Peroxisome Proliferator-activated Receptor γ-mediated Differentiation
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m300637200
ISSN1083-351X
AutoresRajnish A. Gupta, Pasha Sarraf, Elisabetta Mueller, Jeffrey A. Brockman, Jeffery J. Prusakiewicz, Charis Eng, Timothy M. Willson, Raymond N. DuBois,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoActivation of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) inhibits cell growth and induces differentiation in both adipocyte and epithelial cell lineages, although it is unclear whether this occurs through common or cell-type specific mechanisms. We have identified four human colon cancer cell lines that do no undergo growth inhibition or induce markers of differentiation after exposure to PPARγ agonists. Sequence analysis of the PPARγ gene revealed that all four cell lines contain a previously unidentified point mutation in the ninth α-helix of the ligand binding domain at codon 422 (K422Q). The mutant receptor did not exhibit any defects in DNA binding or retinoid X receptor heterodimerization and was transcriptionally active in an artificial reporter assay. However, only retroviral transduction of the wild-type (WT), but not mutant, receptor could restore PPARγ ligand-induced growth inhibition and differentiation in resistant colon cancer cell lines. In contrast, there was no difference in the ability of fibroblast cells expressing WT or K422Q mutant receptor to undergo growth inhibition, express adipocyte differentiation markers, or uptake lipid after treatment with a PPARγ agonist. Finally, analysis of direct PPARγ target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may disrupt the ability of PPARγ to repress the basal expression of a subset of genes in the absence of exogenous ligand. Collectively, these data argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPARγ to induce terminal differentiation in epithelial, but not adipocyte, cell lineages and argues that the receptor induces growth inhibition and differentiation via cell lineage-specific mechanisms. Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) inhibits cell growth and induces differentiation in both adipocyte and epithelial cell lineages, although it is unclear whether this occurs through common or cell-type specific mechanisms. We have identified four human colon cancer cell lines that do no undergo growth inhibition or induce markers of differentiation after exposure to PPARγ agonists. Sequence analysis of the PPARγ gene revealed that all four cell lines contain a previously unidentified point mutation in the ninth α-helix of the ligand binding domain at codon 422 (K422Q). The mutant receptor did not exhibit any defects in DNA binding or retinoid X receptor heterodimerization and was transcriptionally active in an artificial reporter assay. However, only retroviral transduction of the wild-type (WT), but not mutant, receptor could restore PPARγ ligand-induced growth inhibition and differentiation in resistant colon cancer cell lines. In contrast, there was no difference in the ability of fibroblast cells expressing WT or K422Q mutant receptor to undergo growth inhibition, express adipocyte differentiation markers, or uptake lipid after treatment with a PPARγ agonist. Finally, analysis of direct PPARγ target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may disrupt the ability of PPARγ to repress the basal expression of a subset of genes in the absence of exogenous ligand. Collectively, these data argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPARγ to induce terminal differentiation in epithelial, but not adipocyte, cell lineages and argues that the receptor induces growth inhibition and differentiation via cell lineage-specific mechanisms. The induction of terminal cellular differentiation is a complex process requiring the initiation of a gene expression pattern that ultimately results in both cell cycle withdrawal and the expression of a set of proteins that are necessary to carry out the specialized function of the differentiated