PLAU promotes cell proliferation and migration of head and neck cancer via STAT3 signaling pathway.

It was reported that within the head and neck cancer (HNC) cell line CAL21 the epithelial-mesenchymal transition (EMT) and cell proliferation were promoted by Urokinase-Type Plasminogen Activator (PLAU) proteinase through TNFRSF12A. Additionally, in this paper HNC cell lines refer to Fadu and Tu686. A novel PLAU-STAT3 axis was found to be involved in HNC cell line proliferation and metastasis. PLAU expression in HNC samples was upregulated, besides, the elevated expression of PLAU was linked to the lower overall survival (OS) and disease-free survival (DFS). Ectopic PLAU expression promoted cell proliferation and migration, while PLAU knockdown exhibited opposite results. RNA-seq data identified the JAK-STAT signaling pathway, confirmed by western blotting. A recovery assay using S3I-201, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), indicated that PLAU promoted HNC cell line progression via STAT3 signaling in vitro. The oncogenic role of PLAU in HNC tumor growth in vivo was confirmed using xenograft models. In summary, we identified the tumorigenic PLAU function in the HNC progress. PLAU may represent a potential prognostic biomarker of HNC and the PLAU-STAT3 pathway might be considered a therapeutic target of HNC.

A versatile engineered extracellular vesicle platform simultaneously targeting and eliminating senescent stromal cells and tumor cells to promote tumor regression.

Chemotherapy is an important therapeutic approach for malignant tumors for it triggers apoptosis of cancer cells. However, chemotherapy also induces senescence of stromal cells in the tumor microenvironment to promote tumor progression. Strategies aimed at killing tumor cells while simultaneously eliminating senescent stromal cells represent an effective approach to cancer treatment. Here, we developed an engineered Src-siRNA delivery system based on small extracellular vesicles (sEVs) to simultaneously eliminate senescent stromal cells and tumor cells for cancer therapy. The DSPE-PEG-modified urokinase plasminogen activator (uPA) peptide was anchored to the membranes of induced mesenchymal stem cell-derived sEVs (uPA-sEVs), and Src siRNA was loaded into the uPA-sEVs by electroporation (uPA-sEVs-siSrc). The engineered uPA-sEVs-siSrc retained the basic sEVs properties and protected against siSrc degradation. uPA peptide modification enhanced the sEVs with the ability to simultaneously target doxorubicin-induced senescent stromal cells and tumor cells. Src silencing by uPA-sEVs-siSrc induced apoptosis of both senescent stromal cells and tumor cells. The uPA-sEVs-siSrc displayed preferential tumor accumulation and effectively inhibited tumor growth in a tumor xenograft model. Furthermore, uPA-sEVs-siSrc in combination with doxorubicin significantly reduced the senescence burden and enhanced the therapeutic efficacy of chemotherapy. Taken together, uPA-sEVs-siSrc may serve as a promising therapy to kill two birds with one stone, not only killing tumor cells to achieve remarkable antitumor effect, but also eliminating senescent cells to enhance the efficacy of chemotherapeutic agent in tumor regression.

Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis.

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

A microthrombus-driven fixed-point cleaved nanosystem for preventing post-thrombolysis recurrence via inhibiting ferroptosis.

Thrombus-induced cardiovascular diseases threaten human health. Current treatment strategies often rely on urokinase plasminogen activator (uPA) for its efficacy, yet it has such limiting factors as short half-life, lack of thrombus targeting, and systemic side effects leading to unintended bleeding. In addition, thrombolytic interventions can trigger inflammation-induced damage at thrombus sites, which affects endothelial function. To address these challenges, Fer-1/uPA@pep-CREKA-Lipo (Fu@pep-CLipo) has been developed. This system achieves precise and efficient thrombolysis while enhancing the thrombus microenvironment and mitigating ischemia-reperfusion injury, with exceptional thrombus targeting ability via the strong affinity of the Cys-Arg-Glu-Lys-Ala (CREKA) peptide for fibrin. The Cys-Nle-TPRSFL-DSPE (pep) could respond to the thrombus microenvironment and fixed-point cleavage. The uPA component linked to the liposome surface is strategically cleaved upon exposure to abundant thrombin at thrombus sites. Importantly, the inclusion of Fer-1 within Fu@pep-CLipo contributes to reactive oxygen species (ROS) scavenging and significantly improves the thrombus microenvironment. This innovative approach not only achieves highly efficient and precise thrombolysis but also positively influences the expression of eNOS protein while suppressing inflammatory factors like TNF-α and IL-6. This dual action contributes to improved thrombus inflammatory microenvironment and mitigated ischemia-reperfusion injury.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

