Iron overload suppresses hippocampal neurogenesis in adult mice: Implication for iron dysregulation-linked neurological diseases.

AIMS: Adult hippocampal neurogenesis is an important player in brain homeostasis and its impairment participates in neurological diseases. Iron overload has emerged as an irreversible factor of brain aging, and is also closely related to degenerative disorders, including cognitive dysfunction. However, whether brain iron overload alters hippocampal neurogenesis has not been reported. We investigated the effect of elevated iron content on adult hippocampal neurogenesis and explored the underlying mechanism. METHODS: Mouse models with hippocampal iron overload were generated. Neurogenesis in hippocampus and expression levels of related molecules were assessed. RESULTS: Iron accumulation in hippocampus remarkably impaired the differentiation of neural stem cells, resulting in a significant decrease in newborn neurons. The damage was possibly attributed to iron-induced downregulation of proprotein convertase furin and subsequently decreased maturation of brain-derived neurotrophic factor (BDNF), thus contributing to memory decline and anxiety-like behavior of mice. Supportively, knockdown of furin indeed suppressed hippocampal neurogenesis, while furin overexpression restored the impairment. CONCLUSION: These findings demonstrated that iron overload damaged hippocampal neurogenesis likely via iron-furin-BDNF pathway. This study provides new insights into potential mechanisms on iron-induced neurotoxicity and the causes of neurogenesis injury and renders modulating iron homeostasis and furin expression as novel therapeutic strategies for treatment of neurological diseases.

Synthesis, evaluation, and mechanism of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones as potent reversible SARS-CoV-2 entry inhibitors.

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001-0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to discovery that the synthesized compounds also inhibited Spike's receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced bodyweight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.

Furin as a therapeutic target in cystic fibrosis airways disease.

Clinical management of cystic fibrosis (CF) has been greatly improved by the development of small molecule modulators of the CF transmembrane conductance regulator (CFTR). These drugs help to address some of the basic genetic defects of CFTR; however, no suitable CFTR modulators exist for 10% of people with CF (PWCF). An alternative, mutation-agnostic therapeutic approach is therefore still required. In CF airways, elevated levels of the proprotein convertase furin contribute to the dysregulation of key processes that drive disease pathogenesis. Furin plays a critical role in the proteolytic activation of the epithelial sodium channel; hyperactivity of which causes airways dehydration and loss of effective mucociliary clearance. Furin is also responsible for the processing of transforming growth factor-ß, which is increased in bronchoalveolar lavage fluid from PWCF and is associated with neutrophilic inflammation and reduced pulmonary function. Pathogenic substrates of furin include Pseudomonas exotoxin A, a major toxic product associated with Pseudomonas aeruginosa infection and the spike glycoprotein of severe acute respiratory syndrome coronavirus 2, the causative pathogen for coronavirus disease 2019. In this review we discuss the importance of furin substrates in the progression of CF airways disease and highlight selective furin inhibition as a therapeutic strategy to provide clinical benefit to all PWCF.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

INTRODUCTION: Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. METHODS: We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. RESULTS: We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. CONCLUSION: In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Baicalin inhibited both the Furin/TGFß1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy.

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFß1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFß1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

Hippocampal Iron Accumulation Impairs Synapses and Memory via Suppressing Furin Expression and Downregulating BDNF Maturation.

Brain iron overload is positively correlated with the pathogenesis of Alzheimer's disease (AD). However, the role of iron in AD pathology is not completely understood. Furin is the first identified mammalian proprotein convertase that catalyzes the proteolytic maturation of large numbers of prohormones and proproteins. The correlation between altered furin expression and AD pathology has been suggested, but the underlying mechanism remains to be clarified. Here, we found that the expression of furin in the hippocampus of Alzheimer's model APP/PS1 mice was significantly reduced, and we demonstrated that the reduction of furin was directly caused by hippocampal iron overload using wild-type mice with intrahippocampal injection of iron. In cultured neuronal cells, this suppression effect was observed as transcriptional inhibition. Regarding the changes of furin-mediated activities caused by hippocampal iron overload, we found that the maturation of brain-derived neurotrophic factor (BDNF) was impeded and the expression levels of synaptogenesis-related proteins were downregulated, leading to cognitive decline. Furthermore, iron chelation or furin overexpression in the hippocampus of APP/PS1 mice increased furin expression, restored synapse plasticity, and ameliorated cognitive decline. Therefore, the inhibitory effect of hippocampal iron accumulation on furin transcription may be an important pathway involved in iron-mediated synapse damage and memory loss in AD. This study provides new insights into the molecular mechanisms of the toxic effects of iron in neurons and AD pathophysiology and renders furin as a potential target for treatment of iron overload-related neurodegenerative diseases.

In vitro characterization of the furin inhibitor MI-1851: Albumin binding, interaction with cytochrome P450 enzymes and cytotoxicity.

The substrate-analog furin inhibitor MI-1851 can suppress the cleavage of SARS-CoV-2 spike protein and consequently produces significant antiviral effect on infected human airway epithelial cells. In this study, the interaction of inhibitor MI-1851 was examined with human serum albumin using fluorescence spectroscopy and ultrafiltration techniques. Furthermore, the impacts of MI-1851 on human microsomal hepatic cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6 and 3A4 activities were assessed based on fluorometric assays. The inhibitory action was also examined on human recombinant CYP3A4 enzyme and on hepatocytes. In addition, microsomal stability (60 min) and cytotoxicity were tested as well. MI-1851 showed no relevant interaction with human serum albumin and was significantly depleted by human microsomes. Furthermore, it did not inhibit CYP1A2, 2C9, 2C19 and 2D6 enzymes. In human hepatocytes, CYP3A4 was significantly suppressed by MI-1851 and weak inhibition was noticed in regard to human microsomes and human recombinant CYP3A4. Finally, MI-1851 did not impair the viability and the oxidative status of primary human hepatocytes (up to 100 µM concentration). Based on these observations, furin inhibitor MI-1851 appears to be potential drug candidates in the treatment of COVID-19, due to the involvement of furin in S protein priming and thus activation of the pandemic SARS-CoV-2.

The proprotein convertase furin inhibits IL-13-induced inflammation in airway smooth muscle by regulating integrin-associated signaling complexes.

Furin is a proprotein convertase that regulates the activation and the inactivation of multiple proteins including matrix metalloproteinases, integrins, and cytokines. It is a serine endoprotease that localizes to the plasma membrane and can be secreted into the extracellular space. The role of furin in regulating inflammation in isolated canine airway smooth muscle tissues was investigated. The treatment of airway tissues with recombinant furin (rFurin) inhibited the activation of Akt and eotaxin secretion induced by IL-13, and it prevented the IL-13-induced suppression of smooth muscle myosin heavy chain expression. rFurin promoted a differentiated phenotype by activating ß1-integrin proteins and stimulating the activation of the adhesome proteins vinculin and paxillin by talin. Activated paxillin induced the binding of Akt to ß-parvin IPP [integrin-linked kinase (ILK), PINCH, parvin] complexes, which inhibits Akt activation. Treatment of tissues with a furin inhibitor or the depletion of endogenous furin using shRNA resulted in Akt activation and inflammatory responses similar to those induced by IL-13. Furin inactivation or IL-13 caused talin cleavage and integrin inactivation, resulting in the inactivation of vinculin and paxillin. Paxillin inactivation resulted in the coupling of Akt to α-parvin IPP complexes, which catalyze Akt activation and an inflammatory response. The results demonstrate that furin inhibits inflammation in airway smooth muscle induced by IL-13 and that the anti-inflammatory effects of furin are mediated by activating integrin proteins and integrin-associated signaling complexes that regulate Akt-mediated pathways to the nucleus. Furin may have therapeutic potential for the treatment of inflammatory conditions of the lungs and airways.

Furin inhibits epithelial cell injury and alleviates experimental colitis by activating the Nrf2-Gpx4 signaling pathway.

BACKGROUND AND AIM: Furin is a proprotein convertase reported to have protective effects in several autoimmune diseases. However, the role of furin in ulcerative colitis (UC) remains unclear. We aimed to clarify this role. METHODS: Furin expression was measured in UC and dextran sulfate sodium (DSS)-induced colitis. Gain- and loss-of-function experiments were conducted to evaluate the effect of furin in UC using DSS-treated NCM460 cells. Several ferroptotic parameters, including cell viability, cell death rate, lipid reactive oxygen species level, mitochondrial membrane damage and glutathione peroxidase 4 (Gpx4) expression, were measured. Exogenous furin was used to treat the DSS-induced colitis in mice to confirm the results in vivo. Finally, the activation of nuclear factor erythroid 2-like 2 (Nrf2) was detected to explore the mechanism. RESULTS: Furin expression was aberrant in UC. Furin overexpression attenuated DSS-induced ferroptosis-like injury and upregulated Gpx4 in NCM460 cells, whereas silencing furin had the opposite effects. Exogenous furin treatment alleviated DSS-induced colitis in mice by upregulating Gpx4. Mechanistic experiments revealed that furin activated Nrf2 both in vitro and in vivo. CONCLUSIONS: Furin protects epithelial cells from DSS-induced ferroptosis-like cell injury and alleviates experimental colitis by activating the Nrf2-Gpx4 signaling pathway.

ELA/APELA precursor cleaved by furin displays tumor suppressor function in renal cell carcinoma through mTORC1 activation.

Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment.

Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.

Lipoic acid decreases breast cancer cell proliferation by inhibiting IGF-1R via furin downregulation.

BACKGROUND: Breast cancer is the second most common cancer in the world. Despite advances in therapies, the mechanisms of resistance remain the underlying cause of morbidity and mortality. Lipoic acid (LA) is an antioxidant and essential cofactor in oxidative metabolism. Its potential therapeutic effects have been well documented, but its mechanisms of action (MOA) are not fully understood. METHODS: The aim of this study is to validate the inhibitory LA effect on the proliferation of various breast cancer cell lines and to investigate the MOA that may be involved in this process. We tested LA effects by ex vivo studies on fresh human mammary tumour samples. RESULTS: We demonstrate that LA inhibits the proliferation and Akt and ERK signalling pathways of several breast cancer cells. While searching for upstream dysregulations, we discovered the loss of expression of IGF-1R upon exposure to LA. This decrease is due to the downregulation of the convertase, furin, which is implicated in the maturation of IGF-1R. Moreover, ex vivo studies on human tumour samples showed that LA significantly decreases the expression of the proliferation marker Ki67. CONCLUSION: LA exerts its anti-proliferative effect by inhibiting the maturation of IGF-1R via the downregulation of furin.

Synergistic antitumor effects of 9.2.27-PE38KDEL and ABT-737 in primary and metastatic brain tumors.

Standard treatment, unfortunately, yields a poor prognosis for patients with primary or metastatic cancers in the central nervous system, indicating a necessity for novel therapeutic agents. Immunotoxins (ITs) are a class of promising therapeutic candidates produced by fusing antibody fragments with toxin moieties. In this study, we investigated if inherent resistance to IT cytotoxicity can be overcome by rational combination with pro-apoptotic enhancers. Therefore, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting chondroitin sulfate proteoglycan 4 (CSPG4) with a panel of Bcl-2 family inhibitors (ABT-737, ABT-263, ABT-199 [Venetoclax], A-1155463, and S63845) against patient-derived glioblastoma, melanoma, and breast cancer cells/cell lines. In vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized the different tumors to IT treatment, and improved the IC50 values of 9.2.27-PE38KDEL up to >1,000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that increased levels of intracellular IT, processed (active) exotoxin, and PARP cleavage correlated with the enhanced sensitivity to the combination treatment. Furthermore, we confirmed the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy in orthotopic GBM xenograft and cerebral melanoma metastasis models in nude mice. Our study defines strategies for overcoming IT resistance and enhancing specific antitumor cytotoxicity in primary and metastatic brain tumors.

Proprotein convertase furin inhibits matrix metalloproteinase 13 in a TGFß-dependent manner and limits osteoarthritis in mice.

Cartilage loss in osteoarthritis (OA) results from altered local production of growth factors and metalloproteases (MMPs). Furin, an enzyme involved in the protein maturation of MMPs, might regulate chondrocyte function. Here, we tested the effect of furin on chondrocyte catabolism and the development of OA. In primary chondrocytes, furin reduced the expression of MMP-13, which was reversed by treatment with the furin inhibitor α1-PDX. Furin also promoted the activation of Smad3 signaling, whereas activin receptor-like kinase 5 (ALK5) knockdown mitigated the effects of furin on MMP-13 expression. Mice underwent destabilization of the medial meniscus (DMM) to induce OA, then received furin (1 U/mice), α1-PDX (14 µg/mice) or vehicle. In mice with DMM, the OA score was lower with furin than vehicle treatment (6.42 ± 0.75 vs 9.16 ± 0.6, p < 0.01), and the number of MMP-13(+) chondrocytes was lower (4.96 ± 0.60% vs 20.96 ± 8.49%, p < 0.05). Moreover, furin prevented the increase in ALK1/ALK5 ratio in cartilage induced by OA. Conversely, α1-PDX had no effect on OA cartilage structure. These results support a protective role for furin in OA by maintaining ALK5 receptor levels and reducing MMP-13 expression. Therefore, furin might be a potential target mediating the development of OA.

Luteolin escape mutants of dengue virus map to prM and NS2B and reveal viral plasticity during maturation.

We previously showed that luteolin, a well-known plant-derived component found in the "heat clearing" class of Traditional Chinese Medicine (TCM) herbs, is an uncompetitive inhibitor (Ki 58.6 µM) of the host proprotein convertase furin, an endoprotease that is required for maturation of flaviviruses in the trans-Golgi compartment. Luteolin also weakly inhibited recombinant dengue virus NS2B/NS3 protease (Ki 140.36 µM) non-competitively. In order to further explore the mechanism of inhibition we isolated resistant mutants by continuous passaging of DENV2 in the presence of increasing concentrations of luteolin. Nucleotide sequence analysis of the luteolin-resistant escape mutants revealed nucleotide changes that lead to amino acid substitutions in the prM (T79R) and NS2B (I114M) genes. These mutations were introduced into a DENV2 infectious clone and tested for replication in Huh-7 cells. Interestingly we found that the replication kinetics of prM T19R-NS2B I114M double-mutant (DM) was similar to wild-type virus (WT). On the other hand the prM T79R single mutant (SM1) was attenuated and the NS2B I114M single mutant (SM2) showed enhanced replication. Time of drug addition assay with luteolin showed that the mutant viruses were able to produce more mature virions than WT in the order DM > SM2>SM1>WT. Exogenous addition of furin to purified immature WT or mutant viruses revealed that luteolin blocked the prM cleavage of WT and SM2 at a similar level. On the other hand the SM1 immature virus showed some cleavage while the DM immature virus revealed efficient furin cleavage of prM even in the presence of 50 µM luteolin. Our findings suggest that luteolin inhibition of furin may occur at host/pathogen interface that permits the virus to escape the suppression by mutating key residue that may lead to an altered interface.

A Sema3C Mutant Resistant to Cleavage by Furin (FR-Sema3C) Inhibits Choroidal Neovascularization.

In age-related macular degeneration (AMD), abnormal sub retinal choroidal neovascularization (CNV) is a major cause of blindness. FR-sema3C is a point mutated form of semaphorin-3C that is resistant to cleavage by furin like pro-protein convertases (FPPC). We have found in previous work that FR-sema3C functions as an anti-angiogenic factor. In this study we investigated the possible use of FR-sema3C as an inhibitor of CNV. FR-sema3C inhibits VEGF as well as PDGF-BB signal transduction in endothelial cells and to less extent bFGF induced signal transduction using a mechanism that does not depend upon the binding of VEGF like the drugs that are currently the mainstay treatment for AMD. CNV was induced in eyes of C57 black mice by laser photocoagulation. Intravitreal injection of FR-Sema3C or aflibercept (VEGF-trap) was then used to inhibit CNV formation. Invading choroidal vessels were visualized a week later by injection of FITC-dextran into the circulation, followed by the measurement of the area of the invading blood vessels. Injection of 0.1 µg FR-Sema3C inhibited CNV by 55% (P<0.01) and was as effective as 5 µg aflibercept. FR-sema3C did not display any adverse effects on retinal function following its injection into eyes of healthy mice as assessed by optokinetic reflex (OKR) and Electro-retinogram (ERG) criteria. Furthermore, FR-sema3C did not induce apoptosis in the retina as determined by TUNEL nor was there any discernable structural damage to the retina as assessed by several immuno-histochemical criteria. Our results suggest that FR-sema3C could perhaps be used for the treatment of AMD, and that it may perhaps be of benefit to patients that do not respond well to current treatments relying on VEGF sequestering agents.

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.

Furin-dependent CCL17-fused recombinant toxin controls HTLV-1 infection by targeting and eliminating infected CCR4-expressing cells in vitro and in vivo.

BACKGROUND: Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, there are no therapies to prevent ATL development in high-risk asymptomatic carriers. To develop a therapy targeting HTLV-1-infected cells that are known to express CCR4 frequently, we tested whether truncated Pseudomonas exotoxin (PE38) fused to a CCR4 ligand, CCL17/thymus and activation-regulated chemokine (TARC), selectively eliminates such cells. RESULTS: Our data show that TARC-PE38 efficiently killed HTLV-1-infected cell lines. It also shrank HTLV-1-associated solid tumors in an infected-cell-engrafted mouse model. In HTLV-1-positive humanized mice, TARC-PE38 markedly inhibited the proliferation of HTLV-1-infected human CD4(+)CD25(+) or CD4(+)CD25(+)CCR4(+) cells and reduced the proviral loads (PVLs) in peripheral blood mononuclear cells (PBMCs). Importantly, TARC-PE38 significantly reduced the PVLs in PBMCs obtained from asymptomatic carriers. We show that the cytotoxicity of TARC-PE38 is mediated by the expression of the proprotein convertase, furin. The expression of furin was enhanced in HTLV-1-infected cells and correlated positively with PVLs in HTLV-1-infected individuals, suggesting that infected cells are more susceptible to TARC-PE38 than normal cells. CONCLUSIONS: TARC-PE38 robustly controls HTLV-1 infection by eliminating infected cells in both a CCR4- and furin-dependent manner, indicating the excellent therapeutic potential of TARC-PE38.

Overexpression of TNF-α-converting enzyme in fibroblasts augments dermal fibrosis after inflammation.

TNF-α-converting enzyme (TACE) can cleave transmembrane proteins, such as TNF-α, TNF receptors, and epidermal growth factor receptor (EGFR) ligands, to release the extracellular domains from the cell surface. Recent studies have suggested that overexpression of TACE may be associated with the pathogenesis of inflammation and fibrosis. To determine the roles of TACE in inflammation and fibrosis, TACE transgenic (TACE-Tg) mice, which overexpressed TACE systemically, were generated. As the transgene-derived TACE was expressed as an inactive form, no spontaneous phenotype developed in TACE-Tg mice. However, the transgene-derived TACE could be converted to an active form by furin in vitro and by phorbol myristate acetate (PMA) in vivo. Subcutaneous injection of PMA into mice induced inflammatory cell infiltration 1 day later and subsequent dermal fibrosis 7 days later. Interestingly, the degree of dermal fibrosis at day 7 was significantly higher in TACE-Tg mice than in wild-type mice. Correspondingly, PMA increased the expression of type I collagen in the primary culture of dermal fibroblasts derived from TACE-Tg mice. Furthermore, phosphorylated EGFR was increased in the fibroblasts by the PMA treatment. The collective findings suggest that TACE overexpression and activation in fibroblasts could shed off putative EGFR ligands. Subsequently, the soluble EGFR ligands could bind and activate EGFR on fibroblasts, and then increase the type I collagen expression resulting in induction of dermal fibrosis. These results also suggest that TACE and EGFR on fibroblasts may be novel therapeutic targets of dermal fibrosis, which is induced after diverse inflammatory disorders of the skin.

Protective role of systemic furin in immune response-induced arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune joint disease associated with chronic inflammation of the synovium that causes profound damage of joints. Inflammation results in part from the influx of immune cells secreting inflammatory cytokines and the reduction in the number of Treg cells. We undertook this study to assess the effect of furin, a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of both proteinase maturation and immune cells, in an experimental model of RA. METHODS: The effect of furin and its inhibitor α1-PDX was tested in mice with collagen-induced arthritis (CIA). Joints were processed for histology and protein expression. Levels of cytokines were measured in joint tissue, and Treg cell numbers were measured in spleens. RESULTS: Furin expression and activity were high in the synovial pannus in RA patients and mice with CIA. Systemic administration of furin prevented increases in the arthritis score, joint destruction, and bone loss, in contrast to systemic administration of the furin inhibitor α1-PDX, which enhanced these parameters. By preventing the development of synovial pannus, furin reduced the expression of metalloproteinases in the joints. In contrast, α1-PDX enhanced synovial proliferation and the expression and activity of matrix metalloproteinases. Furthermore, furin reversed the local Th1/Th2 balance and restored the number of Treg cells in the spleen, indicating mediation by immune cells. CONCLUSION: These findings show the protective role of exogenous furin against RA, mediated by an immune response. The data suggest the potential therapeutic use of furin or its derivatives in autoimmune diseases including RA.