Although many patients with chronic viral hepatitis C infection suffer from progressive liver disease, the rate of fibrosis progression is highly variable and some patients do not show any measurable progression. However, our ability to predict which patients progress is very limited. Since transforming growth factor-beta (TGF-beta) is a key mediator of liver fibrogenesis, we assessed the predictive role of TGF-beta for fibrogenesis in chronic hepatitis C. We studied 39 patients with chronic hepatitis C in whom two liver biopsies were taken at least 12 months apart, and who did not receive therapy during this period. TGF-beta was measured by bioassay and by ELISA in serum samples taken at the time of the first biopsies, and TGF-beta was determined semiquantitatively by immunostaining of liver biopsy sections. Fibrosis was scored blinded in the biopsy samples by two pathologists independently. There was a close correlation between TGF-beta serum levels and the rate of fibrosis progression. Patients with no progression of fibrosis had significantly lower (59 ng/mL +/- 22) TGF-beta serum levels than patients with progressive disease (115 ng/mL +/- 20), and a TGF-beta level below 75 ng/mL was predictive for stable disease. Immunohistology for TGF-beta in biopsy samples was also predictive for progressive liver disease with fibrosis progression found in those patients displaying staining of hepatocytes and sinusoidal cells. No such correlation was found with other markers such as procollagen III peptide, viral load or transaminase levels. These results further support the role of TGF-beta in liver fibrogenesis, and offer an opportunity to predict clinical disease progression, which may help in selecting patients who are in need of therapeutic interventions.

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses.

Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) that results in transient suppression of protein translation to allow recovery but leads to cell death when stress cannot be resolved. Central to initiation of the UPR is the activation of the ER transmembrane kinase protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Here we report that the thiol oxidoreductase ERp57 and protein disulfide isomerase-A1 (PDI), which belong to the same family of luminal ER oxidoreductases, have strikingly opposing roles in the regulation of PERK function. In HCT116 colon carcinoma cells, lentiviral depletion of ERp57 resulted in oxidation of PDI and activation of PERK, whereas depletion or chemical inhibition of PDI reduced PERK signaling and sensitized the cancer cells to hypoxia and ER stress. We conclude that oxidized PDI acts as a PERK activator, whereas ERp57 keeps PDI in a reduced state in the absence of ER stress. Thus, our study defines a new interface between metabolic redox signaling and PERK-dependent activation of the UPR and has the potential to influence future cancer therapies that target PERK signaling.

• Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.• Granule permeabilization in neutrophils leads to cathepsin G-mediated death upstream and independent of apoptotic caspases.Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1 2/2 mice.Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1 2/2 mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1 2/2 PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity. (Blood. 2013;121(19):3900-3907)

Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation.