C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway

Abstract

PPARgamma and C/EBPalpha are critical transcription factors in adipogenesis, but the precise role of these proteins has been difficult to ascertain because they positively regulate each other's expression. Questions remain about whether these factors operate independently in separate, parallel pathways of differentiation, or whether a single pathway exists. PPARgamma can promote adipogenesis in C/EBPalpha-deficient cells, but the converse has not been tested. We have created an immortalized line of fibroblasts lacking PPARgamma, which we use to show that C/EBPalpha has no ability to promote adipogenesis in the absence of PPARgamma. These results indicate that C/EBPalpha and PPARgamma participate in a single pathway of fat cell development with PPARgamma being the proximal effector of adipogenesis.

Figure 1

Two models of adipogenesis. Competing models are shown consistent with available data on the relative roles of PPARγ and C/EBPα in adipogenesis. See text for details.

Figure 2

Generation of PPARγ^−/− fibroblasts. (A) Scheme showing strategy used to make PPARγ^−/− cells. PPARγ flox/− (or flox/+) embryos were harvested at day 12.5. Embryonic fibroblasts were plated and passaged repeatedly using a classic 3T3 protocol. After immortalization, cells were split into two aliqouts and infected with one of two different adenoviruses expressing Cre recombinase and GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-X-IRES-GFP). Cells were sorted by fluorescence to pick GFP-expressing cells. (B) Southern blot showing complete loss of floxed alleles in cells that received Ad-Cre-IRES-GFP, whereas floxed alleles were retained by cells that received Ad-X-IRES-GFP. (C) Immunoblot of PPARγ in PPARγ flox/− and −/− cells exposed to a prodifferentiative regimen.

Figure 3

PPARγ restores adipogenesis in PPARγ^−/− cells, but C/EBPα does not. (A) Dishes containing PPARγ flox/− or −/− cells were infected with retroviruses expressing PPARγ2, C/EBPα, or vector only. Cells were exposed to a prodifferentiative regimen with or without troglitazone, and stained with oil red O after 7 d. (B) Microscopic view of cells in A.

Figure 4

Expression of C/EBPα, PPARγ, and other adipocytic markers in PPARγ flox/− and −/− cells. (A) Immunoblot analysis of C/EBPα in PPARγ flox/− and −/− cell lines. (B) Northern analysis of multiple adipocytic markers in PPARγ flox/− and −/− cell lines. (V) Viral message, (E) endogenous message.