Modulation of the adipogenic master regulator PPARγ

Abstract

Obesity is regarded as a major public health problem by the World Health Organization. Obesity is characterized by an increase in adipose tissue, both through an increase in the amount of fat stored per adipocyte, and the generation of new adipocytes through differentiation of pre-adipocyte (adipogenesis), a process in which the transcription factor PPARγ plays a significant role. In this thesis we described different aspects of PPARγ, adipose tissue and adipogenesis. PPARγ is necessary for adipogenesis, maintenance of adipose tissue and is able to regulate many “adipocyte genes” that are necessary for lipid and glucose metabolism. Many aspects of PPARγ are under intense study, here we focused on the modulation of the expression and activity this factor. It is known that PPARγ is subject to post-translational modifications (PTM) such as phosphorylation, ubiquitination, acetylation and sumoylation. All of these marks have an effect on the activity of PPARγ. However, not much was known about the opposite process: de-modification. We have identified PPM1B to be a phosphatase for PPARγ. PPM1B was capable of directly binding to PPARγ and dephosphorylating a serine residue at position 112. In general, S112 phosphorylation lowers PPARγ activity, subsequently leading to decreased target gene expression. Knocking down PPM1B indicated that this was indeed the case for a subset of genes. This led to the conclusion that PPARγ (de)phosphorylation at S112 had subtle effects which could be seen in terms of specific target gene expression, but not on adipogenesis as a whole. Interestingly, a second phosphorylation site was recently identified on PPARγ, but no link has been made between S273 phosphorylation and PPM1B. Adipogenesis occurs in at least two waves; genes such as C/EBPβ, C/EBPδ, KLF5, CREB, SREBP-1c and Krox20 come up early in adipogenesis and ultimately lead to the expression of PPARγ and C/EBPα, which can than regulate genes that have a direct role in glucose and lipid metabolism. We investigated which other proteins would be capable of regulating the PPARG gene and thus adipogenesis. Consequently we developed an siRNA based knockdown screen and subsequently found Baf57, a subunit of the SWI/SNF chromatin remodeling complex, and Phf12 a “chromatin-associated” factor that has mainly been implicated in transcriptional repression, to regulate PPARG expression and adipogenesis. Knockdown of Baf57/Phf12 impairs adipogenesis; transcriptome analysis reveals that the genes that are inhibited are to a large extent the same as the genes that are inhibited by PPARγ knockdown. The genes that are affected are the so-called late adipocyte genes, rather than the early genes mentioned above. Further analysis indicated that Baf57/Phf12 directly binds to the PPARG promotor to induce its expression in both human and murine adipocyte cell lines. Finally we discuss PPARγ as a potential target for clinical intervention. Modulation of PPARγ may be used in order to ameliorate obesity if done properly. Full agonists of PPARγ lead to many severe side effects in humans, but subtle modulation through post translational modifications (PTM) or PPARγ associated proteins may provide an alternative answer for treatment of obesity