SIGNIFICANCE STATEMENT: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. BACKGROUND: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. METHODS: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. RESULTS: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. CONCLUSIONS: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Single-cell transcriptome analysis reveals keratinocyte subpopulations contributing to psoriasis in corneum and granular layer.

BACKGROUND: Psoriasis is a chronic, inflammatory skin disease that is common and relapses easily. While the importance of keratinocyte proliferation in psoriasis development is well-documented, the specific functional subpopulations of epidermal keratinocytes associated with this disease remain enigmatic. MATERIALS AND METHODS: Therefore, in our analysis of single-cell transcriptome data from both normal and psoriatic skin tissues, we observed significant increases in certain keratinocytes in the stratum corneum (KC) and stratum granulosum (KG) within psoriatic skin. Furthermore, we identified upregulated expression of specific secreted factors known to promote inflammatory responses. Additionally, we conducted a KEGG pathway enrichment analysis on these identified subsets. RESULTS: In the stratum corneum, the expression of FTL was upregulated in HIST1H1C+ KC. S100P+ KC displayed a significant increase in the expression of both S100P and S100A10, whereas PRR9+ KC showed upregulated expression of DEFB4B, S100A8, and S100A12. SLURP1+ KC was characterized by elevated expression levels of IL-36G, SLURP1, and S100A12. Meanwhile, in the stratum granulosum, KRT1+ KG highly expressed SLURP1, S100A7, S100A8, and S100A9, while DEFB4B expression was upregulated in PI3+ KG. Our findings indicated that subsets within the stratum corneum primarily participate in pathways related to MAPK, NOD-like receptors, HIF-1, cell senescence, and other crucial processes. In contrast, subsets in the stratum granulosum were predominantly associated with pathways involving MAPK, NOD-like receptors, HIF-1, Hippo, mTOR, and IL-17. CONCLUSION: These findings not only uncover the keratinocyte subsets linked to psoriasis but also unveil the molecular mechanisms and related signaling pathways that drive psoriasis development. This knowledge opens new horizons for the development of innovative clinical treatment strategies for psoriasis.

Morin inhibits osteosarcoma migration and invasion by suppressing urokinase plasminogen activator through a signal transducer and an activator of transcription 3.

Osteosarcoma, the most common primary bone cancer that affects adolescents worldwide, has the early metastatic potential to be responsible for high mortality rates. Morin has a multipurpose role in numerous cancers, whereas little is known about its role in osteosarcoma migration and invasion. Therefore, we hypothesized that morin suppresses the invasive activities and the migratory potential of human osteosarcoma cells. Our results showed that morin reduced migration and invasion capabilities in human osteosarcoma U2OS and HOS cells. Moreover, morin inhibited the urokinase plasminogen activator (uPA) expression through a signal transducer and an activator of transcription-3 (STAT3) phosphorylation. After STAT3 overexpression, the decrease of the migratory potential and uPA expression caused by 100 µM of morin in U2OS cells was countered, indicating that STAT3 contributes to the antimetastatic property of morin in human osteosarcoma cells by reducing uPA. In conclusion, morin may be a potential candidate for the antimetastatic treatment of human osteosarcoma.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

Potential hemostatic compounds targeting urokinase plasminogen activator explored from three Euphorbiaceae species: Euphorbia maculata, Euphorbia humifusa, and Acalypha australis, with bio-affinity ultrafiltration UPLC-MS.

INTRODUCTION: Numerous species of the Euphorbiaceae family, including Euphorbia maculata, Euphorbia humifusa, and Acalypha australis, have been used to manage bleeding disorders. However, few investigations have demonstrated their hemostatic potential, and their procoagulant compounds remain elusive. OBJECTIVE: This study aimed to determine the most active procoagulant extracts from the three species' crude extract (CE) and fractions in order to screen out the active compounds and to analyze their possible mechanisms of action. METHODS: An integrative approach, comprising prothrombin time and activated partial thromboplastin time evaluations and urokinase-type plasminogen activator (uPA) inhibitory assessment, followed by bio-affinity ultrafiltration paired with UPLC/QTOF-MS targeting uPA and docking simulations, was used. RESULTS: The extracts with highest procoagulant activity were the CE for both E. maculata (EMCE) and E. humifusa (EHCE) and the n-butanol fraction (NB) for A. australis (AANB). The most promising ligands, namely, isoquercetin, orientin, rutin, and brevifolin carboxylic acid, were selected from these lead extracts. All of these compounds exhibited pronounced specific binding values to the uPA target and showed tight intercalation with the crucial side chains forming the uPA active pocket, which may explain their mode of action. The activity validation substantiated their hemostatic effectivity in inhibiting uPA as they had better inhibition constant (Ki) values than the reference drug tranexamic acid. CONCLUSION: Collectively, the integrative strategy applied to these three species allowed the elucidation of the mechanisms underlying their therapeutic effects on bleeding disorders, resulting in the fast detection of four potential hemostatic compounds and their mode of action.

Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

Decreased fertility in female mice lacking urokinase plasminogen activator.

It is well established that mouse ovarian granulosa cells secrete urokinase plasminogen activator (uPA) under gonadotropin stimulation. The synthesis and secretion of the enzyme correlate well with the time of follicular rupture in vivo. Moreover, uPA is secreted by the trophoblast at the time of implantation. In the present study, we have analyzed whether the absence of uPA could influence follicular growth, ovulation, and embryo implantation. Our data show fewer preantral follicles in uPA-/- ovaries but no decrease in hormonally induced ovulation. However, we observed a significant decrease in the number of implanted embryos in uPA-/- animals and, therefore, a lower number of pups per family. Adding uPA to the epithelial and stromal uterine cell culture medium strongly upregulates the expression of prostaglandin-endoperoxide synthase 2 (Ptgs2), the enzyme required for prostaglandin production and embryo implantation. The uPA inhibitor amiloride abrogated this increase.

Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

circPSD3 is a promising inhibitor of uPA system to inhibit vascular invasion and metastasis in hepatocellular carcinoma.

BACKGROUND: Vascular invasion is a major route for intrahepatic and distant metastasis in hepatocellular carcinoma (HCC) and is a strong negative prognostic factor. Circular RNAs (circRNAs) play important roles in tumorigenesis and metastasis. However, the regulatory functions and underlying mechanisms of circRNAs in the development of vascular invasion in HCC are largely unknown. METHODS: High throughput sequencing was used to screen dysregulated circRNAs in portal vein tumor thrombosis (PVTT) tissues. The biological functions of candidate circRNAs in the migration, vascular invasion, and metastasis of HCC cells were examined in vitro and in vivo. To explore the underlying mechanisms, RNA sequencing, MS2-tagged RNA affinity purification, mass spectrometry, and RNA immunoprecipitation assays were performed. RESULTS: circRNA sequencing followed by quantitative real-time PCR (qRT-PCR) revealed that circRNA pleckstrin and Sect. 7 domain containing 3 (circPSD3) was significantly downregulated in PVTT tissues. Decreased circPSD3 expression in HCC tissues was associated with unfavourable characteristics and predicted poor prognosis in HCC. TAR DNA-binding protein 43 (TDP43) inhibited the biogenesis of circPSD3 by interacting with the downstream intron of pre-PSD3. circPSD3 inhibited the intrahepatic vascular invasion and metastasis of HCC cells in vitro and in vivo. Serpin family B member 2 (SERPINB2), an endogenous bona fide inhibitor of the urokinase-type plasminogen activator (uPA) system, is the downstream target of circPSD3. Mechanistically, circPSD3 interacts with histone deacetylase 1 (HDAC1) to sequester it in the cytoplasm, attenuating the inhibitory effect of HDAC1 on the transcription of SERPINB2. In vitro and in vivo studies demonstrated that circPSD3 is a promising inhibitor of the uPA system. CONCLUSIONS: circPSD3 is an essential regulator of vascular invasion and metastasis in HCC and may serve as a prognostic biomarker and therapeutic target.

