The Role of Krüppel Like Factor 4 in Idiopathic Pulmonary Fibrosis




Grimsley, John Robert

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Idiopathic Pulmonary Fibrosis (IPF) is a fatal interstitial lung disease (ILD) with no known cause and is characterized by a progressive build up fibrotic tissue in the diseased lung. Krüppel-Like Factor 4 (KLF4), a transcription factor with key roles in the cell cycle, cellular differentiation and development, was found to be over-expressed in primary IPF fibroblasts. In this study we sought to investigate the potential role of KLF4 in IPF by investigating the effect of its over-expression on fibroblast differentiation and proliferation status in the normal human pulmonary fibroblasts cell line, MRC5. In addition, we investigated the localization of KLF4 in vivo using normal and IPF tissue (LTRC). The in vivo localization of KLF4 in IPF and normal tissue was confirmed by double-immunohistochemistry (IHC) in combination with either alpha-SMA or PCNA (Abcam). KLF4 over-expression in MRC5 cells was achieved using a doxycyclineinducible lentiviral system (Addgene) and confirmed by quantitative real time PCR (QRTPCR) and western blot. The effect of KLF4 over-expression on markers of proliferation and activation was monitored by Q-RTPCR. KLF4 in the IPF lung was localized on the perimeter of the fibroblastic foci and in the parenchyma in areas of advanced fibrosis, however, it absent with in the fibrotic foci. Co-localization of KLF4 and PCNA was observed, however, alpha-SMA and KLF4 were not observed to colocalize. Following KLF4 induction in vitro a 6-fold increase in KLF4 mRNA and a 3-fold increase in protein were observed. A decrease (2-fold) in alpha-SMA expression at both the gene and protein level was detected. A 3-fold increase in collagen 1A1 (3-fold) mRNA was observed, but of all the selected proliferation markers (PCNA, cyclin D1 and p21 and p53) decreased. The in vivo distribution of KLF4, suggests that it may be involved in the advancement of fibrosis and expansion of the foci. In vitro overexpression studies in MRC5 cells revealed a profoundly negative effect on the cell cycle, which appears to be in contrast to IPF, however, in keeping with the dual roles of KLF4 as both a tumor suppressor and tumor promoter. Of particular interest was an increase in collagen production, the main ECM component of IPF, as the the result of KLF4 overexpression. KLF4 operated in a strongly context dependent manner, and IPF is a particularly context-driven disease where the ECM acts almost as another pathogenic entity. Therefore, while direct comparison between in vivo IPF fibroblasts and in vitro MRC5 cells cannot be made, these data indicate a potential role for KLF4 in the pathogenesis of IPF. Further characterization of this role through experimentation in primary adult IPF fibroblasts is warranted.



Idiopathic Polmoneory Fibrosis, Fibrosis, KLFY, Fibroblust, Kruppel