, resulting in a final collagen concentration of 1 mg/mL and 2.5 × 10⁵ cells per well of a six-well plate. Rosiglitazone, LG268, or both were added to the collagen/cell suspension of specific experimental wells. Following incubation at 37°C for 60 minutes, the collagen gelled and 1 mL of DMEM/LH was added on top of the cell-containing collagen plug. After 24 hours of incubation in DMEM/LH, IL-1β was added to the media to induce MMP production and subsequent collagen degradation. Approximately 24 hours after the addition of IL-1β, the media were removed from each well and weighed to quantify the extent of collagen degradation.

Luciferase reporter plasmids incorporating four copies of the putative overlapping DR-1/AP-1 site of the MMP-1 (MMP1-ENDOG-Luc) or MMP-13 (MMP13-ENDOG-Luc) promoters were constructed using the pGL3-basic plasmid (Promega Corporation). Control reporters were constructed in a similar fashion, with four scrambled copies of the DR-1/AP-1 element of MMP-1 (MMP1-SCRAM-Luc) or MMP-13 (MMP13-SCRAM-Luc). SW-1353 cells were plated in six-well plates at a density of 1.5 × 10⁵ cells per well. The next day, cells were transiently transfected in six-well plates with 2 μg/well of the PPRE-tk-luciferase plasmid [31], or the custom DR-1/AP-1-luciferase plasmids described above, using 5 μL/well of Lipofectamine 2000 (Invitrogen Corporation) in accordance with the manufacturer's instructions. Four to six hours after transfection, cells were washed twice with HBSS followed by the addition of 2 mL of DMEM/LH media containing the indicated NHR ligand. After 24 hours of ligand treatment, IL-1β was added to the media for an additional 24 hours. The cells were then washed three times with cold 1× PBS, and lysates were harvested using 1× Passive Lysis Buffer (Promega Corporation). Protein concentration was determined using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Inc.), and equal amounts of total protein were loaded for each sample. Luciferase activity was measured in relative light units using an Lmax II luminometer (Molecular Devices Corporation, Sunnyvale, CA, USA).

The chromatin immunoprecipitation (ChIP) protocol was adapted from the 'fast ChIP method' [32]. SW-1353 cells were grown to confluence in 150-mm plates (approximately 10⁷ cells). Crosslinking was performed by adding 40 μL of 37% formaldehyde per milliliter of cell culture media directly to the culture media, and the plates were rocked gently at room temperature for 10 minutes. Crosslinking was quenched by adding 141 μL of 1 M glycine per milliliter media and gently rocking for 5 minutes at room temperature. Cells were washed twice with ice-cold 1 × PBS, scraped, and collected in 15-mL conical tubes on ice. Cells were pelleted by centrifugation at 2,000 g for 5 minutes at 4°C, resuspended in 1 mL of ChIP buffer (150 mM NaCl, 50 mM Tris HCl pH 7.5, 5 mM EDTA [ethylenediaminetetraacetic acid], 0.5% NP40, 1% Triton X-100) with protease inhibitors (complete mini tabs; Roche, Nutley NJ, USA), and lysed on ice for 10 minutes. Nuclei were collected by centrifugation at 12,000 g for 1 minute at 4°C and then washed twice by aspirating the supernatant and resuspending with 1 mL of ChIP buffer. Chromatin was sonicated on ice with 15 × 15 second pulses at power setting #40 on a Sonics Vibro-Cell VC 130PB-1 ultrasonoic processor (Newtown, CT. USA). Debris was cleared by centrifugation at 12,000 g for 10 minutes at 4°C, and the supernatant was split into 200-μL aliquots in 1.5-mL microcentrifuge tubes for immunoprecipitation (IP). Two micrograms of specific antibodies to the HA epitope tag (Abcam, Cambridge, UK), acetylated histone H4 (Upstate, now part of Millipore Corporation), PPARγ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or normal IgG was added to each tube, and tubes were rotated overnight at 4°C. Twenty microliters of protein A/G agarose (Santa Cruz Biotechnology, Inc.) per IP was washed three times, resuspended 1:1 with ChIP buffer, and distributed (40 μL per IP) to 1.5-mL microcentrifuge tubes. IP reactions were centrifuged at 12,000 g for 10 minutes at 4°C, and then 180 μL of supernatant was transferred to the protein A/G agarose tubes and rotated for 45 minutes at 4°C. Beads were collected by centrifugation at 2,000 g for 30 seconds at 4°C and then washed five times by removing the supernatant and resuspending in ice-cold ChIP buffer. After washing, the pellet was resuspended in 100 μL of 10% Chelex-100 (Fisher Scientific Co., Pittsburgh, PA, USA), boiled for 10 minutes, and then cooled on ice. One microliter of proteinase K (20 μg/μL) was added to the cooled solution, vortexed, incubated at 55°C for 30 minutes, boiled for 10 minutes, and centrifuged at 12,000 g for 1 minute. Eighty microliters of supernatant was transferred to a new microcentrifuge tube, and 120 μL of water was added back to the original tube, vortexed, and centrifuged as before, and 120 μL of supernatant was transferred to the previous 80 μL. Samples were stored at -20°C or immediately quantified using real-time PCR with primers flanking the DR-1/AP-1 site or with negative-control primers flanking an upstream control region (-3 kb for MMP-1 and -1 kb for MMP-13), normalized to IgG-precipitated DNA, and expressed as a fold-change over untreated cells.

