Cell Specific Matrix Metalloproteinase-1 Regulates Lung Metastasis Synergistically with Smoke Exposure.

MMP1, a matrix metalloproteinase that degrades the extracellular matrix, is produced not only by cancer cells but also synthesized in stromal and inflammatory cells during tumorigenesis, invasion and lung metastasis. However, the function of MMP1 expression from host cells, especially tumor-associated macrophages (TAMs), and cells in the lung parenchyma remains to be elucidated. Here we demonstrate that in vitro macrophages co-cultured with tumor cells drastically enhance MMP1 expression, which is further exacerbated upon cigarette smoke exposure. In addition, in vivo, macrophage specific MMP1 was found to have a causative role in primary tumor development and lung metastasis, which was enhanced under smoke exposure as demonstrated in a transgenic mouse model that expressed human MMP1 specifically in macrophages (Mac-MMP1). In contrast, MMP1 from lung cells (Lung-MMP1) reduced colonization to the lung despite the fact that collagen deposition decreased in the Lung-MMP1 mouse tumors. These results demonstrate that the varying cellular source of MMP1 in tumors leads to the complexity observed in the tumor microenvironment. Furthermore, macrophage-specific inhibition of MMP1 secretion may be a potential therapy to aid in the reduction of lung metastasis.

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production.

BACKGROUND: Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood. METHODS: Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT-PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied. RESULTS: Lung metastases of B16BL6 cells were significantly higher by 2.5-5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin. CONCLUSION: Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.

Correlation of lung surface area to apoptosis and proliferation in human emphysema.

Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function. To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area. In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.

The role of collagenase in emphysema.

The extracellular matrix is essential for the integrity of the lung and when disrupted can lead to the architectural changes seen in emphysema. The etiology of emphysema is believed to be due to an imbalance in the proteases and antiproteases within the lung. Studies have focused on elastolytic enzymes as the primary agents in disease pathogenesis, however, recent data suggest that collagenases may also be involved in the destruction of lung tissue in emphysema. It is hoped that this expanded understanding of the pathophysiology of emphysema will lead to improved therapy in the treatment of the disease.

ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis.

Matrix metalloproteinase-1 (MMP-1), or interstitial collagenase, has been hypothesized to contribute to the progression of the human atherosclerotic lesions by digesting the fibrillar collagens of the neointimal ECM. The apolipoprotein E knockout (apoE0) mouse model develops complex atherosclerotic lesions, but mice do not possess a homologue for MMP-1. To provide an in vivo evaluation of the role of MMP-1 in atherogenesis, we created a transgenic mouse model that expresses this enzyme specifically in the macrophage, under the control of the scavenger receptor A (SCAV) enhancer/promoter. The MMP-1 transgenic mice were crossed into the apoE0 background and fed an atherogenic diet for 16-25 weeks. Surprisingly, the transgenic mice demonstrated decreased lesion size compared with control littermates. The lesions of the transgenic animals were less extensive and immature, with fewer cellular layers and a diminished content of fibrillar collagen. There was no evidence of plaque rupture. Our data suggest that remodeling of the neointimal extracellular matrix by MMP-1 is beneficial in the progression of lesions.

Human collagenase (matrix metalloproteinase-1) expression in the lungs of patients with emphysema.

Pulmonary emphysema is believed to result from an imbalance between proteolytic enzymes and their inhibitors. Multiple studies have examined the presence of various proteases within the bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease (COPD). However, to date extensive examination of the lung parenchyma for the expression of destructive enzymes has not yet been determined. The following study examines the lung parenchyma of 23 patients with emphysema and 8 normal control samples for the expression of matrix matalloproteinase-1 (MMP-1), MMP-12, and MMP-9. We report here that interstitial collagenase (MMP-1) RNA, protein, and activity are present in the lung parenchyma of patients with emphysema and not in the lung of normal control subjects. In contrast, metalloelastase (MMP-12) expression is absent in these samples. Immunohistochemistry studies localized MMP-1 to the Type II pneumocyte in patients with emphysema and not normal control subjects or smokers without emphysema. This observation demonstrates that the lung is altered in emphysema such that the Type II pneumocyte secretes MMP-1 and suggests that MMP-1 may be an important enzyme involved in the destruction of the lung in the human disease. In addition, the induction of a proteolytic enzyme within the Type II pneumocyte suggests that the cells within the lung itself are capable of producing degradative enzymes in this disease process.

Disruption of the myocardial extracellular matrix leads to cardiac dysfunction.

MMP activity with disruption of structural collagen has been implicated in the pathophysiology of dilated cardiomyopathy. To examine the role of this enzyme in cardiac function, a transgenic mouse was created that constitutively expressed human collagenase (MMP-1) in the heart. At 6 months of age, these animals demonstrated compensatory myocyte hypertrophy with an increase in the cardiac collagen concentration due to elevated transcription of type III collagen. Chronic myocardial expression of MMP-1 produced loss of cardiac interstitial collagen coincident with a marked deterioration of systolic and diastolic function at 12 months of age. This is the first animal model demonstrating that direct disruption of the extracellular matrix in the heart reproduces the changes observed in the progression of human heart failure.

Collagenase induction promotes mouse tumorigenesis by two independent pathways.

Although progress has been made in the understanding of the role of metalloproteinases in tumor progression during metastasis, little is known about their contributions, if any, to tumor formation. Accumulating evidence identified an increased presence of several matrix metalloproteinases in human cancers, but the precise role for interstitial collagenase in tumor formation or progression has not been well defined. Transient induction of collagenase was observed in wild-type mouse skin after treatment with the tumor-promoting agents 12-O-tetradecanoylphorbol-13-acetate (TPA) and chrysarobin, which promote tumorigenesis through protein kinase C-dependent and -independent pathways, respectively. Transgenic mice that constitutively express interstitial collagenase within the epidermis of the skin have an increased susceptibility to tumorigenesis and produced tumors at lower doses of TPA as compared with wild-type mice. Similarly, the transgenic mice showed increased tumorigenesis when promoted with chrysarobin. These results demonstrate that collagenase overexpression can contribute to tumorigenesis via protein kinase C-dependent and -independent pathways. Significantly, compared with wild-type mice, the transgenic mice demonstrated an elevated expression of c-fos in the skin at baseline, before tumor promotion, suggesting a molecular mechanism for the increased tumor susceptibility in collagenase transgenic mice. These findings further support the importance of MMP deregulation in tumorigenesis and suggest that the role of MMP family members is not limited to metastasis but may also contribute to initial tumor development.

Epidermal expression of collagenase delays wound-healing in transgenic mice.

A vital characteristic of skin is its ability for wound repair in response to injury. A transient elevation of matrix metalloproteinases (MMP) in the epidermal and dermal compartments of healing wounds implicates the MMP family of enzymes in the regulation of events important to injury repair. Transgenic mice expressing human interstitial collagenase (MMP-1) in the epidermis were used to perturb the regulation of this proteinase in order to examine the role of epidermal collagenase during wound healing. The relative healing potential of collagenase transgenic mice and wild-type littermates was assessed by measurements of the wound area during closure of full-thickness wounds. Transgenic mice exhibited a 2-3 d delay in the time required to reach 50% closure of 6 mm wounds. Histologic analysis of the transgenic wound bed revealed the retarded migration of the epithelium across the open wound. The results are consistent with the hypothesis that control of collagenase (MMP-1) expression is important for re-epithelialization during wound healing and indicate that collagenase regulation is critical to the kinetics of normal wound closure.

Expression of plasma phospholipid transfer protein mRNA in normal and emphysematous lungs and regulation by hypoxia.

The lung is the major site expressing plasma phospholipid transfer protein (PLTP) mRNA in humans and mice, suggesting that this protein might have an important role in maintaining normal function of this organ. In the lung of human collagenase transgenic mice, an emphysematous animal model, PLTP mRNA was 3-fold higher than in control mice. However, the mRNA in other tissues was not changed. To further assess the expression and function of PLTP, we measured PLTP mRNA level in lung tissue of two emphysematous patients and found that the mRNA was 4-fold higher than in control subjects. In situ hybridization on mouse lung suggested positive staining in alveolar type II epithelial cells. In addition, immortalized rat alveolar pre-type II epithelial cells and freshly isolated mature rat alveolar type II epithelial cells both highly expressed PLTP mRNA, and the former cells actively secreted PLTP activity into the medium. To examine the possible mechanisms leading to high levels of PLTP expression in vivo, we exposed the pre-type II cells to hypoxia and demonstrated induction of PLTP mRNA and a coordinate increase in secreted PLTP activity. Thus, the PLTP gene is highly expressed in alveolar type II epithelial cells and is induced during hypoxia and in emphysema. These observations suggest that a hypoxic stimulus occurring in emphysema may be a novel mechanism that contributes to enhanced expression of PLTP.

Tissue, temporal and inducible expression pattern of haptoglobin in mice.

Haptoglobin (Hp) is a member of the acute phase plasma proteins previously thought to be synthesized solely by the adult liver. The present study analyzes the tissue and temporal expression pattern of endogenous haptoglobin in the mouse and acute phase inducibility in various tissues. The liver is found to be the major site of haptoglobin expression but significant expression levels were also observed in the lung and skin. Acute phase induction by bacterial lipopolysaccharide (LPS) demonstrated that haptoglobin was induced not only in the liver but also in other tissues, including lung, skin, spleen, and kidney. Temporal analyses demonstrated that haptoglobin is expressed during embryogenesis in the liver and is inducible in various tissues surveyed throughout development. Transgenic mice that harbored a 1.05-kilobase (kb) region of the human haptoglobin promoter linked to two different reporter genes gave rise to lung-specific expression in the majority of transgenic lines with minimal liver expression. However, when induced with lipopolysaccharide, the 1.05-kb fragment contained the necessary elements for a response comparable to endogenous expression levels. In conclusion, these studies demonstrate that haptoglobin is not an adult liver specific gene, and its role as an acute phase reactant may well be more diverse than previously suspected.

Collagenase expression in transgenic mouse skin causes hyperkeratosis and acanthosis and increases susceptibility to tumorigenesis.

In a series of transgenic mice, the human tissue collagenase gene was expressed in the suprabasal layer of the skin epidermis. Visually, the mice had dry and scaly skin which upon histological analysis revealed acanthosis, hyperkeratosis, and epidermal hyperplasia. At the ultrastructural level, intercellular granular materials were absent in the transgenic skin epidermis but contact was maintained through the intact desmosomes. Despite a diversity of underlying etiologies, similar morphological hyperproliferative changes in the epidermis are observed in the human skin diseases of lamellar ichthyosis, atopic dermatitis, and psoriasis. Subsequent experiments demonstrate that when the transgenic mouse skin was treated once with an initiator (7,12-dimethyl-benz[a]anthracene) and then twice weekly with a promoter (12-O-tetradecanoylphorbol-13-acetate), there was a marked increase in tumor incidence among transgenic mice compared with that among control littermates. These experiments demonstrate that by overexpressing the highly specific proteolytic enzyme collagenase, a cascade of events leading to profound morphological changes which augment the sensitivity of the skin towards carcinogenesis is initiated in the epidermis.

Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.

Transgenic mice were generated that expressed a human collagenase transgene in their lungs under the direction of the haptoglobin promoter. Histological analysis demonstrated disruption of the alveolar walls and coalescence of the alveolar spaces with no evidence of fibrosis or inflammation. This pathology is strikingly similar to the morphological changes observed in human emphysema and therefore implicates interstitial collagenase as a possible etiological agent in the disease process. Although elastase has been proposed as the primary enzyme responsible for emphysematous lung damage, this study provides evidence that other extracellular matrix proteases could play a role in emphysema. In addition, these transgenic mice are a defined genetic animal model system to study the pathogenesis of emphysema.

Evolution of collagen IV genes from a 54-base pair exon: a role for introns in gene evolution.

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.