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The multifaceted role of cathepsins in liver disease

  • Author Footnotes
    † The three authors contributed equally to this work.
    Paloma Ruiz-Blázquez
    Footnotes
    † The three authors contributed equally to this work.
    Affiliations
    Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC), Barcelona, Spain
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  • Author Footnotes
    † The three authors contributed equally to this work.
    Valeria Pistorio
    Footnotes
    † The three authors contributed equally to this work.
    Affiliations
    Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC), Barcelona, Spain

    University of Naples Federico II, Naples, Italy
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  • Author Footnotes
    † The three authors contributed equally to this work.
    María Fernández-Fernández
    Footnotes
    † The three authors contributed equally to this work.
    Affiliations
    Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC), Barcelona, Spain
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  • Anna Moles
    Correspondence
    Corresponding author. Address: Institute of Biomedical Research of Barcelona. Spanish National Research Council (IIBB-CSIC). 6th Floor Rosselló St, 161, 08036 Barcelona. Spain; Tel.: 933638300.
    Affiliations
    Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC), Barcelona, Spain

    IDIBAPS, Barcelona, Spain

    CiberEHD, Spain
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  • Author Footnotes
    † The three authors contributed equally to this work.
Open AccessPublished:July 06, 2021DOI:https://doi.org/10.1016/j.jhep.2021.06.031

      Summary

      Proteases are the most abundant enzyme gene family in vertebrates and they execute essential functions in all living organisms. Their main role is to hydrolase the peptide bond within proteins, a process also called proteolysis. Contrary to the conventional paradigm, proteases are not only random catalytic devices, but can perform highly selective and targeted cleavage of specific substrates, finely modulating multiple essential cellular processes. Lysosomal protease cathepsins comprise 3 families of proteases that preferentially act within acidic cellular compartments, but they can also be found in other cellular locations. They can operate alone or as part of signalling cascades and regulatory circuits, playing important roles in apoptosis, extracellular matrix remodelling, hepatic stellate cell activation, autophagy and metastasis, contributing to the initiation, development and progression of liver disease. In this review, we comprehensively summarise current knowledge on the role of lysosomal cathepsins in liver disease, with a particular emphasis on liver fibrosis, non-alcoholic fatty liver disease and hepatocellular carcinoma.

