Research interests

Molecular Genetics of Peroxisome Proliferator-Activated Receptors

PPARs (peroxisome proliferator-activated receptors) are orphan nuclear hormone receptors that regulate diverse biological processes. Our laboratory studies PPAR function in trophoblast differentiation, adipose tissue biology, and metabolic disease.

PPARγ and Trophoblast Differentiation

The placenta is critical for survival and growth of all mammalian embryos. Abnormal placental function underlies diverse pregnancy and fetal disorders, including spontaneous miscarriages, preeclampsia, and intrauterine growth retardation. We previously showed that PPARγ is essential for placental development. We integrate two approaches to study the mechanism of PPARγ action in the placenta: identification and characterization of PPARγ target genes, and analysis of its role in trophoblast stem (TS) cell differentiation.

We recently demonstrated that Muc1 is a PPARγ target gene, whose expression in trophoblasts depends on the integrity of both PPARγ and its obligatory co-receptor retinoid X receptor α (RXRα), and is augmented by PPARγ agonists. The MUC1 protein is an established component of luminal epithelia, such as those of the milk or salivary ducts. MUC1 localization to trophoblasts that surround maternal blood pools in the placenta invokes anatomical and functional analogies between these trophoblast structures and luminal epithelia.

Muc1 transcription provides an excellent model for studying the complexity of gene regulation by nuclear receptors. A weak PPARγ response element (PPRE) in the Muc1 promoter acts as a basal silencer, and its derepression by PPARγ allows an upstream enhancer to induce Muc1 robustly and specifically. Surprisingly, Muc1 induction by PPARγ ligands is suppressed by physiological concentrations of diverse RXR agonists (rexinoids) in both TS cells and reporter assays. This effect contrasts sharply with the typical ability of rexinoids to activate PPAR target genes, and suggests that Muc1 regulation by PPARγ -RXR heterodimers involves unique interactions with transcriptional co-activators. To further resolve the complexity of Muc1 regulation we are currently pursuing the identity of the PPRE-bound basal repressor, and dissecting the molecular basis of the atypical rexinoid response of Muc1.

Pparg is induced robustly during TS cell differentiation, and its deficiency interferes severely with formation of the spongiotrophoblast and giant cell lineages. The physiological significance of these findings is corroborated by aberrant histology and marker expression of spongiotrophoblasts and giant cells in Pparg-/- placentas in vivo. These observations indicate that PPARγ is essential for terminal differentiation of multiple trophoblast lineages. We are currently screening TS cells for PPARγ target genes that mediate this function.

Molecular Analysis of Lipodystrophy

The epidemic of obesity and its associated Syndrome X, which includes type 2 diabetes, hypertension, and atherosclerosis, is a leading cause of death in developed countries. Adipose tissue is a critical regulator of glucose and lipid homeostasis, and its central role in type 2 diabetes and Syndrome X is demonstrated in lipodystrophy, in which acute fat degeneration results in typical obesity-associated metabolic disorders. Phenotypic convergence of the diametrically opposed obesity and lipodystrophy suggests that the physiological status of adipose tissue in obesity is a functional equivalent of its physical degeneration in lipodystrophy. Therefore, molecular characterization of lipodystrophic fat tissue should provide important insights into the degenerative responses of obese adipose tissue.

Over the past two years, we have been studying a novel mouse model, in which lipodystrophy, dyslipidemia, and hyperinsulinemia are conferred dominantly by a targeted allele of Pparg, termed Ppargldi. We use this mutant strain to pursue the etiology of lipodystrophy, its cellular and molecular manifestations, and how it causes the metabolic syndrome.

We originally generated the Ppargldi allele in an attempt to substitute the endogenous Pparg gene with recombinant inducible PPARγ. This feat was executed by inserting a knock-in cassette containing the Tet-controlled transactivator (tTA) and a tetracycline-regulated Pparg cDNA into the Pparg gene. The basal lipodystrophic outcome was not originally expected, and our ongoing analyses pinpoint the tTA protein as a major contributor to this phenotype. We hypothesize that tTA elicits lipodystrophy by interfering with key cellular regulators of adipocyte homeostasis.