A dual role for ERK-1/2 in the regulation of plasmin activity and cell migration in metastatic NSCLC-H1299 cells.

Occupational and environmental exposure of various toxins or cigarette smoke causes non-small cell lung carcinoma (NSCLC); a devastating disease with a very low survival rate after metastasis. Increased activity of plasmin is a hallmark in NSCLC metastasis. It is accepted that metastatic cells exhibit higher plasmin activity than cells from primary tumors. Mechanisms behind this elevation, however, are barely understood. We compared plasmin activity and cell migration of A549 cells derived from a primary lung tumor with metastatic H1299 lung cells isolated from lymph nodes. Surprisingly, we found higher plasmin activity and migration for A549 cells. mRNA levels of the plasminogen activator inhibitor-1 (PAI-1) were higher in H1299 cells and activity of extracellular-regulated kinases-1/2 (ERK-1/2) was increased. An inhibitor of ERK-1/2 decreased PAI-1 mRNA levels and increased plasmin activity or cell migration in H1299 cells. Transforming growth factor-ß (TGF-ß) decreased plasmin activity and migration in A549 cells but enhanced both in H1299 cells. The cytokine massively increased PAI-1 and decreased urokinase plasminogen activator (uPA) levels in A549 cells but strongly induced uPA and only weakly PAI- 1 expression in H1299 cells. Consequently, TGF-ß enhanced plasmin activity and cell migration in H1299. Additionally, TGF-ß activated ERK-1/2 stronger in H1299 than in A549 cells. Accordingly, an ERK-1/2 inhibitor completely reversed the effects of TGF-ß on uPA expression, plasmin activity and migration in H1299 cells. Hence, we provide first data indicating TGF-ß-promoted increased plasmin activity and suggest that blocking TGF-ß-promoted ERK-1/2 activity might be a straightforward approach to inhibit NSCLC metastasis.

Macrophages and Urokinase Plasminogen Activator Receptor System in Multiple Myeloma: Case Series and Literature Review.

The microenvironment plays an essential role in multiple myeloma (MM) development, progression, cell proliferation, survival, immunological escape, and drug resistance. Mesenchymal stromal cells and macrophages release tolerogenic cytokines and favor anti-apoptotic signaling pathway activation, while the urokinase plasminogen activator receptor (uPAR) system contributes to migration through an extracellular matrix. Here, we first summarized the role of macrophages and the uPAR system in MM pathogenesis, and then we reported the potential therapeutic effects of uPAR inhibitors in a case series of primary MM-derived adherent cells. Our preliminary results showed that after uPAR inhibitor treatments, interleukein-6 (mean ± SD, 8734.95 ± 4169.2 pg/mL vs. 359.26 ± 393.8 pg/mL, pre- vs. post-treatment; p = 0.0012) and DKK-1 levels (mean ± SD, 7005.41 ± 6393.4 pg/mL vs. 61.74 ± 55.2 pg/mL, pre- vs. post-treatment; p = 0.0043) in culture medium were almost completely abolished, supporting further investigation of uPAR blockade as a therapeutic strategy for MM treatment. Therefore, uPAR inhibitors could exert both anti-inflammatory and pro-immunosurveillance activity. However, our preliminary results need further validation in additional in vitro and in vivo studies.