cell. Transcription factors often serve as driving forces for cellular differentiation. For example, muscle cell differentiation is critically dependent on the MyoD family of basic helix-loop-helix transcription factors (1Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (247) Google Scholar). Expression of MyoD family members initiates a cascade of temporal-specific gene expression patterns ultimately leading to growth arrest and differentiation. Cancer is essentially a state of de-differentiation in which the malignant cells do not undergo the normal maturation process that leads to cessation of cell growth. The ability of transcription factors to initiate terminal differentiation pathways has been exploited as a form of differentiation therapy for cancer that can serve as an alternative to more toxic chemotherapeutic regimens. For example, ligand activation of the retinoic acid receptor α-promyelocytic leukemia fusion protein using all-trans retinoic acid has been successfully used in the treatment of acute promyelocytic leukemia (2Warrell Jr., R.P. Frankel S.R. Miller Jr., W.H. Scheinberg D.A. Itri L.M. Hittelman W.N. Vyas R. Andreeff M. Tafuri A. Jakubowski A. et al.N. Engl. J. Med. 1991; 324: 1385-1393Crossref PubMed Scopus (1214) Google Scholar). Peroxisome proliferator-activated receptor γ (PPARγ) 1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXT, retinoid X receptor; WT, wild-type; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; luc, luciferase; CEA, carcino-embryonic antigen; PGJ2, prostaglandin J2. is a ligand-activated transcription factor that is capable of initiating terminal differentiation pathways. PPARγ and related subtypes PPARα and PPARδ are members of the nuclear hormone receptor gene superfamily (3Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1706) Google Scholar) that form functional heterodimers with members of the retinoid X receptor (RXR) family of nuclear receptors (4Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). PPARs play fundamental roles in metabolic homeostasis, primarily as regulators of fatty acid storage and catabolism (5Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1678) Google Scholar). Putative endogenous ligands for PPARγ include both polyunsaturated fatty acids and the eicosanoids 15-deoxyΔ12,14-PGJ2 (6Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2740) Google Scholar, 7Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1872) Google Scholar), 13-hydroxyoctadecadienoic acid, and 15-hydroxyeicosatetraenoic acid (8Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar), but their respective roles in PPARγ signaling in vivo remains unclear. High affinity synthetic ligands that selectively activate PPARγ include the thiazolidinediones, a class of insulin sensitizing drugs currently in use for the treatment of insulin-resistant diabetes mellitus (9Willson T.M. Lehmann J.M. Kliewer S.A. Ann. N. Y. Acad. Sci. 1996; 804: 276-283Crossref PubMed Scopus (57) Google Scholar). PPARγ appears to play a dominant role in the differentiation of adipocytes. Early experiments established that ectopic expression of PPARγ in fibroblasts resulted in conversion of the cells to adipocytes (10Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar). More recent studies using mice null for the PPARγ gene have confirmed this essential role in adipogenesis (11Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar, 12Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). The cellular response induced by PPARγ during adipogenesis involves both cell cycle withdrawal and the expression of lipogenic-related genes such as the fatty acid-binding protein aP2 (13Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). The growth arrest pathway is characterized by a G1 cell cycle arrest and the induction of cyclin-dependent kinase inhibitors p18 and p21 (14Morrison R.F. Farmer S.R. J. Biol. Chem. 1999; 274: 17088-17097Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). PPARγ has also been shown to restrict S phase entry by inhibiting the DNA binding