Gene expression profiling reveals a diverse array of pathways inhibited by nuclear receptor SHP during adipogenesis

Abstract

Orphan receptor small heterodimer partner (SHP, NROB2) has been shown to be a metabolic regulator in pathways associated with several major aspects of the metabolic syndrome. However, the significance and transcriptional regulatory role of SHP in adipocyte differentiation remain unclear. Transcriptional profiles of 3T3-L1 preadipocytes and early differentiating preadipocytes in response to SHP were systemically surveyed using Affymetrix Genome Array representing well-characterized 14,000 genes. Analysis revealed about 963 genes that were up- or down-regulated by more than 2-fold during differentiation and/or by the overexpression of SHP. These genes were organized into 4 clusters that demonstrated concerted changes in expression of genes controlling various aspects of the cellular events and metabolism. Quantitative PCR was employed to further characterize gene expression and led to the identification of several key regulators and stimulators of the adipogenic program as potential new SHP targets. Overexpression of SHP inhibited the differentiation process as well as the accumulation of neutral lipids within the cells. Our data suggests that SHP may function as a molecular switch that governs adipogenesis and a potent adipogenic suppressor that maintains preadipocytes in an undifferentiated state through inhibition of the adipogenic transcription factors and stimulators. Developing SHP agonist may promise a future treatment for obesity.

Keywords: SHP; adipocyte differentiation; gene expression profiling; microarray; nuclear receptor.

Figure 1

Affymetrix microarray analysis of SHP-regulated genes during early adipogenesis. A-D: Hierarchical clustering analysis highlighting modulated genes during adipogenesis. 3T3-L1 cells were transduced with GFP (G) control or SHP (S) adenovirus for three days, treated with differentiation cocktail for two days, and gene expression was analyzed using Affymetrix GeneChip® Mouse Genome 430A 2.0 Array platform. Day 0 (G0 and S0), unstimulated cells; day 2 (G2 and S2), cells differentiated for 2 days. Fold changes of expression levels for genes in each cluster: A, G2/G0>2, S0/S2=1, S2/G2<-2; B, G2/G0<2, S0/S2=1, S2/G2>2; C, G2/G0>2, S0/G0>2, S2/G2<2; D, G2/G0<2, S0/G0<2, S2/G2>2.

Figure 2

Gene ontology analysis of signaling pathways during early adipogenesis. A-B: Diagrammatic representation of GO analysis carried out by using EASE. GO subgroups were collapsed into related groups to facilitate visualization of the data. A: Genes that were at least two fold up-regulated at G2 as compared to G0 (G2/G0>2) and two fold down-regulated at S2 as compared to G2 (S2/G2<2) were shown. B: Genes that were at least two fold down-regulated at G2 as compared to G0 (G2/G0<2) and two fold up-regulated at S2 as compared to G2 (S2/G2>2) were shown. The analysis is based on biological process, cellular component and molecular function of the genes.

Figure 3

Effects of differentiation and SHP on gene expression in 3T3-L1 cells. 3T3-L1 fibroblasts were induced to undergo differentiation process with the hormonal cocktail in the absence or presence of SHP transduction (MOI: 50) for two days. RNA samples were prepared from cells at the indicated time points after treatment (day 0 and day 2) and analyzed using quantitative real-time PCR for the indicated genes. Values shown are the mean ± SEM of three determinations. See Materials and Methods for details.

Figure 4

Genes regulated by SHP in PPAR signaling pathway. PPAR signaling pathway from KEGG (Kyoto Encyclopedia of Genes and Genomes) was analyzed. Genes with differential expression pattern from day 0 (G0 and S0) to day 2 (G2 and S2) during 3T3-L1 cell differentiation were indicated by colors. Different color represents different expression level for each gene. Red, upregulated; blue, down-regulated. Genes that were markedly down-regulated by SHP at day 2 (S2) as compared to G2 were highlighted by red. These include CD36, Δ6 desaturase, SCD-1, LPL, CPT-1, MCAD, PGAR, and PPARγ2. G, GFP; S, SHP.

Figure 5

Effect of SHP on adipocyte differentiation in 3T3-L1 cells. A. 3T3-L1 cells were induced to differentiation without or with SHP adenovirus transduction (MOI: 50), as described in the M&M section. Total RNAs were collected at the indicated time (day 0, 2, 5 and 8) and used for Real-time PCR analysis. B. Oil-red O staining of neutral lipids at day 8 post-cell differentiation. Neutral lipid accumulation was largely detected in GFP-infected cells, but dramatically diminished in SHP-overexpressed cells. Ad, adenovirus.