RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

Tatsuya Kosaka,Takuya Waki,Keisuke Iwai,Takashi Fujita,Haruka Nishimura,Motoki Tanabe,Yuko Kurosaka,Rino Fukui,Yuuki Takakura,Mio Matsui,Barry I Hudson

doi:10.1371/journal.pone.0108819

Abstract

RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

Highlights

Advanced glycation end products (AGEs) are permanently modified protein derivatives formed in the presence of reducing sugars, such as glucose and fructose by non-enzymatic glycation, oxidation and dehydration reactions [1]
Reactivity against RAGE antibody was only weakly observed in proliferative chondrocytes, and negligibly in cancellous bone, periosteum and bone collar. These results suggested that RAGE regulates chondrocyte in the prehypertrophic to hypertrophic stages
Using a specific primer pair to detect endogenous RAGE, real-time PCR analysis demonstrated that RAGE by RAGE overexpression with or without AGE was significantly upregulated, and down regulated by DN-RAGE (Fig.1J). These results suggested that extracellular AGE stimulation influences chondrocyte function, DN-RAGE functions as dominant negative form and that AGE regulated RAGE expression

Summary

Introduction

Advanced glycation end products (AGEs) are permanently modified protein derivatives formed in the presence of reducing sugars, such as glucose and fructose by non-enzymatic glycation, oxidation and dehydration reactions [1]. In diabetic vascular complications including bone disease, AGEs are known to accumulate in various tissues at an extremely accelerated rate [2,3]. RAGE is a type I transmembrane protein belonging to the immunoglobulin superfamily and is composed of an extracellular region, a hydrophobic transmembrane-spanning domain and a short cytoplasmic tail [5]. Deletion of the cytoplasmic domain of RAGE imparts a dominant negative (DN)-effect on RAGE-dependent activation of cell signaling both in vitro and in vivo [9,10,11]. RAGE signaling axes induce the nuclear translocation of Nuclear factor (NF)-kB, a hallmark of the pro-inflammatory signal transduction cascade [12]. RAGE is involved in a broad range of inflammatory, degenerative and hyper proliferative diseases, including sepsis, rheumatoid arthritis, diabetic nephropathy/angiopathy, atherosclerosis, cancer and neurological disorders [13,14]

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