activity of E2F/DP (15Altiok S. Xu M. Spiegelman B.M. Genes Dev. 1997; 11: 1987-1998Crossref PubMed Scopus (337) Google Scholar). Current evidence suggests that PPARγ can induce differentiation pathways beyond adipocytes. For example, activating ligands of PPARγ inhibit the proliferation rates of epithelial cells derived from breast, prostate, stomach, and lung (16Mueller E. Sarraf P. Tontonoz P. Evans R.M. Martin K.J. Zhang M. Fletcher C. Singer S. Spiegelman B.M. Mol. Cell. 1998; 1: 465-470Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 17Kubota T. Koshizuka K. Williamson E.A. Asou H. Said J.W. Holden S. Miyoshi I. Koeffler H.P. Cancer Res. 1998; 58: 3344-3352PubMed Google Scholar, 18Chang T.H. Szabo E. Cancer Res. 2000; 60: 1129-1138PubMed Google Scholar, 19Takahashi N. Okumura T. Motomura W. Fujimoto Y. Kawabata I. Kohgo Y. FEBS Lett. 1999; 455: 135-139Crossref PubMed Scopus (251) Google Scholar). In the colon, levels of PPARγ mRNA are nearly equivalent to that found in adipocytes (20Fajas L. Auboeuf D. Raspe E. Schoonjans K. Lefebvre A.M. Saladin R. Najib J. Laville M. Fruchart J.C. Deeb S. Vidal-Puig A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar) with the highest levels of receptor expression observed in the post-mitotic, differentiated epithelial cells facing the lumen (21Lefebvre M. Paulweber B. Fajas L. Woods J. McCrary C. Colombel J.F. Najib J. Fruchart J.C. Datz C. Vidal H. Desreumaux P. Auwerx J. J. Endocrinol. 1999; 162: 331-340Crossref PubMed Scopus (151) Google Scholar). Consistent with this expression pattern, exposure of cultured human colon cancer cells to PPARγ agonists induces growth inhibition associated with a delay in the G1 phase of the cell cycle and an increase in several markers of cellular differentiation (22Sarraf P. Mueller E. Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (929) Google Scholar, 23Brockman J.A. Gupta R.A. Dubois R.N. Gastroenterology. 1998; 115: 1049-1055Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 24Gupta R.A. Brockman J.A. Sarraf P. Willson T.M. DuBois R.N. J. Biol. Chem. 2001; 7: 7Google Scholar). Whether the anti-neoplastic, pro-differentiation effects of PPARγ ligands in the colon operate in vivo is not clear. Agonists of the receptor will reduce pre-malignant intestinal lesions in rats treated with the carcinogen azoxymethane (25Tanaka T. Kohno H. Yoshitani S. Takashima S. Okumura A. Murakami A. Hosokawa M. Cancer Res. 2001; 61: 2424-2428PubMed Google Scholar) but slightly increase colon polyps in Adenomatous polyposis coli mutant mice that are predisposed to intestinal adenomas (26Saez E. Tontonoz P. Nelson M.C. Alvarez J.G. Ming U.T. Baird S.M. Thomazy V.A. Evans R.M. Nat. Med. 1998; 4: 1058-1061Crossref PubMed Scopus (554) Google Scholar, 27Lefebvre A.M. Chen I. Desreumaux P. Najib J. Fruchart J.C. Geboes K. Briggs M. Heyman R. Auwerx J. Nat. Med. 1998; 4: 1053-1057Crossref PubMed Scopus (578) Google Scholar). However, Sarraf et al. (28Sarraf P. Mueller E. Smith W.M. Wright H.M. Kum J.B. Aaltonen L.A. de la Chapelle A. Spiegelman B.M. Eng C. Mol. Cell. 1999; 3: 799-804Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar) have reported that 8% of primary colorectal tumors contained a loss of function point mutation in one allele of the PPARγ gene, emphasizing that the receptor is likely to have a tumor suppressive function in the colon. Four unique mutations in PPARγ were identified in the study; one resulted in a truncated protein that lacked the entire ligand binding domain whereas the other three mutations caused defects in the binding of either synthetic or natural ligands. Although activation of PPARγ will initiate pathways leading to growth arrest in both colon epithelial and adipocyte lineages, it is unknown whether this occurs through similar or distinct mechanisms. For example, in both cell types activation of the receptor eventually leads to a G1 arrest and an increase in cell-specific differentiation markers. Does this occur through the initial regulation of identical target genes in both cell types, and