Identification And Characterization Of Novel Downstream Regulators Of FGF-1 Adipogenic Pathway

Adipose tissue dysfunction links obesity with insulin resistance,type 2 diabetes and other metabolic diseases.Adipose tissue expansion requires the increase of adipocyte size(hypertrophy)and adipocyte number(hyperplasia)and subsequent adipogenesis.Under over nutrition status,adipose tissue lack of ability for generating new adipocytes,but promotes the hypotrophy of inherent adipocytes.Hypertrophied adipocytes secrete more pro-inflammatory cytokines but less anti-inflammatory cytokines,which makes the body to undergo systemic chronic inflammation and insulin resistance.Therefore,understanding the molecular details of adipogenesis and adipose tissue remodeling will give us novel therapeutic opportunities.Adipogenesis is a highly coordinated biological process,in which a series of transcriptional cascades and many transcriptional regulatory factors are involved.Previous work from our group established the canonical FGF-1 signaling pathway as a potent driver of adipogenesis in human preadipocytes.Microarray followed by functional investigations identified BAMBI as a key negative regulator of adipogenesis situated between FGF-1 and the master adipogenic regulator PPAR?,and CPX-1 as a positive regulator of adipogenesis working in parallel to PPAR?.Following pathway analysis established a novel FGF-1-BAMBI-PPAR?/CPX-1 adipogenic pathway.To further explore this FGF-1 adipogenic network and identify novel downstream regulators of FGF-1and BAMBI,we performed RNA-Seq in SGBS PAs and followed by functional investigations,and acquired these specific results:1.We identified 1526 differential expressed genes using RNA-Seq.FGF-1 significantly changed 598 genes,and 131 genes showed PPAR? dependence.BAMBI knockdown only significantly changed 54 genes,in which 20 genes were also downstream of FGF-1,and 12 genes showed PPAR? dependence.Bioinformatics analysis revealed several genes/ gene clusters involved in Wnt,Klf,Hedgehog pathways and ECM components and regulators.2.The most highly represented group of FGF-1 and BAMBI knockdown responsive genes were members of the interferome,accounting for 8%(49/598)of FGF-1 regulated genes with FGF-1 downregulating 86% of these(42/49).3.Five FGF-1-interferome nexus genes(IFI44L,IFI27,ISG15,HERC6 and IFIT1)are negative regulators of adipogenesis.All five genes suit downstream of FGF-1,BAMBI and PPAR? in FGF-1-BAMBI-PPAR?/CPX-1 pathway.Knockdown each of the five genes result in increased adipogenesis,as demonstrated by greater lipid accretion and increased expression of adipogenic markers PPAR?,Adiponectin,AdipoR2 and Glut4.4.ACTA2,ANKRD1 and CPA4 are negative regulators of adipogenesis.In FGF-1-BAMBI-PPAR?/CPX-1 pathway,both ACTA2 and ANKRD1 lie downstream of FGF-1 and BAMBI and independent of PPAR?,while CPA4 is downstream of FGF-1 and independent of BAMBI and PPAR?.Following functional analysis showed knockdown of ACTA2 or ANKRD1 or CPA4 increased lipid accumulation and expression of adipogenic markers PPAR?,Adiponectin,AdipoR2 and Glut4.5.CDK1,ADIRF and HHIP are positive regulators of adipogenesis,and all were upregulated by FGF-1 in a BAMBI and PPAR? independent manner.Functional analysis showed knockdown of all three genes decreased lipid accumulation and expression of adipogenic markers.6.In a male obese cohort,the expression of five FGF-1-interferome nexus genes in subcutaneous fat correlates negatively with adipose tissue resistance(ATIR)and area of visceral adipose tissue(VAT),and positively with hepatic and muscle insulin sensitivity(HIS & MIS).Conversely,expression of five inflammatory genes CD68,CD11 b,CD11c,HO-1and MCP showed inverse correlations that positive correlation with ATIR and VAT,and negative correlation with MIS.Collectively,these findings show that expression of five FGF-1-interferome nexus genes in subcutaneous fat correlates with increased insulin sensitivity and reduced visceral adiposity,and five inflammatory genes showed opposite.7.Expression of CPA4 in subcutaneous fat correlates positively with ATIR,VAT and % liver fat(%LF),and negatively with HIS and MIS,while the expression of ADIRF in subcutaneous fat correlates negatively with ATIR and %LF,positively with MIS.These results suggest expression of CPA4 in subcutaneous fat correlates with reduced insulin sensitivity and increased visceral adiposity,and expression of ADIRF correlates conversely.In this study we successfully identified five FGF-1-interferome nexus genes,ACTA2,ANKRD1 and CPA4 as negative regulators of adipogenesis,and CDK1,ADIRF and HHIP as positive regulators of adipogenesis.Moreover,we found five FGF-1-interferome nexus genes,CPA4 and ADIRF are closely related to insulin sensitivity and ectopic fat.These findings extend our understanding of FGF-1 adipogenic network,and also increase the clinical correlation of adipogenesis and adipogenic regulators.All these provide important theoretical foundation for combating obesity related metabolic diseases.