Plasminogen Activator Receptor Gene Research Articles

The role of Plaur-miR1-5p encoded within the urokinase receptor gene (Plaur) in angiogenesis

Angiogenesis plays a crucial role in tissue and organ regeneration by supplying essential nutrients and oxygen through the development of new blood vessels. Mesenchymal stem/stromal cells release extracellular vesicles that actively contribute to angiogenesis by carrying pro-angiogenic growth factors and microRNAs. MicroRNAs, small non-coding RNA molecules, are central players in angiogenesis, affecting endothelial cell proliferation, specialization, migration, apoptosis, and post-transcriptional gene expression.In the present study, we investigated the impact of extracellular vesicles containing Plaur-miR1- 5p microRNAs on angiogenesis, specifically focusing on its initial stages: vascular cell migration and the formation of capillary-like structures. Recently we discovered Plaur-miR1-5p, which is encoded within the urokinase receptor gene (Plaur). However, the functions of this microRNA remain largely unexplored. Using a vascular ring model embedded in Matrigel, we demonstrate that Plaur-miR1-5p is encapsulated within extracellular vesicles and plays a regulatory role in capillary-like structure formation. Moreover, applying bioinformatic analysis, we have identified potential target genes of Plaur-miR1-5p that participate in the regulation of angiogenesis.This study advances our comprehension of the fundamental processes governing angiogenesis, particularly the involvement of extracellular vesicles and microRNAs. Moreover, it sheds light on the functional aspects ofthe Plaur gene, contributing to a more profound understanding of its role in regulation of angiogenesis.

Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

Increased soluble urokinase plasminogen activator levels modulate monocyte function to promote atherosclerosis.

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

Urokinase Receptor uPAR Overexpression in Mouse Brain Stimulates the Migration of Neurons into the Cortex during Embryogenesis

The regulation of neuronal migration in the cerebral cortex during embryonic development is one of the most important but underinvestigated processes that determines the correct formation of neural networks and the development of cognitive functions. Mutations of the urokinase receptor (uPAR) gene in mice lead to spontaneous seizures; polymorphisms of the PLAUR gene encoding uPAR in humans are associated with autism spectrum disorders. The effect of uPAR overexpression on the radial migration of neurons in the cerebral cortex during mouse embryogenesis has been investigated. The number of neurons migrating from the ventricular zone of proliferating progenitors to the outer layers of the cerebral cortex was evaluated. We found that uPAR overexpression significantly stimulates radial neuronal migration to the outer layers of the differentiating cortex. These data show for the first time uPAR-dependent regulation of neuronal migration in the embryonic period of the cerebral cortex development. Further study of uPAR’s role in regulating the migration trajectory of neuronal precursors will advance our understanding of the mechanisms of brain formation in health and disease.

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients.We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

In vitro activity of probiotic Lactobacillus reuteri against gastric cancer progression by downregulation of urokinase plasminogen activator/urokinase plasminogen activator receptor gene expression.

Gastric cancer (GC) is the third leading cause of cancer death, and most patients represent metastatic phenotype at the time of diagnosis. Urokinase plasminogen activator (uPA) system is well known for its critical roles in cancer cells invasion since uPA/uPA receptor (uPAR) overexpresses in several cancers. Subsequently, suppression of uPA/uPAR gene expression improves patients overall survival and prevents cancer progression. The aim of the current study was to investigate possible effects of live Lactobacillus reuteri as a probiotic in inhibition of GC cells proliferation and invasion. Human gastric adenocarcinoma epithelial cell line (AGS) cells were treated with different ratios of live L. reuteri and were incubated for 24, 48, and 72 h. Viability of cancer cells was measured with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay, and the effects of L. reuteri on uPA/uPAR gene expression were assessed by real-time polymerase chain reaction. Our results showed that L. reuteri inhibits cell proliferation significantly in dose-dependent manner. Expressions of uPA and uPAR were downregulated followed by co-incubation of AGS cells and live L. reuteri compared to untreated-based line level. This study provides strong support in the role of L. reuteri in suppression of GC cell invasion by downregulation of pathways which is involved in extracellular matrix degradation such as uPA and uPAR.

Hypoxia inducible factor-1 mediates upregulation of urokinase-type plasminogen activator receptor gene transcription during hypoxia in cervical cancer cells.

Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia mediates tumor cells to have more invasive property in a variety of cancers. Urokinase plasminogen activator receptor (uPAR) which mediates invasion is considered to be induced by hypoxia. We sought to determine the regulators of uPAR expression during hypoxia in cervical cancer. We showed that cervical cancer cell lines, CaSki and CA, were more invasive under hypoxic condition (1% O2) than under normoxic condition (20% O2) by invasion assays. Using western blot analysis, hypoxia enhanced the endogenous hypoxia-inducible factor (HIF)-1α and uPAR protein expression. uPAR mRNA level was also upregulated by hypoxia using real-time RT-PCR. Overexpression of HIF-1α which is induced by hypoxia activated the transcriptional activity of the uPAR promoter by luciferase assays. HIF-1 protein bound the putative HIF-1 response element on the uPAR promoter using electrophoretic mobility shift analysis, and additional luciferase assays show that this is essential for uPAR transactivation by HIF-1. HIF-1 overexpression enhanced the endogenous uPAR expression and introduction of siRNA for HIF-1α diminishes uPAR expression during hypoxia. These results indicate the upregulation of uPAR by hypoxia in cervical cancer cells is mediated through HIF-1. In cervical cancer tissues, we also demonstrated that uPAR protein expression was detected in cervical cancer but not in normal cervix or cervical intraepithelial neoplasia (CIN) by immunohistopathological staining. Our results provide evidence that regulation of uPAR expression by HIF-1 represents a mechanism for cervical cancer invasion during hypoxia.

Phosphorylation controls a dual-function polybasic nuclear localization sequence in the adapter protein SH2B1β to regulate its cellular function and distribution

An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1β, also localizes SH2B1β to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1β to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1β from the PM and enhances nuclear entry. Release of SH2B1β from the PM and/or nuclear entry appear to be required for SH2B1β enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1β dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.

Urokinase Receptor Gene Expression Inhibited by siRNA Cocktails in Co-culture of Spermatogenic Cells with Sertoli Cells from 3-Week-Old Rat

Objective To study the functions of urokinase receptor (uPAR) during spermatogenesis by adding targeted siRNA cocktails to the co-culture of Sertoli cell with spermatogenic cells. Methods The seminiferous tubule samples from SD rats aged 20-23 d were digested with collagenase for 15-20 min, then were cut into small fragments. Tubular fragments were digested with collagenase again for 5-10 min, then gently resuspended in F12/DMEM supplemented with vitamins and hormones. Cell samples were seeded in plate and cultured at 32°C for 2 weeks. After 48 h, siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were introduced into this cocultured Sertoli/spermatogenic cells. After then for 48 h, expression of uPAR mRNA was analyzed by real-time RT-PCR. Results Some spermatogenic cells took place morphologic changes and generated an obvious flagellum. The expression levels of uPAR mRNA silencing with siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were significantly lower than those in nontransfected group (P Conclusion The meiosis and spermiogenesis could take place in this culture system, and the siRNA cocktails can effectively inhibit the gene expression of uPAR in co-cultured Sertoli/spermatogenic cells.

Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors

Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.

P3. Combination analysis of activator protein-1 family members, Sp1 and an activator protein-2α-related factor binding to different regions of the urokinase receptor (u-PAR) gene in resected colorectal cancers

P3. Combination analysis of activator protein-1 family members, Sp1 and an activator protein-2α-related factor binding to different regions of the urokinase receptor (u-PAR) gene in resected colorectal cancers

A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis

The urokinase receptor, uPAR, which binds to the urinary-type plasminogen activator, controls matrix degradation in the processes of tissue remodeling, cell migration, and invasion. In the present study, we found a new urokinase receptor gene that encodes a 249-amino acid putative protein. Northern blot analysis showed specific expression in the testis of this gene, which we named the spermatogenesis-related gene (SGRG). In situ hybridization revealed a strong expression signal for SGRG in spermatogonia, but not in spermatocytes. Therefore, we conjecture that SGRG may regulate spermatocyte migration through breakdown of extracellular matrix protein barriers in spermatogenesis. Since SGRG is specifically expressed in spermatogonia, it provides an attractive candidate for development of a contraceptive vaccine.

Combination Analysis of Activator Protein-1 Family Members, Sp1 and an Activator Protein-2α-Related Factor Binding to Different Regions of the Urokinase Receptor Gene in Resected Colorectal Cancers

Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2-related factor to promoter region -152/-135 of the metastasis-related u-PAR gene in 60% of in vivo-resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (-190/-171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor-binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions. In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region -190/-171 was done in tumors and normal tissues. In 71 patients, region -152/-135 was also analyzed. U-PAR protein was measured by ELISA. Tumor-specific AP-1 binding to region -190/-171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P<0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P<0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P=0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors. This is the first study differentiating transcription factor-binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.

Inhibition of urokinase receptor gene expression and cell invasion by anti‐uPAR DNAzymes in osteosarcoma cells

The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G)-pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by approximately 93% and approximately 84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis.

