Knockout and Restoration Reveal Differential Functional Roles of PPAR71 and PPAR72 in Chicken Adipogenesis

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is the master regulator of adipogenesis and is expressed as two isoforms, PPAR gamma 1 and PPAR gamma 2. Our previous lentiviral overexpression study showed that PPAR gamma 1 and PPAR gamma 2 differentially regulated proliferation, differentiation, and apoptosis of the immortalized chicken preadipocyte cell line (ICP2). However, we cannot rule out the possibility that the endogenous expression of PPAR gamma isoforms may compromise our findings. In this study, using the dual sgRNA-directed CRISPR/Cas9 system, we generated PPAR gamma (PPAR gamma-/-) and PPAR gamma 2-specific knockout (PPAR gamma 2-/-) ICP2 cell lines and investigated the differences in proliferation and differentiation among PPAR gamma-/-, PPAR gamma 2-/-, and wild-type ICP2 cells. EdU proliferation assay showed that both PPAR gamma 2-specific and PPAR gamma knockouts significantly increased the proliferation rates. Consistently, real-time RT-PCR analysis showed that both PPAR gamma 2-specific and PPAR gamma knockouts significantly upregulated the expression of proliferation marker genes PCNA and cyclinD1. FACS analysis revealed that PPAR gamma knockout significantly increased the number of cells accumulating in the S phase and decreased the number of cells accumulating in the G1/G0 phase. Oil Red O staining and gene expression analysis showed both PPAR gamma 2-specific and PPAR gamma knockouts dramatically reduced capacity for adipogenic differentiation. To corroborate our previous findings, PPAR gamma 1 and PPAR gamma 2 expression were restored in PPAR gamma-/- cells by using the lentiviruses expressing chicken PPAR gamma 1 (LV-PPAR gamma 1) and PPAR gamma 2 (LV-PPAR gamma 2), respectively. Subsequent assays showed that restoration of expression of either PPAR gamma 1 or PPAR gamma 2 suppressed proliferation and stimulated differentiation of the PPAR gamma-/- cells. By comparison, PPAR gamma 2 had stronger anti-proliferative and pro-adipogenic effects than PPAR gamma 1. To understand the molecular mechanism underlying their differential effects on differentiation of the PPAR gamma-/- cells, we performed RNA-seq in the PPAR gamma-/- cells in which individual PPAR gamma isoform expression was restored at 72 h of differentiation. Transcriptomic analysis revealed that restoring PPAR gamma 1 expression caused far more differentially expressed genes (DEGs) than restoring PPAR gamma 2 expression. GO and KEGG pathway enrichment analyses indicated that PPAR gamma 1 and PPAR gamma 2 had distinct and overlapping functions in adipogenesis. Taken together, our results clearly indicate that PPAR gamma 1 and PPAR gamma 2 differentially impact chicken adipogenesis.