Substrate stiffness dictates unique paths towards proliferative arrest in WI-38 cells

Area of Science:

• Cell Biology
• Mechanobiology
• Aging Research

Background:

• Finite replicative potential is a hallmark of somatic cells, leading to replicative senescence via telomere shortening.
• Telomerase reverse transcriptase (hTERT) can overcome replicative arrest.
• Mechanotransduction pathways, like YAP signaling, are activated as cells approach senescence, but their role in replicative lifespan is unclear.

Purpose of the Study:

• To investigate how the mechanical environment, specifically substrate stiffness, influences the replicative lifespan and senescence trajectory of WI-38 human lung fibroblasts.
• To explore the interplay between mechanosensation and telomere-driven replicative arrest.

Main Methods:

• WI-38 cells were cultured on substrates of varying stiffness.
• Proliferation rates, cellular phenotypes, and gene expression were analyzed.
• Bulk and single-cell RNA-sequencing and ATAC-sequencing were employed.
• The effects of ectopic hTERT expression were assessed.

Main Results:

• Matrix softening reduced WI-38 cell proliferation and shortened their proliferative lifespan.
• Softer substrates induced unique cellular phenotypes and a distinct G1 transcriptional state characterized by an AP-1 program.
• Ectopic hTERT expression mitigated or abolished the effects of substrate stiffness on proliferation and senescence.
• The AP-1 transcription factor program on soft substrates was not observed in hTERT-expressing cells.

Conclusions:

• The mechanical environment significantly alters WI-38 cell proliferative lifespan and influences the pathways leading to growth arrest.
• Substrate stiffness can dictate unique cellular responses, including specific transcriptional programs, that contribute to senescence.
• Telomerase activity, through hTERT, can override the impact of mechanical cues on cellular aging and proliferation.