Orphan Nuclear Receptor Small Heterodimer Partner Inhibits Transforming Growth Factor-β Signaling by Repressing SMAD3 Transactivation

Ji Ho Suh,Jiansheng Huang,Yun-Yong Park,Hyun-A Seong,Dongwook Kim,Minho Shong,Hyunjung Ha,In-Kyu Lee,Keesook Lee,Li Wang,Hueng-Sik Choi

doi:10.1074/jbc.m605947200

Abstract

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

Highlights

SHP3 is an atypical member of the orphan nuclear receptor superfamily because its lacks a conventional DBD [1]
small heterodimer partner (SHP) Represses TGF-␤ Signaling—Several nuclear receptors have been reported to interact with Smad3 and regulate TGF␤1-induced Smad signaling (43, 45– 49, 50 –52)
These current observations suggest that the orphan nuclear receptor SHP plays a physiological role in TGF-␤ signaling

Summary

EXPERIMENTAL PROCEDURES

Reagent and Animals—TGF-␤1 was purchased from Sigma. SHP؊/؊ mice have been described previously [63, 65]. HepG2 cells were co-transfected with 150 ng of the reporter gene (CAGA) MLP-luc and the SHP expression plasmid pcDNA3HA-SHP. A, after HepG2 cells were cotransfected with the indicated amounts of pcDNA3HA-SHP, cells were preincubated in serum-free medium for 24 h These quiescent cells were treated with 5 ng/ml TGF-␤1 and assayed for luciferase activity after 12 h. C and D, HepG2 cells and CV-1 cells were co-transfected with 200 ng of expression vectors for pcDNA3FLAG-Smad, pcDNA3FLAG-Smad, and the indicated amounts of pcDNA3HA-SHP. The empty pcDNA3 vector was used to adjust the total DNA amounts, and 1 ␮g of pCMV-␤-galactosidase expression plasmid was used as an internal control. The primer sequence used for human SHP has been described previously [13]

RESULTS

The transcriptional activity of

DISCUSSION