Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats.

Our previous studies had reported that Human Tissue Kallikrein 1 (hKLK1) preserved erectile function in aged transgenic rats, while the detailed mechanism of hKLK1 protecting erectile function in aged rats through activation of cGMP and cAMP was not mentioned. To explore the latent mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 18 months old and divided into four groups: young WTR (yWTR) as the control, aged WTR (aWTR), aged TGR (aTGR) and aged TGRs with HOE140 (aTGRH). Erectile function of all rats was evaluated by cavernous nerve electrostimulation method and measured by the ratio of intracavernous pressure/ mean arterial pressure (ICP/MAP) in rats. Expression levels of cAMP and cGMP were assessed, and related signaling pathways were detected by western blot, immunohistochemistry and RT-PCR. Our experiment results showed erectile function of the aWTR group and aTGRH group was lower compared with those of other two groups. Also, expression levels of cAMP and cGMP were significantly lower than those of other two groups. Moreover, expressions of related signaling pathways including DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP were also downregulated in the corpus cavernosum of rats in aWTR group. Our finding revealed hKLK1 played a protective role in age-related ED. The DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP pathways that were linked to the mechanism hKLK1 could increase the levels of cGMP and cAMP, which might provide novel therapy targets for age-related ED.

Unleashing the therapeutic potential of human kallikrein-related serine proteases.

Tissue kallikreins are a family of fifteen secreted serine proteases encoded by the largest protease gene cluster in the human genome. In the past decade, substantial progress has been made in characterizing the natural substrates, endogenous inhibitors and in vivo functions of kallikreins, and studies have delineated important pathophysiological roles for these proteases in a variety of tissues. Thus, kallikreins are now considered attractive targets for the development of novel therapeutics for airway, cardiovascular, tooth, brain, skin and neoplastic diseases. In this Review, we discuss recent advances in our understanding of the physiological functions and pathological implications of kallikrein proteases, and highlight progress in the identification of kallikrein inhibitors, which together are bringing us closer to therapeutically targeting kallikreins in selected disease settings.

Tissue kallikrein protects cortical neurons against hypoxia/reoxygenation injury via the ERK1/2 pathway.

Systemic or local delivery of human tissue kallikrein gene (hTK) has been shown to be an effective strategy to alleviate cerebral ischemia/reperfusion (I/R) injury, and tissue kallikrein (TK) administration can suppress glutamate- or acidosis-mediated neurotoxicity in vitro. In the present study, the role of TK in hypoxia/reoxygenation (H/R) induced neuronal cell death was investigated. We found that TK administration could remarkably alleviate H/R-induced neuronal injury by reduction of LDH release and promotion of neuron viability. The protective effects of TK could be counteracted by bradykinin B2 receptor (B2R) antagonist HOE140, which could suppress up-regulation of TK on the ERK signal pathway under H/R condition. These results indicate that TK plays an important role in preventing neurons from H/R damage at least partially through the TK-B2R-ERK1/2 pathway.

Role of human tissue kallikrein in gastrointestinal stromal tumour invasion.

BACKGROUND: Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST). METHODS: Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells. RESULTS: Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension. CONCLUSIONS: Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST.

Angiogenesis in cervical cancer is mediated by HeLa metabolites through endothelial cell tissue kallikrein.

High vascularity correlates with poor clinical outcome in cancer of the uterine cervix. We investigated whether human cervical cancer cell (HeLa) metabolites influenced endothelial cell proliferation through the serine protease, tissue kallikrein. The angiogenic potential of tissue kallikrein is proposed due to its proteolytic, mitogenic and invasive properties. Under pre-defined conditions, we examined the regulation of tissue kallikrein simultaneously in both endothelial and HeLa cells using immunochemistry, ELISA, cell proliferation assays and in situ RT-PCR. In an endothelial-cervical carcinoma conditioned-medium model, HeLa metabolites caused a dramatic decrease in endothelial cellular tissue kallikrein and a concomitant proliferation of endothelial cells. ELISA on the conditioned media showed a dose-dependent increase of tissue kallikrein, while in situ RT-PCR demonstrated no change in tissue kallikrein mRNA in both endothelial and HeLa cells when challenged with each other's metabolites. This demonstration of the ability of cervical cancer to simultaneously manipulate both tissue kallikrein processing within endothelial cells and angiogenesis is novel. Should this occur in vivo, the tissue kallikrein released from the endothelial cells into the microenvironment may simultaneously degrade the matrix and elicit a mitogenic effect by promoting angiogenesis. Pre-treatment with TK inhibitors and/or anti-angiogenic therapies may prove to benefit future cervical cancer patients.

