Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses and spreads by metastasis to the liver1. Cancer-associated fibroblasts, the extracellular matrix and type I collagen (Col I) support2,3 or restrain the progression of PDAC and may impede blood supply and nutrient availability4. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers to escape nutrient limitation, remain poorly understood. Here we show that matrix-metalloprotease-cleaved Col I (cCol I) and intact Col I (iCol I) exert opposing effects on PDAC bioenergetics, macropinocytosis, tumour growth and metastasis. Whereas cCol I activates discoidin domain receptor 1 (DDR1)-NF-κB-p62-NRF2 signalling to promote the growth of PDAC, iCol I triggers the degradation of DDR1 and restrains the growth of PDAC. Patients whose tumours are enriched for iCol I and express low levels of DDR1 and NRF2 have improved median survival compared to those whose tumours have high levels of cCol I, DDR1 and NRF2. Inhibition of the DDR1-stimulated expression of NF-κB or mitochondrial biogenesis blocks tumorigenesis in wild-type mice, but not in mice that express MMP-resistant Col I. The diverse effects of the tumour stroma on the growth and metastasis of PDAC and on the survival of patients are mediated through the Col I-DDR1-NF-κB-NRF2 mitochondrial biogenesis pathway, and targeting components of this pathway could provide therapeutic opportunities.

Immunotherapy-based targeting of MSLN+ activated portal fibroblasts is a strategy for treatment of cholestatic liver fibrosis.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

Nondegradable Collagen Increases Liver Fibrosis but Not Hepatocellular Carcinoma in Mice.

Although hepatocellular cancer (HCC) usually occurs in the setting of liver fibrosis, the causal relationship between liver fibrosis and HCC is unclear. in vivo and in vitro models of HCC involving Colr/r mice (that produce a collagenase-resistant type I collagen) or wild-type (WT) mice were used to assess the relationship between type I collagen, liver fibrosis, and experimental HCC. HCC was either chemically induced in WT and Colr/r mice or Hepa 1-6 cells were engrafted into WT and Colr/r livers. The effect of hepatic stellate cells (HSCs) from WT and Colr/r mice on the growth of Hepa 1-6 cells was studied by using multicellular tumor spheroids and xenografts. Collagen type I deposition and fibrosis were increased in Colr/r mice, but they developed fewer and smaller tumors. Hepa 1-6 cells had reduced tumor growth in the livers of Colr/r mice. Although Colr/r HSCs exhibited a more activated phenotype, Hepa 1-6 growth and malignancy were suppressed in multicellular tumor spheroids and in xenografts containing Colr/r HSCs. Treatment with vitronectin, which mimics the presence of degraded collagen fragments, converted the Colr/r phenotype into a WT phenotype. Although Colr/r mice have increased liver fibrosis, they exhibited decreased HCC in several models. Thus, increased liver type I collagen does not produce increased experimental HCC.

Liver fibrosis: Pathophysiology and clinical implications.

Liver fibrosis is a clinically significant finding that has major impacts on patient morbidity and mortality. The mechanism of fibrosis involves many different cellular pathways, but the major cell type involved appears to be hepatic stellate cells. Many liver diseases, including Hepatitis B, C, and fatty liver disease cause ongoing hepatocellular damage leading to liver fibrosis. No matter the cause of liver disease, liver-related mortality increases exponentially with increasing fibrosis. The progression to cirrhosis brings more dramatic mortality and higher incidence of hepatocellular carcinoma. Fibrosis can also affect outcomes following liver transplantation in adult and pediatric patients and require retransplantation. Drugs exist to treat Hepatitis B and C that reverse fibrosis in patients with those viral diseases, but there are currently no therapies to directly treat liver fibrosis. Several mouse models of chronic liver diseases have been successfully reversed using novel drug targets with current therapies focusing mostly on prevention of myofibroblast activation. Further research in these areas could lead to development of drugs to treat fibrosis, which will have invaluable impact on patient survival. This article is categorized under: Metabolic Diseases > Molecular and Cellular Physiology.

Primary Alcohol-Activated Human and Mouse Hepatic Stellate Cells Share Similarities in Gene-Expression Profiles.

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol-induced activation of human and mouse HSCs is not fully understood. We compared the gene-expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA-sequencing analysis. Furthermore, the gene-expression profile of ALD hHSCs was compared with that of alcohol-activated mHSCs (isolated from intragastric alcohol-fed mice) or CCl4-activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol-activated and CCl4-activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor-1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol-activated mHSCs most closely recapitulate the gene-expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up-regulated in primary cultured alcohol-activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony-stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal-associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase-binding protein), and ITGB2 (integrin beta-2), and other genes (compared with CCl4-activated mHSCs). Conclusion: We identified genes in alcohol-activated mHSCs from intragastric alcohol-fed mice that are largely consistent with the gene-expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol-induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD.

Blockade of IL-17 signaling reverses alcohol-induced liver injury and excessive alcohol drinking in mice.

