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

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 ⌬3/⌬3 , 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 ⌬3/⌬3 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 ⌬3/⌬3 fibroblasts exhibited ϳ35 and ϳ85% deposition of versican and HA, without such a structure. The Cspg2 ⌬3/⌬3 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. The extracellular matrix (ECM)3 not only supports cells and imparts architecture characteristic of individual tissues but also regulates cell behavior by storing and distributing cytokines and growth factors to target cells. Proper functioning of the ECM requires an elaborate matrix assembly that involves various ECM molecules, including collagens, proteoglycans, hyaluronan (HA), and glycoproteins. An alteration of the ECM structure may transmit a signal to cells and change their behavior.Versican/PG-M (1, 2) is a large chondroitin sulfate (CS) proteoglycan of the ECM, synthesized mainly by fibroblasts and vascular smooth muscle cells. Its core protein consists of two globular domains G1 and G3 at the N and C termini, respectively, and two CS attachment domains CS␣ and CS␤ between the two globular domains. Up to 23 CS chains are attached to these domains, and the molecular mass of versican reaches 1,000 kDa. The N-terminal G1 domain consists of A, B, and BЈ looped subdomains. The B-BЈ stretch binds HA, and the A subdomain enhances the binding (3). The C-terminal G3 domain binds other ECM molecules (4), including fibrillins (5), fibulin-1 (6), fibulin-2 (7), tenascins (8 -10), type I collagen (11), and fibronectin (11). By interacting with these molecules, versican is incorporated into the ECM and serves as a structural macromolecule.Versican exhibits two distinct expression patterns. Whereas it is constitutively expressed in adult tissues such as dermis and blood vessels and serves as a structural macromolecule of the ECM, versican is transiently expressed at high levels in various embryonic tissues and regulates cell behavior such as adhesion (12-14), migration (1...

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

“…As HA is known to regulate proliferation in a great number of cells (23), it was not surprising to find that inhibition of HA synthesis alters cell cycle genes. On the other hand, the link between HA and p53 is not known, although recently, it was reported that reduction of an HA/versican ECM induced senescence and p53 accumulation in fibroblasts (24).…”

Bral1: Its Role in Diffusion Barrier Formation and Conduction Velocity in the CNS

“…In the case of hyaluronan, it may be degraded by hyaluronidase because Crtl1 could retard the degradation of hyaluronan in proteoglycan aggregates in vitro (Rodriguez and Roughley, 2006). Alternatively, the degradation may be secondary to the lack of the nodal lecticans, including versican, in Bral1-deficient mice, because versican may function to maintain hyluronan levels in the ECM (Suwan et al, 2009).…”

Host stromal versican is essential for cancer‐associated fibroblast function to inhibit cancer growth

“…fibroblasts with lower Vcan expression exhibit decreased levels of proliferation 13 and collagen synthesis (unpublished data). Thus, the decrease in the stromal region could be due to reduced fibroblast growth and diminished collagen biosynthesis caused by loss of Vcan expression.…”

“…Cells were incubated in 0.5 ml of 1.5 M Tris-HCl, pH 7.3, 0.15 M NaCl, 10 mM CaCl 2 and 10 U of protease K for 2 hr at 558C. 13 After centrifugation at 14,000g at 48C for 25 min, the supernatant was applied to a sandwich enzyme-linked immunosorbent assay (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer's instructions.…”

“…The N-terminal G1 domain binds HA and affects HA-mediated signaling. 13 The C-terminal G3 domain binds other ECM molecules such as fibrillin-1, 14 fibulin-1 and -2, 15,16 and tenascins, 15,17 and these supramolecular complexes regulate TGFb and BMP signaling. 18,19 CS chains in the center bind cytokines, 15,20 which may regulate their signaling.…”