Kallikrein protects against microalbuminuria in experimental type I diabetes.

Tissue kallikrein is the main kinin-forming enzyme in mammals, and differences in kinin levels are thought to be a contributing factor to diabetic nephropathy. Here, we determined the role of the kallikrein-kinin system in the pathogenesis of streptozotocin-induced diabetic nephropathy in wild-type and tissue kallikrein-knockout mice. All diabetic mice developed similar hyperglycemia, but the knockout mice had a significant two-fold increase in albuminuria compared to the wild-type mice before and after blood pressure elevation. Ezrin mRNA, a podocyte protein potentially implicated in albuminuria, was downregulated in the kidney of knockout mice. One month after induction of diabetes, the mRNAs of kininogen, tissue kallikrein, kinin B1, and B2 receptors were all increased up to two-fold in the kidney in both genotypes. Diabetes caused a 50% decrease in renal angiotensin-converting enzyme expression and a 20-fold increase in kidney injury molecule-1 reflecting tubular dysfunction, but there was no genotype difference. Our study found an early activation of the kallikrein-kinin system in the kidney and that this has a protective role against the development of diabetic nephropathy. The effect of tissue kallikrein deficiency on microalbuminuria in diabetic mice is similar to the effect of genetically high angiotensin-converting enzyme levels, suggesting that both observations, in part, result from a deficiency in kinins.

Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases.

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.

Critical role of tissue kallikrein in vessel formation and maturation: implications for therapeutic revascularization.

OBJECTIVE: Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity. METHODS AND RESULTS: Here, we demonstrate that tissue kallikrein knockout mice (KLK1-/-) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9-/- mice, whereas its proarteriogenic action was preserved in ApoE-/- mice, an atherosclerotic model of impaired angiogenesis. CONCLUSIONS: These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.

Active Ca(2+) reabsorption in the connecting tubule.

The kidney plays a crucial role in the maintenance of the body calcium (Ca(2+)) balance. Ca(2+) is an essential ion in all organisms and participates in a large variety of structural and functional processes. In mammals, active tubular Ca(2+) reabsorption is restricted to the distal part of the nephron, i.e., the late distal convoluted (DCT2) and the connecting tubules (CNT), where approximately 10-15% of the total Ca(2+) is reabsorbed. This active transcellular transport is hallmarked by the transient receptor potential vanilloid 5 (TRPV5) epithelial Ca(2+) channel, regulated by an array of events, and mediated by hormones, including 1,25-dihydroxyvitamin D(3), parathyroid hormone, and estrogen. Novel molecular mechanisms have been identified, such as the direct regulatory effects of klotho and tissue kallikrein on the abundance of TRPV5 at the apical membrane. The newly discovered mechanisms could provide potential pharmacological targets in the therapy of renal Ca(2+) wasting. This review discusses the three basic molecular steps of active Ca(2+) reabsorption in the DCT/CNT segments of the nephron, including apical entry, cytoplasmic transport, and basolateral extrusion of Ca(2+). In addition, an overview of the recently identified mechanisms governing this active Ca(2+) transport through the DCT2/CNT epithelial cells will be presented.

Role of tissue kallikrein-related peptidases in cervical mucus remodeling and host defense.