For the SUMO IP experiments, cellular proteins were immunoprecipitated following the ExactaCruz system instructions from Santa Cruz Biotechnology, Inc. Briefly, cells were grown to confluency in 150-mm dishes and treated for 1 hour with LG268 (50 nM) and/or rosiglitazone (50 nM) in serum-free DMEM/LH media. The cells were then treated with 1 ng/mL IL-1β for 1 hour and harvested using cold radioimmunoprecipitation assay buffer. Cell lysates were homogenized using QIAshredder columns. IP reactions were performed using 4 μg of anti-SUMO-1 (Santa Cruz Biotechnology, Inc.). IP fractions were resolved using PAGE as described above. The presence of RXR and PPARγ in the IP fractions was detected using 4 μg of RXR (ΔN197) or PPARγ (H-100) antibodies from Santa Cruz Biotechnology, Inc.

The SW-1353 chondrosarcoma cell line is a model for inflammatory cytokine-induced protease production by human chondrocytes [20, 33, 34], and we used these cells to quantify the effects of rosiglitazone treatment on IL-1β-stimulated levels of MMP-1 and MMP-13 mRNA. Cells were incubated for 24 hours in serum-free media containing varying doses of rosiglitazone, followed by IL-1β treatment for 24 hours. Previously, we determined that simultaneous treatment with IL-1β and LG268 leads to modest but significant inhibition of MMP-1 and MMP-13, and a 12- to 24-hour pretreatment is necessary for maximum inhibition of MMP levels in these cells by the RXR ligand, LG268 [21]. The need for pretreatment is consistent with the mechanism involving SUMOylation-dependent anchoring of PPARγ and associated corepressor complexes at a target gene promoter, whereby pretreatment with rosiglitazone prevents the clearance of corepressor complexes by inflammatory stimuli ([14] and see Discussion). Real-time RT-PCR was used to quantify MMP mRNA, and Figure 1 demonstrates a dose-dependent inhibition of MMP-1 and MMP-13 mRNA levels in response to rosiglitazone treatment. Significant inhibition of IL-1β-induced MMP-1 and MMP-13 is seen with 10 nM rosiglitazone, with maximal inhibition at approximately 50 nM. The maximum expression of MMP-1 and MMP-13 mRNA with rosiglitazone treatment is approximately 50% of that seen with IL-1β alone, paralleling results obtained with LG268 [21].

Next, we investigated the specificity of rosiglitazone inhibition against a panel of MMPs. This panel included MMPs that are responsive to IL-1β stimulation (MMP-1, MMP-3, MMP-9, and MMP-13) as well as those that are constitutively expressed in these cells (MMP-2 and MMP-14). Cells were treated with 50 nM rosiglitazone for 24 hours before stimulation with IL-1β. Using real-time RT-PCR analysis, we observed that rosiglitazone treatment significantly inhibited IL-1β induction of MMP-1 and MMP-13 while having either a very modest effect (MMP-9) or no effect (MMP-2, MMP-3, and MMP-14) on other MMP family members (Figure 2). With rosiglitazone, both the maximum level of inhibition of MMP-1 and MMP-13 (50% to 60%) and the pattern of MMP inhibition mirror those previously seen with LG268 treatment [21], suggesting that these compounds may be acting through similar mechanisms.

Considering the parallel effects on MMP production with rosiglitazone and LG268 treatment and the fact that these ligands share a molecular target (RXR:PPARγ heterodimers), we measured the effects on IL-1β-induced MMP-1 and MMP-13 levels when the two ligands were added in combination. SW-1353 cells were treated for 24 hours with 50 nM LG268, 50 nM rosiglitazone, or the combination of both treatments followed by IL-1β stimulation for 24 hours. As shown in Figure 3a, treatment with either LG268 or rosiglitazone effectively reduced MMP-1 and MMP-13 mRNA by approximately 50%, and treatment with both ligands led to significantly greater inhibition (approximately 75%) than either drug alone. This is consistent with the idea that treatment with both ligands might increase the binding of RXR:PPARγ to the DR-1 elements in the MMP-1 and MMP-13 promoters, thereby displacing AP-1 transcription factors and causing greater inhibition of mRNA production.

