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Tyrosine phosphorylation of the LDL receptor-related protein (LRP) and activation of the ERK pathway are required for connective tissue growth factor to potentiate myofibroblast differentiation

MIN YANG^*, HAICHANG HUANG^*,1, JINGZI LI^*, DONGXIA LI and HAIYAN WANG^*

^* Division of Nephrology, First Hospital and Institute of Nephrology, Peking University, Beijing, China; and
FibroGen Inc., South San Francisco, California, USA

¹Correspondence: Division of Nephrology, First Hospital and Institute of Nephrology, Peking University, No. 8 Xi-Shi-Ku St., Beijing 100034, China. E-mail: haichang{at}bjmu.edu.cn

SPECIFIC AIMS

The aim of this study was to investigate the hypothesis that chronic fibrotic disease is partly mediated by CTGF and that it may modulate or amplify the key pathophysiological course of myofibroblast differentiation and persistent activation of myofibroblasts. The molecular mechanisms of CTGF-mediated de novo expression of -SMA were assessed, including TGFß1-induced Smad signaling, as well as tryrosine phosphorylation of the cytoplasmic domain of the low density lipoprotein receptor-associated protein (LRP) and ERK1/2 MAPK signaling pathway.

PRINCIPAL FINDINGS

1. CTGF induces myofibroblast differentiation synergistically with TGFß1 in renal fibroblasts (P<0.001 vs. control group, P<0.01 vs. TGFß1 group)
Incubation of NRK-49F cells with TGFß1 for 48 h induced occurrence of -SMA–positive cells (myofibroblasts) at a percentage of 12% (P<0.001 vs. control group). CTGF plus TGFß1 increased the number of myofibroblasts by 27% (P<0.01 vs. TGFß1 group), whereas CTGF alone did not induce formation of myofibroblasts.

2. -SMA protein levels were increased in cells simultaneously incubated with CTGF and TGFß1 in a CTGF dose-dependent manner compared with TGFß1-treated cells (P<0.01 vs. TGFß1 group, Fig. 1 ), whereas CTGF alone did not induce the expression of -SMA protein

View larger version (22K): [in this window] [in a new window]   Figure 1. Co-stimulation with CTGF and TGFß1 induces expression of -SMA protein in renal fibroblasts (NRK-49F). The level of -SMA expression was assessed by Western blot analysis. Cells were incubated with a fixed concentration of 5 ng/mL TGFß1 in combination with increasing concentrations of CTGF as indicated for 48 h. The cell lysates were probed with antibodies against -SMA and GADPH, respectively.

3. In terms of accumulation of the matrix component fibronectin (FN) in the cell culture medium was increased in CTGF and TGFß1 combinatorial treatment in a CTGF dose-dependent manner when compared with TGFß1 treatment alone(P<0.05 vs. TGFß1 group). Treatment with CTGF alone did not affect FN levels

4. CTGF did not potentate the TGFß1-activated Smad2 signaling pathway
TGFß1 activated all steps of the Smad2 signaling transduction pathway, whereas CTGF promoted myofibroblast differentiation from renal fibroblast without potentiation of TGFß1-mediated Smad2 phosphorylation and its association with Smad4, or the nuclear translocation of activated Smad2.

5. Inhibition of binding of CTGF to the LDL receptor-related protein (LRP) reduced the synergistic -SMA protein expression by CTGF and TGFß1
CTGF stimulated tyrosine phosphorylation of the cytoplasmic portion of LRP, while TGFß1 did not. Inhibition of tyrosine phosphorylation of LRP by an antagonist of ligand binding to LRP (RAP) reduced the increase in the level of -SMA protein mediated by co-stimulation of CTGF and TGFß1 for 24 h (Fig. 2 A), indicating that the effect of CTGF was mediated by LRP signaling.

View larger version (46K): [in this window] [in a new window]   Figure 2. A) Cells were pretreated with GST-RAP or GST fusion proteins followed by 100 ng/mL CTGF and/or 5 ng/mL TGFß1 for 24 h. The expression of -SMA protein was analyzed by immunoblotting. B) TGFß1 preactivated cells were incubated with the inhibitor PD98059 (50 µM) or DMSO for 30 min before CTGF addition. -SMA expression was examined after 24 h by immunoblot analysis.

6. CTGF augmented myofibroblast activation and induced an increase in the expression of -SMA protein in previously TGFß1-activated NRK-49F cells (P<0.01 vs. control group)
Exposition of the TGFß1 pretreated fibroblasts to CTGF induced a prominent increase in -SMA protein level (P<0.01 vs. control group), comparable to the induction by TGFß1.

7. Erk-1/2 activation is required for the CTGF-induced potentiation of -SMA protein expression in previously TGFß1-activated NRK-49F cells
CTGF stimulated Erk-1/2 activation in TGFß1 pretreated cells, but TGFß1 did not. Incubation of TGFß1-pretreated NRK-49F cells with PD98059 prior to CTGF treatment abolished CTGF-induced expression of -SMA protein (P<0.001 vs. control group, Fig. 2B ).

CONCLUSIONS AND SIGNIFICANCE

This study has provide further evidence suggesting that overexpression of CTGF may result in dysregulation of myofibroblast differentiation and activation after persistent or chronic tissue injury, and this could produce excess accumulation of ECM that ultimately leads to tissue scarring and functional organ failure. CTGF alone does not have the potency to induce myofibroblast differentiation. However, it does markedly augment TGFß1-mediated myofibroblast activation of renal interstitial fibroblasts through tyrosine phosphorylation of the LDL receptor-related protein (LRP) and activation of the ERK pathway, not potentiation of TGFß1-induced Smad2 signaling pathway. Understanding the full impact of CTGF on myofibroblast differentiation and activation may help to explore the potential value of CTGF as a therapeutic target for prevention and treatment of chronic fibrotic processes in kidney diseases.

View larger version (19K): [in this window] [in a new window]   Figure 3. Model of CTGF potentiates TGFß1-induced myofibroblast differentiation through combinatorial signaling pathway both of TGFß1-mediated Smad2 and CTGF-associated tyrosine phosphorylation of the LDL receptor-related protein (LRP) and activation of the ERK.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2357fje;

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