Wild-type PPARgamma Research Articles

Altering PPARγ Ligand Selectivity Impairs Adipogenesis by Thiazolidinediones But Not Hormonal Inducers

Peroxisome proliferator-activated receptor gamma (PPARgamma) acts as a ligand-dependent transcription factor with a key role in mediating adipocyte differentiation and insulin sensitivity. Recently, we and others have shown that PPARgamma recruits the nuclear corepressors NCoR and silencing mediator for retinoid and thyroid hormone receptors (SMRT) to modulate adipogenesis. While the synthetic ligands for PPARgamma, the thiazolidinediones (TZD), are widely used in the treatment of type 2 diabetes mellitus, the biologically relevant endogenous PPARgamma ligand involved in adipogenesis remains unidentified. To further understand the role of ligand binding and corepressor interaction in PPARgamma-mediated adipogenesis, a mutation was introduced in the ligand-binding domain (LBD) of murine PPARgamma. PPARgammamut was created via two amino acid substitutions known to be major determinants of ligand selectivity among PPAR isotypes, H323Y and R288M. These mutations alter PPARgamma to the corresponding residues of the PPARalpha. Characterizing the in vitro functional properties of this mutant, we show that PPARgammamut preferentially responds to the PPARalpha agonist, WY-14643, over the TZD, pioglitazone. When expressed in 3T3-L1 preadipocytes using recombinant adenovirus, wild-type PPARgamma leads to adipocyte formation with both hormonal and TZD treatment. PPARgammamut blocks the upregulation of adipocyte-specific proteins by TZD, but surprisingly, not by standard hormonal inducers. Our data suggest that TZDs and the purported endogenous ligand do not interact in the same way with the PPARgamma LBD. We propose that the endogenous ligand has distinct properties that allow for promiscuity within the hydrophobic PPAR ligand-binding pocket, yet fosters appropriate cofactor recruitment and release to allow adipogenesis to proceed.

A Selective Peroxisome Proliferator-Activated Receptor-γ Modulator, Telmisartan, Binds to the Receptor in a Different Fashion from Thiazolidinediones

Angiotensin type 1 receptor blockers are widely used for the treatment of hypertension, and one angiotensin type 1 receptor blocker, telmisartan, specifically activates the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied the impact of PPARgamma mutants on transcriptional control and interaction with cofactors to elucidate differences in the molecular mechanism between telmisartan and other PPARgamma agonists, thiazolidinediones (TZDs). We created several amino acid substitutions in the ligand binding domain of PPARgamma that, based on molecular modeling, may affect the binding of these agents. In transient expression experiments, wild-type PPARgamma-mediated transcription stimulated by telmisartan was more than one third of that stimulated by TZDs. The activation stimulated by TZDs was impaired, whereas activation stimulated by telmisartan was retained, in the H323Y, S342A, and H449A mutants. In the Y473A mutant, the TZD-induced activation was further impaired and lower than that of telmisartan-induced activation. Coexpression of coactivators enhanced the activation by both telmisartan and TZDs, but activation by telmisartan always exceeded that of TZDs in the Y473A mutant. Based on a mammalian two-hybrid assay, the interaction with corepressors was retained in Y473A. Telmisartan and TZDs, but not 9cis retinoic acid, dissociated corepressors from the wild-type PPARgamma. Telmisartan most effectively dissociated corepressors from Y473A. The interaction with coactivators was enhanced by TZD activation of wild-type PPARgamma and both telmisartan and TZD activation of Y473A. Thus, the Y473A mutant is selectively stimulated by telmisartan but not TZDs, suggesting that telmisartan and TZDs have differential effects on the transcriptional control. In conclusion, these PPARgamma mutants could be powerful tools for developing novel therapeutic agents that retain the metabolic efficacy of PPARgamma activation with fewer adverse effects, such as the increase in body weight associated with TZDs.

