Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2-15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B(1) and B(2) receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B(1) and B(2) receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.     

Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer

Abstract Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B(1) and B(2) receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B(1) and B(2) receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.     

The role of human tissue kallikreins 7 and 8 in intracranial malignancies

Recent evidence suggests that many tissue kallikreins are implicated in carcinogenesis. Kallikrein 8 (KLK8) plays a role in the physiology of the central nervous system. Kallikrein 7 (KLK7) takes part in skin desquamation. Both show altered expression in ovarian and breast cancer. In this study, we examined the level of mRNA expression of the KLK7 and KLK8 genes in 73 intracranial tumors using qualitative RT-PCR. The results were correlated with clinical and histomorphological variables and patient outcome. The expression of both genes was also examined in the brain cancer cell lines U-251 MG, D54 and SH-SY5Y and the invasive capacity of glioblastoma cells U-251 MG overexpressing hK7 or hK8 was also investigated in an in vitro Matrigel assay. Follow-up analysis revealed that expression of KLK7 mRNA was associated with shorter overall survival (OS) compared to patients with no KLK7 expression, as determined by Cox proportional hazard regression analysis. Overexpression of hK7 protein by cultivated brain tumor cells significantly enhanced the invasive potential in the Matrigel invasion assay, in contrast to cells overexpressing hK8 protein. Our data suggest that hK7 protein overexpression is associated with a more aggressive phenotype in brain cancer cells.     

美洛昔康对Aβ诱导Alzheimer’s disease大鼠脑内炎症损伤的影响

目的：研究美洛昔康对β-淀粉样蛋白（Aβ）诱导的阿尔茨海默病（AD）模型大鼠脑内炎症损伤的保护作用,并探讨其抑制炎症作用的机制。方法：Aβ1-40海马注射建立AD大鼠模型。免疫组化法观察大鼠海马核因子κBp65（NF-κBp65）和星形胶质细胞（AS）胶质纤维酸性蛋白（GFAP）表达变化;Western-blot法测定大鼠皮层组织GFAP的表达;ELISA法检测大鼠皮层组织肿瘤坏死因子-α（TNF-α）水平变化;RT-PCR法检测大鼠海马组织白细胞介素-1β（IL-1β）mRNA的表达情况。结果：美洛昔康能抑制AD大鼠海马NF-κBp65和GFAP的表达;降低大鼠皮层TNF-α的含量;抑制AD大鼠海马IL-1βmRNA的表达。结论：美洛昔康通过减少AD模型大鼠海马、皮层组织GFAP表达,抑制AS的增生,降低NF-κBp65的活性,减少炎症因子TNF-α和IL-1β的水平,减轻脑内炎症反应。     

Leptin deficiency leads to the regulation of kinin receptors expression in mice

Kinins are vasoactive and pro-inflammatory peptides generated by the cleavage of the kininogen by kallikreins. Two kinin receptors have been described and denominated B1 and B2. Obesity frequently accompanies other pathologies, such as diabetes and hypertention. The clustering of these pathologies is usually known as "metabolic syndrome". Mice lacking leptin gene (ob/ob) are severely obese and hyperphagic. Using quantitative RT-PCR analysis of B1 and B2 mRNAs expression, we described for the first time a correlation between the kallikrein-kinin system (KKS) and severe obesity in mice. The ob/ob mice presented lower expression of B2 mRNA in the white adipose tissue (WAT) and hypothalamus, both primary sites for neuroendocrine regulation of the energetic metabolism. B1 mRNA, however, is overexpressed in these tissues of ob/ob mice. An upregulation of the B1 mRNA has also been seen in liver, abdominal aorta and stomach fundus. However, different from the lean mice, the expression of the B1 mRNA in brown adipose tissue (BAT) and heart is completely abolished. Our data show that kinin receptors are differently modulated in distinct tissues in obesity. These findings suggest a connection between the KKS and obesity, and suggest that kinin receptors could be involved in the ethiopathogenesis of the metabolic syndrome.     

Human tissue kallikrein 9: production of recombinant proteins and specific antibodies

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

The non-peptide kinin receptor antagonists FR 173657 and SSR 240612: preclinical evidence for the treatment of skin inflammation

Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases.     

KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer

Emerging data indicate that serine proteases of the kallikrein family (KLK) are implicated in various human diseases, including carcinoma; however, kallikrein gene expression has never been investigated in lung cancer. Using RT-PCR and Western blotting, we demonstrated the expression of both KLK5 and KLK7, and their respective proteins (hK5 and hK7) in tumoral and nontumoral lung tissues. Quantitative gene expression was then analyzed in a cohort of 56 patients with non-small cell lung cancer by real-time RT-PCR. KLK5 expression is significantly more expressed in squamous cell carcinoma than in matched nonmalignant lung tissue (P=0.02), whereas expression of KLK7 was decreased in adenocarcinoma (P=0.003). Multivariate analysis revealed diverse correlations between the KLK5 and KLK7 expression levels in nonmalignant and malignant tissues, and clinical parameters, including histotype, metastatic status, and grade. Our findings provide new insight into kallikrein gene expression in hormone-independent carcinoma. Altogether, our results suggest that variability in KLK5 and KLK7 gene expression might be involved in lung tumorigenesis and useful for clinical purposes.     