does PPARγ require common co-regulator interactions in both instances? Here we report the detection of an identical exonic mutation (K422Q) in the PPARγ gene in four distinct colon cancer cell lines that are refractory to the decrease in cell growth or increase in differentiation markers normally induced by activators of PPARγ. Only introduction of the WT, but not mutant, receptor was able to restore PPARγ ligand sensitivity in the resistant colon cancer cell lines. In contrast, there was no difference in the ability of WT or K422Q receptor to induce adipocyte differentiation. Analysis of direct PPARγ target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may be non-functional because of an inability of the apo-receptor to basally repress certain target genes. These results argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPARγ to induce terminal differentiation in epithelial, but not adipocyte, cell lineages. Receptor Ligands—All synthetic PPAR ligands were from GlaxoSmithKline and dissolved in Me2SO. 15-DeoxyΔ12,14-PGJ2 was purchased from Cayman Chemical. Cell Culture—The HCT 15, COLO 205, HCT 116, HT-29, and NIH 3T3 cell lines were purchased from ATCC. 293-EBNA cells were purchased from Invitrogen. The MOSER S cell line was a gift from M. Brattain (University of Texas Health Sciences, San Antonio, TX). The MIP 101 and Clone A cell lines were a gift from L. B. Chen (Dana Farber Cancer Institute, Boston, MA). The HCA-7 cell line was obtained from S. Kirkland (University of London, London, United Kingdom). Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone), l-glutamine (2 mmol/liter), penicillin (100 units/ml), and streptomycin (100 μg/ml) in a 5% CO2 atmosphere with constant humidity. For all experiments in which a receptor ligand was added, cells were grown in the above media except regular 10% FBS was replaced with 10% charcoal-stripped FBS (Hyclone). Plasmids—Full-length WT PPARγ was cloned into pBLUESCRIPT KS+. This was used as a template to generate PPARγ K422Q using oligonucleotide-directed in vitro mutagenesis (Muta-Gene; Bio-Rad). Both WT and K422Q PPARγ were cloned into pCDNA3.0 (Invitrogen) for use in transient transfection and EMSA experiments. HA-tagged WT and K422Q PPARγ were generated by PCR using Pfu Turbo Taq polymerase (Stratagene) and the proper non-tagged cDNA as a template. The 5′ primer contained a XhoI site, the full-length HA epitope, and a partial region of PPARγ starting at codon 2. The 3′ primer contained a HpaI site and a partial region of PPARγ starting at codon 479 (stop codon). Each amplicon was digested and cloned into the XhoI/HpaI site of the retroviral expression vector pMSCVpuro (Clontech). All plasmids were sequenced to avoid unwanted mutations. Western Blot Analysis—Cells were harvested in ice-cold 1× phosphate-buffered saline, and cell pellets were lysed in radioimmune precipitation assay buffer. Centrifuged lysates (50 μg) from each cell line were fractionated on a 4–20% gradient SDS-polyacrylamide gel and electrophoretically transferred to a polyvinlylidene difluoride membrane (PerkinElmer Life Sciences). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% powdered milk. The following primary antibodies were used: monoclonal anti-HA antibody clone HA.11 (1:1000; Babco), monoclonal anti-PPARγ (1:500; Santa Cruz Biotechnology, Inc.), and monoclonal anti-keratin 18 and 20 antibodies (1:1000; NeoMarkers). This was followed by incubation with donkey anti-mouse horseradish peroxidaseconjugated secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:50,000 for 1 h. Detection of immunoreactive polypeptides was accomplished using an enhanced chemiluminescence system (Amersham Biosciences). Mutation Detection—Mutations in the PPARγ gene in the COLO 205, MIP 101, and Clone A cell lines were detected using a combination of denaturing gradient gel electrophoresis and direct sequencing as described previously (28Sarraf P. Mueller E. Smith W.M. Wright H.M. Kum J.B. Aaltonen L.A. de la Chapelle A. Spiegelman B.M. Eng C. Mol. Cell. 1999; 3: 799-804Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 29Zhou X.P. Smith W.M. Gimm O. Mueller E. Gao X. Sarraf P. Prior T.W. Plass C. von Deimling A. Black P.M. Yates A.J. Eng C. J. Med. Genet. 