ECM stiffness, mechanotransduction, and cell cycling

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SUMMARY Mechanobiology--how cells and tissues sense and respond to mechanical influences--is a rapidly growing field of increasing importance to the understanding of physiology and fibrosis-associated pathologies including cancer, lung and liver fibrosis, and especially cardiovascular disease. This application studies how cells sense and respond to mechanical cues contained within the stiffness of the extracellular matrix (ECM). Mechanical information in the ECM is relayed through integrin-adhesions and Rho family GTPases, but how these early signaling events drive cell fate and function remains poorly understood. Unraveling these connections is a major challenge in the field. We are addressing this gap in understanding by examining how changes in ECM stiffness are transduced into the signaling events that control cell cycling. By combining molecular analyses with cell culture on deformable substrata (hydrogels), we recently showed that focal adhesion kinase (FAK), p130Cas, and Rac comprise a discrete signaling module that functions as a positive regulator of stiffness-sensitive cyclin D1 expression and cell cycling into S phase. But signaling in non- transformed cells is rarely linear and uni-directional: negative regulation commonly complements positive signaling to provide tight control of fate. These negative signals and pathways are often not well understood, and this is certainly the case for stiffness-regulated mechanotransduction. We therefore used RNASeq to search for ways that cells might limit stiffness-sensing to prevent over-stimulation. This analysis identified the long noncoding RNA, MALAT1, as a novel negative regulator of stiffness-dependent cell cycling: MALAT1 stimulates entry into S phase, but ECM stiffness reduces the expression level of MALAT1. Curiously, stiffness- stimulated Rac activity mediates both the induction of cyclin D1 and the repression of MALAT1. We now propose to examine the relationships between ECM stiffness, MALAT1 and cyclin D1, and their upstream activators. Aim 1 will examine the impact of MALAT1 on the G1 phase cyclin-cdks, assess crosstalk between MALAT1 and cyclin D1, and determine how changes in ECM composition and integrin display may affect rigidity-dependent regulation of MALAT1, cyclin D1 and cell cycling. Aim 2 looks upstream of cyclin D1 and MALAT1 and will determine how distinct components in the integrin-adhesion that share an ability to activate Rac can differentially regulate MALAT1. Finally, Aim 3 will test the relevance of our findings in vivo by analyzing smooth muscle cell proliferation in a mouse model of tissue stiffening and smooth muscle cell proliferation after vascular injury.