Overview
Nuclear hormone receptors are transcription factors that regulate the expression of genes in response to small lipid-soluble signaling molecules, including hormones, vitamins and fatty acid and sterol derivatives. Our lab is investigating the functions of a subfamily of nuclear hormone receptors, termed PPARs (peroxisome proliferators activated receptors). PPARs control embryonic development and metabolism, and are associated with type II diabetes, inflammation and cardiovascular disease. Better understanding of the mechanisms of PPAR action may therefore yield insights into new interventions and treatments for these diseases. Our specific areas of study are the essential roles of PPARgamma in fat cell formation, death, and regeneration, and of PPARgamma and PPARdelta in placental development. Our approach focuses on identifying and determining the biological functions and transcriptional regulation of novel PPAR target genes that mediate these processes.
Research details
Molecular Genetics of Peroxisome Proliferator-Activated Receptors
PPARs (peroxisome proliferator-activated receptors) are orphan nuclear hormone receptors that regulate diverse key biological processes. Our laboratory studies PPAR function in trophoblast differentiation, adipose tissue biology, and metabolic disease.
PPARs in trophoblast differentiation and placental development PPARy and PPARδ play non-redundant roles in placental development. PPARy is essential for trophoblast differentiation, whereas PPARδ is vital for placental growth and structural integrity. To understand the molecular mechanisms of PPAR action in the placenta, we pursue PPAR target genes in whole placentas and cultured trophoblasts.
Canonical PPARy target genes are expected to decrease in Pparg-null placentas in vivo and Pparg-null trophoblast stem (TS) cells in vitro and to increase in response to the PPARy agonist rosiglitazone (rosi) in wild type (wt) but not Pparg-null TS cells. The Muc1 gene, which we previously identified as a PPARy target in trophoblasts, abides stringently by these criteria. Microarray analyses of wt vs. Pparg-null placentas, and of wt vs. Pparg-null TS cell lines differentiated in the presence vs. the absence of rosi, have identified several additional genes that fulfill these criteria. These analyses provide the following insights:
- PPARy induces the in vivo and/or in vitro expression of multiple cathepsin and prolactin family genes that typify sinusoidal trophoblast giant cells (S-TGC). These cells, whose function is currently unknown, originate from syncytiotrophoblast progenitor cells within the labyrinthine layer of the placenta. The downregulation of multiple S-TGC markers implicates PPARy in controlling the differentiation and function of this cell type as a whole.
- Several spongiotrophoblast-specific Ceacam/Psg gene family members depend on PPARy in vivo and/or in vitro. The spongiotrophoblast layer of Pparg-null placentas exhibits histological defects, and differentiation of Pparg-null TS cells into spongiotrophoblasts is compromised. Nevertheless, the spongiotrophoblast layer still forms and expresses several prototypic lineage markers in Pparg-null placentas. This pattern suggests that PPARy may regulate specific functions or sub-specification of the spongiotrophoblast lineage, but not its entire fate.
- Lactate dehydrogenase B (Ldhb) emerges as a trophoblast lineage-specific PPARy target gene, revealing an unexpected facet of energy metabolism that is regulated by PPARy, and linking this facet to placental development. Ldhb exhibits a biphasic expression pattern, whereby it is expressed constitutively and independent of PPARy in undifferentiated trophoblasts in vivo and in vitro, while becoming entirely dependent on PPARy in differentiated trophoblasts. The Ldhb promoter contains 3 putative PPARy-response elements and is induced by PPARy and rosi in heterologous reporter assays, indicating that Ldhb is a direct transcriptional target of PPARy in differentiated trophoblasts. Applying similar rationales, we recently established a Ppard-null TS cell line and are in the process of collecting samples for microarray-based PPAR? target gene screening in vivo and in vitro
PPARy in adipose tissue development
PPARy is necessary and sufficient for adipocyte differentiation (adipogenesis), and Pparg-null mice are completely devoid of adipose tissue. We found that PPARy is dispensable for commitment of cells to the adipocyte lineage and the establishment of adipose primordia, but is obligatory for primordium expansion and differentiation. In Pparg-null chimeric embryos, wt progenitors infiltrate the arrested Pparg-null primordia, where they proliferate, differentiate into adipocytes, and reconstitute the fat pad. Importantly, arrested Pparg-null preadiopcytes resume expansion in the immediate vicinity of infiltrating wt adipocytes, implicating PPARy in the regulation of preadipocyte expansion via non- cell-autonomous, paracrine effectors.
To enhance our insights into the functions of PPARy in early adipogenesis, we complemented our studies of chimeric embryos and neonates in vivo with studies of adipogenesis in mouse embryonic fibroblasts (MEF) in vitro. MEF adipogenesis requires treatment with rosi during the first 48 hours of differentiation, indicating that basal PPARy activity in these cells is rate limiting for the early phase of the process, and its exogenous stimulation is both necessary and sufficient for progression of differentiation. The role of cell-cell interactions is evident in a surprising inhibition of differentiation of adipogenesis-prone 3T3-L1 cells by co-cultured MEF, and the reversal of this inhibition by rosi treatment during the first 48 hours of differentiation. These observations suggest that preadipocytes with limiting PPARy activity, such as MEF, inhibit adipogenesis in a non cell-autonomous fashion, and stimulation of PPARy activity (rosi) is sufficient to relieve this lateral inhibition.
To further dissect the mechanisms of PPARy action in early adipogenesis, we initiated microarray analyses of the effects of PPARy deficiency and stimulation on the early preadipocyte transcriptome in vitro. These analyses identified several novel PPARy target genes related to extracellular signaling and to cell proliferation and death. Mechanistic studies of these genes in whole animal and cell culture models of adipogenesis are underway.
Ppargldi: A novel mouse model of conditional lipodystrophy
The Ppargldi (Lipodystrophy, Dyslipidemia, and Insulin resistance) allele confers dominant heritable lipodystrophy (adipose tissue degeneration), with striking anatomical and metabolic resemblance to protease inhibitor-treated HIV patients. The allele was generated by homologous disruption of the Pparg gene with the Tet-activator (tTA), which unexpectedly elicited mild lipodystrophy, insulin resistance/dyslipidemia, and a tTA-activated Flag-Pparg1 transgene, which surprisingly exacerbated these traits. Importantly, the lipodystrophic pathology of Ppargldi/+ mice is conditional and can be fully suppressed by doxycycline-mediated disruption of tTA. Primary Ppargldi/+ embryonic fibroblasts undergo robust adipogenesis, suggesting that lipodystrophy arises in this mouse independent of adipogenic defects. Microarray analyses of Ppargldi/+ adipose tissue and fibroblast-derived adipocytes reveal cell-autonomous induction of the orphan G protein-coupled receptor, Gpr56, by tTA; Flag-PPARy1 elicits the adipocyte-autonomous induction of multiple innate immunity genes, led by the lymphotropic chemokine gene Cxcl10. These findings invoke novel mechanistic relationships between PPARy, adipose tissue inflammation, and adipocyte function. Analyses of Cxcl10- or lymphocyte-deficient Ppargldi/+ mice suggested that Cxcl10 and its target lymphocyte mitigate hypertrophy of Ppargldi/+ adipocytes but do not impact metabolic homeostasis. We are currently using a similar strategy to determine the potential contribution of the chemokine CCL8 and infiltrating macrophages to the Ppargldi phenotype.
Lab staff
Resarch Associate: Tali Shalom-Barak, D.V.M.
Postdoctoral Fellow: Suyeon Kim, Ph.D.
Research Assistants: Xiaowen Zhang, M.Sc., Aleksandra Aljakna, B.A.
Research Administrative Assistant: Patricia Cherry
Publication listings
Barak Y, Kim S. 2007. Genetic manipulations of PPARs - Effects on obesity and metabolic disease. PPAR Res 12781.
Barak Y, Sadovsky Y, Shalom-Barak T. 2008. PPAR signaling in placental development and function. PPAR Res doi:10.1155/2008/142082.
Kim S, Huang L-W, Snow KJ, Ablamunits V, Hasham MG, Young TH, Paulk AC, Richardson JE, Affourtit J, Shalom-Barak T, Bult CJ, Barak Y. 2007. A novel mouse model of conditional lipodystrophy. Proc Natl Acad Sci USA 104:16627-16632.
Schaiff WT, Knapp FF Jr., Barak Y, Biron-Shental T, Nelson DM, Sadovsky Y. 2007. Ligand-activated peroxisome proliferator activator gamma alters placental morphology and placental fatty acid uptake in mice. Endocrinology 148(8):3625-3645.
McAlpine CA, Barak Y, Matise I, Cormier RT. 2006. Intestinal-specific RRARγ deficiency enhances tumorigenesis in ApcMin/+ mice. Inst J Cancer 119:2339-2346.
Schaiff WT, Barak Y, Sadovsky Y. 2006. The pleiotropic function of PPARγ in the placenta. Mol Cell Endocrinol 249:10-15.
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.
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.
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, 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.
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.
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.