Ectopic expression of porcine peroxisome proliferator-activated receptor δ regulates adipogenesis in mouse myoblasts1

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in regulating adipogenesis. The expression of peroxisome proliferator-activated receptor delta (PPARdelta) precedes that of PPARgamma during adipocyte differentiation in rodents. The current experiment was designed to study the function of porcine PPARdelta and the interaction of PPARdelta and PPARgamma in adipocyte differentiation. Inhibition of myogenesis was observed in mouse myoblasts expressing porcine PPARdelta, similar to myoblasts expressing PPARgamma. Treatment of myoblasts expressing PPARdelta with ligands for both PPARdelta and PPARgamma enhanced lipogenesis and adipogenesis to a greater extent than treatment with a PPARgamma ligand alone, suggesting that both genes were involved in regulating lipogenesis and adipogenesis. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts coexpressing PPARdelta with either wild type or mutated PPARgamma (Ser 112 was mutated to Ala; the mutated PPARgamma is more active than the wild type) compared with myoblasts expressing PPARgamma alone. Adipocyte differentiation in myoblasts coexpressing PPARdelta and mutated PPARgamma was greater than in myoblasts coexpressing PPARdelta and wild type PPARgamma, confirming that Ser 112 is important for the function of PPARgamma. Taken together, our results demonstrate that overexpression of PPARdelta inhibits myotube formation and also enhances adipocyte differentiation. However, the complexity and interaction of PPARdelta and PPARgamma in adipogenesis are not clearly understood.

Porcine peroxisome proliferator-activated receptor γ induces transdifferentiation of myocytes into adipocytes1

Peroxisome proliferator-activated receptor gamma2 (PPARgamma) is a nuclear transcription factor that regulates adipocyte differentiation and lipogenic genes during adipogenesis. The activity of rodent PPARgamma is regulated by phosphorylation of serine 112. The current experiment was designed to study the ability of porcine PPARgamma to stimulate transdifferentiation of myoblasts to adipocytes by overexpressing wild-type PPARgamma or mutated PPARgamma (serine 112 was mutated to alanine) in mouse myoblast cells. The expression of adipogenic marker genes (adipocyte fatty acid binding protein, lipoprotein lipase, and glycerol-3 phosphate dehydrogenase) in cells stably expressing mutated porcine PPARgamma was greater than in cells with wild-type PPARgamma, indicating that the mutated PPARgamma has greater adipogenic capability than the wild-type PPARgamma. Under treatment with a ligand, both wild-type and mutant porcine PPARgamma-expressing C2C12 myoblasts differentiated into adipocytes in 10 d. The expression of myogenic marker genes (myogenin, myogenic regulatory factor-4) was suppressed in cells transfected with the mutated PPARgamma or wild-type PPARgamma. Moreover, wild-type and mutant PPARgamma were able to inhibit myogenesis without addition of a ligand. Our results suggest that porcine wild-type PPARgamma and mutated PPARgamma can both convert myoblast cells into adipocytes, and also that the ability to transdifferentiate was greater in cells containing the mutated PPARgamma than in cells containing the wild-type PPARgamma. Therefore, the existence of serine 112 in PPARgamma may have a role in regulating adipocyte differentiation.

15-Lipoxygenase-2 gene regulation by its product 15-(S)-hydroxyeicosatetraenoic acid through a negative feedback mechanism that involves peroxisome proliferator-activated receptor γ

An inverse relationship exists between the expression of 15-lipoxygenase-2 (15-LOX-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) in normal prostate epithelial cells (PrECs) compared with their expression in prostate carcinoma cells (PC-3). The reason for this difference, however, is unknown. We hypothesized that this inverse expression partly involves the 15-LOX-2 promoter and 15-S-hydroxyeicosatetraenoic acid (15-(S)-HETE), a product of 15-LOX-2 that binds to PPARgamma. We identified an active steroid nuclear receptor half-site present in the 15-LOX-2 promoter fragment F-5 (-618/+177) that can interact with PPARgamma. After forced expression of wild-type PPARgamma, 15-(S)-HETE (1 microM) decreased F-5 reporter activity in PrECs whereas forced expression of 15-LOX-2 resulted in 15-(S)-HETE production which enhanced F-5 activity in PC-3. In contrast, the expression of dominant-negative PPARgamma reversed the transcriptional activation of F-5 by enhancing it 202-fold in PrEC or suppressing it in PC-3; the effect in PC-3 was positively increased 150-fold in the presence of 15-(S)-HETE (1 microM). Peroxisome proliferator-activated receptor gamma interacted with 15-LOX-2 promoter sequences in pulldown experiments using biotinylated 15-LOX-2 (-560/-596 bp) oligonucleotides. In gelshift analyses PPARgamma and orphan receptor RORalpha were shown to interact with the F-5 fragment in PC-3 cells. These data suggest that crosstalk mechanisms exist between the 15-LOX-2 gene and PPARgamma to counterbalance expression and help explain the inverse relationship of these genes in normal versus cancer cells.