      Keywords

      Introduction

      Proteases are the most abundant enzyme gene family in vertebrates and they execute essential functions in all living organisms, controlling both homeostatic and pathological processes. Their main function is to hydrolase the peptide bond within proteins in a process called proteolysis. They were first identified as non-specific hydrolases degrading intake proteins from the diet in the gastric juice. However, recent advances in the field have demonstrated that proteases are not only random catalytic devices, as they can finely tune several biological processes through efficient, highly selective, and limited cleavage of specific substrates. Specific proteolytic processing of targeted substrates is responsible for activation and inactivation of enzymes; hormones and growth factors; spatial migration of proteins; extracellular matrix remodelling and generation of neoproteins (proteolytic cleavage products with roles that are distinct from those of the original protein).
      • Bond J.S.
      Proteases: history, discovery, and roles in health and disease.
      Therefore, proteases participate in the initiation, modulation and termination of essential biological events, such as the cell-cycle, apoptosis, cell migration, tissue remodelling and morphogenesis, angiogenesis, antigen presentation, immune cell activation, neuronal growth, and bone formation.
      It is increasingly recognised that proteases do not operate alone but in signalling cascades and complex networks. Considering the wide range of cellular processes requiring proteolysis, it is not surprising that dysregulation of proteases or their signalling pathways underlie many human pathologies such as arthritis, chronic obstructive pulmonary disease, neurodegenerative disorders, cancer, and liver and cardiovascular diseases, among others.
      • Bond J.S.
      Proteases: history, discovery, and roles in health and disease.
      ,
      The degradome is defined as the complete set of proteases of a living organism. To date, the human and mouse degradome comprises 641 and 677 protease genes, respectively, representing approximately 3% of their genome.
      • Bond J.S.
      Proteases: history, discovery, and roles in health and disease.
      Most proteases are broadly expressed in tissues and can be found in several cellular compartments, both intracellularly (cytosol, lysosome, nucleus or secretory vesicles) and extracellularly, contributing to different cellular signalling pathways depending on their tissue and cellular compartmentalisation. There are several excellent papers reviewing the contribution of individual protease families to disease progression. However, not many of them have focused on the role of proteases in liver disease progression. Thus, in this review, we will comprehensively summarise current knowledge on lysosomal proteases, also named cathepsins, in the context of liver disease (Table 1).
      Table 1Role of cathepsins in liver disease.
      NameMEROPS IDProtease familyLiver pathologyChange in expressionCell typeCell compartmentFunctionRef.
      Cathepsin AS10.002SerFibrosis/cirrhosisHepatocyteLysosomeRegulation of autophagy
      • Cuervo A.M.
      Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.
      Cathepsin CC01.070CysHCCExtracellularMetastasis
      • Zhang G.-P.
      • Yue X.
      • Li S.-Q.
      Cathepsin C interacts with TNF-α/p38 MAPK signaling pathway to promote proliferation and metastasis in hepatocellular carcinoma.
      Cathepsin BC01.060CysFibrosis/cirrhosisExtracellularCollagenolytic activity
      • Murawaki Y.
      • Hirayama C.
      Hepatic collagenolytic cathepsin in patients with chronic liver disease.
      ,
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Gebbia N.
      Lysosomal cathepsins B and L and stef in A blood levels in patients with hepatocellular carcinoma and/or liver cirrhosis: potential clinical implications.
      HepatocyteCytosolHepatocyte apoptosis
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.F.
      • Gores G.J.
      Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.
      HSCUnknownHSC activation
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.
      HSCLysosomeRegulation of autophagy
      • Tao Y.
      • Qiu T.
      • Yao X.
      • Jiang L.
      • Wang N.
      • Jia X.
      • et al.
      Autophagic-CTSB-inflammasome axis modulates hepatic stellate cells activation in arsenic-induced liver fibrosis.
      ,
      • Inzaugarat M.E.
      • Johnson C.D.
      • Holtmann T.M.
      • McGeough M.D.
      • Trautwein C.
      • Papouchado B.G.
      • et al.
      NLR family pyrin domain-containing 3 inflammasome activation in hepatic stellate cells induces liver fibrosis in mice.
      NK/NKTUnknownRegulation of inflammation via NF-kB signalling
      • de Mingo Á
      • de Gregorio E.
      • Moles A.
      • Tarrats N.
      • Tutusaus A.
      • Colell A.
      • et al.
      Cysteine cathepsins control hepatic NF-κB-dependent inflammation via sirtuin-1 regulation.
      NAFLDHepatocyteCytosolLipotoxicity
      • Li Z.
      • Berk M.
      • McIntyre T.M.
      • Gores G.J.
      • Feldstein A.E.
      The lysosomal-mitochondrial axis in free fatty acid-induced hepatic lipotoxicity.
      CytosolRegulation of free fatty acid metabolism
      • Thibeaux S.
      • Siddiqi S.
      • Zhelyabovska O.
      • Moinuddin F.
      • Masternak M.M.
      • Siddiqi S.A.
      Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid–binding protein.
      LysosomeRegulation of autophagy
      • Simoes I.C.M.
      • Karkucinska-Wieckowska A.
      • Janikiewicz J.
      • Szymanska S.
      • Pronicki M.
      • Dobrzyn P.
      • et al.
      Western diet causes obesity-induced nonalcoholic fatty liver disease development by differentially compromising the autophagic response.
      ,
      • Inami Y.
      • Yamashina S.
      • Izumi K.
      • Ueno T.
      • Tanida I.
      • Ikejima K.
      • et al.
      Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.
      HCCCancer cellLysosomeAutophagy-related drug resistance
      • Kao C.
      • Chao A.
      • Tsai C.-L.
      • Chuang W.-C.
      • Huang W.-P.
      • Chen G.-C.
      • et al.
      Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation.
      • Wang N.
      • Liu H.
      • Liu G.
      • Li M.
      • He X.
      • Yin C.
      • et al.
      Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis.
      • Xue L.
      • Liu P.
      Daurisoline inhibits hepatocellular carcinoma progression by restraining autophagy and promoting cispaltin-induced cell death.
      CytosolHepatoma cells apoptosis
      • Ullio C.
      • Casas J.
      • Brunk U.T.
      • Sala G.
      • Fabriàs G.
      • Ghidoni R.
      • et al.
      Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells.
      ,
      • Guicciardi M.E.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      cFLIP L prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis.
      UnknownTumour invasiveness and metastasis
      • Wang S.-J.
      • Chao D.
      • Wei W.
      • Nan G.
      • Li J.-Y.
      • Liu F.-L.
      • et al.
      CD147 promotes collective invasion through cathepsin B in hepatocellular carcinoma.
      UnknownPro-tumorigenic signalling
      • Xu Z.-Z.
      • Xiu P.
      • Lv J.-W.
      • Wang F.-H.
      • Dong X.-F.
      • Liu F.
      • et al.
      Integrin αvβ3 is required for cathepsin B-induced hepatocellular carcinoma progression.
      Cathepsin DA01.009AspFibrosis/cirrhosisExtracellularCollagenolytic activity
      • Kucharz E.
      Clinical and experimental studies on collagen metabolism in hepatic disorders.
      ,
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Ruggeri I.
      • et al.
      Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis.
      HepatocyteCytosolHepatocyte apoptosis
      • Heinrich M.
      • Neumeyer J.
      • Jakob M.
      • Hallas C.
      • Tchikov V.
      • Winoto-Morbach S.
      • et al.
      Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation.
      HepatocyteLysosomeRegulation of autophagy
      • Hung T.-M.
      • Yuan R.-H.
      • Huang W.-P.
      • Chen Y.-H.
      • Lin Y.-C.
      • Lin C.-W.
      • et al.
      Increased autophagy markers are associated with ductular reaction during the development of cirrhosis.
      NAFLDHepatocyteCytosolLipotoxicity
      • Basford J.E.
      • Wancata L.
      • Hofmann S.M.
      • Silva R.A.G.D.
      • Davidson W.S.
      • Howles P.N.
      • et al.
      Hepatic deficiency of low density lipoprotein receptor-related protein-1 reduces high density lipoprotein secretion and plasma levels in mice.
      CytosolRegulation of cholesterol metabolism
      • Houben T.
      • Oligschlaeger Y.
      • Hendrikx T.
      • Bitorina A.V.
      • Walenbergh S.M.A.
      • van Gorp P.J.
      • et al.
      Cathepsin D regulates lipid metabolism in murine steatohepatitis.
      LysosomeRegulation of autophagy
      • Wang X.
      • Zhang X.
      • Chu E.S.H.
      • Chen X.
      • Kang W.
      • Wu F.
      • et al.
      Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis.
      HCCCancer cellLysosomeAutophagy-related drug resistance
      • Kao C.
      • Chao A.
      • Tsai C.-L.
      • Chuang W.-C.
      • Huang W.-P.
      • Chen G.-C.
      • et al.
      Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation.
      • Wang N.
      • Liu H.
      • Liu G.
      • Li M.
      • He X.
      • Yin C.
      • et al.
      Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis.
      • Xue L.
      • Liu P.
      Daurisoline inhibits hepatocellular carcinoma progression by restraining autophagy and promoting cispaltin-induced cell death.
      CytosolHepatoma cells apoptosis
      • Guicciardi M.E.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      cFLIP L prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis.
      ExtracellularTumour invasiveness and metastasis
      • Lyu L.
      • Jin X.
      • Li Z.
      • Liu S.
      • Li Y.
      • Su R.
      • et al.
      TBBPA regulates calcium-mediated lysosomal exocytosis and thereby promotes invasion and migration in hepatocellular carcinoma.
      UnknownPro-tumorigenic signalling
      • Scharf J.G.
      • Braulke T.
      • Hartmann H.
      • Ramadori G.
      Regulation of the components of the 150 kDa IGF binding protein complex in cocultures of rat hepatocytes and Kupffer cells by 3’,5’-cyclic adenosine monophosphate.
      ,
      • Alexia C.
      • Fallot G.
      • Lasfer M.
      • Schweizer-Groyer G.
      • Groyer A.
      An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-induced apoptosis.
      Cathepsin FC01.018CysFibrosis/cirrhosisHSCNucleiHSC activation
      • Maubach G.
      • Lim M.C.C.
      • Zhuo L.
      Nuclear cathepsin F regulates activation markers in rat hepatic stellate cells.
      Cathepsin HC01.040CysFibrosis/cirrhosisHSCCytosol/NucleiEpigenetic control of MMP genes in HSCs
      • Yang Z.
      • Liu Y.
      • Qin L.
      • Wu P.
      • Xia Z.
      • Luo M.
      • et al.
      Cathepsin H–mediated degradation of HDAC4 for matrix metalloproteinase expression in hepatic stellate cells.
      Cathepsin LC01.032CysFibrosis/cirrhosisExtracellularCollagenolytic activity
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Gebbia N.
      Lysosomal cathepsins B and L and stef in A blood levels in patients with hepatocellular carcinoma and/or liver cirrhosis: potential clinical implications.
      NAFLDLysosomeRegulation of autophagy
      • Simoes I.C.M.
      • Karkucinska-Wieckowska A.
      • Janikiewicz J.
      • Szymanska S.
      • Pronicki M.
      • Dobrzyn P.
      • et al.
      Western diet causes obesity-induced nonalcoholic fatty liver disease development by differentially compromising the autophagic response.
      ,
      • Inami Y.
      • Yamashina S.
      • Izumi K.
      • Ueno T.
      • Tanida I.
      • Ikejima K.
      • et al.
      Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.
      Cathepsin SC01.034CysFibrosis/cirrhosisNK/NKTUnknownRegulation of inflammation via NF-kB signalling
      • de Mingo Á
      • de Gregorio E.
      • Moles A.
      • Tarrats N.
      • Tutusaus A.
      • Colell A.
      • et al.
      Cysteine cathepsins control hepatic NF-κB-dependent inflammation via sirtuin-1 regulation.
      HSCLysosomalAntigen presentation in HSC
      • Maubach G.
      • Lim M.C.C.
      • Kumar S.
      • Zhuo L.
      Expression and upregulation of cathepsin S and other early molecules required for antigen presentation in activated hepatic stellate cells upon IFN-γ treatment.
      HCCCancer cellExtracellularMetastasis and angiogenesis
      • Zhang Z.
      • Zhang H.
      • Peng T.
      • Li D.
      • Xu J.
      Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma.
      ExtracellularPro-tumorigenic signalling
      • Lee T.K.-W.
      • Cheung V.C.-H.
      • Lu P.
      • Lau E.Y.T.
      • Ma S.
      • Tang K.H.
      • et al.
      Blockade of CD47-mediated cathepsin S/protease-activated receptor 2 signaling provides a therapeutic target for hepatocellular carcinoma.
      Cathepsin ZC01.013CysHCCCancer cellUnknownEpithelial-mesenchymal transition (EMT)
      • Wang J.
      • Chen L.
      • Li Y.
      • Guan X.-Y.
      Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.
      HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; NAFLD, non-alcoholic fatty liver disease; MMP, matrix metalloproteinases; NK, natural killer.

