Identification of transcription factors and response elements driving expression of Plin5

Abstract

Obesity is a major health problem in the world today, as it results in several metabolic changes leading to elevated risk of various severe conditions such as cancer, diabetes mellitus, coronary heart disease and stroke. The mechanisms leading to development of excess body fat is complex, and there are still many unanswered questions regarding the factors involved.

Lipid droplets (LDs) function as cellular fat stores, and are coated with various proteins such as members of the Perilipin family. This family consists of five members (perilipin 1-5, encoded by the genes Plin1-5) that differ in tissue expression, transcriptional regulation, protein stability and binding affinity to the LDs. The perilipins are thought to have unique roles in the regulation of fatty acid metabolism in various tissues. Previous studies have demonstrated that perilipin 1, 2 and 4 is transcriptionally regulated by direct binding of Peroxisome Proliferator-Activated Receptors (PPARs) to responsive elements in the promoters.

The focus in this thesis has been the transcriptional regulation of perilipin 5 in cultured mouse muscle cells. Perilipin 5 is the latest discovered member of the perilipin family. This family member has been demonstrated by our group and others to be highly expressed in tissues with active fatty acid oxidation, such as heart, liver, striated muscles and brown adipose tissue, and to be induced by PPAR alpha. Preliminary studies in our group suggested that activation of PPAR delta stimulate Plin5 expression in muscle. Based on existing literature, we also hypothesized that the PGC1s and ERRs transcription factors could be important regulators of Plin5.

The main objective for this thesis was to investigate the role of the transcription factors PPARs, PGC1s and ERRs for perilipin 5 expression in cultured mouse muscle cells, and to identify any response elements used by these transcription factors in the Plin5 promoter. Our results confirmed that activation of PPAR alpha regulates the expression of perilipin 5 in muscle cells. However, they also demonstrated that PPAR delta is an important regulator of Plin5 expression. Furthermore, when PPAR alpha is over expressed or PPAR delta is ligand-activated, the relative level of perilipin 5 increase with time, suggesting the involvement of other factors. We found that overexpression of either PGC1 alpha or PGC1 beta enhanced basal expression of perilipin 5 compared to non-transfected cells. Interestingly, cells overexpressing these co-activators, especially PGC1 beta were less responsive to ligand activation of PPARs. This result suggests that overexpression of these coactivators functions to activate the PPARs similar to ligand activation. ERRs did not appear to regulate Plin5 expression. We were also able to confirm a novel functional PPRE in the Plin5 promoter, located downstream of the Plin5 transcriptional start site at position +139 to +151 in intron 1 of the Plin5 gene. This establishes Plin5 as a novel direct PPAR target gene in mouse muscle cells.