Pasha Sarraf

PPARγ Is Required for the Differentiation of Adipose Tissue In Vivo and In Vitro

Abstract The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPARγ, is known to promote fat cell differentiation in vitro. Whether PPARγ is required for this process in vivo has remained an open question because a viable loss-of-function model for PPARγ has been lacking. We demonstrate here that mice chimeric for wild-type and PPARγ null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPARγ for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPARγ gene dosage. These data provide direct evidence that PPARγ is essential for the formation of fat.

Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARγ

Adipocyte differentiation is an important component of obesity and other metabolic diseases. This process is strongly inhibited by many mitogens and oncogenes. Several growth factors that inhibit fat cell differentiation caused mitogen-activated protein (MAP) kinase-mediated phosphorylation of the dominant adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and reduction of its transcriptional activity. Expression of PPARγ with a nonphosphorylatable mutation at this site (serine-112) yielded cells with increased sensitivity to ligand-induced adipogenesis and resistance to inhibition of differentiation by mitogens. These results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.

Differentiation and reversal of malignant changes in colon cancer through PPARgamma.

PPARγ is a nuclear receptor that has a dominant regulatory role in differentiation of cells of the adipose lineage, and has recently been shown to be expressed in the colon. We show here that PPARγ is expressed at high levels in both well- and poorly-differentiated adenocarcinomas, in normal colonic mucosa and in human colon cancer cell lines. Ligand activation of this receptor in colon cancer cells causes a considerable reduction in linear and clonogenic growth, increased expression of carcinoembryonic antigen and the reversal of many gene expression events specifically associated with colon cancer. Transplantable tumors derived from human colon cancer cells show a significant reduction of growth when mice are treated with troglitazone, a PPARγ ligand. These results indicate that the growth and differentiation of colon cancer cells can be modulated through PPARγ.

Terminal Differentiation of Human Breast Cancer through PPARγ

We have previously demonstrated that PPAR gamma stimulates the terminal differentiation of adipocyte precursors when activated by synthetic ligands, such as the antidiabetic thiazolidinedione (TZD) drugs. We show here that PPAR gamma is expressed at significant levels in human primary and metastatic breast adenocarcinomas. Ligand activation of this receptor in cultured breast cancer cells causes extensive lipid accumulation, changes in breast epithelial gene expression associated with a more differentiated, less malignant state, and a reduction in growth rate and clonogenic capacity of the cells. Inhibition of MAP kinase, shown previously to be a powerful negative regulator of PPAR gamma, improves the TZD ligand sensitivity of nonresponsive cells. These data suggest that the PPAR gamma transcriptional pathway can induce terminal differentiation of malignant breast epithelial cells and thus may provide a novel, nontoxic therapy for human breast cancer.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Loss-of-Function Mutations in PPARγ Associated with Human Colon Cancer

The gamma isoform of the peroxisome proliferator-activated receptor, PPAR gamma, regulates adipocyte differentiation and has recently been shown to be expressed in neoplasia of the colon and other tissues. We have found four somatic PPAR gamma mutations among 55 sporadic colon cancers: one nonsense, one frameshift, and two missense mutations. Each greatly impaired the function of the protein. c.472delA results in deletion of the entire ligand binding domain. Q286P and K319X retain a total or partial ligand binding domain but lose the ability to activate transcription through a failure to bind to ligands. R288H showed a normal response to synthetic ligands but greatly decreased transcription and binding when exposed to natural ligands. These data indicate that colon cancer in humans is associated with loss-of-function mutations in PPAR gamma.

APC-dependent suppression of colon carcinogenesis by PPARγ

Activation of PPARγ by synthetic ligands, such as thiazolidinediones, stimulates adipogenesis and improves insulin sensitivity. Although thiazolidinediones represent a major therapy for type 2 diabetes, conflicting studies showing that these agents can increase or decrease colonic tumors in mice have raised concerns about the role of PPARγ in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Pparγ with both chemical and genetic models of this malignancy. Heterozygous loss of PPARγ causes an increase in β-catenin levels and a greater incidence of colon cancer when animals are treated with azoxymethane. However, mice with preexisting damage to Apc, a regulator of β-catenin, develop tumors in a manner insensitive to the status of PPARγ. These data show that PPARγ can suppress β-catenin levels and colon carcinogenesis but only before damage to the APC/β-catenin pathway. This finding suggests a potentially important use for PPARγ ligands as chemopreventative agents in colon cancer.

FUNCTIONAL ANTAGONISM BETWEEN CCAAT/ENHANCER BINDING PROTEIN-ALPHA AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA ON THE LEPTIN PROMOTER

The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass. Recent work has suggested that the antidiabetic agents, the thiazolidinediones (TZ), which are also high affinity ligands of peroxisome proliferator-activated receptor-γ (PPARγ), inhibit leptin expression in rodents. To examine the effects of this class of drug on the leptin gene in adipocytes we performed Northern analysis on primary rat adipocytes cultured in the presence or absence of TZ. TZ reduced leptin mRNA levels by 75%. To determine whether this effect was mediated at the transcriptional level, we isolated 6510 base pairs of 5′-flanking sequence of the leptin promoter and studied reporter constructs in primary rat adipocytes and CV-1 cells. Sequence analysis demonstrated the presence of a consensus direct repeat with a 1-base-pair gap site between −3951 and −3939 as well as a consensus CCAAT/enhancer binding protein (C/EBP) site between −55 and −47. Our functional analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promoter constructs ranging from −6510 to +9 to −65 to +9. In CV-1 cells, which contain endogenous PPARγ, TZ treatment alone had little effect on these constructs. However, TZ treatment did inhibit C/EBPα-mediated transactivation of the leptin promoter. This down-regulation of leptin reporter constructs mapped to a −65 to +9 promoter fragment which binds C/EBPα in gel-mobility shift assays but does not bind PPARγ2 alone or as a heterodimer with 9-cis-retinoic acid receptor. Conversely, the promoter (−5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ. Co-transfection with C/EBPα minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ. These data indicate that PPARγ and C/EBPα can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of leptin expression by thiazolidinediones.

Use of the Peroxisome Proliferator-Activated Receptor (PPAR) γ Ligand Troglitazone as Treatment for Refractory Breast Cancer: A Phase II Study

AbstractPurpose. To evaluate the therapeutic effects of the peroxisome proliferator-activated receptor (PPAR) γ activating ligand, troglitazone, in patients with refractory metastatic breast cancer. Experimental design. Patients with advanced breast cancer refractory to at least one chemotherapy regimen (ER negative tumors) or two hormonal regimens (ER positive tumors) were treated with troglitazone at 800 mg PO QD until disease progression, to determine the percentage of patients free of progression at 6 months. Tumor response, toxicity, and changes in serum tumor markers (CEA, CA27.29) that might reflect alteration in tumor differentiation, were also examined. Results. Twenty-two patients were enrolled before suspension of protocol accrual and treatment when troglitazone was withdrawn from commercial availability following FDA warnings on hepatic toxicity. No objective responses (CR or PR) were observed; only three patients had SD at 8 weeks. Patients came off study for PD (16), DLT (1), FDA withdrawal (2), or other (3) reasons. No patients took troglitazone for more than 20 weeks; all had experienced disease progression or began other systemic therapy within 6 months. All patients with elevated serum tumor markers (CEA and CA27.29) at baseline had rising tumor markers within 8 weeks. Conclusions. While clinical trials among different patient populations might uncover subtle effects on tumor differentiation, PPARγ activation by troglitazone has little apparent clinical value among patients with treatment-refractory metastatic breast cancer.

Targeted Elimination of Peroxisome Proliferator-Activated Receptor γ in β Cells Leads to Abnormalities in Islet Mass without Compromising Glucose Homeostasis

ABSTRACT The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPARγ can reduce cellular proliferation. We show here that activation of PPARγ is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in β cells was eliminated (βγKO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of β-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in β-cell mass, no effect on glucose homeostasis in βγKO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and βγKO mice revealed that PPARγ in β cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPARγ function in β-cell proliferation and also indicate that the mechanisms controlling β-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.