Human tissue kallikrein-related peptidases (KLKs) are 15 hormonally regulated genes on chromosome 19q13.4 encoding secreted serine proteases. Many KLKs are expressed throughout the female reproductive system and found in cervico-vaginal fluid (CVF). Immunohistochemistry was performed to determine KLK localization in the female reproductive system (fallopian tube, endometrium, cervix and vagina tissues). KLK levels were measured in CVF and saliva over the menstrual cycle to study whether KLKs are regulated by hormonal changes during the cycle. In vitro cleavage analysis was performed to establish whether KLKs may play a role in vaginal epithelial desquamation, mucus remodeling or processing of antimicrobial proteins. KLKs were localized in the glandular epithelium of the fallopian tubes and endometrium, the cervical mucus-secreting epithelium and vaginal stratified squamous epithelium. KLK levels peaked in CVF and saliva after ovulation. In vitro cleavage analysis confirmed KLKs 5 and 12 as capable of digesting desmoglein and desmocollin adhesion proteins and cervical mucin proteins 4 and 5B. KLK5 can digest defensin-1alpha, suggesting it may aid in cervico-vaginal host defense. We provide evidence of potential physiological roles for KLKs in cervico-vaginal physiology: in desquamation of vaginal epithelial cells, remodeling of cervical mucus and processing of antimicrobial proteins.

Tissue kallikrein deficiency aggravates cardiac remodelling and decreases survival after myocardial infarction in mice.

BACKGROUND: Tissue kallikrein (TK) is a major kinin-releasing enzyme present in arteries. TK is involved in cardioprotection in the setting of acute myocardial ischaemia but its role in post-ischaemic heart failure (HF), a major cause of delayed mortality after myocardial infarction (MI), is unknown. AIM: To determine whether TK deficiency in the mouse influences survival and cardiac remodelling after MI. METHODS: MI was induced in 10 week-old male TK-deficient mice and wild-type littermates. Survival was assessed up to 14 months. Cardiac morphological and functional parameters were serially measured by echocardiography. In another experiment, myocardial capillary density and NOS content were evaluated at 3 months. RESULTS: Infarct size was similar in both genotypes. MI resulted in severe cardiac dysfunction. Up to 12 months after MI, TK(-/-) mice displayed an increased mortality rate (P<0.05, relative risk of death=3.41) and aggravation of left ventricular hypertrophy and dilatation by comparison with TK(+/+) (+18% and +27% respectively, both P<0.05). NOS1 and NOS3 were abnormally regulated in the heart of TK(-/-) mice after MI. CONCLUSIONS: TK exerts a protective role in HF in mice. Coronary effects are probably involved. As partial genetic deficiency in TK activity occurs in humans, TK-deficient subjects may be at increased risk of mortality in HF.

Partial human genetic deficiency in tissue kallikrein activity and renal calcium handling.

A loss-of-function polymorphism of the human tissue kallikrein (TK) gene (R53H) induces a major decrease in enzyme activity. Inactivation of the TK gene in mice causes a defect in tubular calcium (Ca) reabsorption. Therefore, this study investigated the Ca phenotype of carriers of the 53H allele. In a crossover study, 30 R53R homozygous and 10 R53H heterozygous young white male individuals were randomly assigned to two 7-d low-Ca diets (10 mmol/d) associated with either a low-sodium (Na)/high-potassium (K) diet or a high-Na/low-K diet to modulate TK synthesis. On the seventh day of each diet, the participants were studied before and during a 2-h infusion of furosemide that functionally excludes the thick ascending limb and increases Ca delivery to distal tubular segments. Urinary kallikrein activity was 50 to 60% lower in R53H participants than in R53R participants. Adaptation of urinary Ca excretion to the contrasted Na/K diets was unaffected in R53H participants. By contrast, R53H participants after furosemide infusion had significantly lower serum ionized Ca concentrations than did R53R participants (P < 0.0001) and tendency toward nonsignificantly higher urinary Ca excretions than did R53R participants (P = 0.14). These effects were more marked under low-Na/high-K diet. Despite nonsignificant differences in urinary Ca excretions between the two groups, these results suggest in R53H individuals an increase in Ca reabsorption in the thick ascending limb under baseline conditions that counteracts a distal tubular defect that is revealed by furosemide infusion. In humans as in mice, TK thus may act as an intrarenal modulator of Ca reabsorption.