      Lysosomal protease cathepsins

      The lysosomes contain more than 50 proteolytic enzymes which show their greatest stability and activity at the acidic pH present within these organelles. Despite cathepsins representing most of the lysosomal hydrolases, there are other proteases present within the lysosomes, such as naspin or asparagine endopeptidase, which will not be discussed in this review.
      The name cathepsin was derived from the Greek verbs cata- (digest) and hepsein (boil). Cathepsins share a common biosynthesis pathway, being synthetised in the ribosome as inactive precursors or pre-pro-enzymes containing an N-terminal signal peptide that directs them to the lumen of the endoplasmic reticulum, where the signal peptide is cleaved in parallel with N-linked glycosylation. Once in the Golgi apparatus, phosphorylation of the high-mannose glycan residues results in the formation of mannose-6-phosphate (M6P), which directs the protein into the endo/lysosomal compartment via the M6P receptor pathway. The acidic environment present in the lysosome triggers the activation of cathepsins, resulting in the cleavage of the pro-region, believed to act as an auto-inhibitor, either into a double-chain (connected through disulphide bridges) or a single chain, both active forms of the enzyme.
      • Turk V.
      • Stoka V.
      • Vasiljeva O.
      • Renko M.
      • Sun T.
      • Turk B.
      • et al.
      Cysteine cathepsins: from structure, function and regulation to new frontiers.
      M6P-independent routes have also been described using alternative receptors to reach the lysosomes,
      • Markmann S.
      • Thelen M.
      • Cornils K.
      • Schweizer M.
      • Brocke-Ahmadinejad N.
      • Willnow T.
      • et al.
      Lrp1/LDL receptor play critical roles in mannose 6-phosphate-independent lysosomal enzyme targeting.
      and re-routing of cathepsins into the extracellular space can occur in the absence of M6P.
      • Linebaugh B.E.
      • Sameni M.
      • Day N.A.
      • Sloane B.F.
      • Keppler D.
      Exocytosis of active cathepsin B. Enzyme activity at pH 7.0, inhibition and molecular mass.
      As proteolytic activity needs to be tightly regulated, cathepsins undergo several regulatory checkpoints at transcriptional, epigenetic, translational and post-translational levels. Additionally, cathepsin activity is regulated through lysosomal compartmentalisation, pH and endogenous protein inhibitors, such as cystatins and stefins.
      Lysosomal cathepsins are not only random catalytic devices within lysosomes but can perform highly selective and targeted cleavage of specific substrates, resulting in functional post-translational modifications of proteins.
      Despite cathepsins still being mainly referred to as lysosomal proteases, they can be found in other cellular locations such as the cytosol, nucleus, mitochondria and pericellular surroundings, where they perform non-canonical physiological and pathological roles. Cathepsins (Cts) can be classified into distinct families depending on their structure and catalytic sites: serine (CtsA and G), aspartate (CtsD and E) and cysteine (CtsB, C/DPP1, F, H, K, L, O, S, V, W and Z/X).
      • Rawlings N.D.
      • Barrett A.J.
      • Bateman A.
      MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.
      Of all cathepsins, B, D and L are the most abundant. Most cathepsins are ubiquitously expressed in tissues, but some of them are tissue-specific such as K (osteoclasts and epithelial cells) and S, E and W (mainly expressed in immune cells). In addition, some cathepsins can be differentially regulated during disease progression.
      • Yadati T.
      • Houben T.
      • Bitorina A.
      • Shiri-Sverdlov R.
      The ins and outs of cathepsins: physiological function and role in disease management.
      Cathepsins display endo and/or exopeptidase activities and present improved performance within acidic pH ranges. However, they can also display activity outside their optimal pH range and even at neutral pH for a limited period of time.
      • Turk V.
      • Stoka V.
      • Vasiljeva O.
      • Renko M.
      • Sun T.
      • Turk B.
      • et al.
      Cysteine cathepsins: from structure, function and regulation to new frontiers.
      Due to this and their diverse cellular location, they can participate in numerous cellular processes such as apoptosis, autophagy, matrix remodelling, chemotaxis and antigen presentation, all of which are essential for cellular/tissue homeostasis. Therefore, it is not surprising that cathepsins contribute to the development of several human pathologies, including a range of liver diseases that will be discussed in the following sections.

      Role of cathepsins in liver fibrosis/cirrhosis

      Liver fibrosis is characterised by a dysregulation of the normal wound healing response due to repetitive injury, leading to an abnormal accumulation of electrodense extracellular matrix. Liver fibrosis can in some cases resolve, but in others progress into cirrhosis and end-stage liver disease. Due to their functional and spatial plasticity, cathepsins can participate in several cellular processes during the initiation, development and resolution of liver fibrosis.

      Cathepsins’ collagenolytic activity during liver fibrosis/cirrhosis

      The first report on cathepsins’ role in human liver fibrosis dates back to the early 80s and describes increased collagenolytic cathepsin activity in patients with chronic active hepatitis and cirrhosis.
      • Murawaki Y.
      • Hirayama C.
      Hepatic collagenolytic cathepsin in patients with chronic liver disease.
      Since then, several other groups have positively correlated plasma levels of CtsB, L
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Gebbia N.
      Lysosomal cathepsins B and L and stef in A blood levels in patients with hepatocellular carcinoma and/or liver cirrhosis: potential clinical implications.
      ,
      • Yamamoto H.
      • Murawaki Y.
      • Kawasaki H.
      Collagenolytic cathepsin B and L activity in experimental fibrotic liver and human liver.
      and D
      • Kucharz E.
      Clinical and experimental studies on collagen metabolism in hepatic disorders.
      ,
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Ruggeri I.
      • et al.
      Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis.
      with the development of liver fibrosis/cirrhosis in patients with chronic liver disease, proposing that the collagenolytic activity of these cathepsins could increase tissue remodelling and hence contribute to disease progression. In addition, a recent multicentre combined cross-sectional study in patients with varying degrees of liver fibrosis identified 50 urinary peptides associated with liver fibrosis and 10 putative proteolytic cleavage sites, 2 of them for cathepsins.
      • Bannaga A.S.
      • Metzger J.
      • Kyrou I.
      • Voigtländer T.
      • Book T.
      • Melgarejo J.
      • et al.
      Discovery, validation and sequencing of urinary peptides for diagnosis of liver fibrosis—a multicentre study.

      Cathepsins’ contribution to cell death during liver fibrosis/cirrhosis

      The participation of cathepsins in apoptosis is perhaps their most widely accepted function. During apoptosis, lysosomal membrane permeabilisation (LMP) enables translocation of cathepsins into the cytosol, where they cleave different members of the Bcl-2 family, modulating their activity and contributing to apoptosis upstream of the mitochondria. In the context of liver disease, Gores group have made several valuable research contributions, describing cytosolic CtsB as a member of the canonical apoptosis signalling pathway in experimental models of tumour necrosis factor (TNF)-α-induced hepatocyte injury
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.F.
      • Gores G.J.
      Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.
      and bile duct ligation-induced liver fibrosis
      • Canbay A.
      • Guicciardi M.E.
      • Higuchi H.
      • Feldstein A.
      • Bronk S.F.
      • Rydzewski R.
      • et al.
      Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis.
      using CtsB knock-out mouse. In this context, they proposed two mechanisms for CtsB release from the lysosome via LMP, one involving caspase-8
      • Werneburg N.
      • Guicciardi M.E.
      • Yin X.-M.
      • Gores G.J.
      TNF-α-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.
      and the other involving Bax (Bcl2-associated X, apoptosis regulator)
      • Werneburg N.W.
      • Guicciardi M.E.
      • Bronk S.F.
      • Kaufmann S.H.
      • Gores G.J.
      Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins.
      using hepatoma and cholangiocarcinoma cell lines (Fig. 1). In addition to CtsB, CtsD is also thought to contribute to TNF-α-induced apoptosis upstream of the mitochondria via proteolytic cleavage of BH3-interacting domain death agonist (Bid) into truncated Bid (tBid), resulting in cytochrome C release from the mitochondria and activation of caspases
      • Heinrich M.
      • Neumeyer J.
      • Jakob M.
      • Hallas C.
      • Tchikov V.
      • Winoto-Morbach S.
      • et al.
      Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation.
      (Fig. 1).
      Cathepsins contribute to the initiation, development and progression of several liver diseases, participating in multiple biological processes such as apoptosis, autophagy, lipid metabolism, extracellular matrix remodelling, activation of hepatic stellate cells, inflammation and metastasis among others.
      Figure thumbnail gr1
      Fig. 1Contribution of CtsB/D to apoptosis during liver fibrosis.
      TRAIL
      • Werneburg N.W.
      • Guicciardi M.E.
      • Bronk S.F.
      • Kaufmann S.H.
      • Gores G.J.
      Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins.
      or TNF- α bind
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.F.
      • Gores G.J.
      Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.
      • Canbay A.
      • Guicciardi M.E.
      • Higuchi H.
      • Feldstein A.
      • Bronk S.F.
      • Rydzewski R.
      • et al.
      Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis.
      • Werneburg N.
      • Guicciardi M.E.
      • Yin X.-M.
      • Gores G.J.
      TNF-α-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.
      to their respective death receptors triggering activation of JNK or caspase-8, respectively. Activation of caspase-8 results in LMP and CtsB leakage from the lysosome.
      • Werneburg N.
      • Guicciardi M.E.
      • Yin X.-M.
      • Gores G.J.
      TNF-α-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.
      pJNK phosphorylates and activates Bim, which activates Bax. Then Bax permeabilises the lysosomes releasing CtsB into the cytosol.
      • Werneburg N.W.
      • Guicciardi M.E.
      • Bronk S.F.
      • Kaufmann S.H.
      • Gores G.J.
      Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins.
      Proteolytic cleavage of Bid into tBid by CtsB/D activates Bax or Bak leading to cytochrome C release from the mitochondria and subsequent activation of caspase-9 and caspase-3, culminating in apoptosis.
      • Werneburg N.
      • Guicciardi M.E.
      • Yin X.-M.
      • Gores G.J.
      TNF-α-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.
      ,
      • Heinrich M.
      • Neumeyer J.
      • Jakob M.
      • Hallas C.
      • Tchikov V.
      • Winoto-Morbach S.
      • et al.
      Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation.
      Some cellular components displayed in the figure have been adapted from Smart Servier Medical Art under Creative Commons Attribution 3.0 Unported License. Bak, Bcl2 antagonist/killer 1; Bax, Bcl2-associated X, apoptosis regulator; Bid, BH3 interacting domain death agonist; Bim, Bcl2-interacting mediator of cell death; Cts, cathepsin; LMP, lysosomal membrane permeabilisation; tBid, truncated Bid; TNF, tumour necrosis factor; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

      Cathepsins drive HSC activation during liver fibrosis/cirrhosis

      CtsB can also participate in fibrosis by contributing to hepatic stellate cell (HSC) activation and its inhibition results in decreased liver fibrosis and inflammation in a carbon tetrachloride mouse model.
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.
      Mechanistically, inhibition of CtsB using Ca074-Me in mouse HSCs results in defective Akt phosphorylation after platelet-derived growth factor (PDGF) stimulation, leading to a decrease in HSC proliferation and migration and providing a suitable explanation for the decreased fibrosis observed after carbon tetrachloride treatment. Auto and paracrine activation between different cathepsins has often been described and it also occurs between CtsB and D in HSCs. Alternatively, acid sphingomyelinase (ASMase) has been proposed as an activator for CtsB and D in HSCs. However, their direct relationship has not been well defined yet. In that respect, partial ASMase inhibition using pharmacological (imipramine or amitriptyline) or genetic approaches (ASMase heterozygous mice) reduced CtsB/D levels, mimicking the effect of Ca074-Me, by reducing HSC proliferation and liver fibrosis after both bile duct ligation and carbon tetrachloride challenges in mice
      • Moles A.
      • Tarrats N.
      • Morales A.
      • Domínguez M.
      • Bataller R.
      • Caballería J.
      • et al.
      Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis.
      (Fig. 2). Of note, the complete genetic ablation of ASMase, displayed by ASMase knock-out mice, results in a compensatory mechanism increasing the levels of CtsB and liver fibrosis, demonstrating a strong and dependent relationship between ASMase and CtsB.
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsin B overexpression due to acid sphingomyelinase ablation promotes liver fibrosis in Niemann-Pick disease.
      Extracellular CtsB has also been involved in N-Acetyl-L-cysteine desensitisation of HSC towards PDGF-BB by proteolysis of PDGF receptor-β, resulting in diminished liver fibrosis in a rat thioacetamide model.
      • Okuyama H.
      • Shimahara Y.
      • Kawada N.
      • Seki S.
      • Kristensen D.B.
      • Yoshizato K.
      • et al.
      Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.
      In addition to CtsB, other cathepsins have also been linked to the activation of HSC. On the one hand, nuclear CtsF has been related to transcriptional regulation of rat HSC activation markers
      • Maubach G.
      • Lim M.C.C.
      • Zhuo L.
      Nuclear cathepsin F regulates activation markers in rat hepatic stellate cells.
      by an unknown mechanism. On the other hand, loss of nuclear CtsH has been linked to decreased degradation and stabilisation of class IIa histone deacetylases, resulting in the regulation of matrix metalloproteinases gene expression and the activation of HSCs.
      • Yang Z.
      • Liu Y.
      • Qin L.
      • Wu P.
      • Xia Z.
      • Luo M.
      • et al.
      Cathepsin H–mediated degradation of HDAC4 for matrix metalloproteinase expression in hepatic stellate cells.
      Figure thumbnail gr2
      Fig. 2Participation of CtsB/D in HSC activation during liver fibrosis.
      CtsB and D can be activated by ASMase or by each other within the lysosomes of activated HSCs. In addition, they can also auto-activate. They can be secreted into the extracellular space by HSCs and be recycled back into the lysosome through internalisation into early and late endosomes.
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.
      Pharmacological inhibition of CtsB (Ca074-Me)
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.
      and ASMase (imipramine)
      • Moles A.
      • Tarrats N.
      • Morales A.
      • Domínguez M.
      • Bataller R.
      • Caballería J.
      • et al.
      Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis.
      results in blocking of Akt phosphorylation by PDGF-BB and decreased transcription of proliferative genes, which partially explains the decrease in HSC activation and liver fibrosis observed after Ca074-Me
      • Moles A.
      • Tarrats N.
      • Fernández-Checa J.C.
      • Marí M.
      Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.
      administration in WT mice during chronic CCl4 and in ASMase heterozygous mice after BDL and CCl4 challenges.
      • Moles A.
      • Tarrats N.
      • Morales A.
      • Domínguez M.
      • Bataller R.
      • Caballería J.
      • et al.
      Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis.
      Some cellular components displayed in the figure have been adapted from Smart Servier Medical Art under Creative Commons Attribution 3.0 Unported License. ASMase, acid sphingomyelinase; BDL, bile duct ligation; CCl4, carbon tetrachloride; Cts, cathepsin; HSC, hepatic stellate cell; PDGF, platelet-derived growth factor; PDK-1, pyruvate dehydrogenase kinase 1; PI3K, phosphatidylinositol-3-kinase; WT, wild-type.

