Filamin A-mediated Down-regulation of the Exchange Factor Ras-GRF1 Correlates with Decreased Matrix Metalloproteinase-9 Expression in Human Melanoma Cells

Abstract

The actin-binding protein filamin A (FLNa) is associated with diverse cellular processes such as cell motility and signaling through its scaffolding properties. Here we examine the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs). The lack of FLNa in human M2 melanoma cells was associated with constitutive and phorbol ester-induced expression and secretion of active MMP-9 in the absence of MMP-2 up-regulation. M2 cells displayed stronger MMP-9 production and activity than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon was associated with destabilization and ubiquitylation of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and elevated levels of MMP-9 in M2A7 cells, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, reduced MMP-9 expression in M2 cells. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway partly through a Ras-GRF1 mechanism to modulate the production of MMP-9.Matrix metalloproteinases (MMPs) 5 play a crucial role in degradation of extracellular matrix (ECM) associated not only with normal growth and development but also with various pathological conditions such as tumor invasion and angiogenesis (1). Melanoma progression, as in many other cancers, is associated with invasion into surrounding tissues, which depends on MMP-mediated proteolytic degradation of the basement membrane and ECM. Among its members, the 72-kDa gelatinase A (MMP-2) and 92-kDa gelatinase B (MMP-9) are thought to be key enzymes for degrading type IV collagen, a major component of the basement membrane. Contributions of these enzymes in both physiological and pathophysiological processes such as tumor cell invasion and metastasis have been well documented (2-6). Although the recent work of Bartolome et al. (7) shows that MMP-2 is the most important contributor to melanoma cell invasion mediated by the chemokine CXCL12, others have found that MMP-9 is predominantly expressed by advanced stage melanoma cells and could have prognostic value in identifying patients at high risk of melanoma progression (8 -11). The expression of MMP-9 can be induced by a variety of mitogens, including epidermal growth factor (EGF), tumor necrosis factor ␣ and phorbol esters. There are a number of transcription factors that bind in the upstream regulatory region of the MMP-9 gene, which helps explain the multiple mechanisms of regulation of MMP-9 gene expression in tumor cell lines (12, 13). Stimulu...

Filamin A controls matrix metalloprotease activity and regulates cell invasion in human fibrosarcoma cells.

“…We did not detect activated MMP9 in our cells (Figs 4,5), nor MMP1 activity using casein zymography (not shown), so while our results strongly suggest that the enhanced matrix degradation is mediated by MMP2 we cannot exclude the involvement of other MMPs in the process. Indeed, while to our knowledge this is the first report linking FLNa expression to MMP activity, in human melanoma cells FLNa has been reported to negatively regulate MMP9 expression via the Ras/Raf-1/Erk pathway (Zhu et al, 2007). Consistent with this, an increase of MMP9 expression was reported in FLNA 2/2 platelets (Jurak Begonja et al, 2011).…”

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly

“…ADAM17 is activated via a p38 MAPK-dependent pathway during platelet storage and FlnA down-regulates MMP9 transcription by negatively modulating the ERK pathway in melanoma cells. 21,46 Whether increased ADAM17 and MMP9 expression is the result of potentiated p38 MAPK and ERK pathways in FlnA-null cells remains to be determined.…”

“…Because ADAM17 cleaves GPIb␣ in platelets 20 and FlnA absence leads to MMP9 overexpression in human melanoma cells, 21 the expression of these 2 metalloproteinases was investigated in platelets and MKs. Expression of both ADAM17 and MMP9 was increased in FlnA-null platelets, compared with controls ( Figure 6C), but normal in FlnA-null MKs ( Figure 6D).…”

Interactions between Filamin A and MMP-9 Regulate Proliferation and Invasion in Renal Cell Carcinoma

“…FLNa interacts with Ras-GRF1 and degrades Ras-GRF1 by ubiquitination. Therefore, Zhu et al suggest that FLNA can reduce the MMP-9 expression and finally decrease the invasion of tumor cells through inhibiting Ras/MAPK/ERK cascade (Zhu et al, 2007). Immunocopresipitation experiment and in vitro study demonstrate that FLNA can bind to the insulin receptor (IR) and block the activation of insulin-dependent MAPK pathway.…”