Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells - PubMed (original) (raw)
Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells
Yan Huang et al. PLoS One. 2012.
Abstract
Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423) as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR) α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05) in high adipogenic cells, while transforming growth factor (TGF)-β was higher (156.1±48.7%, P<0.05) in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05) in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis and marbling in beef cattle.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Adipogenic differention of cloned low and high adipogenic cells.
A) Transmissive scanning of Oil-Red-O stained high and low adipogenic cells. B) Quantification of Oil-Red-O staining by absorbance measurement at 520 nm for high and low adipogenic cells. C) 100× magnified image of stained high adipogenic cells. D) 100× magnified image of stained low adipogenic cells. (*_P<_0.05, Mean ± SE; n = 3).
Figure 2. The mRNA expression of adipogenic and fibrogenic genes in low and high adiogenic cells.
Cloned high and low adipogenic cells were induced adipogenic differentiation with a standard protocol. A) Gene expression before inducing differentiation. B) Gene expression at 6th day of adipogenic differentiation. C) Gene expression after 13 days of differentiation. (*_P<_0.05, & _P<_0.10; Mean ± SE; n = 3).
Figure 3. Transforming growth factor (TGF)-β protein content in low and high adipogenic cells.
A) TGF-β content before adipogenic differentiation. B) TGF-β content after 13 days of adipogenic differentiation. (*_P<_0.05, & _P<_0.10; Mean ± SE; n = 3).
Figure 4. The mRNA expression of adipogenic and fibrogenic genes in sorted low and high adiogenic progenitor cells.
Cloned high and low adipogenic cells were sorted by the surface marker, PDGFRα, and induced adipogenic differentiation with a standard protocol. A) Gene expression before inducing differentiation. B) Gene expression at 6th day of adipogenic differentiation. C) Gene expression after 13 days of differentiation. (*_P<_0.05, & _P<_0.10; Mean ± SE; n = 3).
Figure 5. Adipogenic differentiation of stromal vascular cells over-expressing Zfp423.
A) Zfp423 expression 3 days after transfection with a vector over-expressing Zfp423. B) expression of adipogenic marker genes PPARγ and C/EBPα after 3 days of Zfp423 over-expression. C) Zfp423 expression efficiency 6 days after transfection with a vector over-expressing Zfp423. D) expression of adipogenic marker genes PPARγ and C/EBPα after 6 days of Zfp423 over-expression. (*_P<_0.05, & _P<_0.10; Mean ± SE; n = 3).
Figure 6. Adipogenic differentiation of cloned low adipogenic cells after Zfp423 transfection.
A) adipogenic gene expression in control (eGFP transfected low adipogenic cells), Zfp423 transfected low adipogenic cells, and cells without transfection. B) Transmissive scanning of Oil-Red-O stained cells. C) Quantification of Oil-Red-O staining by absorbance measurement at 520 nm. D) 100× magnified image of high adipogenic cells. E) 100× magnified image of low adipogenic cells. F) 100× magnified image of Zfp423 transfected low adipogenic cells. G) 100× magnified image of eGFP tranfected low adipogenic cells. (abMeans bearing different superscripts indicate significant difference, _P<_0.05, Mean ± SE; n = 3).
Figure 7. Adipogenic differentiation of cloned high adipogenic cells after shZfp423 knockdown.
A) adipogenic gene expression in low adipogenic cells, high adipogenic cells, high adipogenic (HA) with shZfp423 to knockdown Zfp423, HA with eGFP (transfection control). B) Transmissive scanning of Oil-Red-O stained cells. C) Quantification of Oil-Red-O staining by absorbance measurement at 520 nm. DEFG) 100× magnified images showing Oil-Red-O stained adipocytes. (abMeans bearing different superscripts indicate significant difference, _P<_0.05, Mean ± SE; n = 3).
Figure 8. MeDIP analysis of bovine TGFβ and Zfp423.
(*_P<_0.05, Mean ± SE; n = 3).
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