2000; 37: 410-414Crossref PubMed Google Scholar). PPARγ mutations in the HCT 15, MOSER S, HT-29, HCT 116, and HCA-7 cell lines were detected by automated dideoxy sequence analysis of PCR products that span the coding region of PPARγ1 using primers sets described previously (30Yen C.J. Beamer B.A. Negri C. Silver K. Brown K.A. Yarnall D.P. Burns D.K. Roth J. Shuldiner A.R. Biochem. Biophys. Res. Commun. 1997; 241: 270-274Crossref PubMed Scopus (494) Google Scholar). Cell Growth Measurements—The day after initial seeding, cells were exposed to Dulbecco's modified Eagle's medium containing 10% charcoal stripped FBS and either 0.1% Me2SO or the indicated ligand. Cells were exposed to fresh medium and compound every 48 h. Cells were counted after 6 days of treatment using a Coulter counter. Each experiment was done in triplicate. Transient Transfections and Luciferase Assays—CV-1 cells (5.0 × 105/well in 24-well plates) were transfected with a mix containing 0.66 μg/ml PPRE3-tk-luciferase (PPRE3-tk-luc) (a gift of R. Evans, Salk Institute, La Jolla, CA), 0.010 μg/ml pRL-SV40 (Promega), and 0.66 μg/ml pCNDA3.0/PPARγ WT or pCDNA3.0/PPARγ K422Q in Opti-MEM (Invitrogen) for 5 h. HCT 15-pMSCV, HCT 15-PPARγ WT, or HCT 15-PPARγ K422Q cell lines were transfected with 0.66 μg/ml PPRE3-tk-luc, 0.010 μg/ml pRL-SV40, and 0.66 μg/ml pCDNA3.0. In each case, FuGENE 6 (Roche Applied Science) was added to the transfection mix at a lipid:DNA ratio of 3.5:1. The transfection mix was replaced with complete medium containing either vehicle or the indicated ligand. After 24–36 h, cells were harvested in 1× luciferase lysis buffer. Relative light units from firefly luciferase activity were determined using a luminometer (MGM Instruments) and normalized to the relative light units from Renilla luciferase using the dual luciferase kit (Promega). EMSA—EMSAs were done based on methods reported by Schulman et al. (31Schulman I.G. Shao G. Heyman R.A. Mol. Cell. Biol. 1998; 18: 3483-3494Crossref PubMed Google Scholar). PPAR and RXR receptors were synthesized using a T7 Quick TNT in vitro transcription/translation kit (Promega). 1.0 μl of the PPAR receptor and 0.10, 0.50, 0.75, or 1.0 μl of RXRα were added to a final reaction buffer volume of 20 μl that contained 1× binding buffer (20 mm HEPES, pH 7.5, 75 mm KCl, 2.0 mm dithiothreitol, 0.1% Nonidet P-40, 7.5% glycerol), 2.0 μg of poly(dI-dC), and 0.02 pmol of an 32P-labeled oligonucleotide containing a PPRE derived from the acyl-CoA oxidase promoter (GTCGACAGGGGACC AGGACA A AGGTCA CGTTCGGGAGT). After 20 min of incubation, the reactions were resolved on 5% nondenaturing acrylamide gels. Viral Infection of Cell Lines—Phoenix-Ampho cells (purchased from ATCC with prior approval of G. Nolan (Stanford University, Palo Alto, CA)) were transiently transfected with pMSCVpuro, pMSCV/HAPPARγ WT, and pMSCV/HA-PPARγ K422Q using FuGENE 6 at a lipid:DNA ratio of 3.5:1. Approximately 72 h post-transfection, viral supernatants were collected, filtered, supplemented with 2 μg/ml of polybrene (Sigma), and used to infect exponentially growing HCT 15 or NIH 3T3 cells. After 48 h, cells were split 1:5 into media containing 4 μg/ml (HCT 15 cells) or 2 μg/ml (NIH 3T3 cells) puromycin (Sigma) to select for infected cells. After selection, all stable cell lines were grown in media containing 2 μg/ml puromycin prior to any experiments. Flow Cytometry—HCT 15-pMSCV, HCT 15-PPARγ WT, and HCT 15-PPARγ K422Q cell lines were treated with 0.1% Me2SO or the indicated receptor ligand for 48 h. The DNA content of nuclei was determined by staining nuclear DNA with propidium iodide (50 μg/ml) followed by measuring the relative DNA content of nuclei using a Facsort fluorescence-activated sorter (BD Biosciences). The proportion of nuclei in each phase of the cell cycle was determined using MODFIT DNA analysis software (BD Biosciences). Tumor