Biochemical and Biophysical Research Communications
Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-α-induced NF-κB activity by binding to TNFR1
Research highlights
► Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. ► The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-α and was blocked TNF-α-activated NF-κB activity. ► IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-α. ► Competition between the L-domain of IGFBP-5 and TNF-α blocked TNF-α-induced NF-κB activity. ► This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-α inhibitor.
Introduction
Insulin-like growth factor binding protein-5 (IGFBP-5) is a secreted, multifunctional protein that functions in both an IGF-dependent and an IGF-independent manner in bone formation, differentiation, proliferation, and cell death [1], [2]. IGFBP-5 is found in both the nucleus and cytoplasm, although the functional relevance of these different locations is unclear. However, a recent study reported that IGFBP-5 might have different functions depending on its subcellular localization [3]. IGFBP-5 has three domains: an N-terminal domain, an L-domain, and a C-terminal domain. The N-terminal domain of IGFBP-5 possesses an IGF-I binding site and a signal peptide, while the C-terminus contains a nuclear localization signal and a heparin-binding site [4], [5]. Protein degradation and posttranslational modifications such as phosphorylation and glycosylation occur in the L-domain [6]. Truncated forms of IGFBP-5 have been detected in serum [7] and conditioned media [8]. In concert with these findings, it has been reported that IGFBP-5 is cleaved by several proteases including MMPs and ADAM family proteases, as well as unknown proteases [8], [9], [10]. However, the functional roles of truncated forms of IGFBP-5 have not been evaluated.
Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine mostly produced by activated macrophages and T lymphocytes [11]. Binding of TNF-α to tumor necrosis factor receptor 1 (TNFR1) triggers a signaling cascade that results in cell death or cell survival, depending on the interacting proteins. TNFR1 can activate cell proliferation via activation of the NF-κB signal pathway or cell death via activation of the Fas-associated death domain (FADD) protein and caspases [12]. Increased expression of TNF-α is a hallmark of several diseases including inflammatory diseases and certain types of cancer [11], [13].
In this study, we investigated the functional mechanisms underlying IGFBP-5-induced TNFR1 gene expression. We found that IGFBP-5 competed with TNF-α for binding to TNFR1 via its L-domain and inhibited the TNF-α-TNFR1 downstream signaling pathway. IGFBP-5 is therefore a potential therapeutic molecule that can be used to treat diseases characterized by dysregulation of TNF-α such as rheumatoid arthritis and cancer.
Section snippets
Plasmids and construction of truncation mutants
Human IGFBP-5 and TNFR1 were obtained by reverse transcription-PCR from HEK293 cells and cloned into a TA vector (Invitrogen, Carlsbad, CA, USA). Flag-TNFR1 was constructed by PCR using TNFR1 in a TA vector. The resulting PCR product was cloned into a pcDNA3 vector containing Flag (kindly provided by Prof. H.S. Choi, Chonnam University, South Korea). To construct glutathione S-transferase (GST)-fused TNFR1, TNFR1 excluding the signal peptide was subcloned into pGEX-2T vector (Amersham). Myc-
Exogenous expression of IGFBP-5 up-regulates TNFR1 mRNA expression
To study the functional mechanisms underlying the effects of IGFBP-5 on cells, IGFBP-5-Myc was stably transfected into HEK293 cells. cDNA microarray analyses of cells stably expressing IGFBP-5-Myc (BP5/293) as well as the empty vector (Vec/293) were performed to obtain global gene expression profiles. The TNFR1 gene was one of the many genes whose expression was upregulated by the overexpression of IGFBP-5-Myc (data not shown). We performed reverse transcription-PCR (RT-PCR) to verify the
Discussion
In this study, we demonstrated that IGFBP-5 induces the expression of TNFR1. In vivo and in vitro binding assays revealed that IGFBP-5 interacts physically with TNFR1. We used IGFBP-5 truncation mutants to more precisely delineate the TNFR1-interacting region and found that IGFBP-5 interacts with TNFR1 through the N- and L-domains. We also determined that the binding of BP5-L to TNFR1 was inhibited by TNF-α in a dose-dependent manner. Taken together, our data suggest that the L-domain of
Acknowledgments
This research was supported by a Science Research Center (Molecular Therapy Research Center) grant from the Korea Science and Engineering Foundation (Grant Number: R11-2000-080-11001-0).
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- 1
These authors contributed equally to this work.
- 2
Present address: CELLTRION Inc., Songdo-dong, Yeonsu-gu, Incheon 406–840, Republic of Korea.