Transcriptional control of preadipocyte determination by Zfp423

Abstract

The worldwide epidemic of obesity has increased the urgency to develop a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARgamma and several other transcription factors, but the molecular basis for preadipocyte determination is not understood. Using a new method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of Pparg in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. Short hairpin RNA (shRNA)-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte Pparg expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is markedly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates Pparg expression, in part, through amplification of the BMP signalling pathway, an effect dependent on the SMAD-binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Figure 1. The C2H2 zinc finger protein Zfp423 is enriched in preadipocytes

Oil-red-O (ORO) staining of preadipocyte cell lines (a) and non-adipogenic cells lines (b) at six days following the induction of adipogenesis with DMI. (c) Zfp423 expression in sub-confluent Swiss 3T3 subclones and existing preadipocyte and fibroblast cell lines. (n=3 replicates per cell line). In this, and other figures, bars represent mean ± standard deviation from the mean. (d) Western Blot of endogenous Zfp423 protein levels in fibroblast cell lines grown under non-differentiating conditions.

Figure 2. Zfp423 regulates preadipocyte PPARγ gene expression and adipocyte differentiation in vitro

(a) Transcription factor gene expression in undifferentiated NIH 3T3 fibroblasts ectopically expressing Zfp423. (b) ORO staining of control and Zfp423-expressing cells 8 days following the induction of adipocyte differentiation. (c) Expression of adipocyte selective genes in the differentiated cultures shown in (b). (d) Western blot of Zfp423 and PPARγ protein levels in 3T3-L1 preadipocytes expressing shGFP (control) or shRNA against Zfp423 (shZfp423). Tubulin protein levels serve as a loading control. (e) Transcription factor gene expression in undifferentiated preadipocytes expressing shGFP or shZfp423. (f) ORO staining six days after the induction of adipocyte differentiation in 3T3-L1 cells expressing shGFP or shZfp423. (g) Expression of adipocyte selective mRNAs in cultures shown in (f). * denotes p<0.05 in Student’s T-test. n=3 replicates

Figure 3. Zfp423 amplifies the pro-adipogenic actions of BMP proteins through its SMAD protein interaction domain

(a) Schematic illustrating the protein domains and zinc-finger (ZF) motifs of full length Zfp423 and Zfp423 lacking the SMAD binding domain (Zfp423 ΔSBD). ZF 14-20 serves as the well-characterized SMAD interaction domain. (b) Western blot of ectopic full length Zfp423 or Zfp423 ΔSBD protein expression in NIH 3T3 cells. Tubulin protein levels serve as a loading control. (c) PPARγ2 mRNA levels in undifferentiated cells expressing Zfp423 or Zfp423 ΔSBD. (d) Expression of adipocyte selective mRNAs in cultures following differentiation with DMI and Rosiglitazone and ORO staining of differentiated cultures. (e) PPARγ2 mRNA levels in undifferentiated NIH 3T3 cells expressing a control vector or full length Zfp423 following 48 hours of incubation with increasing doses of BMP4. (f) PPARγ2 gene expression in undifferentiated, BMP4 treated cells expressing control, full length Zfp423, or Zfp423 ΔSBD. (g) Expression of adipocyte selective mRNAs in cultures following BMP4-induced differentiation, and oil-red O staining of lipid accumulation in differentiated cultures. * denotes p<0.05 in Student’s T-test. n=3 replicates.

Figure 4. Impaired brown and white adipocyte differentiation in Zfp423 deficient embryos

(a-c) Hematoxylin and eosin staining of transverse sections of the interscapular region of E18.5 Zfp423^+/+ (a) and Zfp423^-/- (b,c) embryos. (d,e) Higher magnification images of the interscapular BAT shown in (a,b). FABP4 staining of primitive white adipocytes in the subcutaneous region of E18.5 Zfp423^+/+ mice (f) and E18.5 Zfp423^-/- (g) mice (40x magnification) (h) Quantitation of FABP4⁺; lipid⁺; UCP1^- cells in the subcutaneous regions of wild-type and Zfp423 knockout mice.