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Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs

Nadia A. Hasaneen^*,, Stanley Zucker^*,, Jian Cao, Christian Chiarelli, Reynold A. Panettieri and Hussein D. Foda^*,,1

^* Department of Medicine and Research, VAMC Northport, Northport, New York, USA;
State University of New York at Stony Brook, Stony Brook, New York, USA; and
Pulmonary, Allergy and Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA

¹Correspondence: Pulmonary and Critical Care Medicine, SUNY at Stony Brook, Health Science Center, Stony Brook, NY 11794-8172, USA. E-mail: hussein.foda{at}med.va.gov

SPECIFIC AIMS

Airway smooth muscle (ASM) proliferation is a structural change that occurs in the airway of asthmatic patients. Structural changes are often referred to as airway remodeling. It is recognized that ASM migration may be important to the smooth muscle thickening that occurs in airway remodeling.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases that degrade extracellular matrix. These proteases are secreted by airway smooth muscle cells and other cells in the airway and play a role in airway remodeling. MMP has been reported to play a critical role in cell migration. TN-C is an extracellular matrix glycoprotein prominent in embryonic and adult tissues that are actively remodeling and is often coexpressed with MMPs.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a 58 kDa transmembrane glycoprotein belonging to the Ig superfamily. EMMPRIN stimulates several cells to produce MMP-1, MMP-2, MMP-3, MT1-MMP, and MT2-MMP, and can also increase the activation of MMP-2. We recently reported that mechanical stretch increased EMMPRIN gene expression and protein synthesis in human pulmonary microvascular endothelial cells, followed by increased up-regulation and release of MMP-1, MMP-2, and MT1-MMP.

Cells in the lung are constantly exposed to forces of stretch and relaxation, which play a critical role in the airway remodeling. In this study we wanted to determine whether 1) mechanical strain of HASM induces their proliferation and migration; 2) strain-induced HASM proliferation and migration was MMP dependent; and 3) the mechanisms by which mechanical strain induces MMP release and activation.

PRINCIPAL FINDINGS

1. Mechanical strain induces HASM proliferation in a MMP-dependent fashion
Exposure of HASM cells to cyclic strain for 3 and 5 days caused a significant increase their proliferation rate (P<0.05 and P<0.001, respectively) vs. HASM cells not exposed to strain, as measured by cell counting and thymidine incorporation (Fig. 1 ). This increased proliferation was accompanied by a significant increase in pro- and activated MMP-2 in the conditioned media and an increase in MT1-MMP in cell lysates. There was also an increase in MMP-1 and MMP-3 in conditioned media with no change seen in TIMP-1 or TIMP-2 release.

View larger version (22K): [in this window] [in a new window]   Figure 1. Mechanical strain increases HASM cells proliferation; inhibition by the MMP inhibitor Prinomastat. A) [H3] Thymidine incorporation studies of HASM cells exposed to 17–18% cyclic strain for 1, 3, and 5 days demonstrated a significant increase in [H3] thymidine incorporation on days 3 and 5 when compared with HASM cells not exposed to strain. There was also a significant increase in [H3] thymidine incorporation of HASM not exposed to strain after 5 days. B) Cell count of HASM cells exposed to strain and non-strain conditions for 5 days using Coulter counter confirmed that strain induced HASM cell proliferation and showed that Prinomastat (300 ng/mL) inhibited strain-induced HASM cell proliferation. Values are means ± SEM; n = 6 experiments, run in triplicate. *P< 0.05, **P< 0.01 comparing strain vs. non-strain conditions and strain ± Prinomastat.

To verify the role of MMPs in cyclic strain-induced proliferation, HASM cells were exposed to conditions of cyclic strain and non-strain in the presence of the broad spectrum MMP inhibitor (Prinomastat) or vehicle (DMSO). Our results show that Prinomastat abrogated the strain-induced increase in HASM proliferation while inhibiting MMP-2 activation.

2. Role of EMMPRIN
Immunoblot analysis of cell lysates from cells exposed to cyclic strain and non-strain conditions using antibodies directed against EMMPRIN showed EMMPRIN to be significantly increased in HASM cells exposed to cyclic strain. EMMPRIN was increased in a time-dependent fashion beginning on day 1. We then examined HASM cells transfected with an EMMPRIN-positive vector. Our results show that EMMPRIN-transfected HASM displayed increased MMP-1 and MMP-2 release in conditioned media and an increase in MT1-MMP in cells compared with the GFP-transfected HASM (control).

Zymography and immunoblot analysis of the conditioned medium and cell lysates demonstrated that strain-induced increases in MMP-1, MMP-2, and MT1-MMP were markedly suppressed when anti-EMMPRIN antibody was present.

3. Role of MMPs in HASM cell migration and invasion
HASM cells exposed to cyclic strain showed a significant cell migratory response compared with HASM cells not exposed to strain. This migratory response was enhanced in the presence of PDGF-BB.

Mechanical strain increased HASM cell migration toward PDGF-BB (20 ng/mL) by 2.2 ± 0.3-fold compared with HASM cells not exposed to stretch (P<0.001). Prinomastat abrogated the stretch-induced increase in migration in a dose-dependent fashion. Mechanical strain significantly increased HASM cell ability to invade Matrigel by 2.1 ± 0.3-fold (P<0.05). Prinomastat significantly inhibited this stretch-induced invasion of HASM by 50% (P<0.05).

To examine the potential role of MT1-MMP in HASM migration, HASM cells were transfected with a plasmid encoding MT1-MMP. Our results indicate that with increased cell surface expression of MT1-MMP, cells increased their ability to degrade and migrate over fibronectin. These MT1-MMP-transfected HASM cells displayed increased ability to migrate toward PDGF-BB when compared with GFP (control) transfected cells (P<0.001). Prinomastat inhibited the migration of MT1-MMP-transfected HASM.

4. Cyclic strain of HASM leads to an increase in Tenascin-C Release
TN-C was significantly increased in the conditioned media of HASM cells exposed to cyclic strain compared with media from the control cells not exposed to cyclic strain. Prinomastat inhibited this cyclic strain-induced increase in Tenacin-C, suggesting an MMP-dependent mechanism for the increased TN-C release seen with cell strain.

CONCLUSIONS AND SIGNIFICANCE

Airway smooth muscle proliferation and migration are key components of airway remodeling in asthma. Understanding the mechanisms of airway smooth muscle proliferation and migration is important since it may help develop strategies to prevent or reverse airway remodeling in asthma. In this study we report that cyclic mechanical strain induced HASM proliferation and migration in an MMP-dependent fashion. Strain-induced MMPs release was mediated via an increased expression and release of EMMPRIN. One possible mechanism for MMP-induced proliferation may be the increase in availability of TN-C caused by the increase in MMP production.

Mechanical strain-induced HASM proliferation was accompanied by increased expression, synthesis, and activation of several MMPs: MMP-1, MMP-2, MMP-3, and MT1-MMP. Prinomastat, an MMP inhibitor, abrogated the proliferation of HASM in response to mechanical strain. Mechanical strain has been reported to induce proliferation in human pulmonary epithelial H441 cells and canine ASM. Based on the inhibitory effects of Prinomastat, we propose that cyclic mechanical strain-induced proliferation of HASM requires the release and activation of MMPs.

MMPs can induce HASM cell proliferation by their action on extracellular matrix (ECM). ECM can store inflammatory mediators and growth factors such as TNF-, TGF-ß, IGF, and HBEGF, which can be released by MMPs and mediate smooth muscle proliferative and synthetic capacity. We hypothesized that MMPs released by mechanical stretch could induce HASM proliferation by changing growth factor and matrix protein availability that may have growth-promoting effects. Our results show that the increase in MMP release was accompanied by an increase in the release of one of those factors, the matricellular protein TN-C. Prinomastat inhibited this increase in TN-C. TN-C, which is often coexpressed with MMPs in a variety of actively remodeling tissues, has been reported to potentiate smooth muscle growth and survival. This released TN-C may then act to support growth and potentiate the effect of mitogenic growth factors released by MMPs.

Airway smooth muscle cell migration is recognized as a potentially important component of airway remodeling associated with injury and inflammation. Extracellular matrix (ECM) degradation by MMPs is known to facilitate tumor cell migration and invasion. It was recently suggested that MT1-MMP plays a critical role in cell migration. MT1-MMP’s ability to increase cell migration is independent of its role in MMP-2 activation. We sought to examine whether cyclic strain increased MMP release and activation by HASM cells was involved in their increased migration and invasion abilities. Our results show that HASM cells exposed to cyclic strain increased both migration and invasion capabilities of these cells. Prinomastat inhibited the strain-induced increase in migration and invasion of HASM by 50%. When HASM cells were transfected with MT1-MMP, PDGF-BB-induced chemotaxis was increased as well as HASM’s capacity to degrade and migrate over fibronectin. Prinomastat inhibited the migration of MT1-MMP transfected cells and also inhibited the activation of MMP-2 induced by MT1-MMP transfection. These results suggest that among MMPs, MT1-MMP induced by cyclic strain of HASM plays an important role in the migration and invasion ability of these cells. However, MMP release and activation is not the sole cause for cyclic strain-induced increase in migration and invasion: mechanical strain may also lead to cytoskeleton rearrangement that may be important for cell movement.

Our results show that strain-induced MMPs’ release was preceded by up-regulation of EMMPRIN; when the HASM cells were transfected with EMMPRIN cDNA, transfected cells increased their production of MMP-1, MMP-2, and MT1-MMP compared with GFP-transfected HASM cells. The strain-induced increase in MMP-1, MMP-2, and MT1-MMP was attenuated by an anti-EMMPRIN blocking antibody, indicating that the MMP induction observed in this system is mostly because of the effect of EMMPRIN. These results support the concept that mechanical strain-induced up-regulation of EMMPRIN is responsible for the increased MMP production seen in this system. These results are consistent with the fact that EMMPRIN is known to induce MMP-1, MMP-2, MMP-3, and MT1-MMP expression in human fibroblast.

We propose the following signal pathway that may link mechanical stress to the increased MMP release and activation and HASM proliferation. EMMPRIN, which is up-regulated by cell strain, causes an increase in MMP-1, MMP-2, and MT1-MMP production. The augmented presence of MT1-MMP on the cell surface leads to an increased ability for the cells to migrate. This increased availability of MT1-MMP may also lead to increased MMP-2 activation in response to cyclic strain. MMPs induce HASM cell proliferation through the release of matrix-bound growth factors and cytokines such as TGF-ß, TNF-, heparin binding-EGF (HB-EGF), and IGFs; it also induces the degradation of type 1 collagen by MMPs, which then promotes TN-C expression at the transcriptional level (Fig. 2 ).

View larger version (14K): [in this window] [in a new window]   Figure 2. Schematic diagram.

Our results may have important implications in understanding airway remodeling even though these findings were identified in an in vitro model of airway smooth muscle strain, which has inherent limitations.

Cyclic mechanical strain may play an important role in airway remodeling by increasing the proliferation and migration of HASM cells in an MMP-dependent fashion. MMP release and activation in response to cyclic strain is preceded by increased expression and release of EMMPRIN. Based on the results presented, MMP inhibitors should be considered as one of the therapeutic options for attenuating airway remodeling in asthma.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3350fje;

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