Matrix Metalloproteinase-2-deficient Fibroblasts Exhibit an Alteration in
the Fibrotic Response to Connective Tissue Growth Factor/CCN2 because of an
Increase in the Levels of Endogenous
Fibronectin |
| |
Authors: | Cristian A Droppelmann Jaime Guti??rrez Cecilia Vial and Enrique Brandan |
| |
Institution: | Centro de Regulación Celular y Patología, Centro de Regeneración y Envejecimiento, Departamento de Biología Celular y Molecular, Millenium Institute for Fundamental and Applied Biology, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile |
| |
Abstract: | Matrix metalloproteinase-2 (MMP-2) is an important extracellular matrix
remodeling enzyme, and it has been involved in different fibrotic disorders.
The connective tissue growth factor (CTGF/CCN2), which is increased in these
pathologies, induces the production of extracellular matrix proteins. To
understand the fibrotic process observed in diverse pathologies, we analyzed
the fibroblast response to CTGF when MMP-2 activity is inhibited. CTGF
increased fibronectin (FN) amount, MMP-2 mRNA expression, and gelatinase
activity in 3T3 cells. When MMP-2 activity was inhibited either by the
metalloproteinase inhibitor GM-6001 or in MMP-2-deficient fibroblasts, an
increase in the basal amount of FN together with a decrease of its levels in
response to CTGF was observed. This paradoxical effect could be explained by
the fact that the excess of FN could block the access to other ligands, such
as CTGF, to integrins. This effect was emulated in fibroblasts by adding
exogenous FN or RGDS peptides or using anti-integrin αV
subunit-blocking antibodies. Additionally, in MMP-2-deficient cells CTGF did
not induce the formation of stress fibers, focal adhesion sites, and ERK
phosphorylation. Anti-integrin αV subunit-blocking antibodies
inhibited ERK phosphorylation in control cells. Finally, in MMP-2-deficient
cells, FN mRNA expression was not affected by CTGF, but degradation of
125I-FN was increased. These results suggest that expression,
regulation, and activity of MMP-2 can play an important role in the initial
steps of fibrosis and shows that FN levels can regulate the cellular response
to CTGF.Extracellular proteolysis is an essential physiological process that
controls the immediate cellular environment and thus plays a key role in
cellular behavior and survival
(1). The members of the matrix
metalloproteinase
(MMP)2 family of
zinc-dependent endopeptidases are major mediators of extracellular proteolysis
by promoting the degradation of extracellular matrix (ECM) components and cell
surface-associated proteins (2,
3). Each one of these enzymes
is negatively regulated by tissue inhibitors of metalloproteinases (TIMPs)
(4) and is secreted as a
zymogen (pro-MMPs) that is activated in the extracellular space
(5–7).
This mechanism is an important form of regulation of gelatinase activity and
in consequence, highly significant for ECM homeostasis. Among the members of
the MMP family, the metalloproteinase type 2 (MMP-2 or gelatinase A) is known
to be a key player in many physiological and pathological processes, such as
cell migration, inflammation, angiogenesis, and fibrosis
(8–11).Fibrotic disorders are typified by excessive connective tissue and ECM
deposition that precludes normal healing of different tissues. ECM
accumulation can be explained in two ways: increasing expression and
deposition of connective tissue proteins and/or decreasing degradation of ECM
proteins (12). Transforming
growth factor type β, a multifunctional cytokine, is strongly
overexpressed, and it is associated to the pathogenesis of these diseases
(13,
14). It stimulates the
expression of connective tissue growth factor (CTGF/CCN2)
(15), a cytokine that is
responsible for transforming growth factor type β fibrotic activity
(16,
17). The role of CTGF in
fibrosis has gained attention in recent years
(16,
18–22).
CTGF overexpression is known to occur in a variety of fibrotic skin disorders
(23,
24), renal
(25), hepatic
(26), and pulmonary fibrosis
(27) and in muscles from
patients with Duchenne muscular dystrophy
(28).On the other hand, several pathologies involving fibrosis show an increase
in MMP expression, including gelatinase A. Augmented expression of MMP-2 was
found in submucous (29), skin
(30), liver
(31), and lung fibrosis
(32,
33) and dystrophic myotubes
from fibrotic muscles of Duchenne muscular dystrophy
(34). It has been shown that
transforming growth factor type β induces an increase in the amount of
MMP-2 in fibroblasts (35) and
that CTGF induces MMP-2 expression in cultured renal interstitial fibroblasts
(36). The putative role
assigned to MMP-2 in fibrotic disorders is related to tissue regeneration
because of the capacity of this enzyme to degrade basal lamina
(37–39).
Because MMP-2 expression is up-regulated in these pathologies but still a high
ECM deposition is observed, we propose that this accumulation could be
explained by a diminution of the MMP-2 enzymatic activity.In this article, we demonstrate that CTGF increases fibronectin (FN)
amount, MMP-2 expression, and gelatinase activity in 3T3 fibroblasts. More
significantly, we show that MMP-2-deficient cells have an increased basal
amount of FN and show a response to CTGF that is opposite to that of control
cells. This paradoxical effect could be explained by the increase in the FN
amount that blocks the integrins (at least integrins with αV
subunit), which can act like CTGF receptors. |
| |
Keywords: | |
|
|