To determine whether the inhibitory effects of rosiglitazone and the combination treatment with LG268 were due, at least in part, to effects on the rate of transcription, we performed real-time RT-PCR analysis of MMP-1 and MMP-13 hnRNA levels [21, 34, 35]. Similar to the mRNA results, treatment with LG268 or rosiglitazone alone resulted in equivalent decreases in IL-1β-stimulated MMP-1 and MMP-13 hnRNA levels (Figure 3b), indicating an effect at the transcriptional level. With combined treatment, hnRNA levels for both MMP-1 and MMP-13 were significantly lower when compared with cells treated with a single compound, paralleling the effects seen on mRNA. This inhibition with combined treatment appears to be additive, again suggesting that the compounds may be acting through similar mechanisms.

Figure 3 shows a decrease in expression of MMP-1 and MMP-13 at the transcriptional level in cells treated with LG268 and rosiglitazone. To determine whether this inhibition extended to the level of MMP protein production and enzymatic activity, we performed Western blot analysis of MMP-1 and MMP-13 protein levels in conditioned media and an in vitro collagen destruction assay looking at the breakdown of type I collagen matrix by IL-1β-stimulated SW-1353 cells [30, 36]. A marked increase in MMP-1 and MMP-13 protein was detected in IL-1β-treated cells (Figure 4a, lane 2). Pretreatment with rosiglitazone or LG268 reduced the amount of MMP-1 and MMP-13 protein detected (Figure 4a, lanes 6 and 7), and combined pretreatment with rosiglitazone and LG268 together had a greater effect than either compound alone (Figure 4a, lane 8). In the collagen destruction assay, after treatment with LG268 and rosiglitazone either alone or together for 24 hours, IL-1β was added for an additional 24 hours and liberated culture media that had been trapped within the collagen matrix were harvested and quantified by weighing [30, 36]. Figure 4b shows that treating the cells with IL-1β resulted in substantial destruction of the collagen matrix, as indicated by the liberation of medium trapped within the matrix. The figure also shows that either LG268 or rosiglitazone decreased collagen destruction by IL-1β-stimulated SW-1353 cells by approximately 50% to 60%. When the drugs were added together, there was even less collagen breakdown than that seen with a single compound, resulting in only 20% of the matrix degradation seen with IL-1β alone. This finding indicates that dual treatment with rexinoids and PPARγ ligands may be an attractive avenue of investigation for the therapeutic inhibition of collagen destruction in arthritis (see Discussion).

The previous figures show that LG268 and rosiglitazone have an inhibitory effect on both the production and activity of MMP-1 and MMP-13 in IL-1β-stimulated chondrocytic cells. We next wanted to investigate the possible mechanisms behind this inhibition. RXR:PPARγ heterodimers can regulate gene expression through binding to PPRE/DR-1 sites in the promoters of target genes [37]. Therefore, to determine whether RXR:PPARγ heterodimers function as expected in the SW-1353 cell line, we used a luciferase reporter assay to test the response of a canonical PPRE/DR-1 element to treatment with rosiglitazone and LG268. We obtained a luciferase reporter construct, driven by three copies of the consensus PPRE from the rat acyl-CoA oxidase promoter, which is known to be activated by treatment with PPARγ and RXR ligands [31]. SW-1353 cells were transfected with the reporter construct and then treated with LG268 and rosiglitazone either alone or together for 24 hours. The cells were then treated with IL-1β for an additional 24 hours and cell lysates were assayed for luciferase activity. Figure 5a demonstrates that treatment with either LG268 or rosiglitazone led to an approximately twofold increase in luciferase levels as compared with untreated cells, and treatment with both drugs led to even greater activation, approximately fourfold over untreated cells. The addition of IL-1β appeared to have minimal effect on reporter expression. These findings support the conclusions that (a) LG268 and rosiglitazone are each able to activate a consensus PPRE/DR-1 element in the SW-1353 cells and (b) combination treatment leads to synergistic activation of this element, presumably because both partners of the RXR:PPARγ heterodimer are liganded/activated.

