Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose

Jean-Claude Dussaule,Leighton R James,Hyung-Suk Kim,Heather Doherty,Nobuyo Maeda,Catherine Le

doi:10.1371/journal.pone.0070441

Abstract

Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of Ctgf mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.

Highlights

Connective tissue growth factor (CTGF) (CCN2) is a pleiotropic growth factor belonging to the 30–40 kDa CCN family of proteins which includes Cyr61, NOV and WISP1
Characteristic of CTGF-Knockout Line CTGF gene knockout (CTGF2/2) mice were generated by replacing exons 3 to 5 of Ctgf with bacterial neomycin resistance (Neo) through gene-targeting in 129 SvEv-Tac derived embryonic stem (ES) cells as described in the methods
Quantitative reverse transcription PCR (RT-PCR) performed using total RNA from tail and kidney cortex revealed that Ctgf expression in heterozygous was approximately 50% of the level in wildtype mice (Figure 2A and B)

Summary

Introduction

CTGF (CCN2) is a pleiotropic growth factor belonging to the 30–40 kDa CCN family of proteins which includes Cyr, NOV and WISP1. This family of proteins exhibits a diverse array of cellular effects (mitogenesis, apoptosis, regulation of extracellular matrix (ECM) physiology, osteogenesis, embryogenesis, angiogenesis and tumorigenesis), are critical for growth, development and differentiation and are responsive to various intra- and extracellular stimuli (please see references [1,2,3,4] for detailed review). In addition to the reported positive association between Ctgf expression and extracellular matrix proteins, recent data derived from a Col1a2 promoter insertional transgenic mice model directly link increased Ctgf overexpression with fibrosis in multiple organs including kidney [8]. It is worth noting that, whereas a polymorphism in ctgf promoter has been linked with systemic sclerosis [9,10,11] and infectious hepatic fibrosis [12], these observations have not been consistent [13,14,15]

Methods

Results

Conclusion