Growth in Athymic Mice—Athymic mice (Harlan Sprague-Dawley, Inc.) were injected subcutaneously in the dorsal flanks with 5 × 106 cells of the HCT 15 cells expressing WT or K422Q PPARγ in a volume of 0.10 ml of 1× phosphate-buffered saline. Dosing was started 10–15 days post-injection for each cell line when the mean tumor volumes were ∼75 mm3. Mice were then orally gavaged five times/week with either vehicle (0.5% methylcellulose in 0.05 n HCl) or 10 mg/kg of rosiglitazone (in a total volume of 0.10 ml per mouse). Rosiglitazone was formulated daily by first dissolving the compound in 0.1 n HCL that had been pre-warmed to 40 °C followed by the addition of an equal volume of 1% methylcellulose. The size of each tumor was determined by direct measurement of tumor dimensions. The volume was calculated according to the equation (V = [L × W2] × 0.5), where V = volume, L = length, and W = width. Adipogenesis Assay—Virally infected NIH 3T3 cells were exposed at confluence to dexamethasone (1 μm) for 24 h followed by treatment with vehicle or rosiglitazone for 7 days with media changed every 48 h. Cells were then fixed and stained with Oil Red O (Sigma). Northern Hybridization Analysis—Northern blot analysis was performed as described previously (32Gupta R.A. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13275-13280Crossref PubMed Scopus (359) Google Scholar). The indicated cell lines were treated with either 0.1% Me2SO or 2.0 μm rosiglitazone. Total RNA (20 μg) from each sample was fractionated on a 1.2% agarose-formaldehyde gel and transferred to a Hybond-NX nylon membrane (Amersham Biosciences). Filters were pre-hybridized for 4 h at 42 °C in Ultrahyb (Ambion). Hybridization was conducted in the same buffer in the presence of a 32P-radiolabeled cDNA fragment of the indicated gene. Blots were washed 4 × 15 min at 50 °C in 2× SSC, 0.1% SDS and once for 30 min in 1× SSC, 0.1% SDS. Membranes were then exposed to a phosphorimager, screen and images were analyzed using a Cyclone Storage Phosphor System and Optiquant software (Hewlitt-Packard). Synthesis of cDNA Probes for Northern Blots—A partial cDNA fragment for adipophilin was generated by PCR with M13 forward and reverse primers using a sequence-validated human IMAGE cDNA clone (Research Genetics) as a template. Partial cDNA fragments for Gob-4 and TSC-22 were generated using reverse transcriptase PCR and gene-specific primers corresponding to base pairs (each from the translational start site) 283–550 for Gob-4 and 1–425 for TSC-22. The template for these PCR reactions was a random primed cDNA library of MOSER S colon carcinoma cells treated with either 0.1% Me2SO or 1 μm rosiglitazone. The Gob-4 PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen), and the TSC-22 product was cloned into pPCR-Script (Stratagene). All plasmids were sequenced to confirm gene identity. The aP2 cDNA fragment was obtained from Youfei Guan (Vanderbilt University, Nashville, TN). PPARγ Ligand Sensitivity and PPARγ Gene Mutations in a Panel of Human Colorectal Cancer Cell Lines—In our initial survey of the biological response of human colorectal carcinoma cells to PPARγ agonists, we noticed that some cell lines were resistant to the growth inhibitory effects of PPARγ ligands. A panel of eight cell lines (four sensitive and the four that were resistant) was chosen for further study. All eight cell lines expressed relatively equivalent levels of PPARγ protein (Fig. 1). The ability of the high affinity, PPARγ subtype-selective agonist rosiglitazone to induce growth inhibition in each of the eight lines was tested. Four of these cell lines (MOSER S, HCT 116, HCA-7, and HT-29) were growth-inhibited in the presence of a PPARγ agonist, whereas the other four (HCT 15, MIP 101, Clone A, and COLO 205) were not affected (Table I).Table IPPARγ ligand sensitivity and PPARγ receptor mutations in a panel of human colorectal cancer cell linesCell line% GrowthaCell lines were treated for 6 days with 0.1% Me2SO or 1.0 μm rosiglitazone, and the number of cells was counted using a Coulter