Date Published: October 2, 2014
Publisher: Public Library of Science
Author(s): Tatsuya Kosaka, Rino Fukui, Mio Matsui, Yuko Kurosaka, Haruka Nishimura, Motoki Tanabe, Yuuki Takakura, Keisuke Iwai, Takuya Waki, Takashi Fujita, Barry I. Hudson.
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.
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 . In diabetic vascular complications including bone disease, AGEs are known to accumulate in various tissues at an extremely accelerated rate , . 3 classes of AGE receptors: RAGE (receptor for AGE), a complex of OST-48/80KH/galectin-3, class A scavenger receptor have been identified –. 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 . 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–. RAGE signaling axes ultimately induce the nuclear translocation of Nuclear factor (NF)-κB, a hallmark of the pro-inflammatory signal transduction cascade . 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 , .
Damage to the cartilage is a major problem, especially in joint disease. AGEs are one of the candidates that act as adverse mediators in joint diseases , . RAGE has been shown to be an initiator of inflammatory response in immune responsive cells such as macrophages . Although RAGE knockout mice show skeletal abnormalities after birth , , the biological importance of RAGE expression in chondrocytes remains to be clarified. Hence in this work, we examined the precise mechanisms, 1; regulatory mechanism of chondrocytes functions by RAGE, 2; involvement of most conceivable inflammatory NF-κB or Rho family GTPases signals in chondrocytes. We showed for the first time that RAGE activation in chondrocytes functions as a mediator for suppression of chondrocyte differentiation via NF-κB-independent and Rho family GTPases-dependent mechanisms.