Matrix biology : journal of the International Society for Matrix Biology · 2024
A CNA-35-based high-throughput fibrosis assay reveals ORAI1 as a regulator of collagen release from pancreatic stellate cells.
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Schleinhege Rieke, Neumann Ilka, Oeckinghaus Andrea, Schwab Albrecht, Pethő Zoltán
Аннотация
RATIONALE: Pancreatic stellate cells (PSCs) produce a collagen-rich connective tissue in chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Ca2+-permeable ion channels such as ORAI1 are known to affect PSC proliferation and myofibroblastic phenotype. However, it is unknown whether these channels play a role in collagen secretion.
METHODS: Using the PSC cell line PS-1, we characterized their cell-derived matrices using staining, mass spectroscopy, and cell migration assays. We developed and validated a high-throughput in vitro fibrosis assay to rapidly determine collagen quantity either with Sirius Red or, in the optimized version, with the collagen-binding peptide CNA-35-tdTomato. We assessed collagen deposition upon stimulating cells with transforming growth factor β1 (TGF-β1) and/or vitamin C without or with ORAI1 modulation. Orai1 expression was assessed by immunohistochemistry in the fibrotic tumor tissue of a murine PDAC model (KPfC).
RESULTS: We found that TGF-β1 and vitamin C promote collagen deposition from PSCs. We used small interfering RNA (siRNA) and the inhibitor Synta-66 to demonstrate that ORAI1 regulates collagen secretion of PSCs but not NIH-3T3 fibroblasts. Physiological levels of vitamin C induce a drastic increase of the intracellular [Ca2+] in PSCs, with Synta-66 inhibiting Ca2+ influx. Lastly, we revealed Orai1 expression in cancer-associated fibroblasts (CAFs) in murine PDAC (KPfC) samples.
CONCLUSION: In conclusion, our study introduces a robust in vitro assay for fibrosis and identifies ORAI1 as being engaged in PSC-driven fibrosis.
METHODS: Using the PSC cell line PS-1, we characterized their cell-derived matrices using staining, mass spectroscopy, and cell migration assays. We developed and validated a high-throughput in vitro fibrosis assay to rapidly determine collagen quantity either with Sirius Red or, in the optimized version, with the collagen-binding peptide CNA-35-tdTomato. We assessed collagen deposition upon stimulating cells with transforming growth factor β1 (TGF-β1) and/or vitamin C without or with ORAI1 modulation. Orai1 expression was assessed by immunohistochemistry in the fibrotic tumor tissue of a murine PDAC model (KPfC).
RESULTS: We found that TGF-β1 and vitamin C promote collagen deposition from PSCs. We used small interfering RNA (siRNA) and the inhibitor Synta-66 to demonstrate that ORAI1 regulates collagen secretion of PSCs but not NIH-3T3 fibroblasts. Physiological levels of vitamin C induce a drastic increase of the intracellular [Ca2+] in PSCs, with Synta-66 inhibiting Ca2+ influx. Lastly, we revealed Orai1 expression in cancer-associated fibroblasts (CAFs) in murine PDAC (KPfC) samples.
CONCLUSION: In conclusion, our study introduces a robust in vitro assay for fibrosis and identifies ORAI1 as being engaged in PSC-driven fibrosis.