SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells.Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively.Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

SERPINB3 (SB3) is a serine protease inhibitor overexpressed in several malignancies of epithelial origin, including primary liver cancer, where it inhibits apoptosis through poorly defined mechanisms. In the present study we analyze the effect of SB3 on hepatoma cell death elicited by a panel of chemotherapeutic agents. We report that SB3 shields cells from the toxicity of drugs with a pro-oxidant action such as doxorubicin, cisplatin and EM20-25. The rapid rise in ROS levels prompted by these compounds causes opening of the mitochondrial permeability transition pore (PTP), irreversibly committing cells to death. We find that a fraction of SB3 locates in mitochondrial inner compartments, and that this mitochondrial fraction increases under conditions of oxidative stress. Mitochondrial SB3 inhibits ROS generation and the ensuing PTP induction and cell death through an inhibitory interaction with respiratory Complex I. These findings identify a novel mechanism of action of SB3 that contributes to tumor cell resistance to anti-neoplastic drugs

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-β1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. “In vitro” experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.

SERPIN B3/B4, members of the serpin superfamily, are fundamental for the control of proteolysis through a known inhibitory function of different proteases. Several studies have documented an important role of SERPIN B3 in the modulation of inflammation, programmed cell death and fibrosis. To confirm the role of SERPIN B3 in lung fibrosis and overall investigate its influence on epithelial dysfunction, a stratified controlled trial randomly assigning bleomycin (BLM) treatment was performed on both SERPIN B3 transgenic (TG) and wild-type (WT) mice. TG and WT animals were killed 48 h (group T48 h) and 20 days (group T20d) after BLM treatment. Lung fibrosis was assessed by histology and hydroxyproline measurement. Architectural remodeling, inflammation, epithelial apoptosis and proliferation were quantified. Moreover, the profibrogenetic cytokine transforming growth factor (TGF)-b, cathepsin K, L and S were also investigated. No significant differences were observed between TG and WT mice of group T48 h in any parameters. In group T20d, less inflammation and a significant increase in epithelial proliferation were detected in treated TG than WT mice despite a similar apoptotic index, thus resulting in a different apoptosis/proliferation imbalance with a significant gain of epithelial proliferation. Moreover, TG mice showed higher TGF-b expression and more extended fibrosis. General linear model analysis, applied on morphological data, showed that interaction between SERPIN B3 expression and treatment was mainly significant for fibrosis. This study provides in vivo evidence for a role of SERPIN B3 in inhibiting inflammation and favoring epithelial proliferation with increased TGF-b secretion and thus the likelihood of consequent fibrogenesis.Laboratory Investigation (2011) 91, 945-954;

Aims/hypothesis Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. Methods Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n=17) and non-healing (NH, n=11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. Results Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in nondiabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. Conclusions/interpretation We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.