Lipodystrophy is often described as “disordered adipocyte differentiation.” However, three key observations challenge the generality of this statement. First, protease inhibitor-treated HIV patients develop an indistinguishable form of lipodystrophy, in which the condition affects mature adipose tissue. Second, Ppargldi/+ mice and a phenotypically similar transgenic mouse strain, which expresses SREBP-1c constitutively in adipocyte nuclei, exhibit a form of lipodystrophy in which brown adipose tissue differentiates normally prior to adopting its characteristic hypertrophic configuration. Third, and most important, adipogenesis of primary embryonic fibroblasts derived from both lipodystrophic mouse strains is normal. We therefore suggest that this form of lipodystrophy is independent of and subsequent to adipogenesis.

Our ongoing microarray analyses show profound alterations of gene expression in lipodystrophic adipose tissue. Most of the differentially expressed genes have not been studied previously in fat. Several key patterns emerge:

1. Lipodystrophic and obese adipose tissues share multiple gene expression aberrations, which provide a molecular counterpart to the metabolic convergence of both conditions. One of these mutually dysregulated genes encodes a transcription factor that underlies a rare congenital diabetes syndrome in humans.
2. Multiple genes associated with inflammation and immunity are robustly upregulated in lipodystrophy.
3. Several secreted factors are differentially expressed in lipodystrophic versus obese adipose tissue.
4. Only a few of the genes whose expression is altered in lipodystrophic fat in vivo are also differentially expressed in cultured “lipodystrophic” adipocytes. These regulatory events define the immediate cellular targets of the lipodystrophic allele. They include suppression of two brown adipocyte-specific thermogenic genes, and a surprising induction of a T-cell trophic chemokine; this chemokine may comprise an initiating pro-inflammatory step in the etiology of lipodystrophy.

We continue to analyze both global expression patterns and key individual genes in order to expand our insights into adipose tissue biology and disease.

Publications

• Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Koder A, Chien KR, Evans RM. 1999. PPARγ is required for placental, cardiac, and adipose tissue development. Mol Cell 4:585-595.
• Miles PDG, Barak Y, He W, Evans RM, Olefsky JM. 2000. Improved insulin sensitivity in mice heterozygous for PPARγ deficiency. J Clin Invest 105:287-292.
• Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM. 2001. PPAR-γ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med 7:48-52.
• Barak Y, Liao D, He W, Ong ES, Nelson MC, Olefsky JM, Boland R, Evans RM. 2002. Effects of PPARδ on placentation, adiposity, and colorectal cancer. Proc Natl Acad Sci USA 99:303-308.
• Chawla A, Lee CH, Barak Y, He W, Rosenfeld JM, Liao D, Han J, Kang H, Evans RM. 2003. VLDL transcriptionally activates PPARδ in macrophages. Proc Natl Acad Sci USA 100:1268-1273.
• He W*, Barak Y*, Hevener A*, Olson P, Liao D, Le J, Nelson M, Ong, E, Olefsky JM, Evans RM (*Co-first authors). 2003. Adipose-specific PPARγ knockout causes insulin resistance in fat and liver, but not in muscle. Proc Natl Acad Sci USA 100:15712-15717.
• Hevener AL*, He W*, Barak Y*, Le J, Bandyopadhyay G, Olson P, Wilkes J, Evans RM, Olefsky JM (*Co-first authors). 2003. Muscle-specific Pparγ deletion causes insulin resistance. Nat Med 9:1491-1497.
• Shalom-Barak T, Nicholas JM, Wang Y, Zhang X, Ong ES, Young TH, Gendler SJ, Evans RM, Barak Y. 2004. PPARγ controls Muc1 transcription in trophoblasts. Mol Cell Biol 24:10661-10669.
• Schaiff WT, Barak Y, Sadovsky Y. 2006. The pleiotropic function of PPARγ in the placenta. Mol Cell Endocrinol 249:10-15.

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