Page 65 - F. Cell biology

Inhibition of TGF-β-induced Fibroblast-Myofibroblast Trans-differentiation by JIB-04

Kyeong-Min Park , Wan-Young Kim , Sun-Young Lee , Ho-Shik Kim , Seok Jun Shin 2
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Department of Biochemistry and Internal Medicine , College of Medicine, The Catholic University of Korea
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Abstract

TGF-β1 plays a critical role in fibroblast-myofibroblast trans-differentiation during development of renal fibrosis. Recently, histone demethylase (KDM) has been reported to contribute to TGF-

β1-induced expression of fibrotic genes. In this study, we analyzed the effect of JIB-04, a chemical inhibitor of KDM4A and KDM4B, on TGF-β1-induced trans-differentiation of NRK-49F cells,

normal rat kidney interstitial fibroblast cells. Pretreatment of JIB-04 attenuated TGF-β1-induced expression of profibrotic genes such as snail, PAI-1, and α-SMA at the levels of mRNA and

protein. By confocal microscopy, JIB-04 also reduced expression of myofibroblast markers such as vimentin and fibronectin in TGF-β1-treated cells. In addition, JIB-04 inhibited TGF-β1-

induced phosphorylation of smad2 and smad3. Collectively, these results suggest that JIB-04 may suppress TGF-β1-induced fibroblast-myofibroblast trans-differentiation during renal fibrosis

by preventing phosphorylation of smad2 and smad3.

Introduction

Renal fibrosis is a major mechanism of renal failure, and is the final-stage phase of the (a) (b)

chronic kidney disease (CKD). The CKD affects about 10 percent of the world's population,

snail α-SMA
and is particularly vulnerable to older people aged 70 or older. So far, there is only a way 2.00 9.00

8.00
1.80
to slow down the renal fibrosis, but there is no treatment to target fibrosis, so it is very 1.60 7.00
1.40
6.00
important to study the pathways and epigenetic mechanism related to renal fibrosis. The Relative epression mRNA/GAPDH 1.20 Relative epression mRNA/GAPDH 5.00
1.00
4.00
main cause of renal fibrosis is accumulation of extracellular matrix (ECM). Many kinds of 0.80 3.00
0.60
2.00
0.40
cells are involved in the fibrosis process, especially the accumulation of ECM that is a 0.20 1.00
0.00
0.00
control TGF-β TGF-β+ jib-04 jib-04 control TGF-β TGF-β+ jib-04 jib-04
leading cause of fibrosis occurs in interstitium. So, the Renal fibroblasts can be observed Collagen 3 PAI-1

4.00 6.00
well in the process of renal interstitial fibrosis, which is the pathway of trans-differentiation 3.50

3.00 5.00
from fibroblast to myofibroblast. Especially when myofibroblasts are differentiated α-SMA, 2.50 4.00

which is used as a representative fibrosis molecular marker. Therefore, it is shown that the Relative epression mRNA/GAPDH 2.00 Relative epression mRNA/GAPDH 3.00

1.50
molecular mechanism of fibrosis can be determined by experimenting with such markers. 1.00 2.00

0.50
1.00
0.00
Typically, TGF-β1 is one of the cytokines involved in the process of trans-differentiation control TGF-β TGF-β+ jib-04 jib-04 0.00 control TGF-β TGF-β+ jib-04 jib-04

from fibroblast to myofibroblast. TGF-β1 is one of the transforming growth factor beta (c) (d)

superfamily, cytokine involved in cell growth, cell proliferation, cell differentiation and

apoptosis. Among the roles involved by TGF-β1, the process by which the fibroblast cell

becomes myofibroblast cell is one of the cell differentiations that eventually results in

fibrosis. The process of leading to fibrosis by TGF- β1 is largely divided into smad

dependent pathways and non-smad dependent pathways. It is generally known to go

through the smad dependent Pathway. When TGF-β1 binds to the TGF-β1 receptor, smad2

and smad3 are phosphorylated and activated. The phosphorylated smad2 and smad3 join

with the smad4 to form a complex, and then move from the cytoplasm to the nucleus. The

smad complex in nucleus becomes involved in the transcription process of the genes

related to fibrosis.

JIB-04 is a histone demethylase inhibitor, and according to recent papers, there are reports

that it is related to TGF-β and histone demethylase. TGF-β1 induced fibrosis is regulated by

epigenetic mechanisms. So, we did the experiment in anticipation that JIB-04, the histone

demethylase inhibitor, could prevent fibrosis induced by TGF-β.

Result

(a)

Figure2. Inhibitory effect of JIB-04 on TGF-β1 induced of indicated proteins and genes in

NRK-49F cell. NRK-49F cell were pretreated JIB-04 0.5uM and treated TGF-β1 for 24 hours.

(a) Western blotting and (b) Real-time PCR analyzed EMT markers and Fibrosis marker

such as snail, PAI-1, α -SMA, collagen Ⅲ. (c) Immunofluorescence show that JIB-04

prevents fibrosis through TGF-β1 such as vimentin and fibronectin. (d) Zen 2012 blue

edition was used to analyze the data of Immunofluorescence, magnificent Ⅹ400. *p<0.05

versus control; **p<0.05 versus TGF-β1.

Conclusion

(b) (c)

Figure1. JIB-04 acts on TGF-β1/Smad pathway in NRK-49F cell. (a) JIB-04 0.5uM was

treated 1 hour in advance and TGF-β1 2ng/ml was treated 24 hours a day to confirm

Western blot. (b) When TGF-β1 was treated, p-smad3 was moved to the nucleus and the

movement to the nucleus was inhibited by JIB-04 on Immunofluorescence. (c) Zen 2012

blue edition was used to analyze the data, magnificent Ⅹ400. *p<0.001 versus control;

**p<0.001 versus TGF-β1.

Reference

Lefty-1 alleviates TGF-β1-induced fibroblast–myofibroblast transdifferentiation in NRK-49F cells, 2015, Lijun Zhang, Drug Des Devel Ther.
Epigenetic Reprogramming of TGF-β Signaling in Breast Cancer, 2019, Sudha Suriyamurthy, Cancers (Basel)

Mechanisms of Renal Fibrosis, 2017, Benjamin D. Humphreys ,Annual Review of Physiology

Suramin Inhibits Renal Fibrosis in Chronic Kidney Disease, 2011, Na Liu, Journal of the American Society of Nephrology

Physiology of the Renal Interstitium, 2015, Michael Zeisberg, Clin J Am Soc Nephrol