Hic-5 is required for activation of pancreatic stellate cells and development of pancreatic fibrosis in chronic pancreatitis

Chronic pancreatitis (CP) is an inflammatory disease of the exocrine pancreas that increases the risk of developing pancreatic cancer. The pathological hallmarks are acinar cell injury and progressive pancreatic fibrosis, leading to loss of pancreatic function, and there is currently no therapy to reverse fibrosis. Activated pancreatic stellate cells (PSCs) are key contributors to extracellular matrix (ECM) deposition in CP-associated fibrosis. Quiescent PSCs in normal pancreas contain vitamin A droplets; upon injury/inflammation they activate, upregulate α-smooth muscle actin (α-SMA), and secrete ECM proteins (collagen I/III). Transforming growth factor-β (TGF-β) is a major activator of PSCs via TGF-β1-dependent Smad2 profibrotic signaling. PSCs share morphological and functional features with hepatic stellate cells (HSCs). Prior work showed hydrogen peroxide-inducible clone-5 (Hic-5; TGFβI1), an adhesion scaffold protein involved in cytoskeletal control and ECM remodeling, regulates HSC activation and liver fibrosis. Whether Hic-5 regulates PSC activation and pancreatic fibrosis in CP remained unknown. The study tests the hypothesis that Hic-5 is required for PSC activation and promotes pancreatic fibrosis development in CP via TGF-β/Smad2 signaling.

- PSCs are established mediators of pancreatic fibrosis in CP, transitioning from quiescent vitamin A–rich cells to myofibroblast-like cells with α-SMA expression and ECM secretion under TGF-β signaling. - HSCs in liver fibrosis are activated by TGF-β/Smad pathways; Hic-5 regulates HSC activation and liver fibrosis, with Hic-5 deficiency attenuating fibrosis and modulating TGF-β1/Smad signaling (Lei et al., 2016). - Hic-5 (TGFβI1) is a LIM domain adhesion scaffold protein localized to focal adhesions and the nucleus, implicated in ECM deposition/remodeling and fibrotic diseases (glomerulosclerosis, intestinal fibrosis, liver fibrosis) through effects on myofibroblast differentiation and ECM regulation. - These prior findings support investigating Hic-5 in PSC activation and CP-related pancreatic fibrosis.

- Animal model of CP: Age- and sex-matched C57BL/6 wild type (WT) and Hic-5 knockout (Hic-5 KO) mice (8–12 weeks) were maintained SPF. CP was induced by intraperitoneal injections of caerulein (50 µg/kg) hourly for 6 hours, twice weekly (Monday and Thursday) for 6 weeks; controls received PBS. - Human samples and cells: Human pancreatic tissue arrays were obtained commercially. Human primary PSCs were purchased and cultured in stellate cell medium per manufacturer. - Mouse PSC isolation and culture: PSCs were isolated from WT and Hic-5 KO mice by collagenase digestion and Nycodenz density gradient, washed, and cultured in DMEM with 10% FBS and antibiotics. Cells were used before first passage. Time-course activation was assessed from 2 hours to 5 days in culture. - Gene expression: Total RNA from pancreas tissues or cultured cells was extracted (TRIzol). RT-qPCR performed with GAPDH normalization and 2^−ΔΔCT method; each sample in duplicate. - Protein analysis: Western blotting on tissue/cell lysates (RIPA). Primary antibodies: Hic-5, GAPDH, α-SMA, Collagen I, Smad2, phospho-Smad2. Detection by chemiluminescence; densitometry quantified band intensities. - Histology: Pancreata fixed, paraffin-embedded; H&E, Masson’s trichrome, and Sirius Red staining performed. Fibrotic areas quantified with ImageJ as percentage of total pancreatic area. - Immunohistochemistry: Antigen retrieval in citrate buffer; anti–Hic-5 primary antibody with DAB detection and hematoxylin counterstain. - Immunofluorescence: Cells/tissues fixed, permeabilized, blocked; primary antibodies to α-SMA and Hic-5; secondary Alexa fluorophores; nuclei stained with Hoechst. - TGF-β/Smad signaling assays: PSCs treated with TGF-β (5 ng/mL); phospho-Smad2 and total Smad2 measured by Western blot at indicated times (e.g., 10, 30, 60 min in human PSCs). - siRNA knockdown in human PSCs: Two distinct Hic-5 siRNAs transfected; knockdown efficiency verified by Western blot. Effects on α-SMA and COL1A1 mRNA/protein assessed; morphology evaluated by immunofluorescence; p-Smad2 response to TGF-β measured. - Statistics: Student’s t-test; data as mean ± SD from triplicate measurements in three independent experiments; p < 0.05 significant.

