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TGF-?1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts.

ABSTRACT: TGF-?1 reprograms metabolism in renal fibroblasts, inducing a switch from oxidative phosphorylation to aerobic glycolysis. However, molecular events underpinning this are unknown. Here we identify that TGF-?1 downregulates acetyl-CoA biosynthesis via regulation of the pyruvate dehydrogenase complex (PDC). Flow cytometry showed that TGF-?1 reduced the PDC subunit PDH-E1? in fibroblasts derived from injured, but not normal kidneys. An increase in expression of PDH kinase 1 (PDK1), and reduction in the phosphatase PDP1, were commensurate with net phosphorylation and inactivation of PDC. Over-expression of mutant PDH-E1?, resistant to phosphorylation, ameliorated effects of TGF-?1, while inhibition of PDC activity with CPI-613 was sufficient to induce ?SMA and pro-collagen I expression, markers of myofibroblast differentiation and fibroblast activation. The effect of TGF-?1 on PDC activity, acetyl-CoA, ?SMA and pro-collagen I was also ameliorated by sodium dichloroacetate, a small molecule inhibitor of PDK. A reduction in acetyl-CoA, and therefore acetylation substrate, also resulted in a generalised loss of protein acetylation with TGF-?1. In conclusion, TGF-?1 in part regulates fibroblast activation via effects on PDC activity.

SUBMITTER: Smith ER

PROVIDER: S-EPMC7578649 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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TGF-β1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts.

Smith Edward R ER Hewitson Timothy D TD

Scientific reports 20201021 1

TGF-β1 reprograms metabolism in renal fibroblasts, inducing a switch from oxidative phosphorylation to aerobic glycolysis. However, molecular events underpinning this are unknown. Here we identify that TGF-β1 downregulates acetyl-CoA biosynthesis via regulation of the pyruvate dehydrogenase complex (PDC). Flow cytometry showed that TGF-β1 reduced the PDC subunit PDH-E1α in fibroblasts derived from injured, but not normal kidneys. An increase in expression of PDH kinase 1 (PDK1), and reduction in ...[more]

PMID: 33087819

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Project description:Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100 mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5 mM 2-O-α-d-glucopyranosyl-l-ascorbic acid (VitC) ± 2 ng/ml of T1 or T3. After 4 h or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 h, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

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TGF-?1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts.

Publications

TGF-β1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts.

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