      Cathepsins’ activity in autophagy-associated signalling pathways during liver fibrosis/cirrhosis

      Given that cathepsins are lysosomal proteases, it is not surprising that they participate in autophagy. A recent study, using long-term exposure to arsenic (NaAsO2) to induce liver fibrosis, demonstrated that increased cytosolic CtsB due to upregulated autophagic flux results in NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in rat HSCs, defining an autophagic-CtsB-NLRP3 signalling pathway.
      • Tao Y.
      • Qiu T.
      • Yao X.
      • Jiang L.
      • Wang N.
      • Jia X.
      • et al.
      Autophagic-CTSB-inflammasome axis modulates hepatic stellate cells activation in arsenic-induced liver fibrosis.
      In agreement with this study, lysosomal release of CtsB into the cytosol in conjunction with the production of reactive oxygen species has been reported to partially mediate the activation of NLRP3 upon lipopolysaccharide/ATP stimulation, promoting mouse HSC activation and liver fibrosis.
      • Inzaugarat M.E.
      • Johnson C.D.
      • Holtmann T.M.
      • McGeough M.D.
      • Trautwein C.
      • Papouchado B.G.
      • et al.
      NLR family pyrin domain-containing 3 inflammasome activation in hepatic stellate cells induces liver fibrosis in mice.
      In addition, the lysosomotropic compound, GNS561, decreases human HSC activation by impairing cathepsin activity, resulting in defective autophagy, maturation of transforming growth factor-β1 and decreased cell viability.
      • Bestion E.
      • Jilkova Z.M.
      • Mège J.-L.
      • Novello M.
      • Kurma K.
      • Pour S.T.A.
      • et al.
      GNS561 acts as a potent anti-fibrotic and pro-fibrolytic agent in liver fibrosis through TGF-β1 inhibition.
      Cathepsins do not operate alone but in signalling cascades and regulatory circuits that are regulated in a timely manner in infiltrated and liver-resident cells, depending on cellular demands.
      Non-cysteine cathepsins such as aspartate CtsD and serine CtsA have also been related to autophagy. In this regard, CtsD expression and autophagy have been positively correlated with the ductular reaction observed in preclinical models and human cirrhotic patients,
      • Hung T.-M.
      • Yuan R.-H.
      • Huang W.-P.
      • Chen Y.-H.
      • Lin Y.-C.
      • Lin C.-W.
      • et al.
      Increased autophagy markers are associated with ductular reaction during the development of cirrhosis.
      while CtsA has been associated with the degradation of lysosome-associated membrane protein 2a (LAMP2a), limiting the rate of chaperone-mediated autophagy in rat liver lysosomes.
      • Cuervo A.M.
      Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.

      Role of cathepsins in the inflammatory response associated with liver fibrosis/cirrhosis

      Cathepsins have been implicated in several inflammatory mechanisms contributing to liver fibrosis. Kahraman et al. demonstrated an association between tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and cytosolic CtsB. They described TRAIL being predominantly expressed in natural killer (NK)/NKT cells during mouse bile duct ligation, suggesting CtsB could potentially play a role in the cytotoxic response induced by NK/NKT cells during liver fibrosis. However, the exact role of CtsB within NK/NKT cells remains unknown.
      • Kahraman A.
      • Barreyro F.J.
      • Bronk S.F.
      • Werneburg N.W.
      • Mott J.L.
      • Akazawa Y.
      • et al.
      TRAIL mediates liver injury by the innate immune system in the bile duct-ligated mouse.
      De Mingo et al., reported that CtsB and CtsS regulated sirtuin-1, a prototype mammalian NAD(+)-dependent protein deacetylase that modulates the transcription factor NF-κB. Inhibition of CtsB/S resulted in decreased NF-κB-dependent hepatic inflammation and fibrosis, accompanied by an increase in the levels of sirtuin-1, in toxin or dietary mouse models of liver fibrosis.
      • de Mingo Á
      • de Gregorio E.
      • Moles A.
      • Tarrats N.
      • Tutusaus A.
      • Colell A.
      • et al.
      Cysteine cathepsins control hepatic NF-κB-dependent inflammation via sirtuin-1 regulation.
      In addition, CtsS is upregulated by interferon-γ in rat HSCs and has been proposed to participate in HSC antigen presentation, most likely via CD74 processing.
      • Maubach G.
      • Lim M.C.C.
      • Kumar S.
      • Zhuo L.
      Expression and upregulation of cathepsin S and other early molecules required for antigen presentation in activated hepatic stellate cells upon IFN-γ treatment.
      Finally, the cytoprotective effect of albumin against TNF-α-induced inflammatory injury has recently been linked to its ability to prevent CtsB leakage from the lysosome into the cytosol, avoiding cytochrome C release from the mitochondria and hence apoptosis.
      • Duran-Güell M.
      • Flores-Costa R.
      • Casulleras M.
      • López-Vicario C.
      • Titos E.
      • Díaz A.
      • et al.
      Albumin protects the liver from tumor necrosis factor α-induced immunopathology.

      Role of cathepsins in NAFLD

      Non-alcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is caused by an underlying state of systemic metabolic dysfunction and is the leading cause of chronic liver disease worldwide. The first report on the involvement of cathepsins in fatty liver disease dates back to 1958.
      • Martini E.
      • Dianzani M.U.
      Activation of cathepsin in fatty liver.
      Since then, many papers have established a direct link between the disturbance of lysosomal metabolism, cathepsin levels and the development of NAFLD.

      Cathepsins contribution to lipotoxicity during NAFLD

      Excess lipid accumulation in non-adipose tissue disrupts cell function, resulting in a process of cell death called lipotoxicity. The role of cathepsins in lipotoxicity has been reported by several groups. Different authors associate free fatty acid (FFA) toxicity with cathepsins, however, their role remains controversial. While FFA-induced apoptosis seems to be independent of lysosomal disruption, as CtsB-deficient hepatocytes demonstrate no attenuation of the FFA-mediated toxicity,
      • Malhi H.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.
      Li and co-workers demonstrated that FFA-induced apoptosis was dependent on CtsB leakage into the cytosol by LMP. Hence, CtsB knock-out mice display preserved mitochondrial function, reduced oxidative stress and ameliorated liver damage during NAFLD.
      • Li Z.
      • Berk M.
      • McIntyre T.M.
      • Gores G.J.
      • Feldstein A.E.
      The lysosomal-mitochondrial axis in free fatty acid-induced hepatic lipotoxicity.
      CtsB inhibition using Ca074-Me has also been reported to reduce hepatic inflammation and improve hepatic function in mice after methionine-choline-deficient diet feeding, by limiting NLRP3/caspase-1 activation and interleukin (IL)-1β and IL-18 secretion.
      • Tang Y.
      • Cao G.
      • Min X.
      • Wang T.
      • Sun S.
      • Du X.
      • et al.
      Cathepsin B inhibition ameliorates the non-alcoholic steatohepatitis through suppressing caspase-1 activation.
      Interestingly, LDL receptor-related protein-1 (LRP1) participates in cholesterol efflux via HDL secretion and plays a role in CtsD endocytosis.
      • Basford J.E.
      • Wancata L.
      • Hofmann S.M.
      • Silva R.A.G.D.
      • Davidson W.S.
      • Howles P.N.
      • et al.
      Hepatic deficiency of low density lipoprotein receptor-related protein-1 reduces high density lipoprotein secretion and plasma levels in mice.
      Therefore, it is not surprising that dysregulation of cholesterol efflux and CtsD endocytosis, due to the absence of LRP1 in mouse hepatocytes, results in lipotoxicity after palmitate-induced steatosis.
      • Hamlin A.N.
      • Basford J.E.
      • Jaeschke A.
      • Hui D.Y.
      LRP1 protein deficiency exacerbates palmitate-induced steatosis and toxicity in hepatocytes.
      Due to the wide variety of cellular locations, functions and time-dependent activities exhibited by cathepsins during liver disease, design and development of potential therapies based around them needs careful consideration.

      Involvement of cathepsins in lipid metabolism during NAFLD

      In recent years, CtsD has been studied as a potential biomarker for NAFLD but its clinical applicability remains controversial. In that respect, plasma CtsD levels have been positively correlated with non-alcoholic steatohepatitis (NASH) development
      • Walenbergh S.M.A.
      • Houben T.
      • Rensen S.S.
      • Bieghs V.
      • Hendrikx T.
      • van Gorp P.J.
      • et al.
      Plasma cathepsin D correlates with histological classifications of fatty liver disease in adults and responds to intervention.
      in adults, but negatively correlated in children.
      • Walenbergh S.M.A.
      • Houben T.
      • Hendrikx T.
      • Jeurissen M.L.J.
      • van Gorp P.J.
      • Vreugdenhil A.C.E.
      • et al.
      Plasma cathepsin D levels: a novel tool to predict pediatric hepatic inflammation.
      In addition, plasma CtsD activity has also been positively correlated with metabolic parameters of type 2 diabetes.
      • Ding L.
      • Houben T.
      • Oligschlaeger Y.
      • Bitorina A.V.
      • Verwer B.J.
      • Tushuizen M.E.
      • et al.
      Plasma cathepsin D activity rather than levels correlates with metabolic parameters of type 2 diabetes in male individuals.
      However, a recent paper reported limited applicability of CtsD as a biomarker for NASH,
      • Kamarajah S.K.
      • Khoo S.
      • Chan W.
      • Sthaneshwar P.
      • Nik Mustapha N.R.
      • Mahadeva S.
      Limited applicability of cathepsin D for the diagnosis and monitoring of non-alcoholic steatohepatitis.
      so further investigation is needed to clarify its potential clinical use. In addition to their potential roles as biomarkers, cathepsins have also been directly implicated in lipid metabolism during NAFLD. CtsB-deficient mice display reduced de novo lipogenesis, liver inflammation and fibrosis after fructose-palmitate-cholesterol feeding; however, the direct targets of CtsB remain unclear.
      • Fang W.
      • Deng Z.
      • Benadjaoud F.
      • Yang C.
      • Shi G.-P.
      Cathepsin B deficiency ameliorates liver lipid deposition, inflammatory cell infiltration, and fibrosis after diet-induced nonalcoholic steatohepatitis.
      Liver fatty acid-binding protein is reported to be a substrate for CtsB and its cleavage regulates liver FFA uptake and VLDL secretion after oleic acid exposure in the rat hepatocyte cell line McA-RH7777.
      • Thibeaux S.
      • Siddiqi S.
      • Zhelyabovska O.
      • Moinuddin F.
      • Masternak M.M.
      • Siddiqi S.A.
      Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid–binding protein.
      In addition, pharmacological inhibition of CtsD increases cholesterol conversion into bile acids, improving dyslipidaemia in experimental mouse models of NASH.
      • Houben T.
      • Oligschlaeger Y.
      • Hendrikx T.
      • Bitorina A.V.
      • Walenbergh S.M.A.
      • van Gorp P.J.
      • et al.
      Cathepsin D regulates lipid metabolism in murine steatohepatitis.
      Recently, extracellular inhibition of CtsD has also been reported to slow down NASH progression in high-fat diet-fed rats via an unknown mechanism.
      • Khurana P.
      • Yadati T.
      • Goyal S.
      • Dolas A.
      • Houben T.
      • Oligschlaeger Y.
      • et al.
      Inhibiting extracellular cathepsin D reduces hepatic steatosis in Sprague–Dawley rats.

      Participation of cathepsins in autophagy during NAFLD

      Despite the aforementioned detrimental roles of cathepsins during NAFLD, therapies based on their inhibition require careful consideration, as cathepsins fulfil important homeostatic roles in autophagy. Patients with NAFLD display abnormal autophagy and decreased expression of liver CtsB, D and L.
      • Fukuo Y.
      • Yamashina S.
      • Sonoue H.
      • Arakawa A.
      • Nakadera E.
      • Aoyama T.
      • et al.
      Abnormality of autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease.
      The decreased cathepsin expression detected during NAFLD seems to be a consequence rather than a cause of the disrupted autophagic functions. The current literature indicates that defective lysosomal acidification results in decreased lysosomal cathepsin levels (CtsB, D and L)
      • Simoes I.C.M.
      • Karkucinska-Wieckowska A.
      • Janikiewicz J.
      • Szymanska S.
      • Pronicki M.
      • Dobrzyn P.
      • et al.
      Western diet causes obesity-induced nonalcoholic fatty liver disease development by differentially compromising the autophagic response.
      ,
      • Inami Y.
      • Yamashina S.
      • Izumi K.
      • Ueno T.
      • Tanida I.
      • Ikejima K.
      • et al.
      Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.
      and proteolytic activation (CtsD),
      • Wang X.
      • Zhang X.
      • Chu E.S.H.
      • Chen X.
      • Kang W.
      • Wu F.
      • et al.
      Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis.
      as well as reduced activity of the transcription factor EB,
      • Chen X.
      • Chan H.
      • Zhang L.
      • Liu X.
      • Ho I.H.T.
      • Zhang X.
      • et al.
      The phytochemical polydatin ameliorates non-alcoholic steatohepatitis by restoring lysosomal function and autophagic flux.
      leading to impaired autophagic flux during NAFLD. Thus, several experimental therapies have been proposed to ameliorate NASH by restoring lysosomal acidification, autophagic flux and cathepsins activity.
      • Chen X.
      • Chan H.
      • Zhang L.
      • Liu X.
      • Ho I.H.T.
      • Zhang X.
      • et al.
      The phytochemical polydatin ameliorates non-alcoholic steatohepatitis by restoring lysosomal function and autophagic flux.
      ,
      • Qiu T.
      • Pei P.
      • Yao X.
      • Jiang L.
      • Wei S.
      • Wang Z.
      • et al.
      Taurine attenuates arsenic-induced pyroptosis and nonalcoholic steatohepatitis by inhibiting the autophagic-inflammasomal pathway.

      Role of cathepsins in HCC

      Several types of cancer display cathepsin-dependency, and their inhibition reduces cancer progression, drug resistance and metastasis. Therefore, strategies based on cathepsin inhibition are currently being considered as potential anti-cancer therapies for hepatocellular carcinoma (HCC).
      • Varshosaz J.
      • Fard M.M.
      • Mirian M.
      • Hassanzadeh F.
      Targeted nanoparticles for Co-delivery of 5-FU and nitroxoline, a cathepsin B inhibitor, in HepG2 cells of hepatocellular carcinoma.
      ,
      • Fuchs N.
      • Meta M.
      • Schuppan D.
      • Nuhn L.
      • Schirmeister T.
      Novel opportunities for cathepsin S inhibitors in cancer immunotherapy by nanocarrier-mediated delivery.
      The role of cathepsins in liver cancer has not been explored as extensively as in other types of cancer. Nevertheless, their importance in driving different cellular processes that contribute to cancer progression is clear. Despite cathepsins also seeming to contribute to the development of cholangiocarcinoma,
      • Terada T.
      • Ohta T.
      • Minato H.
      • Nakanuma Y.
      Expression of pancreatic trypsinogen/trypsin and cathepsin B in human cholangiocarcinomas and hepatocellular carcinomas.
      their contribution to the development of HCC has been more widely explored so we will describe it in more detail.
      Improving our biological understanding of how cathepsin signalling networks control liver disease progression is essential to design novel and targeted drugs for the treatment of liver disease.

      Cathepsins as prognostic markers in HCC

      Overexpression of CtsB,
      • Herszényi L.
      • István G.
      • Cardin R.
      • De Paoli M.
      • Plebani M.
      • Tulassay Z.
      • et al.
      Serum cathepsin B and plasma urokinase-type plasminogen activator levels in gastrointestinal tract cancers.
      D,
      • Leto G.
      • Tumminello F.M.
      • Pizzolanti G.
      • Montalto G.
      • Soresi M.
      • Ruggeri I.
      • et al.
      Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis.
      L,
      • Ruan J.
      • Zheng H.
      • Rong X.
      • Rong X.
      • Zhang J.
      • Fang W.
      • et al.
      Over-expression of cathepsin B in hepatocellular carcinomas predicts poor prognosis of HCC patients.
      S
      • Zhuo
      Cathepsin S is aberrantly overexpressed in human hepatocellular carcinoma.
      and Z
      • Wang J.
      • Chen L.
      • Li Y.
      • Guan X.-Y.
      Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.
      has been reported in human HCC and linked to poor prognosis.
      • Tumminello F.M.
      • Leto G.
      • Pizzolanti G.
      • Candiloro V.
      • Crescimanno M.
      • Crosta L.
      • et al.
      Cathepsin D, B and L circulating levels as prognostic markers of malignant progression.
      In addition, single nucleotide polymorphisms A4383C and C76G in the CtsB gene have been associated with tumour size and a high risk of HCC.
      • Chen T.-P.
      • Yang S.-F.
      • Lin C.-W.
      • Lee H.-L.
      • Tsai C.-M.
      • Weng C.-J.
      A4383C and C76G SNP in Cathepsin B is respectively associated with the high risk and tumor size of hepatocarcinoma.
      Cathepsins have also been identified as part of proteomic signatures that distinguish HCC from non-malignant tissues (CtsB)
      • Lee N.P.
      • Chen L.
      • Lin M.C.
      • Tsang F.H.
      • Yeung C.
      • Poon R.T.
      • et al.
      Proteomic expression signature distinguishes cancerous and nonmalignant tissues in hepatocellular carcinoma.
      and plasma (CtsA).
      • Du Z.
      • Liu X.
      • Wei X.
      • Luo H.
      • Li P.
      • Shi M.
      • et al.
      Quantitative proteomics identifies a plasma multi-protein model for detection of hepatocellular carcinoma.
      Due to cathepsin compartmentalisation in malignant tissues, targeted therapies based on cathepsin-cleavable drugs are currently being explored in HCC to safely deliver therapies into HCC cells, reducing undesirable toxic effects.
      • Wang Q.
      • Zhong Y.-J.
      • Yuan J.-P.
      • Shao L.-H.
      • Zhang J.
      • Tang L.
      • et al.
      Targeting therapy of hepatocellular carcinoma with doxorubicin prodrug PDOX increases anti-metastatic effect and reduces toxicity: a preclinical study.

      Contribution of cathepsins to autophagy and apoptosis during HCC

      Autophagy plays a dual and contradictory role in HCC development, both preventing carcinogenesis (cytotoxicity) at early stages and promoting tumour progression (cytoprotection) in advanced ones.
      • Yang S.
      • Yang L.
      • Li X.
      • Li B.
      • Li Y.
      • Zhang X.
      • et al.
      New insights into autophagy in hepatocellular carcinoma: mechanisms and therapeutic strategies.
      Several recent reports have intimately linked a reduction of drug resistance-associated autophagy with increased cancer cell death. In that respect, reduced lysosomal CtsB or D, and hence lysosomal proteolysis, has been associated with increased cytotoxicity in response to bortezomib (a 26S proteasome inhibitor),
      • Kao C.
      • Chao A.
      • Tsai C.-L.
      • Chuang W.-C.
      • Huang W.-P.
      • Chen G.-C.
      • et al.
      Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation.
      water-soluble yeast β-D-glucan
      • Wang N.
      • Liu H.
      • Liu G.
      • Li M.
      • He X.
      • Yin C.
      • et al.
      Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis.
      and different bioactive phytochemicals,
      • Xue L.
      • Liu P.
      Daurisoline inhibits hepatocellular carcinoma progression by restraining autophagy and promoting cispaltin-induced cell death.
      owing to impaired autophagic flux in HCC cells. Direct participation of cathepsins in apoptotic cell death during HCC has also been reported. Thus, LMP-induced leakage of CtsB into the cytosol contributes to apoptosis – mediated by TNF-α/cycloheximide,
      • Ullio C.
      • Casas J.
      • Brunk U.T.
      • Sala G.
      • Fabriàs G.
      • Ghidoni R.
      • et al.
      Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells.
      TRAIL
      • Guicciardi M.E.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      cFLIP L prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis.
      and JNK inhibition
      • Desideri E.
      • Ciriolo M.R.
      Inhibition of JNK increases the sensitivity of hepatocellular carcinoma cells to lysosomotropic drugs via LAMP2A destabilization.
      – by proteolytically activating Bid (by cleavage into tBid)
      • Droga-Mazovec G.
      • Bojič L.
      • Petelin A.
      • Ivanova S.
      • Romih R.
      • Repnik U.
      • et al.
      Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues.
      and triggering apoptosis upstream of the mitochondria in hepatoma cells. Increased lysosomal sphingosine
      • Ullio C.
      • Casas J.
      • Brunk U.T.
      • Sala G.
      • Fabriàs G.
      • Ghidoni R.
      • et al.
      Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells.
      and cytosolic tBid levels
      • Guicciardi M.E.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      cFLIP L prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis.
      as well as LAMP2a destabilisation
      • Desideri E.
      • Ciriolo M.R.
      Inhibition of JNK increases the sensitivity of hepatocellular carcinoma cells to lysosomotropic drugs via LAMP2A destabilization.
      have been proposed as LMP inducers. However, the exact mechanism of LMP in hepatoma cells is not yet fully understood (Fig. 3). Of note, Bid is the only member of the Bcl-2 family to be identified as a substrate of cysteine cathepsins in HepG2 cells.
      • Droga-Mazovec G.
      • Bojič L.
      • Petelin A.
      • Ivanova S.
      • Romih R.
      • Repnik U.
      • et al.
      Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues.
      Lastly, an intriguing positive correlation between patient survival and intranuclear inclusions containing cathepsins and autophagic proteins has recently been reported in HCC.
      • Schwertheim S.
      • Westerwick D.
      • Jastrow H.
      • Theurer S.
      • Schaefer C.M.
      • Kälsch J.
      • et al.
      Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival.
      However, further studies are required to confirm and explain their biological function in cancer cells.
      Figure thumbnail gr3
      Fig. 3Participation of CtsB in apoptosis in hepatoma cells.
      Leakage of CtsB into the cytosol contributes to apoptosis upstream of mitochondria by proteolytic cleavage of Bid into tBid.
      • Droga-Mazovec G.
      • Bojič L.
      • Petelin A.
      • Ivanova S.
      • Romih R.
      • Repnik U.
      • et al.
      Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues.
      Two different mechanisms triggering LMP have been reported in hepatoma cell lines: i) TRAIL binding to its death receptor triggers caspase-8 activation which cleaves Bid into tBid resulting in LMP.
      • Guicciardi M.E.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      cFLIP L prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis.
      ii) TNF-α/CHX stimulation results in increased generation of sphingosine in the lysosome increasing LMP.
      • Ullio C.
      • Casas J.
      • Brunk U.T.
      • Sala G.
      • Fabriàs G.
      • Ghidoni R.
      • et al.
      Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells.
      In addition, pJNK stabilises LAMP2a into the lysosomal membrane protecting lysosomes from LMP, hence JNK inhibition is reported to trigger LMP.
      • Desideri E.
      • Ciriolo M.R.
      Inhibition of JNK increases the sensitivity of hepatocellular carcinoma cells to lysosomotropic drugs via LAMP2A destabilization.
      Some cellular components displayed in the figure have been adapted from Smart Servier Medical Art under Creative Commons Attribution 3.0 Unported License. Bak, Bcl2 antagonist/killer 1; Bax, Bcl2-associated X, apoptosis regulator; Bid, BH3 interacting domain death agonist; CHX, cycloheximide; Cts, cathepsin; LAMP2a, lysosomal associated membrane protein 2a; LMP, lysosomal membrane permeabilisation; tBid, truncated Bid; TNF, tumour necrosis factor; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

      Cathepsins-related tumour invasiveness and metastasis

      Cathepsins play important roles in extracellular matrix remodelling, invasion and migration, actively contributing to the metastatic potential of several types of cancer. Specifically, in HCC, cathepsins have been associated with increased tumour invasiveness due to a range of cellular mechanisms. Increased CtsB transcription by activation of the β-catenin signalling pathway is mediated by CD147 in hepatoma cells and results in increased collective invasion.
      • Wang S.-J.
      • Chao D.
      • Wei W.
      • Nan G.
      • Li J.-Y.
      • Liu F.-L.
      • et al.
      CD147 promotes collective invasion through cathepsin B in hepatocellular carcinoma.
      Extracellular cathepsins also play an important role in promoting tumour invasiveness. In this regard, increased extracellular secretion of CtsB and D promotes invasiveness and migration of hepatoma cells mediated by tetrabromobisphenol A.
      • Lyu L.
      • Jin X.
      • Li Z.
      • Liu S.
      • Li Y.
      • Su R.
      • et al.
      TBBPA regulates calcium-mediated lysosomal exocytosis and thereby promotes invasion and migration in hepatocellular carcinoma.
      In addition, hepatitis B spliced protein can directly interact with CtsB, promoting hepatoma cell invasion and motility due to increased secretion and activation of proteolytic enzymes and activation of the MAPK/Akt signalling pathway.
      • Chen W.-N.
      • Chen J.-Y.
      • Jiao B.-Y.
      • Lin W.-S.
      • Wu Y.-L.
      • Liu L.-L.
      • et al.
      Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion.
      CtsC has also been reported to promote metastasis via TNF-α/p38 MAPK activation.
      • Zhang G.-P.
      • Yue X.
      • Li S.-Q.
      Cathepsin C interacts with TNF-α/p38 MAPK signaling pathway to promote proliferation and metastasis in hepatocellular carcinoma.
      Moreover, CtsS has been associated with metastasis and angiogenesis via the VEGF-A/MEK1/ERK1/2 signalling pathway
      • Zhang Z.
      • Zhang H.
      • Peng T.
      • Li D.
      • Xu J.
      Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma.
      and its silencing supresses proliferation, invasion and angiogenesis of hepatoma cells.
      • Fan Q.
      • Wang X.
      • Zhang H.
      • Li C.
      • Fan J.
      • Xu J.
      Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro.
      Finally, overexpression of CtsZ upregulates epithelial-mesenchymal transition, contributing to the metastatic potential of HCC.
      • Wang J.
      • Chen L.
      • Li Y.
      • Guan X.-Y.
      Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

      Cathepsins’ involvement in pro-tumorigenic signalling pathways during HCC

      Cathepsins can participate in signalling pathways that contribute to mitogenesis and tumour growth. In that respect, CtsD has been proposed to cleave
      • Scharf J.G.
      • Braulke T.
      • Hartmann H.
      • Ramadori G.
      Regulation of the components of the 150 kDa IGF binding protein complex in cocultures of rat hepatocytes and Kupffer cells by 3’,5’-cyclic adenosine monophosphate.
      and participate in the endocytosis of IGFBPs (insulin-like growth factor-binding proteins), decreasing their expression and contributing to mitogenesis and drug resistance in hepatoma cells.
      • Alexia C.
      • Fallot G.
      • Lasfer M.
      • Schweizer-Groyer G.
      • Groyer A.
      An evaluation of the role of insulin-like growth factors (IGF) and of type-I IGF receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against drug-induced apoptosis.
      A CtsS/protease-activated receptor 2 axis has been reported to drive tumour initiation, growth and chemoresistance upon CD47 activation in HCC tumour-initiating cells.
      • Lee T.K.-W.
      • Cheung V.C.-H.
      • Lu P.
      • Lau E.Y.T.
      • Ma S.
      • Tang K.H.
      • et al.
      Blockade of CD47-mediated cathepsin S/protease-activated receptor 2 signaling provides a therapeutic target for hepatocellular carcinoma.
      Besides, CtsB has been proposed to activate the PI3K/Akt signalling pathway and promote HCC proliferation by binding integrin αvβ3.
      • Xu Z.-Z.
      • Xiu P.
      • Lv J.-W.
      • Wang F.-H.
      • Dong X.-F.
      • Liu F.
      • et al.
      Integrin αvβ3 is required for cathepsin B-induced hepatocellular carcinoma progression.
      Lastly, molecular fingerprinting in a murine autochthonous tumour model suggests the importance of CtsS in HCC-derived endothelial cells, pointing towards its possible role in the neovascularisation of the tumour.
      • Ryschich E.
      • Lizdenis P.
      • Ittrich C.
      • Benner A.
      • Stahl S.
      • Hamann A.
      • et al.
      Molecular fingerprinting and autocrine growth regulation of endothelial cells in a murine model of hepatocellular carcinoma.

      Future perspectives

      Mounting evidence describes a pathological landscape for the participation of cathepsins in several cellular processes essential for the initiation, development and progression of different liver diseases. In addition, several cathepsins have been proposed as potentially useful biomarkers for liver disease detection or prognostic assessment, but further validation is required before their possible translation from bench to bedside can be considered. Cathepsin activities are regulated in a timely manner in infiltrating and liver-resident cells depending on the cellular demands and type of disease. Thus, they participate in multiple biological processes such as apoptosis, autophagy, lipid metabolism, extracellular matrix remodelling, activation of HSCs, inflammation, and metastasis among others. Due to the wide variety of cellular locations, functions and time-dependent activities exhibited by cathepsins, design and development of potential therapies will require careful consideration. Despite our growing understanding of the roles played by cathepsins in liver disease, our knowledge of their specific targets and signalling networks remains limited. It is only by increasing our biological understanding of cathepsin signalling networks that we will be able to design novel and target-specific approaches for the treatment of liver disease.

      Abbreviations

      ASMase, acid sphingomyelinase; Bid, BH3 interacting domain death agonist; Cts, cathepsin; FFA, free fatty acid; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; IL-, interleukin-; LMP, lysosomal membrane permeabilisation; LRP1, LDL receptor-related protein-1; MAFLD, metabolic dysfunction-associated fatty liver disease; NAFLD, non-alcoholic fatty liver disease; NK, natural killer; NLRP3, NLR family pyrin domain containing 3; PDGF, platelet-derived growth factor; tBid, truncated Bid; TNF, tumour necrosis factor; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

      Financial support

      AM’s research was supported by a “Research Challenges" R+D+i Project ( RTI2018-097475-A-100 ) and a Ramon y Cajal contract ( RYC-2016-19731 ) awarded by FEDER/Ministerio de Ciencia e Innovación - Agencia Estatal de Investigación . PRB is supported by a FPU PhD studentship (FPU19/05357) funded by Ministerio de Ciencia, Innovación y Universidades. VP held a PhD studentship funded by University of Naples Federico II . MFF salary was supported by JAE Intro fellowship (JAEINT_20_02409) from the Spanish National Research Council (CSIC) and FI AGAUR ( 2021 FI_B 00249 ).

      Authors’ contributions

      PRB, VP, MFF and AM wrote the manuscript. PRB, VP and MFF created table 1. AM generated the figures. AM conceptually created, designed and revised the manuscript.

      Conflict of interest

      The authors have no conflict of interest to disclose.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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