Proline Oxidase, a Proapoptotic Gene, Is Induced by Troglitazone: EVIDENCE FOR BOTH PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ-DEPENDENT AND -INDEPENDENT MECHANISMS

Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor gamma (PPARgamma) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARgamma ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARgamma, troglitazone enhanced the binding of PPARgamma to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARgamma activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARgamma-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARgamma-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARgamma, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARgamma, increased activation was observed by ligand stimulation, indicating that both PPARgamma-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARgamma-dependent and -independent mechanisms.

Novel high-affinity PPARγ agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAF1/CIP1

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists demonstrate antitumor activity likely through transactivating genes that regulate cell proliferation, apoptosis, and differentiation. The PAX8/PPARgamma fusion oncogene, which is common in human follicular thyroid carcinomas appears to act via dominant negative suppression of wild-type PPARgamma, suggesting that it may be a tumor suppressor gene in thyroid cells. We have identified a novel high-affinity PPARgamma agonist (RS5444) that is dependent upon PPARgamma for its biological activity. This is the first report of this molecule and its antitumor activity. In vitro, the IC50 for growth inhibition is approximately 0.8 nM while anaplastic thyroid carcinoma (ATC) tumor growth was inhibited three- to fourfold in nude mice. siRNA against PPARgamma and a pharmacological antagonist demonstrated that functional PPARgamma was required for growth inhibitory activity of RS5444. RS5444 upregulated the cell cycle kinase inhibitor, p21WAF1/CIP1. Silencing p21WAF1/CIP1 rendered cells insensitive to RS5444. RS5444 plus paclitaxel demonstrated additive antiproliferative activity in cell culture and minimal ATC tumor growth in vivo. RS5444 did not induce apoptosis but combined with paclitaxel, doubled the apoptotic index compared to that of paclitaxel. Our data indicate that functional PPARgamma is a molecular target for therapy in ATC. We demonstrated that RS5444, a thiazolidinedione (Tzd) derivative, alone or in combination with paclitaxel, may provide therapeutic benefit to patients diagnosed with ATC.

Evaluation of Peroxisome Proliferator-Activated Receptor-γ Expression in Benign and Malignant Thyroid Pathologies

Impairment of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) function through a dominant negative PAX-8/PPAR-gamma fusion gene or other events resulting in wild-type PPAR-gamma downregulation has been implicated in malignant thyroid cell transformation. The aim of our study was to perform a systematic evaluation of PPAR-gamma mRNA and protein expression in normal thyroid tissue as opposed to benign thyroid pathologies of different functional status and thyroid malignancy, to gain further insights into a putative physiological role of PPAR-gamma in the thyroid and to define whether PPAR-gamma could serve as a marker of thyroid cell differentiation. Ten cold benign (CTN) and 10 toxic (TTN) thyroid nodules and corresponding normal thyroid tissues, 10 follicular thyroid cancers (FTC), 10 papillary thyroid cancers (PTC) and 8 Graves' disease (GD) thyroids were studied by real-time polymerase chain reaction (PCR), immunohistochemistry and reverse transcriptase (RT)-PCR (PAX-8/PPAR-gamma fusion gene). PPAR-gamma mRNA expression was demonstrated in all samples. When comparing benign nodular and normal thyroid tissue of the same patient no significant difference in PPAR-gamma mRNA expression was observed. PPAR-gamma mRNA levels were similar in CTN and FTC. In contrast, PPAR-gamma mRNA expression was downregulated in 9 of 10 PTC and all GD samples, whereby at least 4 fold downregulation (compared with normal and benign nodular thyroid tissues) was observed in the latter. Immunohistochemistry showed an increased, patchy PPAR-gamma nuclear staining in CTNs and TTNs and only faint staining in the corresponding normal thyroid tissues. A diffuse and weak PPAR-gamma staining pattern was observed in all GD samples. No PAX-8/PPAR-gamma rearrangements were detected in any of the 68 thyroid tissue samples. In conclusion PPAR-gamma mRNA and protein expression levels are not concordant in benign thyroid nodular disease. Furthermore there is no clear-cut association of PPAR-gamma mRNA expression with follicular thyroid tumorigenesis. Absence of a PAX-8/PPAR-gamma fusion gene in the series of 68 thyroid samples is in agreement with the suggestion of PAX-8/PPAR-gamma rearrangement being restricted to a subset of follicular thyroid cancers. The marked downregulation of PPAR-gamma in GD warrants further investigation and could be linked, for example, with changes in apoptosis.

Follicular Thyroid Tumors with the PAX8-PPARγ1 Rearrangement Display Characteristic Genetic Alterations

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

A Dominant Negative Peroxisome Proliferator-activated Receptor-γ Knock-in Mouse Exhibits Features of the Metabolic Syndrome

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARgamma mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARgamma L466A mutation, shown previously to inhibit wild-type PPARgamma action in vitro. Homozygous PPARgamma L466A knock-in mice die in utero. Heterozygous PPARgamma L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARgamma function and the metabolic syndrome.

The peroxisome proliferator activated receptor gamma Pro12Ala polymorphism is associated with a lower hirsutism score and increased insulin sensitivity in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and chronic anovulation. The genetic background of the insulin resistance frequently associated with PCOS is unclear. To examine the influe nce of the Pro12Ala polymorphism of the peroxisome proliferator activated receptor gamma (PPARgamma), which is thought to play a role in the regulation of insulin sensitivity, on endocrine and metabolic parameters in PCOS patients. PPARgamma alleles were analysed in 102 PCOS patients (age 27 +/- 5.3 years) and 104 age matched control women. PCOS was defined by the NIH-criteria as the presence of chronic oligo- or anovulation and hyperandrogenism. Family history and clinical parameters were evaluated by personal interview and physical examination, parameters of insulin resistance [homeostasis model assessment (HOMA) and Matsuda-index] were evaluated with a glucose tolerance test. Seventy-nine (77.5%) PCOS patients were carriers of the wild-type PPARgamma allele (Pro/Pro), while 23 (22.5%) had at least one Ala allele (X/Ala), with an equal distribution in controls. X/Ala PCOS women were more insulin-sensitive, evidenced by lower fasting insulin, HOMA index and insulin secretion. Differences in insulin resistance did not depend on body mass index. The genotype had no influence on lipid status, leptin, adiponectin, ghrelin, or family history of type 2 diabetes. A significantly lower proportion of Pro/Ala patients had hirsutism and they had on average lower hirsutism scores than Pro/Pro patients. No relationship was found between the Pro/Ala polymorphism and other signs of hyperandrogenism. The Pro12Ala polymorphism of the PPARgamma gene is associated with increased insulin sensitivity and lower hirsutism scores in PCOS women.

The PAX8/PPAR gamma fusion oncogene as a potential therapeutic target in follicular thyroid carcinoma.

Follicular thyroid carcinoma (FTC) accounts for approximately 20% of all thyroid cancers, and up to 40% of the deaths associated with this disease. Current treatment approaches include surgery, followed by radioactive iodine therapy. However, a significant proportion of locally advanced and metastatic FTC fails to concentrate iodine. Because traditional chemotherapeutic agents have not been shown to alter outcomes in this disease, novel therapeutic strategies are needed for advanced disease. Recently, a genomic rearrangement has been identified in up to 50% of FTC, involving a translocation event between chromosome regions 3p25 and 2q13. This translocation fuses the thyroid-specific transcription factor PAX8 gene with the PPARgamma gene, a ubiquitously expressed transcription factor. We have confirmed that this Pax8/PPARgamma fusion gene (designated PPFP) is an oncogene, which accelerates cell growth, reduces rates of apoptosis and permits anchorage independent and contact uninhibited growth of a thyroid cell line. The action of PPFP arises, at least in part, through its activity as a dominant-negative inhibitor of the wild-type PPARgamma transcription factor. Although the mechanism by which PPFP impairs PPARgamma activity remains unknown at this time, it is likely to be mediated by competition for the genomic PPARgamma response elements, the endogenous ligand, or various cofactors, including the Retinoid X Receptor (RXR). Consequently, modulation of PPFP activity might be possible through the use of PPARgamma agonists, RXR-agonists, or specific modulators of PPFP itself. Alternatively, modulation of several down-stream regulatory pathways may become possible, as the consequences of PPARgamma inhibition become better known. PPFP represents a potential novel target for the management of advanced FTC.

