Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

Phimon Atsawasuwan,Yoshiyuki Mochida,Michitsuna Katafuchi,Masaru Kaku,Keith S.K Fong,Katalin Csiszar,Mitsuo Yamauchi

doi:10.1074/jbc.m803142200

Phimon Atsawasuwan, Yoshiyuki Mochida + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m803142200

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Abstract

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Highlights

Lysyl oxidase (LOX)2 is a copper-dependent amine oxidase that initiates the process of covalent intra- and intermolecular cross-linking in collagen and elastin [1]
Several studies have reported that collagen synthesis/expression significantly increased when osteoblasts or chondrocytes were cultured in the presence of by lathyrogens such as ␤-aminopropionitrile (BAPN) (8 –11), which is suggestive of such functions
Mature LOX Binds to Mature transforming growth factor-␤ (TGF-␤)1—The binding of LOX to transforming growth factor-␤1 (TGF-␤1) and bone morphogenetic proteins (BMPs) (BMP-2, -4, -6, and -7) was investigated by co-expressing those proteins with two types of tag, HA (ϳ1 kDa) for LOX and V5 (ϳ5 kDa) for other proteins followed by IP-Western blot (WB) analysis (Fig. 1A)

Summary

Introduction

Lysyl oxidase (LOX)2 is a copper-dependent amine oxidase that initiates the process of covalent intra- and intermolecular cross-linking in collagen and elastin [1]. SDS sample buffer and subjected to WB analysis with either expression level of LOX in the cultured medium was evaluanti-LOXi or anti-TGF-␤1 antibody.

Results

Conclusion