Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes

Abstract

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBPβ is expressed during the early phases of differentiation. Here, we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBPβ by A-C/EBP protein inhibitor. Signals from EGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2 h of transfections, whereas for A-C/EBP gene transfections, equivalent signals appeared in 48 h. Following transient transfections, the expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocyte pathways were analyzed. Expectedly, the mRNA and protein expression profiles of adipocyte marker genes showed lower expression in both A-C/EBP protein- and gene-transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between A-C/EBP protein- and A-C/EBP gene-transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfrα, Pdgfrβ, Ly6A, CD34, and Itgb1, showed high expression in A-C/EBP protein samples, whereas only Ly6A and CD34 were expressed in A-C/EBP gene-transfected samples. Pdgfrα and Pdgfrβ, two known cell fate markers, were expressed in A-C/EBP protein-transfected samples, suggesting a possible reversal of differentiation. Our study provides evidences for the immediate and efficient knockdown of C/EBPβ protein to understand time-dependent preadipocytes differentiation.

Aging and homeostasis of the hypodermis in the age-related deterioration of skin function

“…Their osteogenic potential is impaired with aging, whereas their adipogenic potential is maintained [ 87 , 88 ]. A combination of intracellular and extracellular stimuli induces a cascade of transcription factors, including members of the C/EBP family and PPARγ [ 89 – 91 ]. These transcription factors act sequentially to alter the expression of over 2500 genes, which leads to the development of the fat cell phenotype [ 92 , 93 ].…”

Potential key factors involved in regulating adipocyte dedifferentiation

“…Notably, several marker genes have been identified as either adipocyte progenitors or mature adipocytes. For example, stem cell antigen 1 (Sca‐1), platelet‐derived growth factor receptor α, and delta‐like homolog 1 (DLK1) have been reported as adipocyte progenitor markers, while Adiponectin, Perilipin, and fatty acid‐binding protein 4 (FABP4) have been identified as mature adipocyte markers (Gao et al, 2012; Gustafson & Smith, 2010; Marangoni et al, 2015; Sharma et al, 2020). During adipogenesis, several transcriptional factors such as the peroxisome proliferative activated receptor gamma (PPARγ) and CCAAT/enhancer‐binding proteins (C/EBPs) control the differentiation process (Figure 1).…”

miR-381-3p Inhibits Intramuscular Fat Deposition through Targeting FABP3 by ceRNA Regulatory Network

“…To date, the deposition of IMF is achieved through the proliferation and differentiation of intramuscular preadipocytes [ 7 ]; the proliferation of preadipocytes is regulated by cell cycle regulators including cyclin-dependent kinases (CDKs) [ 8 ], CDK inhibitors [ 9 ], and other transcription factors [ 10 ]. The differentiation of preadipocytes also involves several regulatory factors such as the peroxisome proliferator-activated receptor γ ( PPARγ ) [ 11 ], CCAAT/enhancer binding protein ( C/EBPα ) family [ 12 ], fatty acid binding protein 3 ( FABP3 ) [ 13 ], and lipoprotein lipase (LPL) [ 14 ]. The above candidate genes related to pig fat content have also been studied in terms of their potential use as molecular markers for the selection of pork quality traits [ 15 ].…”