Secretion Of Cathepsin Research Articles

Neutrophils Release Metalloproteinases during Adhesion in the Presence of Insulin, but Cathepsin G in the Presence of Glucagon.

In patients with reperfusion after ischemia and early development of diabetes, neutrophils can attach to blood vessel walls and release their aggressive bactericide agents, which damage the vascular walls. Insulin and 17β-estradiol (E2) relieve the vascular complications observed in metabolic disorders. In contrast, glucagon plays an essential role in the pathophysiology of diabetes. We studied the effect of hormones on neutrophil secretion during adhesion to fibronectin. Amino acid analysis revealed that proteins secreted by neutrophils are characterized by a stable amino acid profile enriched with glutamate, leucine, lysine, and arginine. The total amount of secreted proteins defined as the sum of detected amino acids was increased in the presence of insulin and reduced in the presence of glucagon. E2 did not affect the amount of protein secretion. Proteome analysis showed that in the presence of insulin and E2, neutrophils secreted metalloproteinases MMP-9 and MMP-8 playing a key role in modulation of the extracellular matrix. In contrast, glucagon induced the secretion of cathepsin G, a key bactericide protease of neutrophils. Cathepsin G can promote the development of vascular complications because of its proinflammatory activity and ability to stimulate neutrophil adhesion via the proteolysis of surface receptors.

Interferon-γ treatment in vitro elicits some of the changes in cathepsin S and antigen presentation characteristic of lacrimal glands and corneas from the NOD mouse model of Sjögren's Syndrome.

Inflammation and impaired secretion by lacrimal and salivary glands are hallmarks of the autoimmune disease, Sjögren’s Syndrome. These changes in the lacrimal gland promote dryness and inflammation of the ocular surface, causing pain, irritation and corneal damage. The changes that initiate and sustain autoimmune inflammation in the lacrimal gland are not well-established. Here we demonstrate that interferon-γ (IFN-γ) is significantly elevated in lacrimal gland and tears of the male NOD mouse, a model of autoimmune dacryoadenitis which exhibits many ocular characteristics of Sjögren’s Syndrome, by 12 weeks of age early in lacrimal gland inflammation. Working either with primary cultured lacrimal gland acinar cells from BALB/c mice and/or rabbits, in vitro IFN-γ treatment for 48 hr decreased expression of Rab3D concurrent with increased expression of cathepsin S. Although total cellular cathepsin S activity was not commensurately increased, IFN-γ treated lacrimal gland acinar cells showed a significant increase in carbachol-stimulated secretion of cathepsin S similar to the lacrimal gland in disease. In vitro IFN-γ treatment did not increase the expression of most components of major histocompatibility complex (MHC) class II-mediated antigen presentation although antigen presentation was slightly but significantly stimulated in primary cultured lacrimal gland acinar cells. However, exposure of cultured human corneal epithelial cells to IFN-γ more robustly increased expression and activity of cathepsin S in parallel with increased expression and function of MHC class II-mediated antigen presentation. We propose that early elevations in IFN-γ contribute to specific features of ocular disease pathology in Sjögren’s Syndrome.

Cathepsin K Inhibitors for Osteoporosis: Biology, Potential Clinical Utility, and Lessons Learned.

Cathepsin K is a cysteine protease member of the cathepsin lysosomal protease family. Although cathepsin K is highly expressed in osteoclasts, lower levels of cathepsin K are also found in a variety of other tissues. Secretion of cathepsin K from the osteoclast into the sealed osteoclast-bone cell interface results in efficient degradation of type I collagen. The absence of cathepsin K activity in humans results in pycnodysostosis, characterized by increased bone mineral density and fractures. Pharmacologic cathepsin K inhibition leads to continuous increases in bone mineral density for ≤5 years of treatment and improves bone strength at the spine and hip. Compared with other antiresorptive agents, cathepsin K inhibition is nearly equally efficacious for reducing biochemical markers of bone resorption but comparatively less active for reducing bone formation markers. Despite multiple efforts to develop cathepsin K inhibitors, potential concerns related to off-target effects of the inhibitors against other cathepsins and cathepsin K inhibition at nonbone sites, including skin and perhaps cardiovascular and cerebrovascular sites, prolonged the regulatory approval process. A large multinational randomized, double-blind phase III study of odanacatib in postmenopausal women with osteoporosis was recently completed. Although that study demonstrated clinically relevant reductions in fractures at multiple sites, odanacatib was ultimately withdrawn from the regulatory approval process after it was found to be associated with an increased risk of cerebrovascular accidents. Nonetheless, the underlying biology and clinical effects of cathepsin K inhibition remain of considerable interest and could guide future therapeutic approaches for osteoporosis.

Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome–plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.

STAT3 and STAT6 Signaling Pathways Synergize to Promote Cathepsin Secretion from Macrophages via IRE1α Activation

Tumor-associated macrophages play critical roles during tumor progression by promoting angiogenesis, cancer cell proliferation, invasion, and metastasis. Cysteine cathepsin proteases, produced by macrophages and cancer cells, modulate these processes, but it remains unclear how these typically lysosomal enzymes are regulated and secreted within the tumor microenvironment. Here, we identify a STAT3 and STAT6 synergy that potently upregulates cathepsin secretion by macrophages via engagement of an unfolded protein response (UPR) pathway. Whole-genome expression analyses revealed that the TH2 cytokine interleukin (IL)-4 synergizes with IL-6 or IL-10 to activate UPR via STAT6 and STAT3. Pharmacological inhibition of the UPR sensor IRE1α blocks cathepsin secretion and blunts macrophage-mediated cancer cell invasion. Similarly, genetic deletion of STAT3 and STAT6 signaling components impairs tumor development and invasion invivo. Together, these findings demonstrate that cytokine-activated STAT3 and STAT6 cooperate in macrophages to promote a secretory phenotype that enhances tumor progression in a cathepsin-dependent manner.

Abstract 3853: Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates

Abstract Thrombocytopenia and neutropenia are common adverse events in cancer patients treated with antibody-drug conjugates (ADCs). We used human hematopoietic stem cells (HSC) in vitro and various ADCs to investigate the potential mechanisms of thrombocytopenia and neutropenia. Two ADCs, AGS-16C3F and T-DM1, were selected for the investigation of megakaryocytes (MKs) as they both induced thrombocytopenia in patients. AGS-16C3F is an ENPP3-targeting antibody conjugated to microtubule-disrupting agent MMAF (licensed from Seattle Genetics) via a non-cleavable linker (mcMMAF) tested in a phase 1 trial in kidney cancer patients. T-DM1 (KADCYLA) is an anti-HER2 antibody conjugated to the maytansine derivative DM1, approved for the treatment of advanced Her-2 positive breast cancer. HSCs were differentiated to MKs and percentage of CD41 positivity from FACS analysis was used to measure the effect of ADCs on MK differentiation. Results showed that while MKs do not express ENPP3 or Her-2, both AGS-16C3F (IC50 = 40nM) and T-DM1 inhibited MK differentiation. MKs express FcγRIIA to which T-DM1 (IgG1) and, to lesser degree, AGS-16C3F (IgG2) can bind; however, blockage of FcγRIIA failed to rescue MKs from cytotoxicity induced by either T-DM1 or AGS-16C3F suggesting that binding to FcγRIIA is not critical for MK cytotoxicity. AGS-16C3F is internalized into MKs via macropinocytosis and causes significant disorganization of tubulin structure as observed by confocal microscopy. HSCs were also differentiated to neutrophils which were characterized by the percentage of CD66b positivity in FACS analysis. Several antibodies to targets absent in neutrophils were conjugated to both MMAE and MMAF and were tested for cytotoxicity during neutrophil differentiation. Our results showed that ADCs containing MMAE via a cleavable linker were more cytotoxic to neutrophil differentiation than the same ADC containing MMAF via a non-cleavable linker, in agreement with clinical results. Different from MKs, results showed that neutrophils have low macropinocytosis activity. Secretion of cathepsins increased during neutrophil differentiation, and a membrane impermeable cathepsin inhibitor blocked ADC cytotoxicity to neutrophils, suggesting that extracellular protease cleavage is critical for neutrophil cytotoxicity in vitro. Neutrophils express high levels of FcγRIIA and saturation of this receptor by control IgG inhibited ADC binding to neutrophils and at least partially rescued neutrophils from ADC cytotoxicity, suggesting that FcγRIIA can play a role in neutropenia induced by ADCs, particularly with IgG1 isotype. In conclusion, applying these in vitro data to clinical experience suggests that thrombocytopenia may be mediated by macropinocytosis of ADC in MK and neutropenia by secreted proteases and interactions with their FcγRs. Understanding these toxicities may help design new ADCs with improved toxicity profile. Citation Format: Hui Zhao, Sara Gulesserian, Sathish K. Ganesan, Zhilan Zeng, Jimmy Ou, Veronica Robles, Josh Snyder, David R. Stover, Fernando Donate. Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3853.

Effect of L-arginine and L-name on lysosomal cysteine proteases activity and lysosomal membranes permeability in rat aorta

Aim. To study the effect of L-arginine and its analogue N-nitro-L-arginine methyl ester (L-NAME) alone and in combination on lysosomal cysteine proteolysis and lysosomal membranes state in rat aorta.Methods. The study was performed on male Wistar rats kept under standard vivarium conditions and divided into three control and three experimental groups of 8 animals each. The experimental samples included groups with L-arginine and/or L-NAME administration. The indicators were studied in the rat aorta homogenate precipitating and non precipitating fractions. Acid phosphatase activity was determined by a standardized method of «end point», the cathepsins B, L and H activity was studied by spectrofluorimetric method.Results. When simulating the changes of nitric oxide synthesis level using L-arginine, the increase of the total cathepsins activity was detected, acid phosphatase lability coefficient was reduced, what is characterized by general lysosomal membranes stabilization. L-NAME group, in contrast, is characterized by a decrease in the cathepsin B and H activity indicators, differences from arginine group were observed in the cathepsin H in lysosomal and general fractions, lysosomal membrane is labile. Combined drugs administration reduces the total cathepsins activity, while there is an increase of the acid phosphatase total activity, all indicators suggest lysosomal membranes labilization.Conclusion. L-arginine at a dose of 500 mg/kg causes increase in the total cathepsins B, L and H activity in rat aorta due to lysosomal fraction; L-arginine action leads to lysosomal membranes stabilization; L-NAME group in cathepsin H shows a decrease in the cathepsins secretion level with decreased total activity due to both factions; combined administration of arginine + L-NAME group in cathepsin B is characterized by an increase in secretion due to lysosomes membrane labilization.

Secondary amyloidosis in autoinflammatory diseases and the role of inflammation in renal damage.

The release of proinflammatory cytokines during inflammation represents an attempt to respond to injury, but it may produce detrimental effects. The inflammasome is a large, multiprotein complex that drives proinflammatory cytokine production in response to infection and tissue injury; the best-characterized inflammasome is the nod-like receptor protein-3 (NLRP3). Once activated, inflammasome leads to the active form of caspase-1, the enzyme required for the maturation of interleukin-1beta. Additional mechanisms bringing to renal inflammatory, systemic diseases and fibrotic processes were recently reported, via the activation of the inflammasome that consists of NLRP3, apoptosis associated speck-like protein and caspase-1. Several manuscripts seem to identify NLRP3 inflammasome as a possible therapeutic target in the treatment of progressive chronic kidney disease. Serum amyloid A (SAA), as acute-phase protein with also proinflammatory properties, has been shown to induce the secretion of cathepsin B and inflammasome components from human macrophages. SAA is a well recognised potent activator of the NLRP3. Here we will address our description on the involvement of the kidney in autoinflammatory diseases driven mainly by secondary, or reactive, AA amyloidosis with a particular attention on novel therapeutic approach which has to be addressed in suppressing underlying inflammatory disease and reducing the SAA concentration.

