Uniformed Services University of the Health Sciences
4301 Jones Bridge Road
Bethesda, Maryland 20814
Phone Office: (301) 295 3587
Phone Lab: (301) 295 9415
Chronic diseases and even aging itself are known to damage the body by dys-regulated inflammatory processes. Dysregulated expression of the pro-inflammatory cytokine and chemokine genes including interleukin-8 (IL-8) are known to contribute to chronic inflammatory diseases. The expressions of such genes are known to be regulated by post-transcriptional mechanisms. Recently, microRNAs (miRNAs) have been proven to be key post-transcriptional regulators of gene expression by directing their target mRNAs towards degradation and/or translational repression. The mis-regulation of specific miRNAs has been demonstrated in a variety of diseases in humans including cancer, heart disease and diabetes. Since each miRNA governs the expression of multiple genes, the most effective way to overcome the effects of a mis-regulated miRNA is to modulate its expression in diseased cells. Thus, miRNAs have emerged as important therapeutic targets in the frontier of biomedical research. Recent studies indicate that even a single miRNA can induce a therapeutic response in an animal model of disease. My laboratory is currently focused on studying mechanisms specific for the pro-inflammatory disease phenotype in Cystic Fibrosis as well as general mechanisms that cause dys-regulation of inflammation, a phenomenon characteristic of a milieu of diseases including cancer, cardiovascular diseases, and immune system disorders. My laboratory is involved in investigating such mechanisms.
Mechanism of Regulation of inflammation in Cystic Fibrosis: Cystic Fibrosis (CF) is the most common life limiting recessive disease in the U.S., and is due to mutations in the CFTR gene. CF mutations, of which the most common is F508-CFTR, cause a massive pro-inflammatory phenotype in the lung, which is characterized by high levels of interleukin-8 (IL-8). The disease phenotype seems to be intrinsic to the CF condition, since fetal CF lung epithelium secretes massive levels of IL-8, in vivo, even in the absence of detectable infection. The problem is that the mechanism by which IL-8 gene expression is dysregulated in CF is not known. However, we have shown that CF lung epithelial cells in culture not only secrete large amounts of IL-8 protein, but also have high levels of very stable IL-8 mRNA. Thus, for a reason yet to be elucidated, IL-8 mRNA is degraded very slowly in CF lung epithelia. On this basis, our approach has been to investigate the mechanism by which IL-8 mRNA is rendered aberrantly stable in CF lung epithelial cells. The rationale is that understanding this dysfunctional regulatory mechanism may lead to more focused anti-inflammatory strategies for CF therapy.
Recently, a novel class of endogenous non-coding RNA molecules known as microRNAs (miRNAs) has emerged as important targets in the frontier of biomedical research. These small ~22 nucleotides long RNAs have been proven to be key regulators of gene expression by directing their target mRNAs towards degradation and/or translational repression. The mis-regulation of specific miRNAs has been demonstrated in a variety of diseases in humans including cancer, heart disease and diabetes. CF lung epithelial cells in culture exhibit mis-expression of specific miRNAs, including miR-155, compared to control cells, both in culture and in ex vivo bronchial biopsies of CF patients. The failure of CFTR channel activity, either by chemical inhibition or by mutation, results in aberrantly enhanced expression of miR-155.
Regulation of microRNA biogenesis and function in inflammation: The goal of this project is to determine the mechanism by which biogenesis and function of miR-155 is regulated by inflammation and how miR-155 is a potent inducer of inflammation. The function and expression of mature miRNAs is controlled by mechanisms that regulate the processing of primary (pri-) and precursor (pre-) miRNAs. Our objective is to investigate the mechanisms of alterations in processing of miR-155 and how that leads to its aberrant expression and dysfunction in inflammatory response. We are using two model systems; pro-inflammatory CF lung epithelial cells characterized by hyper-expression of the pro-inflammatory chemokine interleukin-8 (IL-8), as well as macrophage cells stimulated with inflammatory stimuli. We have found significant elevated expression of miR-155 in CF cells compared to controls and uncovered enhanced processing of the miR-155 precursor as the basis for this increase. Roberto Gherzi's laboratory (Italy) has shown increased expression of miR-155 in LPS-stimulated macrophages. Furthermore, we found that CF cells express negligible endogenous levels of the inflammation-associated RNA-binding protein (RBP) Tristetraprolin (TTP), which accelerates mRNA degradation through recognition of adenine and uridine (AU)-rich sequences. Moreover, over-expression of TTP not only suppresses IL-8 expression through destabilization of IL-8 mRNA but also inhibits miR-155 processing. It has also been shown that KH-type splicing regulatory protein (KSRP), another AU-interacting RBP associated with inflammation, also enhances miR-155 processing in CF cells. Therefore, expression and function of miR-155 is likely to be regulated by the inflammatory RBPs, TTP and KSRP, and that reciprocally miR-155 is also a potent inducer of inflammatory genes. We are in the processing of determining (i) the mechanism by which KSRP enhances miR-155 processing, (ii) how the AU-binding protein TTP regulates miR-155 expression, and (iii) how miR-155 regulates the pro-inflammatory IL-8 gene expression.
Utilizing an innovative combination of in vitro techniques for mapping RNA secondary structures, cell culture-based assays, immunoprecipitation as well as in vivo affinity purification techniques, this project is targeted towards providing new insights into miRNA biogenesis and highlight how variations in these maturation pathways might underlie select human diseases.