The tissue kallikrein-kinin system protects against cardiovascular and renal diseases and ischemic stroke independently of blood pressure reduction

Tissue kallikrein (hK1) cleaves low-molecular-weight kininogen to produce kinin peptide, which binds to kinin receptors and triggers a wide spectrum of biological effects. Tissue kallikrein levels are reduced in humans and in animal models with hypertension, cardiovascular and renal diseases. Transgenic mice or rats over-expressing human tissue kallikrein or kinin B2 receptor are permanently hypotensive, and somatic kallikrein gene delivery reduces blood pressure in several hypertensive rat models. Moreover, kallikrein gene delivery or kallikrein protein infusion can directly improve cardiac, renal and neurological function without blood pressure reduction. Kallikrein has pleiotropic effects in inhibiting apoptosis, inflammation, proliferation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in different experimental animal models. Kallikrein's effects can be blocked by kinin B2 receptor antagonists. Mechanistically, tissue kallikrein/kinin leads to increased nitric oxide levels and Akt activation, and reduced reactive oxygen species formation, TGF-beta1 expression, MAPK and nuclear factor-kappaB activation. Our studies indicate that tissue kallikrein, through the kinin B2 receptor and nitric oxide formation, can protect against oxidative damage in cardiovascular and renal diseases and ischemic stroke. These novel findings suggest that kallikrein/kinin may serve as new drug targets for the prevention and treatment of heart failure, renal disease and stroke in humans.     

Human kallikrein 10 expression in normal tissues by immunohistochemistry.

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern.     

The human kallikrein protein 5 (hK5) is enzymatically active,glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Binding of kallikreins to protease inhibitors is an important mechanism for regulating their enzymatic activity and may have potential clinical applications. Human kallikrein gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). This kallikrein was shown to be differentially expressed at the mRNA and protein levels in diverse malignancies. Our objective was to study the enzymatic activity and the interaction of recombinant hK5 protein with protease inhibitors. Recombinant hK5 protein was produced in yeast and mammalian expression systems and purified by chromatography. HPLC fractionation, followed by ELISA-type assays, immunoblotting and radiolabeling experiments were performed to detect the possible interactions between hK5 and proteinase inhibitors in serum. Enzymatic deglycosylation was performed to examine the glycosylation pattern of the protein. The enzymatic activity of hK5 was tested using trypsin and chymotrypsin-specific synthetic fluorogenic substrates. In serum and ascites fluid, in addition to the free ( approximately 40 kDa) form, hK5 forms complexes with alpha(1)-antitrypsin and alpha(2)-macroglobulin. These complexes were detected by hybrid ELISA-type assays using hK5-specific coating antibodies and inhibitor detection antibodies. The ability of hK5 to bind to these inhibitors was further verified in vitro. Spiking of serum samples with 125I-labeled hK5 results in the distribution of the protein in two higher molecular mass (bound) forms, in addition to the unbound form. The hK5 mature enzyme is active and shows trypsin, but not chymotrypsin-like, activity. The pro-form of hK5 is not active. Recombinant hK5 shows a higher than predicted molecular mass due to glycosylation. hK5 is partially complexed with alpha(1)-antitrypsin and alpha(2)-macroglobulin in serum and ascites fluid of ovarian cancer patients. The recombinant protein is glycosylated and its mature form shows trypsin-like activity.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Kinin receptors in cultured rat microglia

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.     

Quantitative RT-PCR analysis and immunohistochemical localization of the kallikrein-related peptidases 13 and 14 in lung

Expression of the KLK13 and KLK14 genes was examined at the mRNA and protein levels in a cohort of 57 patients with non-small-cell lung cancer (NSCLC). The mRNA levels, assessed by real-time RT-PCR, were significantly different in malignant tissues compared to adjacent non-malignant tissues (KLK13, p=0.006; KLK14, p=0.022). KLK13 and KLK14 mRNA overexpression in tumors (1/3 of the patients) was associated with a positive nodal status in multivariate analysis (p=0.018 and p=0.069, respectively). KLK13 and KLK14 were localized in the cytoplasm of epithelial cells of normal bronchus and NSCLC, as determined by immunohistochemistry. Moreover, positive staining was significantly associated with adenocarcinoma histotype (KLK13, p=0.014) and tumor size (KLK14, p=0.048). Although the results are marginally significant, patients with high KLK13 expression at the mRNA or protein level had lower overall survival.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

An overview of kinin mediated events in cancer progression and therapeutic applications

Kinins are bioactive peptides generated in the inflammatory milieu of the tissue microenvironment, which is involved in cancer progression and inflammatory response. Kinins signals through activation of two G-protein coupled receptors; inducible Bradykinin Receptor B1 (B1R) and constitutive receptor B2 (B2R). Activation of kinin receptors and its cross-talk with receptor tyrosine kinases activates multiple signaling pathways, including ERK/MAPK, PI3K, PKC, and p38 pathways regulating cancer hallmarks. Perturbations of the kinin-mediated events are implicated in various aspects of cancer invasion, matrix remodeling, and metastasis. In the tumor microenvironment, kinins initiate fibroblast activation, mesenchymal stem cell interactions, and recruitment of immune cells. Albeit the precise nature of kinin function in the metastasis and tumor microenvironment are not completely clear yet, several kinin receptor antagonists show anti-metastatic potential. Here, we showcase an overview of the complex biology of kinins and their role in cancer pathogenesis and therapeutic aspects.