Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin

doi:10.1016/j.biocel.2007.06.022

The International Journal of Biochemistry & Cell Biology

Volume 39, Issue 12, 2007, Pages 2324-2338

https://doi.org/10.1016/j.biocel.2007.06.022 Get rights and content

Abstract

Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.

Introduction

Pulmonary fibrosis is the final consequence of a heterogeneous group of disorders known as interstitial lung diseases (Pardo & Selman, 2002). Lung fibrotic remodeling is characterized by fibroblast/myofibroblast activation and unbalanced extracellular matrix accumulation resulting in extensive structural disorganization of the lung. The irreversible changes in the lung architecture result in progressive organ dysfunction and usually a fatal outcome.

Several studies have shown that matrix metalloproteases (MMPs) play an important role in the pathogenesis of pulmonary fibrosis, and the presence of several of these proteases even in the advanced stages of fibrosis highlights the dynamic nature of scarring within the lung. However, the precise role of MMPs in the molecular mechanisms that characterize the fibrotic response is not completely understood (Pardo & Selman, 2006).

MMPs are a family of peptidases with multiple substrate affinities. They are not only collectively capable of cleaving all components of ECM but can also process bioactive mediators, such as growth factors, cytokines, chemokines, and cell-surface receptors (Nagase, Visse, & Murphy, 2006).

From the 23 members of the human MMPs family, MMP9 (gelatinase B), is one of the enzymes shown to be elevated in several human and experimental interstitial lung diseases (Fukuda, Ishizaki, Kudoh, Kitaichi, & Yamanaka, 1998; Hayashi et al., 1996, Pardo et al., 2000, Pérez-Ramos et al., 1999, Selman et al., 2000; Yaguchi, Fukuda, Ishizaki, & Yamanaka, 1998). In idiopathic pulmonary fibrosis (IPF) MMP9 is highly expressed in the lungs and BAL fluids and localized mainly to epithelial cells, neutrophils, and alveolar macrophages (Selman et al., 2000, Suga et al., 2000).

Experimental models of lung fibrosis, including those induced by paraquat plus hyperoxia, silica inhalation, or bleomycin instillation, have also exhibited an increase in MMP9 activity which has been associated with the disruption of the alveolar epithelial basement membrane (Cisneros-Lira, Gaxiola, Ramos, Selman, & Pardo, 2003; Pardo et al., 2003, Ruiz et al., 2003).

However, despite the putative profibrotic role of MMP9 in lung injury, MMP9 null mice develop similar fibrosis to wild-type littermates, after bleomycin instillation, although the lungs of the MMP9 deficient mice showed minimal alveolar bronchiolization (Betsuyaku, Fukuda, Parks, Shipley, & Senior, 2000).

In order to further understand the role of MMP9 in lung fibrosis, we have analyzed the effect of MMP9 overexpression in bleomycin driven lung fibrosis, using transgenic mice (TG) expressing human MMP9 in macrophages (hMMP9 TG), and following the model for 16 weeks. Our results indicate that hMMP9 TG mice develop reduced lung fibrosis compared to wild-type mice and suggest a possible mechanism related to insulin-like growth factor binding protein-3 cleavage by MMP9.

Section snippets

Generation of transgenic mice

Transgenic mice expressing human MMP9 were generated as follows. The 2.4 kb cDNA of human MMP9 (a gift from G. Goldberg, Washington University) was cloned into a vector containing the splice site and polyadenylation sequence of the β-globin gene, and placed under the control of the Scavenger Receptor A enhancer-promoter (SREP; a gift from Dr. Glass, UC, San Diego), a promoter that allows targeted expression of genes specifically in differentiated macrophages of transgenic mice (Lemaitre et al.,

Evidence of human MMP9 in transgenic mice

Levels of mouse and human MMP9 gene expression were assessed by quantitative real-time PCR in total RNA isolated from lungs of bleomycin or saline-treated mice. Mouse MMP9 mRNA was significantly increased at 1, 4 and 8 weeks after bleomycin as compared with saline-treated controls in both hMMP9 TG and WT mice (p < 0.05; Fig. 1A). No significant difference in the gene expression level was found between the TG and WT mice at any time point. Confirming the genotype of the hMMP9 TG mice, human MMP9

Discussion

In the present study we analyzed the effect of the overexpression of MMP9 a metalloprotease mediating diverse biological functions on bleomycin induced pulmonary fibrosis. Following the model for 4 months, we demonstrated that overexpression of MMP9 in macrophages induces a retarded and diminished lung fibrotic reaction. This effect was preceded by a decrease in neutrophil infiltration at 1 week, and decreased lymphocytes at 1 month post-bleomycin treatment. Interestingly, a lower level of

Acknowledgements

This work was supported by a grant from CONACYT 41043-M and by Universidad Nacional Autonoma de Mexico SDI.PTID.05.6. S.C. was funded by CONACYT.

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^☆: This work was submitted in partial fulfillment of the requirements to obtain the PhD degree for S.C. at Universidad Nacional Autonoma de Mexico.

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Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin☆

Abstract

Introduction

Section snippets

Generation of transgenic mice

Evidence of human MMP9 in transgenic mice

Discussion

Acknowledgements

Am. J. Pathol.

Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

J. Biol. Chem.

Blood

Arch. Biochem. Biophys.

Am. J. Pathol.

Enhanced insulin-like growth factor molecules in idiopathic pulmonary fibrosis

Am. J. Respir. Crit. Care Med.

Matrix metalloproteinase-9 in lung remodeling

Am. J. Respir. Cell Mol. Biol.

Cytokine-binding proteins in the lung

Am. J. Physiol.

Cigarrete smoke exposure potentiates bleomycin-induced lung fibrosis in guinea pigs

Am. J. Physiol. Lung Cell Mol. Physiol.

Matrix metalloproteinases in cancer: From new functions to improved inhibition strategies

Int. J. Dev. Biol.

Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures

J. Biol. Chem.

Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases

Lab. Invest.

Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis

Exp. Lung Res.

Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis

Am. J. Pathol.

Insulin-like growth factor binding protein-5 is a target of matrix metalloproteinase-7: Implications for epithelial-mesenchymal signaling

Cancer Res.

Insulin-like growth factors and their binding proteins: Biological actions

Endocrinol. Rev.

Tissue inhibitor of metalloproteinase-1 deficiency amplifies acute lung injury in bleomycin-exposed mice

Am. J. Respir. Cell Mol. Biol.

Differences in the fibrogenic response after transfer of active transforming growth factor-beta1 gene to lungs of “fibrosis-prone” and “fibrosis-resistant” mouse strains

Am. J. Respir. Cell Mol. Biol.