Abstract
Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not c-K(A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K(B)-Ras through a PI-3 kinase:AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras.
Highlights
MMP-21 belongs to an evolutionarily conserved proteinase family that requires Zn2ϩ for enzymatic activity
Several groups have shown that the Ras isoforms possess different potencies in their respective abilities to activate Raf-1 and PI-3 kinase [27, 28]
Using immortalized N-Ras and K-Ras knockout fibroblasts, we have shown that, in the absence of short-term mitogenic stimulation, the expression of matrix metalloproteinase 2 (MMP-2) is regulated through a c-K(B)-Ras dependent process
Summary
MMP-21 belongs to an evolutionarily conserved proteinase family that requires Zn2ϩ for enzymatic activity. Tumor cells expressing oncogenic Ras proteins possess higher potential to metastasize, in part because of the up-regulation of MMP-2 expression (14 –17). The most common forms of oncogenic Ras have mutations at either codon 12, 13, or 61 [18] These mutations result in a higher proportion of Ras proteins with GTP in their binding site. Introduction of oncogenic H-Ras into fibroblasts results in increased expression of MMP-2 [14, 17]. Blocking Rasdependent signaling decreased both expression of MMP-2 and invasion of Src-transformed cells, indicating a role for Ras in regulating MMP-2 expression [19]. The N-Ras and K-Ras knockout fibroblasts provide excellent tools to study the role the K- and N-Ras isoforms in regulating the expression of MMP-2
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
