Non-canonical Hedgehog signaling via GLI2 promotes pancreatic stellate cell activation and fibrosis in chronic pancreatitis

Excessive Hedgehog (Hh) signaling activity contributes to fibrosis in multiple organs, however, its role in pancreatic stellate cell (PSC) activation and fibrosis development during chronic pancreatitis (CP) remains unclear. We demonstrate that GLI2 is upregulated in activated PSCs from CP patients and animal models. Specific deletion of Gli2, but not Smo, significantly alleviated fibrosis in CP models, indicating that CP is driven by non-canonical Hh signaling. Experiments on culture-activated primary PSCs reveal early nuclear translocation and increased GLI2 expression promptly after in vitro culture, with GLI2 being essential for PSC activation. SMO inhibition only reduces GLI2 activation in the short term, while activation of GLI2 by TGF-ß/SMAD3 signaling explains the SMO-independent mechanisms leading to Hh activation in PSCs. Altogether, our data demonstrate the activation of the non-canonical Hh pathway in PSCs and indicate that GLI2 represents a promising therapeutic target for CP. Overall design: To elucidate the molecular mechanisms underlying the antifibrotic effects of GLI2, we performed bulk RNA sequencing (RNA-seq) of primary PSCs isolated from Gli2fl/fl:Pdgfrb-CreERT2 (Gli2?PSC) mice and Gli2fl/fl mice. The genetically modified mice were intraperitoneally injected with tamoxifen at a dose of 100 mg/kg body weight for 5 consecutive days. After a 1-week interval, PSCs were isolated and then cultured in vitro for 4 days, as GLI2 reached its peak abundance by day 4 of culture. We then performed gene expression profiling analysis from data obtained from RNA-seq of D4 PSCs isolated from Gli2?PSC and Gli2fl/fl mice.