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Altered Expression of Developmental Gene Sine Oculis Homeobox Homolog 1 (Six1) in Idiopathic Pulmonary Fibrosis
Cory Wilson1, Tinne CJ Mertens1, Scott Collum1, Weizhen Bi1, Ashrith Guha2, Rajarajan A Thandavarayan2, Keshava Rajagopal3, Soma Jyothula3 and Harry Karmouty Quintana1, 1 Department of Biochemistry and Molecular Biology, McGove rn Medical School, UTHealth, 2 Houston Methodist Hospital, 3 Department of Internal Medicine McGovern Medical School, UTHealth
Idiopathic pulmonary fibrosis (IPF) is a spontaneously occurring chronic and irreversible lung disease. Treatment for IPF is extremely limited, eventually leading to the need for lung transplantation. The pathophysiology of lung fibrosis is not fully understood, but it is hypothesized to be driven by the loss of alveolar epithelial cell integrity, specifically the type II epithelial cells (AECII), that leads to altered repair and fibroproliferative injury. Recapitulation of developmental genes, like Sonic hedgehog (Shh), have been reported to play a role in the development of IPF, however the role these genes play in IPF is poorly understood.
In this project we have looked at the developmental gene Sine Oculis Homeobox Homolog 1 (Six1). Six1 is a transcription factor that is only normally expressed in utero and is essential for normal lung development and has also been shown to be aberrantly expressed in lung and breast cancer. We have preliminary data that demonstrate increased Six1 expression in the AECII of IPF patients with the hypothesis that increased Six1 expression levels in AECII modulates Shh expression to promote aberrant repair processes that contribute to lung fibrosis.
In order to study the role of Six1 in the epithelium, we utilized transgenic mice lacking Six1 in surfactant protein C (SPC) Cre expressing cells that target type II lung epithelial cells; termed Six1f/f SPC Cre. These mice were exposed to a chronic model of fibrosis using bleomycin (BLM) 0.035 U/g, intraperitoneal (IP) injections twice weekly for 4 weeks or PBS (control). Flash frozen and paraffin-embedded (FFPE) lung tissue was collected after surgery. Immunohistochemistry and histological analysis were performed to evaluate and identify markers of fibrosis. In vivo lung mechanics studies were performed using the forced oscillation technique using the Flexivent. Using human explant tissue from IPF patients, along with COPD patients and healthy controls, RT-qPCR and Western blots were performed to evaluate the expression profiles of Six1 in human disease.
We demonstrate a significant reduction in fibrosis in Six1f/f SPC Cre mice when compared to the control SPC Cre mice. These are consistent with improved in vivo lung function measured using the Flexivent, including increased static compliance and lowered tissue resistance in the Six1f/f SPC Cre mice compared to SPC Cre controls. We demonstrate a reduction in fibrosis and improved lung function in the Six1f/f SPC Cre mice when treated with delayed tamoxifen on day 15 after fibrosis has been established. The human IPF samples showed an increase in Six1 protein expression and transcript levels compared to both COPD and control samples.
These observations suggest that the developmental gene Six1 could play a novel role in the pathogenesis of pulmonary fibrosis and could provide a unique drug target for future development.