After demonstrating that a canonical PPRE/DR-1 reporter construct responded as expected, we reasoned that if RXR:PPARγ were binding to the DR-1/AP-1 site in the MMP-1 and MMP-13 proximal promoters, this DNA element may also be responsive to treatment with combinations of LG268 and rosiglitazone. We used a luciferase reporter assay with a construct driven by four copies of the endogenous DR-1/AP-1 element from either the MMP-1 or MMP-13 promoter. Figure 5b shows that neither the MMP-1 (MMP1-ENDOG-Luc) nor the MMP-13 (MMP13-ENDOG-Luc) construct was responsive to treatment with rosiglitazone or LG268. The figure also shows that treatment with IL-1β reduced expression of both constructs, which does not reflect the response of endogenous MMP-1 and MMP-13, whose expression is induced by IL-1β (Figures 1 and 3). Previous studies have shown that reporter gene expression driven by components of the MMP-1 and MMP-13 promoters often do not mirror expression of the endogenous genes [20, 21]. Although Figure 5 suggests that the putative DR-1 element of the DR-1/AP-1 site does not function as a traditional response element for RXR:PPARγ in these cells, the paradoxical response to IL-1β led us to abandon further studies with the DR-1/AP-1 luciferase constructs in favor of a more direct, in vivo approach, illustrated by the ChIP studies (see below). Taken together, the luciferase reporter data suggest that mechanisms requiring native chromatin conformation are involved in regulating expression of these genes, including histone modification ([21] and see below) and interaction with factors at other promoter elements [20, 21].

If LG268 and rosiglitazone increase the affinity of RXR:PPARγ binding to the DR-1/AP-1 site, thereby interfering with binding to that site by the AP-1 transcription factors, then elevated levels of PPARγ would be expected at the DR-1/AP-1 site in cells treated with LG268 and rosiglitazone when compared with cells treated with IL-1β alone. In that regard, we used ChIP assays to test whether endogenous PPARγ was detectable at the DR-1/AP-1 sites in the MMP-1 and MMP-13 promoters. SW-1353 cells were treated with rosiglitazone, LG268, or both, with or without IL-1β. Sonicated chromatin was immunoprecipitated with anti-PPARγ antibody (catalog number sc-7196 X; Santa Cruz Biotechnology, Inc.) and the enriched DNA was quantified with real-time PCR using primers targeting the DR-1/AP-1 sites of MMP-1 and MMP-13 or a nonspecific upstream region of the promoter as a negative control (see Materials and methods). The data in Figure 6 are representative of at least three independent experiments. We detected a marked increase in PPARγ at the DR-1/AP-1 site at both promoters in cells treated with IL-1β, which appeared to be blocked by ligand treatment at the MMP-1 promoter but only modestly inhibited at the MMP-13 promoter (Figure 6). We saw little effect when rosiglitazone or LG268 was added alone. For all conditions, there was little variation at the upstream control region, demonstrating localization of changes in PPARγ binding at the target site. We also performed these experiments in SW-1353 cells transiently transfected with a plasmid expressing hemagglutinin-tagged PPARγ (HA-PPARγ). Endogenous MMP-1 and MMP-13 mRNA expression was unaffected by the overexpression of HA-PPARγ and responded as seen previously to rosiglitazone, LG268, and IL-1β, as measured by real-time RT-PCR (data not shown). We immunoprecipitated with an antibody to the HA tag and saw similar results (Figure 7). The unexpected increase in PPARγ with IL-1β treatment may suggest a potential role for PPARγ in IL-1β signaling at the DR-1/AP-1 element in the MMP-1 and MMP-13 promoters (see Discussion). We concluded that these findings do not support the competitive binding model, in which one would expect to see an increase in PPARγ at the DR-1/AP-1 site with rosiglitazone or LG268 treatment as compared with treatment with IL-1β alone.

RXR:PPARγ is known to affect the transcription of target genes via interaction with coactivator and corepressor complexes that modify histones in the target gene promoter by acetylating and deacetylating core histone subunits, including histone subunit H4 [12]. LG268 has been shown to prevent histone acetylation at the proximal promoter region of both MMP-1 and MMP-13 in IL-1β-treated SW-1353 cells [21]. We used ChIP assays, as described above, with antibodies to acetylated histone H4 to detect changes in acetylation of histones at the DR-1/AP-1 element in both MMP-1 and MMP-13 promoters in SW-1353 cells treated with rosiglitazone, LG268, or both, with or without IL-1β. At the DR-1/AP-1 element in both promoters, IL-1β treatment led to a marked increase in histone acetylation (Figure 8), consistent with HAT recruitment, H4 acetylation, and subsequent transcriptional activation [13]. This increase in acetylation was blocked by treatment with either rosiglitazone or LG268, consistent with the recruitment of HDACs and subsequent transcriptional repression [13]. Importantly, combined treatment with rosiglitazone and LG268 led to a dramatic decrease in H4 acetylation at both the MMP-1 and MMP-13 promoters, suggesting that decreased acetylation may be a prominent mechanism by which these two ligands decrease transcriptional activity of these genes (see Discussion). We also note that, as seen previously in Figure 6, there was little variation at the upstream control region, demonstrating localization of alterations in H4 acetylation to the DR-1/AP-1 site. These data, considered with the PPARγ ChIP results (Figures 6 and 7), suggest that rosiglitazone and LG268 may be inhibiting the IL-1β-induced transcription of MMP-1 and MMP-13 not by a physical blockade of factor binding but through a mechanism involving interaction with HDAC-containing coregulatory complexes and regulation of histone acetylation [12].

