Anti-renal interstitial fibrosis effect of norcantharidin is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3.

Abstract

Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is known to inhibit renal fibrosis. However, the underlying mechanism is largely unknown. The present study investigates whether NCTD exerts this effect through regulation of the protein phosphatase 2A catalytic subunit (PP2Ac)-Smad3 pathway. HK-2 human renal proximal tubule cells exposed to transforming growth factor (TGF)-β1 were used as an in vitro model of renal fibrosis. The levels of total Smad3, C-terminal-phosphorylated Smad3 (p-Smad3), PP2Ac, and fibronectin (Fn) were evaluated by Western blotting. A PP2Ac overexpression plasmid and the PP2Ac inhibitor okadaic acid (OA) were used for functional analyses. The subcellular localization of Smad3 was visualized by immunofluorescence labeling. The results showed that PP2Ac overexpression increased Smad3 phosphorylation and nuclear translocation in HK-2 cells, while pharmacologic inhibition of PP2Ac with OA had the opposite effect. NCTD suppressed Fn and p-Smad3 expression and TGF-β1-induced nuclear entry of Smad3, but these effects were abrogated by inhibition of PP2Ac. Thus, the anti-renal interstitial fibrosis effect of NCTD is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3. These findings highlight the therapeutic potential of NCTD for the treatment of renal interstitial fibrosis.

• Authors: Han-Wen Luo, Dan-Dan Yin, Zheng Xiao, Lu Wen, Ying-Jun Liao, Cheng-Yuan Tang, Dong Zeng, Heng-Ting Xiao, Ying Li
• Journal: Chemical biology & drug design (Chem Biol Drug Des) Vol. 97 Issue 2 Pg. 293-304 (02 2021) ISSN: 1747-0285 [Electronic] England
• PMID: 32896083 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2020 John Wiley & Sons A/S.

Chemical References

• Bridged Bicyclo Compounds, Heterocyclic
• Fibronectins
• SMAD3 protein, human
• Smad3 Protein
• Transforming Growth Factor beta1
• Okadaic Acid
• norcantharidin
• Protein Phosphatase 2

Topics

• Active Transport, Cell Nucleus (drug effects)
• Bridged Bicyclo Compounds, Heterocyclic (pharmacology)
• Catalytic Domain
• Cell Line
• Fibronectins (metabolism)
• Humans
• Okadaic Acid (pharmacology)
• Phosphorylation (drug effects)
• Protein Phosphatase 2 (antagonists & inhibitors, genetics, metabolism)
• Smad3 Protein (metabolism)
• Transforming Growth Factor beta1 (pharmacology)