Abstract Rationale Idiopathic Pulmonary Fibrosis (IPF) is an insidious, progressive restrictive lung disease contributing to significant loss of life and quality of life for approximately 5 million individuals globally with poorly understood etiology, few therapies, and no known cure. One of the hallmarks of IPF is a phenotypic change of fibroblasts into myofibroblasts, which contribute to differential extracellular matrix (ECM) production, leading to the scarring and stiffening of the lung that drives the progressive pulmonary dysfunction and death. Our lab has previously demonstrated that an increase in tissue stiffness leads to the progressive development of a fibrotic phenotype in fibroblasts, but the underlying intracellular mechanotransduction response to changing tissue stiffness remains uncharacterized. This study aimed to clarify the intracellular signaling cascade in response to stiffness for potential development of future antifibrotic therapies. Methods Primary Human Lung Fibroblasts (HLFs) were cultured on collagen coated substrate with differing stiffnesses (1 kPA, 60 kPA, and tissue culture plastic). After five days, whole cell protein lysate was placed on a Phospho Explorer Antibody Microarray (Full Moon Biosystems) following the manufacturer’s protocol. Fold change protein phosphorylation was calculated and Pathway Enrichment Analysis was conducted in String. Results Pathway enrichment analysis in STRING highlighted significant interactions among 51 proteins influenced by stiffness. The analysis isolated clusters related to cell proliferation, survival, repair, and cell cycle gatekeeping (Fig 1A). HLF cells grown on softer substrates relative to plastic exhibited notable posttranslational modification of proteins involved in DNA regulation and repair with an increase in phosphorylation of p53, Calmodulin, CREB, JUN, CHK2, and SMC1 and dephosphorylation of BRCA1, CDC25A, and CaMK4 (Fig 1B). Furthermore, proliferation, adhesion and migration signals including EGFR, ICAM1, SRC, MEK, and PXN were increasingly phosphorylated while RAF1, MKK7 and PYK2 were dephosphorylated on softer substrates (Fig 1C). Conclusions These findings suggest that tissue stiffness affects cellular survival and proliferation via the DNA damage repair and cell cycle regulation pathways, calcium signaling via calmodulin, and proliferation, adhesion, and migration pathways. These data help define the intracellular response to phenotypic changes previously described in response to substrate stiffness in fibrosis and implicate a complex interplay between tyrosine kinase receptors and tissue stiffness in fibrotic progression. Modulation of cellular mechanosensation may offer a new direction for antifibrotic therapies. In the future, we plan to investigate whether targeting these pathways may provide synergistic benefit with current tyrosine kinase inhibitor therapeutics. This abstract is funded by: This work was supported in part by the NIH, the Pulmonary Fibrosis Foundation, and the Parker B. Francis foundation. MATF is funded by Parker B. Francis Foundation and NIH grant K99HL169903-01A10. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Kelley et al. (Fri,) studied this question.