Idiopathic pulmonary fibrosis (IPF) is a fatal scarring of the lungs and has no available medical therapies. Recently, attention has focused on the role of pulmonary fibroblasts, differentiated into contractile, highly synthetic myo-fibroblasts, in the pathological progression of IPF. In vivo, myo-fibroblasts have been linked to active areas of disease in IPF (""""""""fibroblastic foci"""""""") and TGF? signaling has been shown to be critical in myofibroblastic differentiation. It has been previously demonstrated that the extra-cellular matrix (ECM) regulates the cell's cytoskeleton through its underlying mechanical properties and that this tension can be transmitted to the cell's nucleus. This proposal focuses on a novel hypothesis: cytoskeletal tension transmits fibrotic substrate stiffness to the nuclear membrane and potentiates transcription factor activity by disrupting its interactions with components of the nuclear membrane. Previously, the down-stream effectors of TGF? signaling, r-Smads, have been shown to physically associate with MAN1, an inner nuclear membrane integral protein and this complex antagonizes TGF? signaling. TGF? has been shown in vitro to be a central signaling axis in IPF and has pleiotropic disease-state consequences, including myo-fibroblastic differentiation. The effects of mechanical forces on this r-Smad/MAN1 complex have not been characterized. This proposal will develop a model system to investigate the nuclear membrane deformation associated with the underlying substrate stiffness using innovating genetic tools to disrupt the physical connection between the nuclear membrane and the cytoskeleton. State-of-the-art physical measurements of this system will be conducted using atomic force microscopy and an active micro-rheological technique based on optical trapping technology. Additionally, it will investigate the effects of this deformation on TGF?/rSmad signaling by using proximity ligation assays to look at the stability and spatial localization on the nuclear membrane of the rSmad/MAN1 complex under stress, and genetic and functional assays to measure TGF? signaling. Finally, it will validate the role of substrate stiffness in regulating the r-Smad/MAN1 complex at the nuclear membrane in an advanced, ex vivo model of IPF. This work helps address a possible novel mechanism of the pathobiology of fibrotic disease in the lung. The results of this study should have translational significance by focusing clinical attention on nove pro-fibrotic mechanisms that could form the basis of new therapies for IPF. Additionally, this model of nuclear stiffness as a regulator of transcription factor activity may help inform biomedical research into other disease states, such as cancer/tumor biology and other fibro-proliferative disorders, and development of biomaterial technologies for regenerative medicine approaches. This proposal serves as the lynchpin of a training plan for the applicant and will be the vehicle for imparting technical, scientific and professional skills necessary for his development as a physician-scientist investigating fibrotic diseases.
The goal of this project is to understand how the interactions between a cell and a pathologically stiff environment contribute to the development of fibrotic disease in the lung. Specifically, this proposal focuses on the role of substrate stiffness in mediating mechanical strain on the nucleus and the consequences of that strain on transcription factors thought to be important in pulmonary fibrosis. Understanding the importance of this mechanical connection to the pathology of pulmonary fibrosis will be important in developing new therapies for this devastating condition.
|Chambers, Dwight M; Moretti, Leandro; Zhang, Jennifer J et al. (2018) LEM domain-containing protein 3 antagonizes TGF?-SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol. J Biol Chem 293:15867-15886|