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Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

ABSTRACT: Cells are subjected to dynamic mechanical environments which impart various forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells, leading to elevated tension in fibroblasts among other cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-beta-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (3 GPa) substrates followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Computational meta-analysis of human interstitial lung disease single-cell RNA sequencing datasets confirmed these genes are highly expressed in disease samples and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

INSTRUMENT(S): Orbitrap Eclipse, TripleTOF 6600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Birgit Schilling

PROVIDER: MSV000095880 | MassIVE |

SECONDARY ACCESSION(S): PXD055921

REPOSITORIES: MassIVE

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Project description:Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modelled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-Seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted species-specific responses to telomere dysfunction. We then conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans.

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Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

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