Extracellular Cathepsin Research Articles

Cathepsin L-mediated EGFR cleavage affects intracellular signalling pathways in cancer.

Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors.

Integration of osteoclastogenesis through addition of PBMCs in human osteochondral explants cultured ex vivo

The preservation of tissue specific cells in their native 3D extracellular matrix in bone explants provides a unique platform to study remodeling. Thus far, studies involving bone explant cultures showed a clear focus on achieving bone formation and neglected osteoclast activity and resorption. To simulate the homeostatic bone environment ex vivo, both key elements of bone remodeling need to be represented. This study aimed to assess and include osteoclastogenesis in human osteochondral explants through medium supplementation with RANKL and M-CSF and addition of peripheral blood mononuclear cells (PBMCs), providing osteoclast precursors.Osteochondral explants were freshly harvested from human femoral heads obtained from hip surgeries and cultured for 20 days in a two-compartment culture system. Osteochondral explants preserved viability and cellular abundance over the culture period, but histology demonstrated that resident osteoclasts were no longer present after 4 days of culture. Quantitative extracellular tartrate resistant acid phosphatase (TRAP) analysis confirmed depletion of osteoclast activity on day 4 even when stimulated with RANKL and M-CSF. Upon addition of PBMCs, a significant upregulation of TRAP activity was measured from day 10 onwards. Evaluation of bone loss trough μCT registration and measurement of extracellular cathepsin K activity revealed indications of enhanced resorption upon addition of PBMCs. Based on the results we suggest that an external source of osteoclast precursors, such as PBMCs, needs to be added in long-term bone explant cultures to maintain osteoclastic activity, and bone remodeling.

All Roads Lead to Cathepsins: The Role of Cathepsins in Non-Alcoholic Steatohepatitis-Induced Hepatocellular Carcinoma.

Cathepsins are lysosomal proteases that are essential to maintain cellular physiological homeostasis and are involved in multiple processes, such as immune and energy regulation. Predominantly, cathepsins reside in the lysosomal compartment; however, they can also be secreted by cells and enter the extracellular space. Extracellular cathepsins have been linked to several pathologies, including non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). NASH is an increasingly important risk factor for the development of HCC, which is the third leading cause of cancer-related deaths and poses a great medical and economic burden. While information regarding the involvement of cathepsins in NASH-induced HCC (NASH-HCC) is limited, data to support the role of cathepsins in either NASH or HCC is accumulating. Since cathepsins play a role in both NASH and HCC, it is likely that the role of cathepsins is more significant in NASH-HCC compared to HCC derived from other etiologies. In the current review, we provide an overview on the available data regarding cathepsins in NASH and HCC, argue that cathepsins play a key role in the transition from NASH to HCC, and shed light on therapeutic options in this context.

Sex-specific extracellular matrix remodeling during early adipogenic differentiation by gestational bisphenol A exposure.

Bisphenol A (BPA) is an endocrine disrupting chemical known to promote adipose tissue mass in vivo and adipogenesis in vitro. Whether BPA can affect and reprogram early adipogenic differentiation signals that trigger adipogenic differentiation, remains unknown. We hypothesized that gestational BPA exposure results in a preadipocyte phenotype that leads to accelerated adipogenic differentiation, and that this phenotype is sex specific. Primary ovine fetal preadipocytes were derived from control (C) and BPA-exposed during pregnancy and differentiated in vitro. Gestational BPA enhanced lipid accumulation at early stages of differentiation (48h) and this was evident in females but not male-derived fetal preadipocytes. After an RNA sequencing approach, samples were compared as follows: 2 groups (C vs. BPA); 2 sexes (female (F) vs. male (M)); and 2 time points (0h vs. 48 h). Before differentiation, 15 genes were differentially expressed between the C and the BPA-exposed preadipocytes within sex. In BPA-F, extracellular matrix remodeling genes cathepsin K and collagen 5α3 were upregulated compared to C-F. At 48h, BPA-F had 154 genes differentially expressed vs. C-F and BPA-M had 487 genes differentially expressed vs. C-M. Triglyceride and glycerophospholipid metabolism were the most upregulated pathways in BPA-F. Downregulated pathways were associated with extracellular matrix organization in BPA-exposed preadipocytes. These findings are among the first to demonstrate that gestational BPA can modify the fate of adipocyte precursors by altering pathways associated to extracellular matrix components, an often-disregarded, but required aspect of adipogenic differentiation. This work highlights the need to investigate early adipogenic differentiation changes in other obesogenic chemicals.

