MMP-1 is important in the pathogenesis of COPD with increased expression being a common finding in patients with this disease
[5, 6, 15]. Animal studies have shown that MMP-1 plays a key role in the initiation of the disease process
. Previous studies from our laboratory have identified the distal MMP-1 promoter to be fundamental for the direct activation of the MMP-1 promoter by cigarette smoke
. Studies of genetic variants in the MMP-1 promoter are important because identification of functional polymorphisms will lead to a greater understanding of the regulatory mechanisms involved in both health and disease, and may provide useful knowledge for identifying at-risk individuals to allow for early interventions.
This present study generated the following three important findings. First, the genetic variants in the MMP-1 cigarette smoke-responsive element were further characterized and four novel polymorphisms identified, as well as the confirmation of six previously reported genetic variants. Second, an important interaction between Sp1 binding and the MMP-1 cigarette smoke-responsive element in human small airway epithelial cells exposed to cigarette smoke extract was documented. Sp1 protein binding was influenced by cigarette smoke and this DNA-protein interaction occurred specifically in the cigarette-smoke responsive element and did not appear to occur outside of the core regulatory region. Third, polymorphisms in the cigarette smoke-responsive element were not more prevalent in subjects who had an accelerated decline in lung function over five-years of the Lung Health Study.
MMP-1 is tightly regulated at the level of transcription, post-transcription, and post-translation; it is also known that MMP-1 expression is influenced by genetic variants in the promoter
. In the cigarette smoke-responsive element spanning approximately 1.5 kb, a total of ten polymorphisms were identified via re-sequencing. Four novel polymorphisms were revealed. These data are important because they allow researchers to further analyze the contribution of genetic polymorphisms to the development and progression of multiple diseases associated with MMP-1 expression. In addition to the established role of MMP-1 in COPD, elevated expression of MMP-1 has been associated with a variety of pathological conditions such as rheumatoid and osteoarthritis, atherosclerosis, Alzheimer disease, and cancer
. There is a functional polymorphism in the MMP-1 promoter (rs1799750) described by Rutter et al. that has been associated with numerous malignant processes
. Further understanding of the genetic variants in the MMP-1 promoter, and more specifically the cigarette smoke-responsive element, may provide additional insights into the molecular mechanisms of these diseases and help define the role of smoking as a risk factor.
Understanding the molecular mechanisms controlling MMP-1 expression may identify potential targets for the prevention of diseases and degenerative conditions associated with dysregulation of MMP-1. The first cis-promoter element found to be responsible for regulating MMP-1 gene expression was AP-1
. Previous studies by our laboratory have identified a number of transcription factors, including AP-1, that are important for regulating MMP-1 promoter activity
. Further analysis in the present study revealed two Sp1 sites (−3987 and −3455) in the cigarette smoke-responsive element that have now been analyzed using a ChIP assay to assess the functional importance of these sites in vitro. Our data demonstrates that these are functional binding sites and that binding of Sp1 to the MMP-1 promoter is influenced by cigarette smoke. This is in contrast to the decreased binding of Sp1 to the MMP-1 promoter outside of the cigarette smoke responsive-element in response to cigarette smoke exposure. Not only are these sites functional, they also occur in a region harbouring a number of common SNPs. Although the genetic variants identified did not directly alter the primary transcription factor binding sites we assessed, they may influence the binding of chromatin modifying proteins resulting in a measurable functional effect on promoter activity. Unfortunately, this study is limited by the fact that susceptibility to COPD (i.e. accelerated decline in lung function with smoking) and Sp1 binding to the MMP-1 gene were not measured in the same individuals since the appropriate cells were not available from the LHS participants. However, the findings that the SNPs identified do not directly alter the Sp1 bindings sites at the DNA level and are not associated with rapid decline in lung function are interesting and potentially important. It may be that since Sp1 plays a significant role regulating MMP-1 expression these sites are conserved. These negative results do suggest that the SNPs are not a risk for rapid decline in lung function among smokers.
COPD is a chronic disease of complex phenotype
[20, 21]. There is even speculation that COPD represents a syndrome comprised of many rare diseases
. In the present association study the relationship between polymorphisms in the cigarette smoke-responsive element and one COPD phenotype, rate of decline in lung function, was tested. No significant associations were found despite the sufficient power of the study. Such a finding however is not surprising since the results of the basic studies suggest that MMP-1 expression is critical in the pathogenesis of lung destruction and generation of the emphysema phenotype
. It will be necessary in future detailed human studies to test for an association of the cigarette smoke-responsive element polymorphisms with CT evidence of lung destruction. Unfortunately, CT data was not available on the LHS participants and therefore we were unable to investigate this further in our population. Additionally, our lab has demonstrated that cigarette smoke directly induces the expression of MMP-1 highlighting the importance of MMP-1 in the susceptibility to lung destruction in smokers
. All LHS participants exhibit some degree of airflow limitation, indicating that they are already responsive to the damaging effects of cigarette smoke. Therefore, to test the importance of this element in disease susceptibility it will be necessary to assess these genetic variants in alternative well-characterized cohorts of smokers with and without COPD, as well as normal non-smokers.
We acknowledge several limitations to the present study in addition to the issues outlined above. Although an association between the MMP-1 polymorphisms and rate of decline in lung function was not identified, these polymorphisms may influence other COPD-related phenotypes. We know that lung function normally declines as part of the aging process, while accelerated lung function decline is a hallmark of COPD
[23, 24], therefore, the use of this phenotype seems reasonable for genetic association studies and decline in lung function over time has been a common end point in clinical trials of COPD therapies
[25, 26]. However despite the rigor of the lung function phenotyping in the LHS, the five-year period of follow-up may not be sufficient to clearly separate groups whose decline in lung function varied significantly over time. Indeed, the use of this single COPD phenotype and a short follow-up period are limitations of this study. On the other hand our group has previously identified susceptibility variants in candidate genes using this approach
[27, 28]. The association study was limited to non-Hispanic white subjects but the genetic basis of COPD may differ between racial/ethnic groups as several COPD association studies have shown distinct results within different races. Indeed, it is known that MMP-1 polymorphisms are associated with diseases such as cancer in other ethnic groups
[29, 30]. Therefore, it will be of interest to further investigate the role of these polymorphisms in alternative racial/ethnic populations. Future studies may also include haplotype analysis to fully characterize the genetic architecture of this region.