Activation of Peroxisome Proliferator-activated Receptor γ Inhibits Interleukin-1β-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.

In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses. The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs. The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7. By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

Resistance to thyroid hormone, and peroxisome-proliferator-activated receptor gamma resistance.

Resistance to thyroid hormone (RTH) is usually inherited in a dominant fashion, and is characterized by elevated serum thyroid hormone levels and failure to suppress pituitary secretion of thyroid-stimulating hormone, with variable refractoriness to hormone action in peripheral tissues. Two major forms of the disorder are recognized: asymptomatic individuals with generalized resistance (GRTH) and patients with thyrotoxic features suggesting predominant pituitary resistance (PRTH). In over 100 families with GRTH or PRTH, we have identified heterozygous mutations in the thyroid hormone receptor beta isoform (TRbeta), which localize to three regions (amino acids 234-282, 310-353 and 429-461) of the hormone-binding domain of the receptor. The mutant receptors are transcriptionally impaired, due either to reduced ligand binding or to attenuated interaction with co-activators, and inhibit wild-type TR action in a dominant-negative manner. In the TRbeta crystal structure, most RTH mutations cluster around the hormone-binding pocket, with receptor regions that mediate functions (DNA binding, dimerization, co-repressor recruitment) required for dominant-negative activity being devoid of natural mutations. The pathogenesis of variable tissue resistance is not fully understood, but may be related to the differing tissue distributions of TRalpha and TRbeta, and to variable dominant-negative activity of mutant receptors on different target genes. The nuclear receptor peroxisome-proliferator-activated receptor gamma (PPARgamma) regulates adipogenesis and mediates the action of thiazolidinediones - novel anti-diabetic agents which enhance tissue insulin sensitivity. The PPARgamma gene was screened in 85 subjects with severe insulin resistance, and two different heterozygous receptor mutations (P467L and V290M) were identified in three affected individuals. The PPARgamma mutants are markedly transcriptionally impaired due to altered ligand binding and co-activator recruitment. Analogous to RTH, they inhibit the function of wild-type PPARgamma when co-expressed, and such dominant-negative inhibition is linked to their ability to silence basal gene transcription via aberrant interaction with co-repressors. In addition to insulin resistance, all three affected subjects developed Type II diabetes mellitus and hypertension at an unusually early age. Our findings provide compelling evidence that PPARgamma is important in the control of insulin sensitivity, glucose homoeostasis and blood pressure in humans. Future studies aim to elucidate the mechanism by which this receptor regulates insulin action and vascular tone.

Dominant negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension.

Thiazolidinediones are a new class of antidiabetic agent that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type 2 diabetes. Although these agents can bind and activate an orphan nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), there is no direct evidence to conclusively implicate this receptor in the regulation of mammalian glucose homeostasis. Here we report two different heterozygous mutations in the ligand-binding domain of PPARgamma in three subjects with severe insulin resistance. In the PPARgamma crystal structure, the mutations destabilize helix 12 which mediates transactivation. Consistent with this, both receptor mutants are markedly transcriptionally impaired and, moreover, are able to inhibit the action of coexpressed wild-type PPARgamma in a dominant negative manner. In addition to insulin resistance, all three subjects developed type 2 diabetes mellitus and hypertension at an unusually early age. Our findings represent the first germline loss-of-function mutations in PPARgamma and provide compelling genetic evidence that this receptor is important in the control of insulin sensitivity, glucose homeostasis and blood pressure in man.