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Leukotriene D4 induces MMP-1, which functions as an IGFBP protease in human airway smooth muscle cells
R Rajah1, S E Nunn, D J Herrick
1Division of Endocrinology, Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine 19104, USA.
Abstract:
We have previously demonstrated that the asthma-associated proinflammatory eicosanoid leukotriene D4 (LTD4) is comitogenic with insulin-like growth factors (IGF) in airway smooth muscle (ASM) cells. This synergistic effect of LTD4 and IGF on ASM cell growth involves proteolysis of ASM-produced inhibitory IGF-binding proteins (IGFBP). In this report, we analyzed the conditioned media (CM) from LTD4-treated human ASM cells (ASM-LTD4-CM) by Western ligand blotting and demonstrated a marked LTD4-induced reduction in the levels of the intact IGFBP (predominantly IGFBP-2) secreted by these cells. The IGFBP-2 in the ASM-LTD4-CM was identified as lower-molecular-weight fragments by Western immunoblotting. Incubation with 125I-labeled IGFBP demonstrated that an IGFBP protease was induced in the ASM cells in response to LTD4 treatment. Immunodepletion of ASM-LTD4-CM with anti-matrix metalloproteinase (MMP)-1 antibodies demonstrated a dose-dependent reduction of IGFBP proteolysis. Tissue inhibitor of MMP-1 and Batimastat (synthetic) inhibited proteolysis of IGFBP. Immunoblotting the ASM-LTD4-CM with anti-MMP-1 demonstrated a dose-dependent increase in MMP-1 protein. Similar results were also obtained by immunocytochemistry. Collectively, these observations demonstrate that MMP-1 is an IGFBP protease induced by leukotrienes that plays a significant role in modulating IGF action in ASM cells. A similar mechanism may be applicable in vivo in the airways of patients with asthma.
Insights
Leukotriene D4 (LTD4) induces matrix metalloproteinase-1 (MMP-1) in airway smooth muscle cells, which degrades insulin-like growth factor-binding proteins (IGFBPs). This process enhances insulin-like growth factor (IGF) action, potentially contributing to asthma.
Area of Science:
- Cellular and Molecular Biology
- Respiratory Medicine
- Biochemistry
Background:
- Leukotriene D4 (LTD4), an asthma-associated eicosanoid, acts synergistically with insulin-like growth factors (IGFs) to promote airway smooth muscle (ASM) cell growth.
- This synergy involves the breakdown of inhibitory IGF-binding proteins (IGFBPs) produced by ASM cells.
Purpose of the Study:
- To investigate the mechanism by which LTD4 affects IGFBP levels in human ASM cells.
- To identify the specific protease responsible for IGFBP degradation induced by LTD4.
- To elucidate the role of matrix metalloproteinases (MMPs) in this process.
Main Methods:
- Analysis of conditioned media from LTD4-treated human ASM cells using Western ligand blotting and Western immunoblotting.
- Incubation experiments with 125I-labeled IGFBP to detect protease activity.
- Immunodepletion assays using anti-matrix metalloproteinase (MMP)-1 antibodies.
- Inhibition studies using tissue inhibitor of MMP-1 and Batimastat.
- Immunoblotting and immunocytochemistry to detect MMP-1 protein levels.
Main Results:
- LTD4 treatment significantly reduced intact IGFBP levels (predominantly IGFBP-2) in ASM cell conditioned media, with IGFBP-2 appearing as lower-molecular-weight fragments.
- LTD4 induced an IGFBP protease in ASM cells.
- MMP-1 was identified as the induced IGFBP protease; its activity was dose-dependently reduced by anti-MMP-1 antibodies and inhibited by MMP inhibitors.
- LTD4 treatment led to a dose-dependent increase in MMP-1 protein levels in ASM cells.
Conclusions:
- Matrix metalloproteinase-1 (MMP-1) is an IGFBP protease induced by leukotrienes (LTD4) in airway smooth muscle cells.
- MMP-1 plays a significant role in modulating IGF action in ASM cells by degrading IGFBPs.
- This MMP-1-mediated mechanism may be relevant in the airways of asthma patients in vivo.