Urokinase-type plasminogen activator promotes corneal epithelial migration and nerve regeneration.

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a central role in the pericellular fibrinolytic system, mediates the degradation of extracellular matrix proteins and activation of growth factors, and contributes to the regulation of various cellular processes including cell migration and adhesion, chemotaxis, and angiogenesis. The corneal epithelium responds rapidly to injury by initiating a wound healing process that involves cell migration, cell proliferation, and tissue remodeling. It is innervated by sensory nerve endings that play an important role in the maintenance of corneal epithelial homeostasis and in the wound healing response. We here investigated the role of uPA in corneal nerve regeneration and epithelial resurfacing after corneal injury with the use of uPA-deficient mice. Both the structure of the corneal epithelium and the pattern of corneal innervation in uPA-/- mice appeared indistinguishable from those in uPA+/+ mice. Whereas the cornea was completely resurfaced by 36-48 h after epithelial scraping in uPA+/+ mice, however, such resurfacing required at least 72 h in uPA-/- mice. Restoration of epithelial stratification was also impaired in the mutant mice. Fibrin zymography revealed that the expression of uPA increased after corneal epithelial scraping and returned to basal levels in association with completion of re-epithelialization in wild-type animals. Staining of corneal whole-mount preparations for ßIII-tubulin also revealed that the regeneration of corneal nerves after injury was markedly delayed in uPA-/- mice compared with uPA+/+ mice. Our results thus demonstrate an important role for uPA in both corneal nerve regeneration and epithelial migration after epithelial debridement, and they may provide a basis for the development of new treatments for neurotrophic keratopathy.

FAM3C/ILEI protein is elevated in psoriatic lesions and triggers psoriasiform hyperproliferation in mice.

FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte-specific ILEI overexpression in mice (K5-ILEIind ) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte-specific ILEI deletion ameliorates TPA-induced skin inflammation. A transcriptomic ILEI signature obtained from the K5-ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA-induced K5-ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top "separator" genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI-regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

Involvement of matrix metalloproteinase 1 and urokinase-type plasminogen activator in the PKCα-p38 MAPK pathway-mediated progression of human liver cancer cells.

Our previous studies have shown that the plasminogen activator (PA) and matrix metalloproteinases (MMPs) proteinase systems were highly expressed in highly malignant liver cancer cells and regulated by PKCα. This study investigates whether the PKCα regulation of PA and MMPs systems is conducted through p38 mitogen-activated protein kinase (MAPK) signaling and the pathway is responsible for promoting cell progression. We found that the expressions of p38 MAPK in both highly malignant HA22T/VGH and SK-Hep-1 liver cancer cells were higher than that in other lower malignancy liver cancer cells. Since PKCα activates p38 MAPK in progression of liver cancer, we suspected the PKCα/p38 MAPK signaling pathway to be involved in the regulation of MMPs and PA systems. When SK-Hep-1 cells were treated with SB203580 or DN-p38, only MMP-1 and u-PA mRNA expressions decreased. The p38 MAPK inhibition also decreased the cell migration and invasion. In addition, the mRNA decay assays showed that the higher expressions of MMP-1 and u-PA mRNA in SK-Hep-1 cells were due to the alteration of mRNA stability by p38 MAPK inhibition. Zymography of SK-Hep-1 cells treated with siPKCα vector also showed the decrease of the activity of MMP-1 and u-PA and confirmed changes in mRNA level. Furthermore, only the transfection of MKK6 to the siPKCα-treated SK-Hep-1 stable clone cell restored the attenuation of MMP-1 and u-PA expressions. The treatment of SK-Hep-1 cells with either inhibitor of MMP-1 or u-PA reduced migration, and the reduction was enhanced with both inhibitors. In addition, tumorigenesis was also reduced with both inhibitors. These data suggest a novel finding that MMP-1 and u-PA are critical components in PKCα/MKK6/p38 MAPK signaling pathway which mediates liver cancer cell progression, and that the targeting of both genes may be a viable approach in liver cancer treatment.