Anti-senescence role of heterozygous fumarate hydratase gene knockout in rat lung fibroblasts in vitro.

Zhirui Fan,Jing Cao,Mengmeng Dou,Lifeng Li,Zhenhe Suo,Huixiang Li,Mingzhi Zhang,Jing Zhao,Xiaoli Li,Xiaoming Deng,Meng Zhang

doi:10.18632/aging.101761

Abstract

Abnormalities in tricarboxylic acid (TCA) cycle function were related to a variety of pathological processes. Fumarate hydratase (FH) is a required enzyme in the TCA cycle. To explore the general influence of FH knockout, we isolated FH+/– rat and normal rat lung fibroblasts and cultured these cells in vitro. The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA (α-smooth muscle actin). Sequencing of the PCR (polymerase chain reaction) products flanking the FH gene mutation verified the FH+/– status, and the FH gene and protein expression were confirmed to be reduced in the FH+/– cells. No sign of ageing for the FH+/– cells after 61 passages was observed, but the controls died out at this stage. Flow cytometry revealed increased S-phase and decreased G1/G0 proportions with significantly less early apoptosis in FH+/– cells compared to that in control cells. At the same time, increased glucose consumption, intracellular fumarate production and extracellular lactate secretion were verified in the FH+/– cells. Correspondingly, FH+/– cells showed a lower basal oxygen consumption rate (OCR) but a higher level of reactive oxygen species (ROS) production. Single cell cloning and cell line establishment were successfully performed with the FH+/– cells at the 84th passage. All the above results indicate an important role for FH+/– in the longevity or immortality of the FH+/– cells, in which increased p53 and TERT (telomerase reverse transcriptase) protein expression, decreased p21 and p16 protein expression and negative SA-β-Gal (senescence-associated beta-galactosidase) were verified along with metabolic reprogramming.

Highlights

The tricarboxylic acid (TCA) cycle is a central hub for energy metabolism, macromolecule synthesis and redox balance
All primary rat lung fibroblasts displayed typical spindle-shaped morphology under Haematoxylin and eosin (HE) staining (Figure 1B) and were positive for vimentin expression (Figure 1C), but a few cells were positive for αSMA (Figure 1D), as reported in an previous rat lung fibroblast study [10]
In contrast to normally differentiated cells, which primarily utilize mitochondrial oxidative phosphorylation to generate the energy for cellular bioactivities, rely on www.aging‐us.com www.aging‐us.com sentative ultrastructure images of wild type (WT) and Fumarate hydratase (FH)+/– cells at the 55th passage evaluated by electron microscopy (a,d:2000×; b,e:4000×; c.f:8000×), where a larger cell body with plenty of rough endoplasmic reticulum, mitochondria and ribosomes is shown in the FH+/– cells, but the WT

Summary

Introduction

The TCA cycle is a central hub for energy metabolism, macromolecule synthesis and redox balance. The cycle consists of a series of biochemical reactions occurring in the mitochondrial matrix, which allow aerobic organisms to oxidize fuel sources and provide energy, macromolecules, and redox balance to the cell [1]. Abnormalities in TCA cycle function were related to a variety of pathological processes ranging from cancer to neurological and metabolic disorders. Mutation in the FH gene leads to the abnormal accumulation of fumarate in cells [6]. This increased fumarate can act as an oncometabolite and promote cancer. Homozygous mutations in the FH gene have previously been reported to cause a severe neurological disorder, and heterozygous germline mutations in the FH gene result in a clinical syndrome characterized by hereditary leiomyomatosis and renal cell cancer [7]

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