Chronic alcohol abuse has a detrimental effect on the brain and liver. There is no effective treatment for these patients, and the mechanism underlying alcohol addiction and consequent alcohol-induced damage of the liver/brain axis remains unresolved. We compared experimental models of alcoholic liver disease (ALD) and alcohol dependence in mice and demonstrated that genetic ablation of IL-17 receptor A (IL-17ra-/-) or pharmacological blockade of IL-17 signaling effectively suppressed the increased voluntary alcohol drinking in alcohol-dependent mice and blocked alcohol-induced hepatocellular and neurological damage. The level of circulating IL-17A positively correlated with the alcohol use in excessive drinkers and was further increased in patients with ALD as compared with healthy individuals. Our data suggest that IL-17A is a common mediator of excessive alcohol consumption and alcohol-induced liver/brain injury, and targeting IL-17A may provide a novel strategy for treatment of alcohol-induced pathology.

Activated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice.

BACKGROUND & AIMS: Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2-/-) mice, a genetic model that resembles primary sclerosing cholangitis in patients. METHODS: Mdr2-/- mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2-/- mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2-/- mice aged 12-16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. RESULTS: Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2-/- mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2-/- mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2-/- mice. The development of cholestatic liver fibrosis in Mdr2-/- mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2-/- mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. CONCLUSIONS: Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2-/- mice, and serve as targets for antifibrotic therapy. LAY SUMMARY: Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2-/- mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2-/- mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

The Role of Fibrosis and Liver-Associated Fibroblasts in the Pathogenesis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive types of cancer and lacks effective therapeutic approaches. Most HCC develops in the setting of chronic liver injury, hepatic inflammation, and fibrosis. Hepatic stellate cells (HSCs) and cancer-associated fibroblasts (CAFs) are key players in liver fibrogenesis and hepatocarcinogenesis, respectively. CAFs, which probably derive from HSCs, activate into extracellular matrix (ECM)-producing myofibroblasts and crosstalk with cancer cells to affect tumor growth and invasion. In this review, we describe the different components which form the HCC premalignant microenvironment (PME) and the tumor microenvironment (TME), focusing on the liver fibrosis process and the biology of CAFs. We will describe the CAF-dependent mechanisms which have been suggested to promote hepatocarcinogenesis, such as the alteration of ECM, CAF-dependent production of cytokines and angiogenic factors, CAF-dependent reduction of immuno-surveillance, and CAF-dependent promotion of epithelial-mesenchymal transition (EMT). New knowledge of the fibrosis process and the role of CAFs in HCC may pave the way for new therapeutic strategies for liver cancer.

Ultrastructural characterisation of Bacillus subtilis TatA complexes suggests they are too small to form homooligomeric translocation pores.

Tat-dependent protein transport permits the traffic of fully folded proteins across membranes in bacteria and chloroplasts. The mechanism by which this occurs is not understood. Current theories propose that a key step requires the coalescence of a substrate-binding TatC-containing complex with a TatA complex, which forms pores of varying sizes that could accommodate different substrates. We have studied the structure of the TatAd complex from Bacillus subtilis using electron microscopy to generate the first 3D model of a TatA complex from a Gram-positive bacterium. We observe that TatAd does not exhibit the remarkable heterogeneity of Escherichia coli TatA complexes but instead forms ring-shaped complexes of 7.5-9nm diameter with potential pores of 2.5-3nm diameter that are occluded at one end. Such structures are consistent with those seen for E. coli TatE complexes. Furthermore, the small diameter of the TatAd pore, and the homogeneous nature of the complexes, suggest that TatAd cannot form the translocation channel by itself. Biochemical data indicate that another B. subtilis TatA complex, TatAc, has similar properties, suggesting a common theme for TatA-type complexes from Bacillus.

TatAc, the third TatA subunit of Bacillus subtilis, can form active twin-arginine translocases with the TatCd and TatCy subunits.

Two independent twin-arginine translocases (Tat) for protein secretion were previously identified in the Gram-positive bacterium Bacillus subtilis. These consist of the TatAd-TatCd and TatAy-TatCy subunits. The function of a third TatA subunit named TatAc was unknown. Here, we show that TatAc can form active protein translocases with TatCd and TatCy.

Targeting of lumenal proteins across the thylakoid membrane.

The biogenesis of the plant thylakoid network is an enormously complex process in terms of protein targeting. The membrane system contains a large number of proteins, some of which are synthesized within the organelle, while many others are imported from the cytosol. Studies in recent years have shown that the targeting of imported proteins into and across the thylakoid membrane is particularly complex, with four different targeting pathways identified to date. Two of these are used to target membrane proteins: a signal recognition particle (SRP)-dependent pathway and a highly unusual pathway that appears to require none of the known targeting apparatus. Two further pathways are used to translocate lumenal proteins across the thylakoid membrane from the stroma and, again, the two pathways differ dramatically from each other. One is a Sec-type pathway, in which ATP hydrolysis by SecA drives the transport of the substrate protein through the membrane in an unfolded conformation. The other is the twin-arginine translocation (Tat) pathway, where substrate proteins are transported in a folded state using a unique mechanism that harnesses the proton motive force across the thylakoid membrane. This article reviews progress in studies on the targeting of lumenal proteins, with reference to the mechanisms involved, their evolution from endosymbiotic progenitors of the chloroplast, and possible elements of regulation.

Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.