Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-α-induced NF-κB activity by binding to TNFR1

https://doi.org/10.1016/j.bbrc.2011.01.064Get rights and content

Abstract

IGFBP-5 is known to be involved in various cell phenomena such as proliferation, differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-α, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-κB activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-α in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-α resulted in inhibition of TNF-α-induced NF-κΒ activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-α inhibitor.

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).

References (20)

There are more references available in the full text version of this article.

Cited by (17)

  • Mesenchymal stromal cells mitigate experimental colitis via insulin-like growth factor binding protein 7-mediated immunosuppression

    2016, Molecular Therapy
    Citation Excerpt :

    The pathogenesis of CD is due to a dysfunctional interaction between the intestinal microflora and the mucosal immune system. In this context, the presence of overactive mucosal effector T-cells and/or underactive mucosal Treg cells leads to improper modulation of even normal effector T-cells.32 The results of our in vitro and in vivo studies indicated that MSCs-secreted IGFBP7 could suppress the proliferation of CD4+ and CD8+ T-cells and thereby alleviate experimentally-induced colitis.

  • Insulin-like growth factor binding proteins 4-6

    2015, Best Practice and Research: Clinical Endocrinology and Metabolism
    Citation Excerpt :

    A cell surface receptor for IGFBP-5 was reported a number of years ago but no sequence or clone was identified [37]. IGFBP-5 has recently been reported to bind to the TNF-α receptor via its linker domain, thereby inhibiting TNF-α actions [38]. IGFBP-5 also affects breast cancer cell adhesion via interaction with the α2β1 integrin [39].

View all citing articles on Scopus
1

These authors contributed equally to this work.

2

Present address: CELLTRION Inc., Songdo-dong, Yeonsu-gu, Incheon 406–840, Republic of Korea.

View full text