The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.     

The molecular basis of urokinase inhibition: from the nonempirical analysis of intermolecular interactions to the prediction of binding affinity

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a crucial role in angiogenesis process. In addition to its physiological role in healthy organisms, angiogenesis is extremely important in cancer growth and metastasis, resulting in numerous attempts to understand its control and to develop new approaches to anticancer therapy. The α-aminoalkylphosphonate diphenyl esters are well known as highly efficient serine protease inhibitors. However, their mode of binding has not been verified experimentally in details. For a group of average and potent phosphonic inhibitors of urokinase, flexible docking calculations were performed to gain an insight into the active site interactions responsible for observed enzyme inhibition. The docking results are consistent with the previously suggested mode of inhibitors binding. Subsequently, rigorous ab initio study of binding energy was carried out, followed by its decomposition according to the variation–perturbation procedure to reveal stabilization energy constituents with clear physical meaning. Availability of the experimental inhibitory activities and comparison with theoretical binding energy allows for the validation of theoretical models of inhibition, as well as estimation of the possible potential for binding affinity prediction. Since the docking results accompanied by molecular mechanics optimization suggested that several crucial active site contacts were too short, the optimal distances corresponding to the minimum ab initio interaction energy were also evaluated. Despite the deficiencies of force field-optimized enzyme-inhibitor structures, satisfactory agreement with experimental inhibitory activity was obtained for the electrostatic interaction energy, suggesting its possible application in the binding affinity prediction. Figure The comparison of an arrangement of inhibitors within uPA active. Amino acid residues form S1 pocket that binds the variable part of inhibitor molecules     

Isolation and characterization of plasminogen activators from hyperlastic and malignant prostate tissue

Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen framents in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates $\text{pyro-Glu-gly-Arg}\text{-p}\text{NA and H-}\text{D}\text{-Ile-Pro-Arg}\text{-p}\text{NA}$ were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase.     

uPA、PAI-1的表达和MVD计数与浸润性乳腺癌侵袭性的关系

目的：探讨尿激酶型纤溶酶原激活剂（uPA）、纤溶酶原激活物抑制剂（PAI-1）的表达和血管形成与浸润性乳腺癌侵袭性的关系。方法：应用免疫组化SP法检测80例浸润性乳腺癌、20例良性乳腺肿瘤组织中uPA、PAI-1的表达情况并进行微血管计数。结果：uPA和PAI-1在浸润性乳腺癌组的阳性表达显著高于良性肿瘤组,且其高表达率与乳腺癌的临床病理参数密切相关（P〈0.05）;微血管计数在乳腺癌组和良性肿瘤组分别为30.87±7.64、20.28±8.72,两组比较有显著性差异（P〈0.01）。uPA与PAI-1在浸润性乳腺癌中的表达呈正相关（P〈0.05）。结论：uPA、PAI-1的高表达与浸润性乳腺癌的浸润、转移相关,uPA、PAI-1的高表达和MVD计数可作为评价浸润性乳腺癌侵袭性、评估预后和确定治疗方案的生物学指标。     

The tyrosine phosphatase SHP-2 controls urokinase-dependent signaling and functions in human vascular smooth muscle cells

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of functional behaviour of human vascular smooth muscle cells (VSMC). uPAR associates with platelet-derived growth factor receptor β (PDGFR-β), which serves as a transmembrane adaptor for uPAR in VSMC, to transduce intracellular signaling and initiate functional changes. The precise and rapid propagation of these signaling cascades demands both strict and flexible regulatory mechanisms that remain unexplored. We provide evidence that the tyrosine phosphatase SHP-2 mediates these processes. uPA regulated SHP-2 phosphorylation, catalytic activity, and its co-localization and association with the PDGFR-β. Active PDGFR-β was required for the uPA-induced SHP-2 phosphorylation. uPAR-directed STAT1 pathway was disturbed in cells expressing SHP-2 inactive mutant. Both, cell proliferation and migration were impaired in VSMC with downregulated SHP-2. Elucidating the underlying mechanisms, we found that uPA induced SHP-2 recruitment to lipid rafts. Disruption of rafts abolished uPA-related control of SHP-2 phosphorylation, its association with PDGFR-β and finally the VSMC functional responses. Our results demonstrate that SHP-2 plays an important role in uPA-directed signaling and functional control of human VSMC and suggest that this phosphatase might contribute to the pathogenesis of the uPA-related vascular remodeling.     

