Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Keittisak Suwan,Kanyamas Choocheep,Sonoko Hatano,Prachya Kongtawelert,Koji Kimata,Hideto Watanabe

doi:10.1074/jbc.m806927200

Keittisak Suwan, Kanyamas Choocheep + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m806927200

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Abstract

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Highlights

The extracellular matrix (ECM)3 supports cells and imparts architecture characteristic of individual tissues and regulates cell behavior by storing and distributing cytokines
We have demonstrated that Cspg2⌬3/⌬3 fibroblasts, whose versican lacks the A subdomain of the G1 domain, grow more slowly than wild type (WT) fibroblasts and acquire cellular senescence
In culture of Cspg2⌬3/⌬3 fibroblasts, the disorganized HA matrix by decreased versican deposition and HA chains released in the conditioned medium, enhance CD44mediated signal transduction, leading to constitutively active ERK1/2 and premature senescence

Summary

Introduction

The extracellular matrix (ECM)3 supports cells and imparts architecture characteristic of individual tissues and regulates cell behavior by storing and distributing cytokines. Versican Is Decreased in the Cspg2⌬3/⌬3 Fibroblasts—Because senescence of Cspg2⌬3/⌬3 fibroblasts was likely caused by the alteration of the ECM, we investigated distributions and levels of various ECM molecules known to regulate cell behavior. To confirm that the decreased versican deposition in the Cspg2⌬3/⌬3 fibroblasts (Fig. 3A, upper middle panel) was not due to a decreased level of confluence, we examined the expression levels of other matrix molecules, such as fibronectin and laminin.

Results

Conclusion