Internalization of Aggrecan G1 Domain Neoepitope ITEGE in Chondrocytes Requires CD44

Wataru Ariyoshi,Cheryl B Knudson,Na Luo,Amanda J Fosang,Warren Knudson

doi:10.1074/jbc.m110.129270

Wataru Ariyoshi, Cheryl B Knudson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.129270

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Abstract

Degradation of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. Little is known concerning the fate of the residual N-terminal G1 domains of cleaved aggrecan; domains that remain bound to hyaluronan. In this study, 68-72-kDa bands representative of aggrecan G1 domains containing ITEGE(373) neoepitope were detected within a hyaluronidase-sensitive pool at the cell surface of bovine articular chondrocytes and within a hyaluronidase-insensitive, intracellular pool. To determine the mechanisms that contribute to this distribution, CD44 expression was knocked down by siRNA or function by CD44-DN. Both approaches prevented the retention and internalization of G1-ITEGE. Inhibition of CD44 transit into lipid rafts blocked the endocytosis of G1-ITEGE but not the retention at the cell surface. Chondrocytes derived from CD44 null mice also exhibited limited potential for retention and internalization of G1-VTEGE. The consequence of a lack of chondrocyte-mediated endocytosis of these domains in cartilage of the CD44 null mice was the accumulation of the degradation fragments within the tissue. Additionally, chondrocytes or fibroblasts derived from CD44 null mice exhibited little capacity for retention and internalization of exogenous G1-ITEGE derived from bovine cartilage explants. Bovine or wild type mouse fibroblasts were able to bind and internalize bovine-derived G1-ITEGE. Although several pathways are available for the clearance of these domains, CD44-mediated cellular internalization is the most prominent.

Highlights

In the present study we demonstrate that aggrecan G1-ITEGE is retained by chondrocytes in culture and that a fraction of the retained G1-ITEGE is internalized via a mechanism that requires CD44
Aggrecan G1-ITEGE Is Internalized by Articular Chondrocytes—Chondrocytes at all times of culture exhibited a background level of ITEGE neoepitope primarily localized at the cell surface (Fig. 1A)
G1-ITEGE that remains with the cells after hyaluronidase and is presumably intracellular was visualized in IL-1␤-treated chondrocytes (Fig. 1D) but was more evident at the 3-day time point (Fig. 1F)

Summary

Introduction

Aggrecan G1-ITEGE Is Internalized by Articular Chondrocytes—Chondrocytes at all times of culture exhibited a background level of ITEGE neoepitope primarily localized at the cell surface (Fig. 1A). To visualize whether the G1-ITEGE was internalized, the chondrocytes were treated either with trypsin (data not shown) or hyaluronidase to enzymatically release the cell-associated matrix pool of neoepitope. The 68 –72-kDa bands were similar in size to the G1-ITEGE domain present in the conditioned medium of IL-1␤-treated explant cultures of bovine articular cartilage (supplemental Fig. 1, lane ϩ).

Results

Conclusion