Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein, Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor.

Nirmal Rajasekaran,Jin-Hee Lee,Hunseok Lee,Kyoung Song,Young-Kee Shin,Özgür Cem Erkin,Yun Wei

doi:10.3390/ijms22115595

Abstract

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.

Highlights

In vertebrates, the transforming growth factor-β (TGF-β) pathway regulates the expansion of epithelial and neural tissues and the immune system and functions in wound repair
We found that the amino–terminal domain of nuclear respiratory factor-1 (NRF1) promotes NRF1 binding to SMAD4 in the nucleus based on multiple assays, including Bimolecular Fluorescence Complementation (BiFC), in vivo proximity ligation assays, and protein immunoprecipitation from HeLa cells
The BiFC fluorescence signal detected for NRF1:SMAD4 binding may result from functional nuclear localization sequence 88–116 of SMAD4 MH1 and the SMAD4 MH2 domain mainly in the nucleus

Summary

Introduction

The transforming growth factor-β (TGF-β) pathway regulates the expansion of epithelial and neural tissues and the immune system and functions in wound repair. The TGF-β receptor complex phosphorylates the transcription factors SMAD2 and SMAD3, which bind to SMAD4, translocate into the nucleus, and associate with diverse DNA-binding cofactors to target genes for regulation. SMAD4, a key downstream component in this system, was first isolated as a tumor-suppressor gene in human pancreatic ductal carcinomas [2,3]. The homozygous deletion or inactivating mutations of SMAD4 play a crucial role in the malignant progression of certain cancer types [4,5]. SMAD4 mutations are rarely observed in other cancer types. The function and significance of positive or negative regulators of SMAD4 are being studied in many cancers [6]

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Conclusion