Maximum inhibition of IL-1β-induced MMP-1 and MMP-13 expression by LG268 requires 12 to 24 hours of pretreatment with LG268 prior to the addition of IL-1β [21], and we see a similar requirement for pretreatment with rosiglitazone (data not shown). A mechanism of SUMOylation-dependent inhibition of inflammatory gene expression by PPARγ, proposed by Pascual and colleagues [14], suggested a possible explanation for the necessity of pretreatment described above. In their model, SUMOylation of PPARγ anchors the nuclear receptor at the target gene promoter and prevents the clearance of associated corepressors by proinflammatory stimuli [14]. Therefore, we sought to determine whether LG268 and rosiglitazone could cause SUMOylation of RXR and PPARγ in SW-1353 cells. We treated SW-1353 cells with LG268, rosiglitazone, or both compounds for 1 hour, followed by no additional treatment or by IL-1β stimulation for 1 hour. Cell lysates were harvested and total cellular protein was subjected to IP with an antibody against SUMO-1. We then probed the immunoprecipitated fractions for RXR or PPARγ using Western blotting. Figure 9a demonstrates that SW-1353 cells contain a minimal amount of singly SUMOylated PPARγ (molecular weight 82 kDa) and that treatment with IL-1β alone may slightly increase this level (lanes 1P and 2P). However, the presence of rosiglitazone induced a higher molecular weight form of the SUMOylated receptor (lanes 4P and 5P). The molecular weight of the induced band is approximately 115 kDa, which suggests that it is a doubly SUMO1-ylated form of PPARγ. Surprisingly, treatment with LG268 alone (lane 3P) also caused a modest increase in doubly SUMOylated PPARγ, similar to that induced by rosiglitazone (lane 4P). This suggests two conclusions: (a) LG268 treatment alone induces a SUMOylated form of PPARγ, which may transrepress inflammatory genes, and (b) liganding one receptor in the heterodimer can cause SUMOylation of the unliganded partner (for example, liganded RXR causes the SUMOylation of unliganded PPARγ). Interestingly, IL-1β treatment partially decreases the levels of the doubly SUMOylated receptor in all pretreatment groups (lanes 6P, 7P, and 8P), suggesting that proinflammatory stimuli may negatively regulate ligand-induced SUMOylation of PPARγ (see Discussion).

We next investigated whether the rexinoid LG268 similarly affected the SUMOylation state of its receptor target, RXR, which has been shown to be a target of SUMOylation [28]. We performed the same Western blot/IP analysis as described above, with the exception of Western blotting with anti-RXR antibody. As shown in Figure 9b, no SUMOylated RXR is present in untreated cells or in cells treated with IL-1β (lanes 1R and 2R). However, treatment with LG268 led to modest increases in both a low- and high-molecular weight forms of SUMO1-RXR (lane 3R).

The figure also shows that treatment with rosiglitazone induced a high-molecular weight form (molecular weight 115 kDa) of SUMO1-RXR (lane 4R), again likely representing SUMO1-ylation of the receptor at multiple sites. Along with the effect seen in lane 3P of Figure 9b, the SUMOylation of RXR induced by rosiglitazone (lane 4R) lends additional support to the hypothesis that the binding of ligand by one nuclear receptor may cause cross-SUMOylation of the unliganded dimer partner. Treatment with both ligands leads to an increase in the effects observed with single agents: a strong upper band and a modest increase in a lower band (lane 5R). Interestingly, when the cells are stimulated with IL-1β after ligand pretreatment, only the singly SUMO1-ylated form of RXR is seen (lanes 6P, 7P, and 8P). This suggests that treatment with a proinflammatory cytokine may activate pathways that cause removal of SUMO1 from conjugated RXR or perhaps degradation of doubly SUMO1-ylated RXR (see Discussion). However, we can conclude that treatment with either LG268 or rosiglitazone leads to the induction of SUMO1-ylated forms of both PPARγ and RXR.