Matrix stiffness induced CREB3L1 activation contributed to skin fibrosis through calcium influx triggered endoplasmic reticulum stress.

BACKGROUND

Skin fibrosis substantially contributes to morbidity and mortality. Fibrotic remodeling, characterized by accumulated and stiffened extracellular matrix, persistently exerts mechanical cues and consistently activates fibroblasts, implying biomechanics as a major driver of fibrosis. However, our understanding towards mechanisms of biomechanics induced fibrosis remained limited.

METHODS

Integrated single-cell sequencing analysis was performed to reveal the atlas of fibrotic skin. ChIP sequencing was performed to reveal the binding sites of CREB3L1. Nanoindenter was used to identify the mechanical properties. Gel contraction assay, wound healing assay, and live cell imaging were conducted to assess the behavior of fibroblasts cultured on hydrogels of different rigidities. Bleomycin induced and mechanical loading induced skin fibrosis models were established on CREB3L1 knockdown and control mice.

RESULTS

We identified a mechanosensitive fibroblast cluster that exerts contraction and ECM deposition functions in fibrotic skin, and its transcriptional identity was maintained by CREB3L1. Elevated expression of CREB3L1 was confirmed in human and mouse fibrotic skin. Further analysis showed that CREB3L1 was required for fibroblast activation, including contraction, migration, and ECM accumulation. More importantly, we revealed that matrix stiffness could alter calcium homeostasis, causing calcium influx and endoplasmic reticulum stress. The ER stress state in turn triggered the cleavage of CREB3L1, releasing its luminal domain that function as a profibrotic transcription factor. Inhibiting CREB3L1 could alleviate skin fibrosis both in vivo and in vitro.

CONCLUSIONS

This study elucidates the molecular mechanism of CREB3L1 mechanosensitive activation in matrix stiffness induced skin fibrosis, and presents a promising therapeutic target for clinical translation.