Epigenetic regulation of cyclooxygenase-2 expression in systemic sclerosis lung fibroblasts
Lead Research Organisation:
University College London
Department Name: Medicine
Abstract
Systemic sclerosis (SSc) is a chronic multi-system disease that has a significant effect on quality of life and leads to premature death. Uncontrolled scarring, termed fibrosis, is one of the major characteristic features of this disease and occurs in the lungs in up to 80% of individuals with SSc. Although treatment for some manifestations of SSc has improved, there is no effective therapy for lung fibrosis, other than transplantation, and lung disease is the major cause of death in SSc. Further studies are therefore needed to understand the mechanisms involved in the development and progression of lung fibrosis in SSc in order to identify and develop new effective therapies.
We have previously shown that lack of an enzyme called cyclooxygenase-2 (COX-2), plays important roles in the development of lung fibrosis associated with SSc. Recently we have found that this deficiency in COX-2 can be overcome by treating cells with drugs, called methyltransferase inhibitors and that this restores normal functions to the cells. These drugs block the chemical modification (methylation) of portions of DNA (genes) and some proteins in the cell nucleus, called histones, that can prevent production of proteins and enzymes like COX-2. Using a unique resource of cells obtained from the lungs of individuals with SSc, we aim to identify the mechanisms by which these methyltransferase inhibitors restore COX-2 production and normal cell function. Specifically we will determine whether this occurs through methylation of DNA or proteins and which genes or proteins are involved.
The discovery that COX-2 production and function in cells, obtained from the lungs of patients with SSc, can be restored by methyltransferase inhibitors is of particular importance since drugs of this type are currently in use for the treatment of some cancers. Therefore greater understanding of the mechanisms involved would provide a strong basis for the use of these drugs in clinical trials for the treatment of SSc.
We have previously shown that lack of an enzyme called cyclooxygenase-2 (COX-2), plays important roles in the development of lung fibrosis associated with SSc. Recently we have found that this deficiency in COX-2 can be overcome by treating cells with drugs, called methyltransferase inhibitors and that this restores normal functions to the cells. These drugs block the chemical modification (methylation) of portions of DNA (genes) and some proteins in the cell nucleus, called histones, that can prevent production of proteins and enzymes like COX-2. Using a unique resource of cells obtained from the lungs of individuals with SSc, we aim to identify the mechanisms by which these methyltransferase inhibitors restore COX-2 production and normal cell function. Specifically we will determine whether this occurs through methylation of DNA or proteins and which genes or proteins are involved.
The discovery that COX-2 production and function in cells, obtained from the lungs of patients with SSc, can be restored by methyltransferase inhibitors is of particular importance since drugs of this type are currently in use for the treatment of some cancers. Therefore greater understanding of the mechanisms involved would provide a strong basis for the use of these drugs in clinical trials for the treatment of SSc.
Technical Summary
Pulmonary fibrosis is a common manifestation of systemic sclerosis (SSc) that is associated with significant morbidity and mortality. Compelling evidence from our group and others demonstrates that failure to up-regulate cyclooxygenase-2 (COX-2) expression plays important roles in the pathogenesis of SSc and other lung fibroses. Our recent data demonstrates that methyltransferase inhibitors restore the responsiveness of SSc lung fibroblasts to inducers of COX-2, and normalisation of cell functions including prostaglandin E2 production and sensitivity to apoptosis. However, this does not associate with changes in COX-2 CpG methylation suggesting an indirect mechanism of methyltransferase inhibition. Additional preliminary data shows increased levels of DNA methyltransferases 1, 3a and 3b in SSc lung and DNA methyltransferase activity in SSc lung fibroblasts compared with controls, as well as, increased repressive methylhistone marks, including histone H3K9me2, both of which could be affected by methyltransferase inhibition. This indicates that DNA and/or histone methylation may play roles in limiting COX-2 expression.
We therefore aim to determine the mechanism/s by which methyltransferase inhibitors restore SSc lung fibroblast expression of COX-2 and cell function. More specifically, we will: (i) determine whether repressive histone methylation marks are associated with the limited capacity of SSc lung fibroblasts to express COX-2. Repressive methylhistone marks will be systematically investigated and those that are increased in SSc compared with controls and reduced by methyltransferase inhibitors will be assessed by ChIP-quantitative RT-PCR to determine their association with COX-2 expression; (ii) Identify and investigate the role of novel methylated genes in the regulation of fibroblast COX-2 expression. Genes that are hypermethylated in SSc lung fibroblasts and in which methylation is reduced by methyltransferase inhibitors will be identified using methylation arrays followed by characterisation of their ability to regulate COX-2 expression; (iii) elucidate changes in miRNA expression in SSc lung fibroblasts and investigate their role in the deregulation of COX-2 expression. Using expression arrays, miRNAs that exhibit low expression in SSc lung fibroblasts compared with controls and which increase following treatment with methyltransferase inhibitors will be identified and their role in regulating COX-2 expression determined using expression plasmids and siRNA.
Such studies will provide fundamental information on the regulation of cyclooxygenase-2 expression in SSc and, if successful, provide a strong rationale for clinical trials of methyltransferase inhibitors, currently used in other clinical conditions, in SSc. In addition, the methylation and miRNA profiling studies will likely identify novel targets for the investigation and potential treatment of SSc.
We therefore aim to determine the mechanism/s by which methyltransferase inhibitors restore SSc lung fibroblast expression of COX-2 and cell function. More specifically, we will: (i) determine whether repressive histone methylation marks are associated with the limited capacity of SSc lung fibroblasts to express COX-2. Repressive methylhistone marks will be systematically investigated and those that are increased in SSc compared with controls and reduced by methyltransferase inhibitors will be assessed by ChIP-quantitative RT-PCR to determine their association with COX-2 expression; (ii) Identify and investigate the role of novel methylated genes in the regulation of fibroblast COX-2 expression. Genes that are hypermethylated in SSc lung fibroblasts and in which methylation is reduced by methyltransferase inhibitors will be identified using methylation arrays followed by characterisation of their ability to regulate COX-2 expression; (iii) elucidate changes in miRNA expression in SSc lung fibroblasts and investigate their role in the deregulation of COX-2 expression. Using expression arrays, miRNAs that exhibit low expression in SSc lung fibroblasts compared with controls and which increase following treatment with methyltransferase inhibitors will be identified and their role in regulating COX-2 expression determined using expression plasmids and siRNA.
Such studies will provide fundamental information on the regulation of cyclooxygenase-2 expression in SSc and, if successful, provide a strong rationale for clinical trials of methyltransferase inhibitors, currently used in other clinical conditions, in SSc. In addition, the methylation and miRNA profiling studies will likely identify novel targets for the investigation and potential treatment of SSc.