Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

PPARγ regulates expression of carbohydrate sulfotransferase 11 (CHST11/C4ST1), a regulator of LPL cell surface binding.

The transcription factor PPARγ is the key regulator of adipocyte differentiation, function and maintenance, and the cellular target of the insulin-sensitizing thiazolidinediones. Identification and functional characterization of genes regulated by PPARγ will therefore lead to a better understanding of adipocyte biology and may also contribute to the development of new anti-diabetic drugs. Here, we report carbohydrate sulfotransferase 11 (Chst11/C4st1) as a novel PPARγ target gene. Chst11 can sulphate chondroitin, a major glycosaminoglycan involved in development and disease. The Chst11 gene contains two functional intronic PPARγ binding sites, and is up-regulated at the mRNA and protein level during 3T3-L1 adipogenesis. Chst11 knockdown reduced intracellular lipid accumulation in mature adipocytes, which is due to a lowered activity of lipoprotein lipase, which may associate with the adipocyte cell surface through Chst11-mediated sulfation of chondroitin, rather than impaired adipogenesis. Besides directly inducing Lpl expression, PPARγ may therefore control lipid accumulation by elevating the levels of Chst11-mediated proteoglycan sulfation and thereby increasing the binding capacity for Lpl on the adipocyte cell surface.

The serine/threonine phosphatase PPM1B (PP2Cß) selectively modulates PPARγ activity.

Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cß] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.

Regulation of expression and secretion of galectin-3 in human monocyte-derived dendritic cells.

Galectin-3 (Gal-3) is a beta-galactoside binding lectin displaying both intracellular and extracellular immune functions. In Schistosoma mansoni infection, Gal-3 has been associated with the induction of a T helper 2 response. Whereas dendritic cells (DCs) play a pivotal role in the regulation of T cell differentiation, little is known about the regulation of Gal-3 expression in DCs. In this study we determined Gal-3 mRNA and protein levels during in vitro differentiation of human monocytes into immature DCs (iDCs), by culturing monocytes in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Gal-3 mRNA levels show a moderate, transient increase during iDC generation, accompanied by elevated cell-associated Gal-3 protein. Our data show that culturing monocytes with IL-4 alone strongly increases Gal-3 mRNA levels, whereas GM-CSF induces a low increase in Gal-3 mRNA. The combined data indicate that GM-CSF reduces IL-4 induced Gal-3 mRNA levels during the generation of iDC. Remarkably, stimulation of monocytes with GM-CSF results in secretion of significant amounts of Gal-3 in the medium, whereas iDCs do not release detectable amounts of Gal-3, indicating a suppressive role of IL-4 on GM-CSF induced Gal-3 secretion. Finally, our data demonstrate that all differentiated cell types tested show a significantly lower capacity to bind Gal-3 on the cell surface than monocytes. In conclusion, Gal-3 expression in iDCs is restricted, and Gal-3 protein is localized mainly intracellular, due to the opposite actions of IL-4 and GM-CSF. By these properties, the DCs may be protected against Gal-3 induced phosphatidylserine (PS) exposure and/or apoptosis.