- Hic-5 is upregulated in CP and localizes to activated PSCs: - Human CP tissues showed strong Hic-5 expression in spindle-shaped stromal cells co-expressing α-SMA; normal pancreas showed Hic-5 mainly in vascular smooth muscle cells. - In caerulein-induced mouse CP, Hic-5 co-localized with α-SMA in PSCs; Western blot and qPCR confirmed increased Hic-5 expression after caerulein vs controls (p<0.05). - During in vitro activation of primary mouse PSCs (2 h to day 5), Hic-5 was absent in quiescent cells but increased at days 3–5, localizing to focal adhesions; α-SMA exhibited a similar induction pattern. - Hic-5 deficiency reduces pancreatic fibrosis in CP: - Hic-5 KO mice had attenuated pancreatic shrinkage after caerulein. - Histology (H&E) showed reduced acinar cell loss; Sirius Red and Masson’s trichrome quantifications showed significantly decreased fibrotic area in Hic-5 KO vs WT mice (p<0.05). - Western blots showed lower α-SMA and collagen I protein in Hic-5 KO pancreata after caerulein; qPCR showed reduced Col1a1 and Col3a1 mRNA (p<0.05). TGF-β1 mRNA did not differ between genotypes (data not shown). - Hic-5 is required for PSC activation in vitro: - Cultured Hic-5 KO mouse PSCs (day 5) displayed reduced α-SMA and collagen I mRNA and decreased α-SMA protein vs WT; morphological features of myofibroblast differentiation were attenuated. TGF-β1 mRNA and inflammatory cytokine levels were not altered by Hic-5 loss (data not shown). - Hic-5 mediates TGF-β/Smad2 signaling in PSCs: - Total Smad2 levels were similar between WT and Hic-5 KO PSCs, but TGF-β–induced phospho-Smad2 was robust in WT and significantly blunted in Hic-5 KO PSCs (p<0.05). - In human PSCs, Hic-5 siRNA (two distinct siRNAs) reduced Hic-5 protein, decreased α-SMA and COL1A1 mRNA and α-SMA protein, altered activation-associated morphology, and significantly reduced TGF-β–induced Smad2 phosphorylation vs control siRNA (p<0.05 to p<0.01). - Overall, Hic-5 acts as a marker and functional mediator of PSC activation and fibrosis progression in CP via TGF-β/Smad2.

The study addresses whether Hic-5 regulates PSC activation and consequent pancreatic fibrosis in CP. Findings show Hic-5 expression is specifically elevated in activated PSCs in human and murine CP and increases during PSC activation in culture. Genetic deficiency of Hic-5 in mice diminishes caerulein-induced pancreatic fibrosis, reduces PSC activation markers (α-SMA, collagen I/III), and preserves pancreatic architecture. Mechanistically, Hic-5 is required for TGF-β–induced Smad2 phosphorylation in PSCs without altering total Smad2 or TGF-β1 expression, positioning Hic-5 as a key intracellular mediator of the TGF-β/Smad profibrotic pathway in PSCs. In human PSCs, siRNA-mediated Hic-5 knockdown attenuates activation, ECM gene expression, and Smad2 phosphorylation, supporting translational relevance. These results link Hic-5 to PSC-driven fibrogenesis in CP and complement prior evidence in HSCs and liver fibrosis, highlighting a conserved role of Hic-5 in myofibroblast differentiation and ECM remodeling. Targeting Hic-5 could reduce PSC activation and fibrosis severity in CP.

Hic-5 is markedly upregulated in activated PSCs and is required for their activation and for the development of pancreatic fibrosis in a murine CP model. Hic-5 deficiency or knockdown suppresses PSC activation markers, reduces ECM production, and impairs TGF-β–induced Smad2 phosphorylation. Hic-5 thus serves as a marker of activated PSCs and a potential therapeutic target for CP-related fibrosis. Future work should evaluate therapeutic Hic-5 inhibition in established CP, develop efficient pancreas-targeted delivery systems (e.g., siRNA), and delineate Hic-5’s broader signaling network in PSCs beyond Smad2.

- The in vivo therapeutic potential of Hic-5 inhibition in established CP was not tested; effective pancreas-targeted delivery systems (e.g., siRNA) are currently lacking and were identified as a need. - Some mechanistic assessments reported no changes in TGF-β1 mRNA, inflammatory cytokines, and MMP-2/MMP-9/TIMP-1 in activated PSCs with Hic-5 deficiency, but detailed datasets were not shown. - The study relies on a single murine CP model (caerulein) and primary PSC culture; generalizability to other CP etiologies and human in vivo contexts remains to be validated. - Quantitative in vivo functional outcomes (e.g., exocrine/endocrine function) were not extensively characterized beyond histology and molecular markers.