Defect of vacuolar protein sorting stimulates proteolytic processing of human urokinase-type plasminogen activator in the yeast Hansenula polymorpha

Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing.     

Induction of Urokinase-Type Plasminogen Activator in Rat Facial Nucleus by Axotomy of the Facial Nerve

Abstract: The response of plasminogen activator activity in the CNS to peripheral nerve axotomy was examined in vivo. After transection of the rat facial nerve, a transient increase in plasminogen activator activity was observed in the facial nucleus on the operated side with maximal activity 3–5 days after lesion. This activity was inhibited by the urokinase-specific inhibitor amiloride but not by antibodies against tissue plasminogen activator. The molecular mass of the induced form of plasminogen activator was estimated to be ∼48 kDa. An in vitro assay of plasminogen hydrolysis also demonstrated an increase in amiloride-sensitive plasminogen activator activity in facial nerve extracts following facial nerve axotomy. These data indicate that the plasminogen activator activity induced in the facial nucleus following axotomy of facial motoneurons is of the urokinase type. It is suggested that the urokinase-type plasminogen activator might play a role in the events accompanying injury and regeneration in the facial nucleus following motoneuron lesion.     

Active site-directed plasmin inhibitors: Extension on the P2 residue

Based on the structure of YO-2 [N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr(O-picolyl)-NH-octyl], active site-directed plasmin (Plm) inhibitors were explored. The picolyl moiety in the Tyr(O-picolyl) residue (namely, the P2 residue) was replaced with smaller or larger groups, such as hydrogen, tert-butyl, benzyl, (2-naphthyl)methyl, and (quinolin-2-yl)methyl. Those efforts produced compound 17 {N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr[O-(quinolin-2-yl)methyl]-NH-octyl} [IC₅₀ = 0.22 and 77 μM for Plm and urokinase (UK), respectively], which showed not only 2.4-fold greater Plm inhibition than YO-2, but also an improvement in selectivity (Plm/UK) by 35-fold. The docking experiments of the Plm-17 complexes disclosed that the amino group of the tranexamyl moiety interacted with the side-chain of Asp753 which formed S1 site.     

Construction and expression of antibody targeted plasminogen activator*

It has been known that antibody-mediated plasminogen activator will be much more specific than its parent molecular. To get a cheaper and more effective medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoclonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator. PCR was used to amplify the region of VL and VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu-PA-32KD(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via NdeI site. The recombinant protein was expressed in BL21 (DE3) plyS, a kind of E. coli. It was shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase. After partial purification: dialysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate. The specific activity was about 47,000 IU/mg, corresponding to 80,000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody. Activation of plasminogen by the chimera followed Michaelis-Menten kinetics, and the K_m was 1.08 uM.     

uPA、PAI-1 的表达和MVD 计数与浸润性乳腺癌侵袭性的关系

目的:探讨尿激酶型纤溶酶原激活剂(uPA)、纤溶酶原激活物抑制剂(PAI-1)的表达和血管形成与浸润性乳腺癌侵袭性的关系。方法:应用免疫组化SP法检测80例浸润性乳腺癌、20例良性乳腺肿瘤组织中uPA、PAI-1的表达情况并进行微血管计数。结果:uPA和PAI-1在浸润性乳腺癌组的阳性表达显著高于良性肿瘤组,且其高表达率与乳腺癌的临床病理参数密切相关(P<0.05);微血管计数在乳腺癌组和良性肿瘤组分别为30.87±7.64、20.28±8.72,两组比较有显著性差异(P<0.01)。uPA与PAI-1在浸润性乳腺癌中的表达呈正相关(P<0.05)。结论:uPA、PAI-1的高表达与浸润性乳腺癌的浸润、转移相关,uPA、PAI-1的高表达和MVD计数可作为评价浸润性乳腺癌侵袭性、评估预后和确定治疗方案的生物学指标。