Transgenic Mice Demonstrate Novel Promoter Regions for Tissue-Specific Expression of the Urokinase Receptor Gene

The urokinase-type plasminogen activator receptor (u-PAR) contributes to cell migration and proteolysis in normal and cancerous tissues. Currently, there are no reports on the regulatory regions directing tissue-specific expression. Consequently, we undertook a study to identify novel promoter regions required for expression of this gene in transgenic mice bearing a LacZ reporter regulated by varying amounts (0.4, 1.5, and 8.5 kb) of upstream sequence. The 0.4-kb u-PAR upstream sequence directed weak and strong LacZ expression in the placenta and epididymis, respectively, both of which are tissues that express endogenous u-PAR. Conversely, transgene expression in the apical cells of the colon positive for endogenous u-PAR protein required 1.5 kb of upstream sequence for optimal expression. Furthermore, chromatin accessibility assays coupled with real-time polymerase chain reaction suggested a putative regulatory region spanning -1295/-1192 driving u-PAR expression in colonic cells. Interestingly, placental transgene expression was augmented with the 8.5-kb upstream fragment compared with the shorter 1.5-kb fragment indicating contributing element(s) between -1.5 and -8.5 kb. Thus, while 0.4 kb of upstream sequence directs u-PAR expression in the epididymis, sequences located between -0.4 and -1.5 kb and between -1.5 and -8.5 kb are required for optimal tissue-specific expression in the colon and the placenta, respectively.

The urokinase receptor mediates basic fibroblast growth factor-dependent smooth muscle cell migration through baboon aortic explants

The urokinase receptor is required for vascular smooth muscle cell migration in vitro, but may not be needed in vivo since smooth muscle cell migration and intimal hyperplasia after arterial injury in mice are not affected by urokinase receptor gene deletion. We have used baboon aortic explants as a bridge between cell culture and in vivo experiments to determine if the urokinase receptor is required for smooth muscle cell proliferation and smooth muscle cell migration in primate vessels. Levels of urokinase receptor in explants increased with time after explantation, while blockade of urokinase receptor with an antibody decreased smooth muscle cell proliferation and smooth muscle cell migration from the explants. A blocking antibody to basic fibroblast growth decreased levels of urokinase and urokinase receptor in explants, and it decreased smooth muscle cell migration and mitogenesis. These results suggest that the factor urokinase receptor plays a positive role in smooth muscle cell migration and proliferation in injured primate arterial tissue, in part mediating the pro-migratory and proliferative effects of basic fibroblast growth factor released by damaged smooth muscle cells.

The role and regulation of urokinase-type plasminogen activator receptor gene expression in cancer invasion and metastasis.

This article reviews the role of urokinase-type plasminogen activator receptor (uPAR) and its protein, mRNA, cDNA, genomic organization, promoter, transcription activation factors, and signal transduction. The uPAR has been implicated in several biological processes including angiogenesis, monocyte migration, cancer metastasis, trophoblast implantation, and wound healing. It is a specific cell surface receptor for its ligand uPA which catalyzes the formation of plasmin from plasminogen to generate the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. The uPAR is a 55-60 kDa glycoprotein organized as three homologous cysteine-rich domains. It attaches to the plasma membrane via a covalent linkage to a glycosyl-phosphatidylinositol (GPI) moiety and appears to play an important role in transmembrane signalling. The 1.4-kb human uPAR cDNA and 21.23-kb genomic DNA have been cloned and the gene contains seven exons. The uPAR promoter region was defined in a 188 bp fragment between bases -141 and +47 relative to the transcription start site. Binding of transcription factors (Sp1, AP-2, NFkappaB and two AP-1) to the uPAR promoter region activates the basal transcription of the gene. There is a strong correlation between uPAR expression and the invasive cancer cell phenotype. uPAR may play a critical role in the process of cancer invasion and metastasis, as antisense uPAR mRNA can inhibit cancer spread in vitro and in vivo. These studies may provide a novel therapeutic target for blocking cancer invasion and metastasis.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

Sp1 mediates constitutive and transforming growth factor β-inducible expression of urokinase type plasminogen activator receptor gene in human monocyte-like U937 cells

Urokinase type plasminogen activator receptor (uPAR) is known to be involved in conversion of plasminogen into plasmin and its expression can be regulated by a variety of biological agents including transforming growth factor β (TGF-β). In the present study, we cloned the promoter region of the human uPAR (huPAR) gene (−653 to +61) and investigated the transcription regulatory mechanism of the expression of the huPAR gene upon treatment with TGF-β in human monocyte-like U937 cells. By deletion and point mutational analysis of the huPAR gene promoter, it was found that the sequence positioned at −70 is required for both constitutive and TGF-β-inducible expression of the huPAR gene in U937 cells. Using electrophoretic mobility shift assay, we could observe that Sp1 formed a DNA–protein complex at the −70 sequence. In addition, antisense oligonucleotide against human Sp1 blocked both constitutive and TGF-β-inducible expression of the luciferase reporter gene driven by the huPAR gene promoter in U937 cells. These results led us to conclude that Sp1 transcription factor mediates constitutive and TGF-β-inducible expression of the huPAR gene in U937 cells through binding to the sequence located at −70.

Transcriptional Induction of the Urokinase Receptor Gene by a Constitutively Active Src: REQUIREMENT OF AN UPSTREAM MOTIF (−152/−135) BOUND WITH Sp1

Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify required cis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of the src transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (-152/-135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the -152/-135 region was not. Nuclear extracts from the src transfectants demonstrated increased Sp1 binding to region -152/-135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding.