Exposure to the phthalate DEHP has been associated with increased obesity in adults and children. Prenatal exposure to DEHP has profound effects on health in later life. The long-term goal of this study is to understand the mechanisms of adipogenic dysregulation resulting from exposure to environmental chemicals. We found that exposure of mouse embryonic fibroblasts (MEFs) to DEHP induces phosphorylation of Proliferating Cell Nuclear Antigen (PCNA) at tyrosine residue 114 (Y114). We generated mutated mice with the Y114 residue replaced with a phenylalanine (114F), which is structurally similar to tyrosine that cannot be phosphorylated. Using this mouse model we found that Y114 phosphorylation of PCNA is essential for adipocyte differentiation. DEHP treatment enhanced adipogenesis in MEFs from wild type (WT) mice, but not from PCNA114F/114F mice, indicating that Y114 phosphorylation is critical for DEHP-induced adipocyte differentiation. To probe the underlying mechanism, we have identified William Syndrome Transcription Factor (WSTF) as a potential tyrosine kinase of Y114.
Specific Aim 1 will test the hypothesis that DEHP stimulates expression/activity of WSTF, resulting in Y114 phosphorylation of PCNA, and consequently promoting adipogenesis in MEFs. To this end, we will examine expression and tyrosine kinase activity of WSTF in response to DEHP, and its association with PCNA on the chromatin. Furthermore, we will introduce a mutant form of PCNA in which Y114 is replaced by a phospho-mimicking residue (phospho-mimetic mutant) to ascertain whether it can rescue adipogenesis in WSTF-deficient MEFs exposed to DEHP.
Specific Aim 2 will assess the metabolic consequences of WT and PCNA114F/114F mice prenatally exposed to DEHP. To this end, mice will be exposed to DEHP during gestation through weaning and then subjected to normal or high-fat diet. Body weight, body composition and energy homeostasis will be determined at selected time points. The causal effect of PCNA Y114 phosphorylation on adipogenesis will be tested by reconstituting phospho-mimetic PCNA in cultured preadipocytes from DEHP-exposed WT and PCNA114F/114F mice. In summary, these studies should establish WSTF-Y114 phosphorylation as a key signaling event downstream of DEHP action. In addition, we will elucidate the mechanisms by which early exposure to DEHP in combination with high fat diet results in an expanded adipose tissue, obesity and altered energy metabolism. Future studies will further dissect the mechanisms through which DEHP regulates the WSTF-PCNA axis. To ensure successful accomplishment of these studies, we have assembled a team of experts in adipocyte biology, energy metabolism, animal models, and environmental xenobiotics.
Obesity is a major risk factor for type 2 diabetes, cardiovascular diseases, and certain types of cancer. In the United States, about two third of the population is classified as obese or overweight. The proposed research is relevant to public health because the discovery unveils a novel mechanism of early exposure to environmental chemicals to induce obesogenicity in later life. Therefore, the proposed research is relevant to NIH's mission that pertains to developing fundamental knowledge that will help reduce the burden of human diseases.
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