Cysteine Peptidase Cathepsin X as a Therapeutic Target for Simultaneous TLR3/4-mediated Microglia Activation

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Extracellular cathepsin Z signals through the α5 integrin and augments NLRP3 inflammasome activation

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1β production. Cathepsin Z has been associated with the development of inflammation and IL-1β production; however, the mechanism of how cathepsin Z leads to IL-1β production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1β release was determined using enzyme-linked immunosorbent assay or IL-1β bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1β expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1β through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

Inhibition of Extracellular Cathepsin D Reduces Hepatic Lipid Accumulation and Leads to Mild Changes in Inflammationin NASH Mice.

Background & AimsThe lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.MethodsLow-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.Results Ldlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.ConclusionWe have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

The Ins and Outs of Cathepsins: Physiological Function and Role in Disease Management.

Cathepsins are the most abundant lysosomal proteases that are mainly found in acidic endo/lysosomal compartments where they play a vital role in intracellular protein degradation, energy metabolism, and immune responses among a host of other functions. The discovery that cathepsins are secreted and remain functionally active outside of the lysosome has caused a paradigm shift. Contemporary research has unraveled many versatile functions of cathepsins in extralysosomal locations including cytosol and extracellular space. Nevertheless, extracellular cathepsins are majorly upregulated in pathological states and are implicated in a wide range of diseases including cancer and cardiovascular diseases. Taking advantage of the differential expression of the cathepsins during pathological conditions, much research is focused on using cathepsins as diagnostic markers and therapeutic targets. A tailored therapeutic approach using selective cathepsin inhibitors is constantly emerging to be safe and efficient. Moreover, recent development of proteomic-based approaches for the identification of novel physiological substrates offers a major opportunity to understand the mechanism of cathepsin action. In this review, we summarize the available evidence regarding the role of cathepsins in health and disease, discuss their potential as biomarkers of disease progression, and shed light on the potential of extracellular cathepsin inhibitors as safe therapeutic tools.

Cathepsin D in the Tumor Microenvironment of Breast and Ovarian Cancers.

Cancer remains a major and leading health problem worldwide. Lack of early diagnosis, chemoresistance, and recurrence of cancer means vast research and development are required in this area. The complexity of the tumor microenvironment in the biological milieu poses greater challenges in having safer, selective, and targeted therapies. Existing strategies such as chemotherapy, radiotherapy, and antiangiogenic therapies moderately improve progression-free survival; however, they come with side effects that reduce quality of life. Thus, targeting potential candidates in the microenvironment, such as extracellular cathepsin D (CathD) which has been known to play major pro-tumorigenic roles in breast and ovarian cancers, could be a breakthrough in cancer treatment, specially using novel treatment modalities such as immunotherapy and nanotechnology-based therapy. This chapter discusses CathD as a pro-cancerous, more specifically a proangiogenic factor, that acts bi-functionally in the tumor microenvironment, and possible ways of targeting the protein therapeutically.

2228Circulating serum extracellular matrix degradation enzyme cathepsin S predicts mortality and improves risk stratification over the GRACE score in patients with non-ST elevation acute coronary syndrom

Abstract Background Blood-based biomarkers may be useful in the identification of residual risk for death or acute myocardial infarction (AMI) in patients with a previous acute coronary syndrome. Cathepsin S (CTSS) is a lysosomal cysteine protease with potent elastolytic and collagenolytic activity, which plays an important role in cardiovascular disease through extracellular matrix degradation, vasa vasorum development and atherosclerotic plaque rupture. The aim of the present study was to determine the prognostic and reclassification value of baseline circulating levels of CTSS after adjustment for the Global Registry of Acute Coronary Events (GRACE) score, which is widely recommended for risk stratification in non-ST-segment elevation acute coronary syndrome (NSTE-ACS). Methods CTSS was measured in blood samples collected from 1,129 consecutive patients with adjudicated NSTE-ACS presenting at an acute chest pain unit for evaluation of a possible acute coronary syndrome. Cardiovascular (CV) death and a composite of all-cause mortality and AMI were evaluated as the primary and secondary endpoints of the study, respectively. The additive prognostic value of CTSS over the GRACE score was estimated by the Net Reclassification Index (NRI) that examines the net upward and downward reclassification into correct pre-defined risk categories. Results After a median follow-up of 21 months, 101 (8.95%) deaths were reported, of which 63 (5.6%) were of cardiac origin. The combined endpoint occurred in 176 (15.6%) patients. Patients with CTSS in the highest tertile presented the greatest risk for all-cause mortality (HR=1.84 for highest versus lowest tertile of CTSS distribution, 95% CI 1.1–3.08, P=0.02) and CV death (HR=2.5 for highest versus lowest tertile of CTSS distribution, 95% CI 1.24–5.05, P=0.011) after adjustment for age, gender, diabetes mellitus, hs-cTnT, hsCRP, revascularization and index diagnosis. Similarly, CTSS was associated with increased risk of cardiovascular death after adjusting for the GRACE Score (adjusted HR for highest versus lowest tertile of CTSS distribution=2.34, 95% CI 1.18–4.64, P=0.015). Further, CTSS predicted the combined endpoint of all-cause death or non-fatal MI independently of the GRACE Score (adjusted HR for highest versus lowest tertile of CTSS distribution=1.67, 95% CI 1.15–2.42, P=0.007). When CTSS was added over the GRACE Score, it conferred significant reclassification value for CV death (NRI=21.4%, P=0.008). Similarly, CTSS correctly reclassified risk for all-cause death or non-fatal MI (P=0.006) in 15.9% of the population. Conclusions Circulating CTSS predicts mortality and improves risk stratification of patients with NSTE-ACS over the GRACE score recommended by clinical guidelines. The clinical application of CTSS as a novel biomarker in NSTE-ACS should be further explored and validated.

Functional analysis and gene expression profiling of extracellular cathepsin Z in red sea bream, Pagrus major

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.

HIV Infection Induces Extracellular Cathepsin B Uptake and Damage to Neurons

HIV-associated neurocognitive disorders prevail in 20–50 percent of infected individuals. Macrophages transmigrate through the blood brain barrier during HIV-1 infection, triggering neuronal dysfunction. HIV-infected macrophages secrete cathepsin B (CATB), and serum amyloid p component (SAPC), inducing neuronal apoptosis by an unknown mechanism. We hypothesized that HIV infection facilitates CATB/SAPC secretion from macrophages followed by neuronal internalization, promoting dysfunction. SK-N-SH neuronal cells were exposed to active recombinant histidine-tagged cathepsin B (His-CATB). His-CATB entry was tracked by intracellular flow cytometry, and neuronal dysfunction was verified by western blot. Macrophage-derived extracellular vesicles (EVs) were tested for the presence of CATB and SAPC. Neurons internalized His-CATB, an effect that was partially decreased by pre-treatment with anti-CATB antibody. Pre-treatment with CATB and SAPC antibodies decreased cleavage of caspase-3 and restored synaptophysin in neurons. Neurons exposed to macrophage-conditioned media differentially internalized His-CATB, dependent on the HIV replication levels. Finally, CATB and SAPC were secreted in EVs. We report for the first time that CATB is secreted from macrophages both free and in EVs, and is internalized by neurons. Moreover, HIV-replication levels modulate the amount of CATB neuronal uptake, and neuronal dysfunction can be decreased with CATB antibodies. In conclusion, the CATB/SAPC complex represents a novel target against HIV-associated neurocognitive disorders.

Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2–8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M−1 s−1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.

Graphene Oxide-Based Targeting of Extracellular Cathepsin D and Cathepsin L As A Novel Anti-Metastatic Enzyme Cancer Therapy.

Overexpression and secretion of the enzymes cathepsin D (CathD) and cathepsin L (CathL) is associated with metastasis in several human cancers. As a superfamily, extracellularly, these proteins may act within the tumor microenvironment to drive cancer progression, proliferation, invasion and metastasis. Therefore, it is important to discover novel therapeutic treatment strategies to target CathD and CathL and potentially impede metastasis. Graphene oxide (GO) could form the basis of such a strategy by acting as an adsorbent for pro-metastatic enzymes. Here, we have conducted research into the potential of targeted anti-metastatic therapy using GO to adsorb these pro-tumorigenic enzymes. Binding of CathD/L to GO revealed that CathD/L were adsorbed onto the surface of GO through its cationic and hydrophilic residues. This work could provide a roadmap for the rational integration of CathD/L-targeting agents into clinical settings.

Cathepsins: Potent regulators in carcinogenesis.

Cathepsins (CTS) are mainly lysosomal acid hydrolases extensively involved in the prognosis of different diseases, and having a distinct role in tumor progression by regulating cell proliferation, autophagy, angiogenesis, invasion, and metastasis. As all these processes conjunctively lead to cancer progression, their site-specific regulation might be beneficial for cancer treatment. CTS regulate activation of the proteolytic cascade and protein turnover, while extracellular CTS is involved in promoting extracellular matrix degradation and angiogenesis, thereby stimulating invasion and metastasis. Despite cancer regulation, the involvement of CTS in cellular adaptation toward chemotherapy and radiotherapy augments their therapeutic potential. However, lysosomal permeabilization mediated cytosolic translocation of CTS induces programmed cell death. This complex behavior of CTS generates the need to discuss the different aspects of CTS associated with cancer regulation. In this review, we mainly focused on the significance of each cathepsin in cancer signaling and their targeting which would provide noteworthy information in the context of cancer biology and therapeutics.

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5(Atg5) protein on pancreatic tumor formation and progression. We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration andinvasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin Land D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

Luteolin Targets the Toll-Like Receptor Signaling Pathway in Prevention of Hepatic and Adipocyte Fibrosis and Insulin Resistance in Diet-Induced Obese Mice.

This study was to investigate the protective role of luteolin on inflammation-mediated metabolic diseases, focusing on the role of luteolin in the modulation of the Toll-like receptor (TLR) signaling pathway. C57BL/6J mice were fed a normal, high-fat, or high-fat + 0.005% (w/w) luteolin diet for 16 weeks. Luteolin improved chronic low-grade inflammation by modulating the TLR signaling pathway, resulting in reduced pro-inflammatory cytokines and macrophage accumulation. A positive relationship was detected between gene expressions of Tlr5, Map2k7, Mapk12, Mapk13, and Mapk9 and lipogenesis in epididymal white adipose tissue (eWAT) of luteolin-treated mice, which was linked to attenuation of hepatic lipotoxicity by increasing free fatty acid (FFA) flux to the WAT. Luteolin prevented fibrosis by decreasing extracellular matrix accumulation and cathepsin gene expressions, while enhancing the hepatic antioxidant system. Emr1 and Ccl7, important markers inducing low-grade inflammation, were affected by advanced age and greater body weight, which were normalized by luteolin treatment. Luteolin improved insulin resistance by normalizing pancreatic islet dysfunction and differentially modulating the plasma glucagon-like peptide-1 and gastric inhibitory polypeptide levels. Our results suggest that luteolin ameliorates diet-induced obesity and its comorbidities. Overall, this study provides novel insights into the effect of luteolin on the links among adiposopathy, insulin resistance, hepatic steatosis, and fibrosis.

Full length histone H3 conjugated electrochemical biosensor for extracellular proteolytic Cathepsin L activity detection

Timely and accurate detection of proteolytic extracellular Cathepsin L (CTSL) activity is vital for assessing the metastatic potential of cancer. In this article, extracellular CTSL activity was detected using biotinylated full length histone H3 (BFLH3) treated extended gate (EG) field effect transistors (FETs) with 104 times the sensitivity of fluorometric CTSL activity assays. Upon active reaction by CTSL, the treated BFLH3 is cleaved after amino acid 21, inducing a significant charge change on the EG surface due to the large size and charge of the cleaved BFLH3. This change in EG surface charge can be detected by FET, enabling the quantification of proteolytic extracellular CTSL activity. Moreover, self-normalized quantification of CTSL activity was achieved in heterogeneous LNCaP and PANC-1 cultured cell media, which accurately represented higher CTSL levels in LNCaP derived from metastatic lesion of human prostatic adenocarcinoma. CTSL activity detection with EG FET allows multiple measurements of CTSL activity with one FET and opens up the possibility of detecting other proteolytic enzymes for cancer diagnosis and prognosis.

Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis.

The incidence of melanoma is increasing, particularly in young women, and the disease remains incurable for many because of its aggressive, metastatic nature and its high rate of resistance to conventional, targeted, and immunological agents. Cathepsins are proteases that are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave or degrade proteins that restrict cancer progression, whereas extracellular cathepsins directly cleave the extracellular matrix and activate proinvasive proteases in the tumor microenvironment. Cathepsin secretion is markedly increased in cancer cells. We investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells. We found that the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) promoted the secretion of cathepsin B and cathepsin L by activating transcription factors (namely, Ets1, Sp1, and NF-κB/p65) that have key roles in the epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance. In some melanoma cell lines, Abl/Arg promoted the Ets1/p65-induced secretion of cathepsin B and cathepsin L in a kinase-independent manner, whereas in other melanoma lines, Abl/Arg promoted the kinase-dependent, Sp1/Ets1/p65-mediated induction of cathepsin L secretion and the Sp1/p65-mediated induction of cathepsin B secretion. As an indication of clinical relevance, the abundance of mRNAs encoding Abl/Arg, Sp1, Ets1, and cathepsins was positively correlated in primary melanomas, and Abl/Arg-driven invasion in culture and metastasis in vivo required cathepsin secretion. These data suggest that drugs targeting Abl kinases, many of which are FDA-approved, might inhibit cathepsin secretion in some melanomas and potentially other aggressive cancers harboring activated Abl kinases.

Cathepsin K in Lymphangioleiomyomatosis: LAM Cell–Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. Onprotease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed inLAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, andboth were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM.