Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration

Abstract

Henderson N, Markwick LJ, Elshaw SR, Freyer AM, Knox AJ, Johnson SR. Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration. Am J Physiol Lung Cell Mol Physiol 292: L1030-L1038, 2007. First published December 22, 2006; doi:10.1152/ajplung.00317.2006.-Increased proinflammatory mediators and ECM deposition are key features of the airways in asthma. Matrix metalloproteinases (MMPs) are produced by airway smooth muscle (ASM) cells and have multiple roles in inflammation and tissue remodeling. We hypothesized that components of the asthmatic airway would stimulate MMP production and activation by ASM and contribute to airway remodeling. We measured human ASM-derived MMP mRNA, protein, and activity by real-time RT-PCR, zymography, Western blotting, and MMP activity assay. Collagen I and thrombin caused a synergistic increase in MMP-2 protein and total MMP activity but paradoxically decreased MMP-2 mRNA. Additionally, collagen I activated MMP-2 in culture supernatants independent of the cell surface. Together, collagen I and thrombin strongly enhanced MMP-14 mRNA and protein but had no effect individually, suggesting increased MMP-14, the activating protease for MMP-2, may be partially responsible for MMP-2 activation. Furthermore, collagen I reduced tissue inhibitor of metalloproteinase-2 protein (TIMP-2). We examined the role of MMPs in functions of ASM related to airway remodeling and found migration and proliferation were MMP dependent, whereas adhesion and apoptosis were not. Ilomastat inhibited migration by 25%, which was also inhibited by TIMPs 1-4 and increased by the MMP-2 activator thrombin. These in vitro findings suggest that the environment within the airways of patients with asthma enhances MMP-2 and -14 protein and activity by a complex interaction of transcriptional and posttranscriptional mechanisms, which may contribute to ASM migration. matrix metalloproteinases; asthma PATIENTS WITH ASTHMA DEVELOP structural airway changes termed remodeling. Starting in infancy, remodeling is categorized by airway smooth muscle (ASM) hypertrophy, hyperplasia, increased subepithelial myofibroblasts, altered ECM deposition, infiltration of ASM by mast cells, increased mucosal vascularity, epithelial shedding, metaplasia, and mucus gland hyperplasia (1,22,43). Remodeling results in bronchial hyperresponsiveness and airflow obstruction and, therefore, increased symptoms and use of asthma medication and health care resources (1, 34). The ASM cell is emerging as a key cell in airway remodeling being increased in both size and number in the airways of patients with asthma (4). Moreover, ASM cells produce ECM components (10), pro-and anti-inflammatory mediators, and growth factors of relevance to airway remodeling, including vascular endothelial growth factor (30), interleukin-8 (52), eotaxin (41), and prostaglandin E 2 (39). Thus the ASM cell is an important modulator of inflammation, inflammatory cell influx, and angiogenesis in the asthmatic ...

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

“…Legumain activates pro-MMP-2 by cleavage at the Asn 109 -Tyr and Asn 111 -Phe peptide bonds (17). Serine protease activation of pro-MMP-2, such as that mediated by thrombin and plasmin, has been well studied, with some reports indicating the involvement of MT1-MMP, which remains controversial (14,15,27,35,43,44). However, the detailed molecular mechanism for pro-MMP-2 activation by serine proteases remains to be elucidated.…”

“…We first speculated that MT1-MMP may be involved in this process because thrombin has been reported to activate pro-MMP-2 in an MT1-MMP-dependent manner (15,27). Thus, we examined MT1-MMP dependence in thrombin-mediated processing of pro-MMP-2 using a metalloprotease inhibitor and MT1-MMP-deficient cells, all of which provided evidence for non-MT1-MMP components at the cell surface.…”

“…7A). Because thrombin has been shown to activate pro-MMP-2 in an MT1-MMP-dependent manner (15,27), we tested whether the increase in processing observed under cell-associated conditions was due to the proteolytic activity of MT1-MMP. However, zymography showed that the processing was unaffected by GM6001 (Fig.…”

“…Factor Xa acts as a potent mitogen for endothelial cells (23), fibroblasts (24), and vascular SMCs (25,26). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13,27,28). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism for MMP-2 activation has yet to be elucidated (15,27).…”

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

“…Some controversial reports suggest the involvement of membrane-type 1 MMP (MT1-MMP) in thrombin-mediated activation of pro-MMP-2 (19,(22)(23)(24). In our previous studies, we demonstrated that thrombin directly cleaved the propeptide on the C-terminal side of Arg 98 and Arg 101 with a preference for Arg 101 ; this was followed by intermolecular autoproteolytic cleavage at the Asn 109 -Tyr peptide bond, resulting in full enzymatic activation (7).…”