Expression Of Plasminogen Activator Research Articles

β-catenin nuclear translocation represses thyroid cancer stem cells differentiating into cells with sodium-iodine symporter functional expression

Objective: To confirm whether β-catenin nuclear translocation in thyroid cancer stem cells can differentiate into thyroid cancer cells without functional membrane expression of sodium-iodine transporter (NIS) and be resistant to iodide 131 treatment. Methods: Thyroid cancer stem cells were firstly isolated as a side population (SP) from human thyroid cancer cell line FTC133. The SP cells from FTC133 were transfected with β-catenin, and then differentiated. The cells were further collected for Western blot, Transwell and MTT assay to investigate the epithelial-mesenchymal transition (EMT) characteristics, tumor growth, invasion, and iodine uptake potency in vitro. Functional NIS expression and iodide uptake in differentiated cells were detected with immunofluorescent staining and iodide uptake assay, respectively. Subcutaneous severe combined immunodeficient (SCID) mice tumor model was induced with differentiated cancer cells to explore the in vivo effect of radioiodine treatment. Further immunohistochemical staining was performed to reveal the changes of functional proteins involved in tumor radioiodine treatment. Results: Side population was isolated from FTC133 accounting for about 0.03%, with high expression of stem cell markers and decreased expression of differentiated cell markers. Western blot showed prominent EMT phenotype in the differentiated cells from β-catenin transfected stem cell model, with absence of epithelial expression of E-cadherin and cytokeratin 18, as well as abnormal expression of vimentin,fibronectin and urokinase-type plasminogen activator. Moreover,compared with cells differentiated from untransfected or empty plasmid transfected stem cells, in vitro proliferation markedly increased 85.4% and 81.0%, respectively (both P<0.01); while in vitro invasion augmented 78.8% and 84.4%, respectively (both P<0.01). Immunofluorescent staining identified that, after transfected with β-catenin, differentiated cells underwent β-catenin nuclear translocation and NIS localization transferred from membrane to plasma, compared with cells from untransfected or empty plasmid transfected stem cells. Cell iodide uptake in vitro decreased about 52.8% and 45.2%, respectively (both P<0.01). Furthermore, in vivo experiment further demonstrated that, cells differentiated from β-catenin transfected stem cells were found with much higher tumor proliferation,tumor growth rate and larger tumor mass after radioiodine 131 treatment (both P<0.05). Conclusion: Induction of β-catenin nuclear translocation in stem cells may generate differentiated thyroid cancer cells that are not sensitive to radioiodine treatment.

TLR5 activation induces expression of the pro-inflammatory mediator Urokinase Plasminogen Activator via NF-κB and MAPK signalling pathways in human dental pulp cells.

To explore the involvement of TLR5 in pulp inflammation and to examine the effects of TLR5 activation with its ligand, FlaB protein, on pro-inflammatory gene expression. TLR5 expression in dental pulp tissues and human dental pulp cells (hDPCs) were determined by immunohistochemistry, immunocytochemistry, Western blots and RT-PCR analyses. To examine the role of TLR5, hDPCs were treated with recombinant FlaB protein (500ngmL-1 ) to activate the receptor or with a small interfering RNA against TLR5 (si-TLR5) to downregulate the receptor. After exposure to FlaB, the expression of inflammation-related proteins was screened using a protein array kit. Western blots or qRT-PCR analyses were performed to identify changes in the expression of uPA (urokinase plasminogen activator), TIMPs (tissue inhibitor of metalloproteinases), and IL-6 and to determine their signalling pathways. Statistical analysis was performed using one-way analysis of variance (anova) with Tukey post hoc test; P<0.05 was considered statistically significant. TLR5 expression was identified in pulp tissues and hDPCs. Inthe protein array analysis, treatment with FlaB significantly increased uPA expression (P<0.01) andsignificantly decreased TIMP1/4 (P<0.05). FlaB treatment also significantlyincreased expression of the inflammatory marker IL-6 (P<0.01). FlaB treatment increased phosphorylation of the NF-κB p65 subunit, JNK, p38 and ERK. Chemical inhibitors of NF-κB (Bay11-7082), p38 (SB202190) or ERK (U0126) decreased the FlaB induction of uPA expression. Downregulation of TLR5 expression by siRNA decreased the FlaB induction of uPA protein and p65 phosphorylation. TLR5 activation with FlaB treatment induced the expression of uPA via the NF-κB and MAPK signalling pathways. Flagellin-bearing oral bacteria may cause pulp inflammation through TLR5. The findings provide new clues to control pulpal diseases by targeting TLR5 signalling pathways.

Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf

To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P &lt; 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

Near-Infrared Light Activated Thermosensitive Ion Channel to Remotely Control Transgene System for Thrombolysis Therapy.

Current antithrombotic therapeutic strategies often suffer from severe post-thrombotic syndromes (PTS), inconvenient daily subcutaneous injections for a long time and short circulation times accompanied by a dose-dependent risk of intracranial hemorrhage. Aiming at noninvasive, on-demand, and sustained antithrombotic therapy, a new thrombolysis approach based on the transgene system has been developed to remotely and precisely control the expression of urokinase plasminogen activator (uPA) by bioengineered cells for antithrombotic therapy both in vitro and in vivo. In this design, the near-infrared (NIR) light could activate the expression of the thermosensitive TRPV1 channel in response to photothermal responsive nanotransducers to trigger the synthetic signaling pathway to secret uPA. By encapsulating bioengineered cells in injectable hydrogel to ensure long-term survival and convenience for injection, the engineered cells could noninvasively and precisely control the production of uPA protein in situ via an NIR laser to significantly enhance the thrombolysis therapeutic effects by spatiotemporally controlling the local temperature, in both the microfluidic blood circulation mimic and the murine tail thrombus model. This novel thrombolysis approach could overcome some key limitations that are associated with conventional antithrombotic therapy, thus opening a new direction for developing remotely and precisely controllable continuous thrombolysis through artificially designed signaling.

Investigations on the Role of the Fibrinolytic Pathway on Outflow Facility Regulation.

PurposeTo understand the role and further dissect pathways downstream of tissue plasminogen activator (tPA) and the fibrinolytic pathway in modulating outflow facility.MethodsOutflow facility of tissue plasminogen activator (Plat) knockout (KO) mice was determined and compared to that of wild-type (WT) littermates. Gene expression of urokinase plasminogen activator (Plau), plasminogen activator inhibitor (Pai-1), plasminogen (Plg), and matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle tissues. Expression of the same genes and outflow facility were measured in KO and WT mice treated with triamcinolone acetonide (TA). Amiloride was used to inhibit urokinase plasminogen activator (uPA) in Plat KO mice, and outflow facility was measured.ResultsPlat deletion resulted in outflow facility reduction and decreased Mmp-9 expression in angle tissues. Plasminogen expression was undetectable in both KO and WT mice. TA led to further reduction in outflow facility and decreases in expression of Plau and Mmp-13 in plat KO mice. Amiloride inhibition of uPA activity prevented the TA-induced outflow facility reduction in Plat KO mice.ConclusionstPA deficiency reduced outflow facility in mice and was associated with reduced MMP expression. The mechanism of action of tPA is unlikely to involve plasminogen activation. tPA is not the only mediator of TA-induced outflow facility change, as TA caused reduction in outflow facility of Plat KO mice. uPA did not substitute for tPA in outflow facility regulation but abrogated the effect of TA in the absence of tPA, suggesting a complex role of components of the fibrinolytic system in outflow regulation.

Expression of urokinase plasminogen activator (uPA) in the leukocytes and tissues of patients with benign and malignant breast lesions.

To show that the expression of urokinase plasminogen activator (uPA) in the leucocytes of patients with benign and malignant breast lesions correlates with its expression in the lesions, and to explore the role of uPA as a tumour marker in breast cancer. Using real time reverse transcriptase - polymerase chain reaction (RT-PCR), we examined the expression of uPA in leukocytes and tissues of three groups of women: a) with breast cancer (BC), b) with benign breast lesions and c) a control group of healthy women. An arbitrary value of 1 was assigned to the level of uPA expressed in the leucocytes of the healthy controls; all other measurements were expressed as a function of this value. The expression of uPA was significantly higher in BC samples compared to benign breast lesion (5.7 versus 3.8 times; p < 0.001). The leukocyte uPA of healthy volunteers was significantly lower than the leukocyte uPA from patients with breast lesions (p < 0.001). Leukocyte uPA level of patients with BC was higher compared to leukocyte uPA of women with benign lesions (p < 0.01). The expression of tissue uPA was the highest in triple-negative breast cancer. Methylation status was similar across tissue and leukocyte samples. Leukocyte uPA can be considered a surrogate of the tissue uPA expressed in BC samples. These results further support the use of leukocyte uPA as a biochemical marker of breast cancer.

Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1.

Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. The mRNA expression of caveola-forming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal samples and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.

Role of Inflammation in Control of Hepatic Lipid Metabolism—New Insights to the Pathogenesis of NASH

Non‐alcoholic fatty liver disease (NAFLD) is a common metabolic disorder whose incidence among US adults exceeds 30%. While NAFLD manifests as benign hepatic steatosis in most patients, in about 20% of patients it assumes a much more aggressive form – non‐alcoholic steatohepatitis (NASH). The mechanisms that control the switch from benign steatosis to NASH are poorly understood, but were suggested to depend on ER stress. To query the role of ER stress in NASH development and establish a proper mouse model for studying the disease, we fed high‐fat diet (HFD) to MUP‐uPA mice, which are prone to liver ER stress due to hepatocyte‐specific urokinase plasminogen activator (uPA) expression. Placing these mice on HFD triggered full‐blown NASH within 3–4 months and most of the NASH‐afflicted mice progressed to develop hepatocellular carcinoma (HCC), the most common form of liver cancer whose incidence and development rates greatly increase with steatohepatitis. Both NASH and HCC development in MUP‐uPA mice depend on ER stress and are associated with persistent activation of sterol response element binding proteins (SREBP) 1 and 2. SREBP1 activation drives de novo lipogenesis (DNL) and cholesterol biosynthesis in HFD‐fed MUP‐uPA mice remains elevated without any sign of feedback inhibition mechanisms that control SREBP2 activation in tissue culture cells. DNL and cholesterol synthesis are also chronically elevated in NASH patients and the hepatic accumulation of free cholesterol was suggested to be a key switch from simple steatosis to NASH. While investigating the mechanism by which ER stress leads to persistent SREBP activation, we uncovered a previously unknown pathway in which caspase‐2 (Casp2) leads to constitutive activation of site 1 protease (S1P), which initiates SREBP cleavage activating protein (SCAP)‐independent SREBP activation. Of note, genetic ablation or pharmacological inhibition of Casp2 in MUP‐uPA mice blocks NASH development by preventing hepatic steatosis. Casp2 ablation in both MUP‐uPA and BL6 mice also prevents HFD‐induced adipocyte hypertrophy and increases energy consumption. None of the effects of Casp2 on liver lipid metabolism are related to cell death. The NAFLD/NASH epidemic was also suggested to be associated with increased consumption of fructose, which is a major constituent of high‐fructose corn syrup (HFCS), the main sweetener and a general food additive in the US. Increased consumption of HFCS has been linked to elevated risk of developing HCC, pancreatic and colon cancer. Indeed, feeding MUP‐uPA mice a high‐fructose diet (HFrD) results in NASH and HCC development even without the peripheral obesity that accompanies HFD consumption. Although fructose was believed to give rise to hepatic steatosis through preferential uptake and direct incorporation into the glycolytic pathway in hepatocytes, recent results indicate that the first site of fructose metabolism is the small intestine. Correspondingly, we found that fructose gives rise to NASH and HCC by first disrupting the intestinal epithelial barrier, evoking an inflammatory response that drives both NASH and HCC development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Plasminogen activator inhibitor-1 augments damage by impairing fibrinolysis after traumatic brain injury.

ObjectivePlasminogen activator inhibitor‐1 (PAI‐1) is the key endogenous inhibitor of fibrinolysis, and enhances clot formation after injury. In traumatic brain injury, dysregulation of fibrinolysis may lead to sustained microthrombosis and accelerated lesion expansion. In the present study, we hypothesized that PAI‐1 mediates post‐traumatic malfunction of coagulation, with inhibition or genetic depletion of PAI‐1 attenuating clot formation and lesion expansion after brain trauma.MethodsWe evaluated PAI‐1 as a possible new target in a mouse controlled cortical impact (CCI) model of traumatic brain injury. We performed the pharmacological inhibition of PAI‐1 with PAI‐039 and stimulation by tranexamic acid, and we confirmed our results in PAI‐1–deficient animals.ResultsPAI‐1 mRNA was time‐dependently upregulated, with a 305‐fold peak 12 hours after CCI, which effectively counteracted the 2‐ to 3‐fold increase in cerebral tissue‐type/urokinase plasminogen activator expression. PAI‐039 reduced brain lesion volume by 26% at 24 hours and 43% at 5 days after insult. This treatment also attenuated neuronal apoptosis and improved neurofunctional outcome. Moreover, intravital microscopy demonstrated reduced post‐traumatic thrombus formation in the pericontusional cortical microvasculature. In PAI‐1–deficient mice, the therapeutic effect of PAI‐039 was absent. These mice also displayed 13% reduced brain damage compared with wild type. In contrast, inhibition of fibrinolysis with tranexamic acid increased lesion volume by 25% compared with vehicle.InterpretationThis study identifies impaired fibrinolysis as a critical process in post‐traumatic secondary brain damage and suggests that PAI‐1 may be a central endogenous inhibitor of the fibrinolytic pathway, promoting a procoagulatory state and clot formation in the cerebral microvasculature. Ann Neurol 2019;85:667–680

Gene expression profile in mouse bacterial chronic rhinosinusitis.

Persistent infection in the paranasal sinuses impairs sinus drainage and leads to bacterial chronic rhinosinusitis. Greater knowledge of the key molecules in the pathology will help to clarify the pathogenesis. Study of the gene expression profile and analysis of the associated pathway is important to identify key molecules. This study investigates the expression of different genes and analyzes the key pathway in the pathological process of bacterial chronic rhinosinusitis. Bacterial chronic rhinosinusitis was induced in mice using a Merocel nasal pack inoculated with Staphylococcus aureus. Three months of mucosa samples were collected for histological and ELISA analysis, and gene expression was tested using DNA microarray. Differentially expressed genes were selected and verified for pathway analysis. The nasal mucosa of mice with chronic rhinosinusitis showed epithelial damage and lamina propria edema in extra cellular matrix with obvious mucosal inflammation. A total of 6,018 genes in bacterial chronic rhinosinusitis group were differentially expressed compared with the control. Among them, plasma, coagulation factors, urokinase plasminogen activator and urokinase receptor plasminogen activator expression were increased. Following gene ontology analysis and reverse transcription-quantitative polymerase chain reaction, coagulation cascades associated cytokines were found to be upregulated in bacterial chronic rhinosinusitis. The present results suggest that, bacterial chronic rhinosinusitis showed severe mucosal inflammation and genes differential expression in the pathogenesis. In this process, the coagulation cascades pathways were upregulated.

Peptide P7 inhibits the bFGF-stimulated proliferation and invasion of SKOV3 cells.

Peptide P7 specifically binds with basic fibroblast growth factor (bFGF) to inhibit the proliferation and invasion of numerous types of cancer cell. However, this effect has remained to be demonstrated in ovarian cancer-derived cell lines. In the present study, the protein P7 was used treat bFGF-stimulated SKOV3 epithelial ovarian cancer cells to explore the therapeutic potential of P7. An MTT and a scratch wound assay were used to respectively evaluate the proliferation and migration of bFGF-stimulated SKOV3 cells treated with P7. Reverse transcription-quantitative polymerase chain reaction analysis was used to detect the gene expression of urokinase-type plasminogen activator (uPA), as well as matrix metallopeptidase (MMP)-2 and −9, which have a role in cell migration/invasion. The morphology and proliferation of SKOV3 cells were not significantly affected by different concentrations of P7. However, P7 had an obvious inhibitory effect on the proliferation and migration of bFGF-stimulated SKOV3 cells. Treatment with P7 significantly lowered the gene expression of uPA, MMP-2 and MMP-9 compared with that in the control group. In conclusion, the present results suggested that P7, which, at least in part, acts through inhibition of bFGF, may have a potential therapeutic application in epithelial ovarian cancer.

Influence of miR-34a on preeclampsia through the Notch signaling pathway.

The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway. The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression. The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression. MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.

Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo.

Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.

Deficiency of Bmal1 disrupts the diurnal rhythm of haemostasis.

Mice deficient in the circadian clock gene BMAL1 (Brain and Muscle ARNT-like protein-1) exhibit a hypercoagulable state and an enhanced arterial and venous thrombogenicity, which aggravates with age. We investigated the effect of BMAL1 deficiency in mice at a different age on the diurnal rhythm of factors involved in coagulation and fibrinolysis. Hepatic, cardiac and brain tissues were isolated from 10- and 25-weeks-old Bmal1-deficient (BMAL1-/-) and wild-type (BMAL1+/+) mice at ZT2 and at ZT14 to analyze the mRNA expression level of genes involved in coagulation and fibrinolysis. Body weight and brain weight were significantly lower in all BMAL1-/- versus BMAL1+/+ mice. Bmal1 deficiency disturbed the diurnal rhythm of plasminogen activator inhibitor-1 (PAI-1) in liver and plasma, but not in cardiac or brain tissues. BMAL1+/+ livers showed diurnal fluctuations in factor (F)VII, FVII, protein S and anti-thrombin gene expression, which were not observed in BMAL1-/- tissues. Interestingly, tissue plasminogen activator (t-PA) expression was significantly upregulated in all BMAL1-/- versus BMAL1+/+ brains at both time points. Plasma t-PA-PAI-1 complex levels were however similar for all groups. Bmal1 deficiency affected the biphasic rhythm of coagulation and fibrinolysis factors predominantly in the liver. In the brain, Bmal1-dependent control of t-PA gene expression was documented for the first time.

Expression of Tissue Plasminogen Activator in Transgenic Tobacco Plants by Signal Peptides Targeting for Delivery to Apoplast, Endoplasmic Reticulum and Cytosol Spaces

Expression of Tissue Plasminogen Activator in Transgenic Tobacco Plants by Signal Peptides Targeting for Delivery to Apoplast, Endoplasmic Reticulum and Cytosol Spaces

Chloroquine attenuates TLR3-mediated plasminogen activator inhibitor-1 expression in cultured human glomerular endothelial cells.

Chloroquine, an antimalarial agent, has been reported to prevent the risk of thrombosis and decrease renal damage in patients with systemic lupus erythematosus (SLE); however, its detailed mechanisms remain unclear. Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis and is involved in fibrin deposition in glomeruli. Since upregulation of glomerular Toll-like receptor 3 (TLR3) signaling reportedly plays a pivotal role in the pathogenesis of lupus nephritis (LN), we examined whether chloroquine affects TLR3-mediated expression of PAI-1 in cultured human glomerular endothelial cells (GECs). We examined the effect of polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, on PAI-1 and tissue plasminogen activator (t-PA) expression in GECs. Then, we analyzed whether pretreatment of chloroquine or dexamethasone inhibits poly IC-induced expression of these proteins using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Poly IC increased PAI-1 expression in a time- and concentration-dependent manner, but did not affect t-PA expression in GECs. RNA interference against TLR3 inhibited poly IC-induced PAI-1 expression. Interestingly, pretreating cells with chloroquine, and also hydroxychloroquine, but not dexamethasone, attenuated poly IC-induced PAI-1 expression in GECs. Considering that TLR3 signaling is implicated in LN pathogenesis, our results suggest that chloroquine exert postulated renoprotective effects by inhibiting PAI-1 expression.

Expression of urokinase-type plasminogen activator and its receptor in squamous cell carcinoma of the oral tongue.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) act in the proteolysis of basement membrane and extracellular matrix structures, facilitating tumor invasion. The purpose of this study was to evaluate the relationship between these proteins and clinicopathological parameters in squamous cell carcinoma of the oral tongue (SCCOT). Sixty cases of SCCOT were submitted to immunohistochemistry and analyzed semiquantitatively at the invasion front and in the tumor core. The results were associated with lymph node metastasis, clinical stage, locoregional recurrence, clinical outcome and histological grade of malignancy. A higher expression of uPA was observed in cases of tumors of high-grade versus low-grade malignancy (p = 0.010). Moreover, the cases with the worst pattern of invasion presented an overexpression of uPA (p = 0.011). The presence of locoregional recurrence was associated with uPAR (p = 0.039), and the expression of both biomarkers was much higher at the invasion front than in the tumor core (p < 0.001). The results suggest uPA and uPAR are involved in the progression and aggressiveness of SCCOT, mainly at the tumor-host interface.

Abstract A25: Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F

Abstract Rationale: Cancers of the lung and bronchus are responsible for nearly 30% of all cancer-related deaths in the US, and mutations in the tumor-suppressor gene TP53 occur in 54% of lung adenocarcinomas and 86% of squamous cell carcinomas. Compared with other cancers, TP53 mutational hotspots in lung cancer more often include missense mutations at codons V157, R158, and A159, with frequencies approaching that of the canonical mutational hotspot R273. The resulting mutant p53 (mutp53) protein often exhibits not only loss of tumor suppressor capability but also gain of oncogenic function (GOF). Our previous work identified a novel, p53-dependent transcriptome including alterations in expression of urokinase plasminogen activator (uPA), a key regulator of the plasminogen activation system. When activated, uPA converts plasminogen to plasmin, which cleaves extracellular matrix components and promotes cell migration. We evaluated uPA expression and cell migration in human lung cancer cell lines expressing wild-type or mutant p53. Methods: Human lung cancer cell lines with wild-type p53 (A549) or with somatic mutations at the endogenous p53 locus (NCI-H1781 and NCI-H2087, V157F; NCI-H441, R158L; NCI-H2110, R158P; NCI-H2009, R273L) were obtained from ATCC. Using RNAi (shRNA and siRNA) cells were depleted of p53 as confirmed by puromycin selection of cells infected with lentivirus, in the case of shRNA. Whole-cell lysates were harvested for protein and RNA purification, and conditioned medium was harvested for measurement of secreted proteins. Immunoblotting and qRT-PCR were used to assess expression of protein and mRNA, respectively. A wound healing assay was performed following siRNA transfection to determine cell migration in cells expressing vs. depleted of wild-type or mutant p53. Results: Silencing of p53 by shRNA and siRNA targeted to TP53 was confirmed by Western blot and qRT-PCR. With p53 depletion, protein and mRNA expression of the uPA pro-enzyme pro-uPA and its activated B- and A-chains increased in the H2087 (V157F mutp53) cell line. No p53-dependent change was seen in uPA expression in H2009 cells (R273L mutp53) despite a similar relative quantity of uPA protein. Secreted uPA also increased as measured in conditioned media from H2087 cells (V157F mutp53). A wound healing assay showed increased migration by mutp53-depleted H2087 cells compared with mutp53-expressing H2087 cells, while A549 cells (wt p53) showed no p53-dependent difference in migration rate. Conclusions: To our knowledge, GOF phenotypes in lung cancer cells carrying V157F mutp53 have not been characterized. We have shown here that mutp53 represses uPA expression in human lung cancer cells with endogenous V157F mutp53, and depletion of p53 in these cells leads to an increase in migration. These findings prompt a reexamination of the signaling pathways associated with uPA. Further study is needed to elucidate the mechanism of the p53-dependent alterations in uPA expression and to establish additional biologic effects of V157F mutp53. Citation Format: Julie A. Barta, Steven B. McMahon. Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A25.

Src mediates TGF-β-induced intraocular pressure elevation in glaucoma.

Glaucoma, a progressive and irreversible optic neuropathy, is one of the leading causes of vision impairment worldwide. Elevation of intraocular pressure (IOP) due to transforming growth factor-β (TGF-β)-induced dysfunction of the trabecular meshwork is a risk factor for glaucoma, but the underlying molecular mechanisms remain elusive. Here, we show that Src kinase is involved in TGF-β-induced IOP elevation. We observed that dasatinib, a potent Src inhibitor, suppressed TGF-β2-induced IOP in rat eyes. Mechanistic analyses in human trabecular meshwork cells showed that TGF-β2 activated Src signaling and concomitantly increased cytoskeletal remodeling, cell adhesion, and extracellular matrix (ECM) accumulation. Src was activated via TGF-β2-induced upregulation of the Src scaffolding protein CasL, which mediates the assembly of focal adhesions, cytoskeletal remodeling, and ECM deposition. Activation of Src suppressed the expression of tissue plasminogen activator, thereby attenuating ECM degradation. Furthermore, the Src inhibitor ameliorated TGF-β2-induced changes in the contractile and adhesive characteristics of trabecular meshwork cells, and ECM deposition. These findings underscore the crucial role of Src activity in TGF-β-induced IOP elevation and identify Src signaling as a potential therapeutic target in glaucoma.

A novel recombinant human plasminogen activator: Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats.

BackgroundThromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in goat mammary glands has never been reported before.MethodsWe constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were transgenic goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 transgenic goats, the F0 transgenic goat BP21 was mated with a normal male goat to generate an F1 transgenic goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients.ResultsWestern blot results showed a specific 39 kDa band. The rhPA expression level in transgenic goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 transgenic goats was 13.3 times higher than that of the reteplase reference material.ConclusionThus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of transgenic goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in transgenic animals. The expression of biopharmaceuticals by transgenic animals can be used for pharmacological research and bioactive analysis, and transgenic goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.