Defining a mechanism for inner nuclear membrane protein SUN2's mechanical regulation of skin fibrosis

About This Grant

PROJECT SUMMARY/ABSTRACT In progression of skin fibrosis, the pathological stiffening of skin, both pro-fibrotic biochemical signaling and mechanical signals from the stiffening extracellular environment drive hyper-activated fibroblasts to secrete excess extracellular matrix (ECM) proteins. However, how these two inputs are integrated to drive fibrosis remains poorly understood, posing a barrier for treatments that can effectively reverse the fibrotic process. Our lab’s preliminary work provides new insight to this unanswered question, having identified a novel role for SUN2, an inner nuclear membrane protein, in driving fibrosis, potentially by regulating both mechanical and biochemical signaling. We have found that SUN2 responds to fibrotic stimuli in vivo, increasing in fibrotic patient and mouse skin samples, and in fibroblasts cultured on stiff substrates modeling fibrotic tissue. Additionally, depleting SUN2 decreases expression of pro-fibrotic TGFβ pathway target genes necessary for driving excess ECM deposition, as well as chromatin accessibility at the promoters of these genes. In vivo, loss of SUN2 is protective against injury-induced skin fibrosis. Given SUN2’s well-characterized function as a Linker of Nucleoskeleton and Cytoskeleton (LINC) complex component, which transduces bi-directional force from the cytoskeleton to the nuclear interior, a compelling hypothesis is that mechanical signals from the stiffening, fibrotic ECM increases SUN2 levels at the nuclear envelope, which amplifies force transduction to the nucleus necessary for upregulation of pro-fibrotic TGFβ target genes. The goal of this proposal is to investigate this hypothesis and define a molecular mechanism for how mechanical signals alter the composition and function of the nuclear envelope to regulate pro-fibrotic biochemical signaling. To accomplish this, I will first recapitulate the mechanical conditions of skin fibrosis in vitro, culturing dermal fibroblasts on substrates of relevant stiffnesses. Then, I will define how ECM stiffness regulates SUN2 levels by performing a series of RT-qPCR and cycloheximide chase experiments, utilizing inhibitors and a CRISPR Cas9 approach to disrupt cellular degradation pathways and further probe this mechanism. To determine how biological SUN2 levels alter the mechanical environment of the nucleus I will perturb SUN2 expression via siRNA or alteration of SUN2’s phosphorylation state and utilize our lab’s Nesprin molecular tension sensor, created to measure tension on LINC complexes. Lastly, to identify how SUN2 regulates pro-fibrotic TGFβ target gene expression in response to ECM stiffness, I will perform various genomics experiments including ATAC-Seq, RNA-Seq, and CUT&RUN to determine how ECM stiffness alters chromatin accessibility and gene expression of TGFβ target genes, and the activity of TGFβ pathway effectors SMAD 2/3. This proposal will address fundamental aspects of cell biology and mechanobiology and will define a novel mechanism for how mechanical signaling and biochemical signaling are integrated to drive fibrosis. Additionally, successful completion of this proposal will provide new therapeutic targets for reversing the fibrotic process in skin.