counter. Each experiment represents the mean of three independent experiments. Data are expressed as the number of cells in the rosiglitazone-treated samples as a percent of the total cell number in the Me2SO-treated samples ± S.E.Codon alteredHCT 15103 ± 4.0K422QMIP 10196.0 ± 2.1K422QClone A98.2 ± 3.0K422QCOLO 20595.3 ± 1.8K422QMOSER44.4 ± 0.5HCT 11666.1 ± 1.1HCA-769.0 ± 2.5HT-2962.3 ± 1.8a Cell lines were treated for 6 days with 0.1% Me2SO or 1.0 μm rosiglitazone, and the number of cells was counted using a Coulter counter. Each experiment represents the mean of three independent experiments. Data are expressed as the number of cells in the rosiglitazone-treated samples as a percent of the total cell number in the Me2SO-treated samples ± S.E. Open table in a new tab There are a number of explanations for why a particular cell line could be resistant to activators of PPARγ despite expressing robust levels of the receptor. Because somatic loss of function mutations have been identified in a subset of colorectal tumors, we sought to determine whether PPARγ ligand resistance in the four cell lines could be because of a loss of function mutation in the PPARγ gene. All four resistant lines contained a monoallelic point mutation in the PPARγ gene at codon 422 resulting in a change from lysine (Lys) to glutamine (Gln) (K422Q); this mutation was not found in the four sensitive cell lines (Table I). The correlation between the K422Q allele and lack of sensitivity to PPARγ ligands provided suggestive, but not definitive, evidence that this mutation caused the HCT 15, MIP 101, Clone A, and COLO 205 cell lines to be resistant to the growth inhibitory effects of PPARγ ligands. Characterization of K422Q Mutant Allele—No previous studies documenting the sequence of the PPARγ gene in various malignancies or from individuals at risk for diabetes or obesity have reported mutations at codon 422 of the receptor. Lys-422 lies within the ninth α-helix (H9) of the ligand binding domain of the receptor. Crystallographic studies of PPARγ/RXRα heterodimers suggest a role for H9 in receptor dimerization (33Gampe R.T. Montana V.G. Lambert M.H. Miller A.B. Bledsoe R.K. Milburn M.V. Kliewer S.A. Willson T.M. Xu H.E. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). However, these studies found no direct role for Lys-422 in any polar interactions found at the dimer interface. X-ray crystallography of PPAR homodimers revealed Lys-422 to be located at the receptor surface and exposed to solvent, suggesting the possibility of involvement in co-factor interactions (Fig. 2A) (34Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1700) Google Scholar). Lys-422 is conserved in the PPARγ cDNAs from all species reported in the NCBI Entrez nucleotide data base, including the six different species shown in Fig. 2B. However, Lys-422 is not conserved in either PPARα or PPARδ, both of which encode a Gln at the homologous amino acid (Gln-413 and Gln-386, respectively) (Fig. 2B). Because codon 422 of PPARγ in the resistant cell lines is mutated to an amino acid (Gln) that is normally present in the homologous positions of WT PPARα and PPARδ, it is unlikely that the K422Q mutation disrupts an important structural interaction common to all three PPARs. In fact, it may be that the Lys at position 422 present in WT PPARγ is responsible for an interaction unique to the γ subtype. As no obvious function has been ascribed to Lys-422, we first characterized what effects the K422Q mutation might have on WT receptor activity. There was no difference in the DNA binding activity of WT PPARγ/RXRα or K422Q PPARγ/RXRα on a PPRE from the acyl-coA oxidase promoter (Fig. 2C). Identical results were observed using RXRβ and RXRγ (data not shown). Transcriptional activity was assayed in cells transfected with either receptor cDNA and the PPRE3-tk-luc reporter vector that contains a luciferase cDNA downstream of three tandem repeats of the PPRE from the acyl-coA oxidase gene (35Kliewer S.A. Forman B.M. Blumb
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