Trophic factors deprivation induces long-term protection of neurons against excitotoxic damage

One of the strategies to induce tolerance of neurons to toxic injury is preconditioning. Preconditioning is caused by a weak damage of cells, which become more resistant to subsequent, more severe damage. We found that preconditioning by deprivation of trophic factors, or deprivation of trophic factor and glucose effectively protects neurons against subsequent toxic effects of glutamate. Deprivation of trophic factors plays a decisive role in the development of resistance, regardless of whether it has been combined with glucose deprivation or not. Neuronal protection is achieved when the deprivation lasts from 30 min to two hours and is kept for a period of from one to five days. Preconditioning is accompanied neuronal secretion of cathepsin B occurs. We suggest that this phenomenon is associated with a more general process of exocytosis of lysosomes triggered by deprivation of trophic factors.

Tensile force on human macrophage cells promotes osteoclastogenesis through receptor activator of nuclear factorκB ligand induction.

Little is known about the effects of tensile forces on osteoclastogenesis by human monocytes in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study we consider the effects of tensile force on osteoclastogenesis in human monocytes. The cells were treated with receptor activator of nuclear factorκB ligand (RANKL) to promote osteoclastogenesis. Then,expression and secretion of cathepsin K were examined. RANKL and the formation of osteoclasts during the osteoclast differentiation process under continual tensile stress were evaluated by Western blot. It was also found that -100kPa or lower induces RANKL-enhanced tartrate-resistant acid phosphatase activity in a dose-dependent manner. Furthermore, an increased tensile force raises the expression and secretion of cathepsin K elevated by RANKL, and is concurrent with the increase of TNF-receptor-associated factor6 induction and nuclear factor κB activation. Overall, the current report demonstrates that tensile force reinforces RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The tensile force is able to modify every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, affecting the fusion of preosteoclasts and function of osteoclasts. However, tensile force increased TNF-receptor-associated factor6 expression. These results are in vitro findings and were obtained under a condition of tensile force. The current results help us to better understand the cellular roles of human macrophage populations in osteoclastogenesis as well as in alveolar bone remodeling when there is tensile stress.

Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

Effect of ultraviolet A radiation on the expression and secretion of cathepsin G by human dermal fibroblasts

Objective To investigate the effect of ultraviolet A (UVA) radiation on the expression and secretion of cathepsin G (CatG) by human dermal fibroblasts.Methods Dermal fibroblasts were isolated from the foreskins of boys,and subjected to primary culture and subculture.After 10 or less passages,the fibroblasts were collected and divided into several groups to be irradiated with 10 J/cm2 UVA followed by 24,48 and 72 hours of additional culture,or be irradiated with 10,20 and 30 J/cm2 UVA followed by 24 hours of additional culture,with those receiving no treatment serving as the control group.Subsequently,cells and culture supernatant were collected,real time PCR and Western blot were performed to detect the expressions of CatG mRNA and protein respectively in these cells,and enzyme-linked immunosorbent assay (ELISA) was conducted to measure the expression of CatG protein in the culture supernatant of these cells.Results Compared with the control group,the fibroblasts irradiated with 10 J/cm2 UVA showed a significant increase at 24,48 and 72 hours in the expressions of CatG mRNA (0.376 ± 0.014 vs.0.183 ± 0.003,0.308 ± 0.022 vs.0.185 ± 0.005,0.296 ± 0.032 vs.0.182 ± 0.004,respectively,all P＜ 0.05) and protein (1.80 ± 0.12 vs.0.96 ± 0.06,1.41 ± 0.17 vs.0.95 ± 0.22,1.27 ± 0.09 vs.1.00 ± 0.14,respectively,all P ＜ 0.05),as well as in the supernatant level of CatG protein ((161.35 ± 7.55) vs.(122.45 ± 6.46) ng/L,(141.76 ± 2.95) vs.(124.17 ± 6.15) ng/L,(139.63 ± 3.04) vs.(121.72 ± 3.17) ng/L respectively,all P ＜0.05),with the strongest increase observed at 24 hours.At 24 hours after 10,20 and 30 J/cm2 of UVA radiation,the expression of CatG mRNA in irradiated fibroblasts was 1.90,2.51 and 3.04 times respectively (all P ＜ 0.05),the expression of CatG protein was 1.88,3.97 and 4.72 times respectively (P ＜ 0.05),and the supernatant level of CatG protein was 1.36,1.50 and 1.66 times respectively (P ＜ 0.05),that in the control group,and there was an increasing trend in all the above three parameters with increasing dose of UVA.Conclusion Acute UVA radiation can promote the expression and secretion of CatG by human dermal fibroblasts. Key words: Fibroblasts; Cathepsins; Ultraviolet rays; Cell aging

Abstract 4837: Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells

Abstract Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837

Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2), urokinase-type plasminogen activator(uPA) and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel mechanism underlying EMMPRIN-2 enhanced tumor progression in head and neck cancer.

V‐ATPase‐mediated secretion and activation of cathepsins in breast cancer cells: investigating the role of vacuolar H+‐ATPases subunit A isoforms in breast cancer metastasis (934.4)

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation synaptic signaling, etc. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231, and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating local tumor invasion. We are studying the role of subunit a isoforms in secretion and activation of cathepsin B and L. The 4 subunit a isoforms are responsible for cellular localization of V‐ATPases. Treatment with concanamycin A (a general V‐ATPase inhibitor) affects the activity of intracellular and secreted cathepsins B and L. Concanamycin A treamtment also affects the secretion of cathepsins B and L. siRNAs were used to reduce mRNA expression of each subunit a isoform. We have previously shown that knockdown of the a4 isoform decreased the level of processed cathepsin L in conditioned media, but not the unprocessed form. Our previous findings also indicate a4 targets V‐ATPases to the cell surface, thus we hypothesize that the a4 isoform will have the largest effect on secretion and activation of cathepsin B and/or L. Following isoform‐specific knockdown, immunoblotting and cathepsin activity assays are being used to analyze cathepsin B and L expression and activity in conditioned media and cell lysates of MB231 cells. This study increases our understanding of the role V‐ATPases are playing in the activation of secreted cathepsins in the tumor microenvironment of MB231 cells.Grant Funding Source: Funded by Denison University Anderson Fellowship

Bone Microenvironment Tissue Surrogates Engineered for Reporting of Metastasized Breast Cancer Osteolytic Activity

ABSTRACTBreast cancer metastasis to bone continues to be a major clinical problem, and patient-to-patient variability in rates of disease progression and metastasis complicate treatment even further. This may be due to differences in the cancer cells, the osteoclasts, or the pre-metastatic niche, but all of these contribute to proteolytic remodeling necessary for osteolytic lesion establishment, primarily through secretion of cathepsin K, the most powerful human collagenase. There is debate about the relative contributions of breast cancer cells and osteoclasts and synergism between the two in altering the biochemical and biomechanical properties of the colonized bone, as these are difficult to parse with animal models. To quantify the relative contributions of breast cancer cells and osteoclasts in bone resorption, we have been developing engineered bone microenvironment tissue surrogates by adapting a poly(ester urethane) urea system embedded with microbone particles. Here, we report their use with MDA-MB-231 breast cancer cells and RAW264.7 derived osteoclasts, to provide temporal, multiscale reporters of bone resorption that can be measured non-destructively: 1) collagen degradation measured by C-terminal collagen fragment release, 2) mineral dissolution by measuring calcium released with the calcium arsenazo assay, and also show their beneficial effects in upregulating cathepsin K expression compared to tissue culture polystyrene controls. These more natural derived bone surrogates may be useful tools in mimicking bone metastatic niche and determining differences between proteolytic activity of different patients’ tumor and bone resident cells in a controlled manner.

Amyloid-β Increases Metallo- and Cysteine Protease Activities in Human Macrophages

Background/Aims: Amyloid-β (Aβ) plays a crucial role in the onset and progression of atherosclerosis. Macrophages are a source of matrix metalloproteinases (MMPs), cysteine proteases and transforming growth factor (TGF)-β1 in the vascular wall. The aims of this study were to analyze the capacity of Aβ peptide (1-40) (Aβ40), Aβ peptide (1-42) (Aβ42) and fibrillar Aβ42 (fAβ42) to modulate the expression and activity of MMP-9, MMP-2 and tissue inhibitor of MMP-1 (TIMP-1) in human monocyte-derived macrophages (HMDM). Additionally, we analyzed whether Aβ internalization alters the secretion of cathepsin S (CatS) and TGF-β1 by macrophages. Methods: HMDM were exposed to native and fibrillar Aβ. MMPs and TIMP-1 expression was analyzed by real-time PCR, and MMP abundance by zymography. Protein levels of precursor and active forms of CatS were analyzed by Western blot and TGF-β1 levels by ELISA. Results: Aβ40, Aβ42 and especially fAβ42 strongly induced MMP-9/MMP-2 levels. Moreover, we showed enhanced active CatS and reduced TGF-β1 protein levels in the secretome of Aβ42 and fAβ42-exposed macrophages. Conclusions: Aβ can regulate the proinflammatory state of human macrophages by inducing metallo- and cysteine protease levels and by reducing TGF-β1 secretion. These effects may be crucial in atherosclerosis progression.

Antiosteoclastogenic Activity of Silicate-based Materials Antagonizing Receptor Activator for Nuclear Factor KappaB Ligand–induced Osteoclast Differentiation of Murine Marcophages

IntroductionThis study investigated whether calcium silicate cement extract exerted antiosteoclastogenic actions in murine RAW 264.7 macrophages cultured with receptor activator for nuclear factor kappaB (RANKL). MethodsThe RAW 264.7 macrophage cell was treated with RANKL to osteoclastogenesis. Then, cell viability, cell death, and cathepsin K expression were examined. ResultsThe silicon (Si)-inhibited RANKL-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that ≥4 mmol/L Si reduced RANKL-enhanced tartrate-resistant acid phosphatase (TRAP) activity in a dose-dependent manner. Furthermore, Si diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and nuclear factor kappaB activation. ConclusionsThe current report shows that silicate abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The Si can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts, and the function of osteoclasts. Therefore, silicate-based materials may be a potential therapeutic agent targeting osteoclast differentiation in bone defects.

Expression of duodenase-like protein in epitheliocytes of Brunner’s glands in human duodenal mucosa

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G- Paneth cells located at the bottom of Lieberkuhn crypts - was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner's glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner's glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its co-localization with cathepsin G in Brunner's glands.

Effects of Rab27a on proliferation, invasion, and anti-apoptosis in human glioma cell

This study aims to investigate the relationship between Rab27a and the characteristics of glioma cell U251 such as proliferation, apoptosis, and invasion and to provide an experimental basis for future therapy in human glioma. Recombinant plasmid of pcDNA3.1-Rab27a was constructed and transfected into U251 cells with the help of Lipofectamine™2000. The expression of Rab27a was detected by Western blot. Cell viability, cell cycle, cell apoptosis, and cell migration were analyzed, respectively, by (3-(4,5)-dimethylthi-azol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay, flow cytometry, and Transwell invasion chamber methods. Meanwhile, the effect of Rab27a on secretion of cathepsin D in U251 cells was also examined. With the help of luciferase reporter assay system, the relationship between miR-124 and gene Rab27a expression was explored. Western blot showed that the expression of Rab27a was significantly increased in pcDNA3.1-Rab27a transfection group (p < 0.01) and that was significantly decreased in Rab27a-shRNA transfection group (p < 0.01) compared with control group. MTT assay, flow cytometry, and Transwell invasion chamber experiment indicated that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were improved significantly in pcDNA3.1-Rab27a transfection group compared with control group and that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were reduced markedly in Rab27a-shRNA transfection group compared with control group. The apoptosis analysis by flow cytometry demonstrated that the ratio of apoptosis in pcDNA3.1-Rab27a transfection group was significantly lower than that in control group (p < 0.05) and the ratio was notably higher in Rab27a-shRNAtransfection group than that in the control group. Cathepsin D activity assay indicated that the release of cathepsin D was enhanced in pcDNA3.1-Rab27a transfection group compared to that in the control group (p < 0.05). Rab27a could increase the glioma cell ability, promote proliferation and invasion, and suppress cell apoptosis. The above-stated effects of Rab27a possibly were exerted by increasing the secretion of cathepsin D and regulated by miR-124. In addition, the inhibition of expression of Rab27a perhaps benefited the therapy for glioma patients.