Acetylation-defective mutant of Pparγ is associated with decreased lipid synthesis in breast cancer cells.

In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPARγ) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Pparγ1 are acetylated. Herein, we demonstrate that Pparγ1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Pparγ1 and the catalytic domain of SIRT1 are both required for the interaction between Pparγ1 and SIRT1. Sirt1 and Pparγ1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Pparγ1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Pparγ1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells

Genetic Variation Determines PPARγ Function and Anti-diabetic Drug Response In Vivo

SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.ISSN:0092-8674ISSN:1097-417

A Novel Subfamily of Three StAR-Related Lipid Transfer Proteins That Are Differentially-Regulated and Function in Intracellular Cholesterol Metabolism

thesis describes the discovery, cloning, and initial characterization of StarD4, sterol-regulated gene encoding a StAR-related lipid transfer (START) protein, and its close homologues, StarD5 and StarD6. StarD4 was identified using cDNA microarrays, as liver StarD4 expression decreased three-fold in mice fed a high cholesterol diet. StarD4 also sterol-regulated in cultured cells, and a functional sterol regulatory element (SRE) identified in its promoter. StarD4 was preferentially activated in mouse liver by SREBP-rather than SREBP-1, supporting a role in cholesterol rather than fatty acid metabolism. X-ray crystal structure of StarD4 was solved, revealing a hydrophobic lipid binding cavity described for other START domains. StarD5 and StarD6 were identified by homology to StarD4, and these three genes constituted a novel subfamily most similar to the cholesterolbinding START domains of StAR and MLN64. StarD4 and StarD5 were ubiquitously expressed with highest mRNA levels in liver, while StarD6 expression was limited to male germ cells of the testis. StarD5 was not activated by SREBP or LXR transcription factors, well-characterized regulators of cholesterol metabolism, but rather by the ER stress response, a recently-described means by which cholesterol regulates gene expression. The nematode C. elegans has one StarD4 subfamily protein, K02D3.2. Reporter studies indicated that gene was not regulated by cholesterol or ER stress, and it was only expressed in hypodermal seam cells of embryos and larvae. Overexpression of StarD4 and StarD5 revealed functional activity in three cell culture assays: (1) StAR-like activation of steroidogenesis by mitochondrial P450 side chain cleavage enzyme, (2) repression of an SREBP-regulated reporter, and (3) activation of an LXR-regulated reporter. The START domains of StAR and MLN64 were active in these assays, while the related phosphatidylcholine transfer protein (PCTP) was an inactive negative control. Based on these results, the novel StarD4 subfamily is likely to play roles in the intracellular transport and metabolism of cholesterol

Pparγ1 Facilitates ErbB2-Mammary Adenocarcinoma in Mice

HER2, which is associated with clinically aggressive disease, is overexpressed in 15–20% of breast cancers (BC). The host immune system participates in the therapeutic response of HER2+ breast cancer. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ and Cxcl5 signaling module, that was recapitulated in human breast cancer. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Herein, endogenous Pparγ1 promoted ErbB2-mediated mammary tumor onset and progression. PPARγ1 increased expression of an EGF-EphA2 receptor tyrosine kinase module and a cytokine/chemokine 1 transcriptional module. The induction of a pro-tumorigenic inflammatory state by Pparγ1 may provide the rationale for complementary coextinction programs in ErbB2 tumors