Regulation of the epithelial calcium channel TRPV5 by extracellular factors.

PURPOSE OF REVIEW: Recent studies have greatly increased our knowledge concerning the regulation of renal calcium handling. This review focuses on newly identified calciotropic factors present in the pro-urine and the mechanisms by which they control the transient receptor potential channel vanilloid subtype 5 (TRPV5) which forms the gatekeeper of active renal calcium reabsorption. RECENT FINDINGS: The antiaging hormone klotho regulates TRPV5 activity via a novel mechanism modifying its glycosylation status, thereby entrapping the channel at the cell surface. Functional characterization of tissue kallikrein knockout mice revealed that these animals exhibit a pronounced hypercalciuria, comparable to the calcium leak observed in TRPV5 knockout mice. Recently, it has been demonstrated that tissue kallikrein stimulates active calcium reabsorption via the bradykinin receptor type 2 pathway involving protein kinase C-dependent activation of TRPV5. Finally, the extracellular pH appears to act as a dynamic switch controlling cell surface expression of TRPV5. SUMMARY: Unraveling the molecular mechanisms of TRPV5 channel regulation by the antiaging hormone klotho, tissue kallikrein and extracellular pH demonstrated the existence of novel regulatory mechanisms of active calcium reabsorption acting from the tubular lumen.

TSG-6 potentiates the antitissue kallikrein activity of inter-alpha-inhibitor through bikunin release.

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-alpha-inhibitor and pre-alpha-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6.HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin.HC complexes (byproducts of TSG-6.HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-alpha and IL-1beta, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-alpha-inhibitor) were also induced by TNF-alpha in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

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.

The kallikrein world: an update on the human tissue kallikreins.

Human tissue kallikreins (hKs) are attracting increased attention owing to their association with various forms of cancer and other diseases. Human tissue kallikrein genes represent the largest contiguous group of proteases within the human genome. There are many areas of kallikrein research that need to be further explored, including their tissue expression patterns, their regulation, identification of specific substrates, their participation in proteolytic cascades, and their clinical applicability as cancer biomarkers and therapeutic targets. In this review, we briefly describe the current status of kallikrein research and identify future avenues that will enhance our understanding of their function and involvement in human diseases.

Proteinase-mediated cell signalling: targeting proteinase-activated receptors (PARs) by kallikreins and more.

Serine proteinases, like trypsin, can play a hormone-like role by triggering signal transduction pathways in target cells. In many respects these hormone-like actions of proteinases can now be understood in terms of the pharmacodynamics of the G protein-coupled 'receptor' responsible for the cellular actions of thrombin (proteinase-activated receptor-1, or PAR1). PAR1, like the other three members of this receptor family (PAR2, PAR3 and PAR4), has a unique mechanism of activation involving the proteolytic unmasking of an N-terminally tethered sequence that can activate the receptor. The selective activation of each PAR by short synthetic peptides representing these sequences has demonstrated that PAR1, PAR2 and PAR4 play important roles in regulating physiological responses ranging from vasoregulation and cell growth to inflammation and nociception. We hypothesise that the tissue kallikreins may regulate signal transduction via the PARs. Although PARs can account for many of their biological actions, kallikreins may also cause effects by mechanisms not involving the PARs. For instance, trypsin activates the insulin receptor and thrombin can act via a mechanism involving its non-catalytic domains. Based on the data we summarise, we propose that the kallikreins, like thrombin and trypsin, must now be considered as important 'hormonal' regulators of tissue function.

Gum mastic inhibits the expression and function of the androgen receptor in prostate cancer cells.

Accumulating evidence suggests that the androgen receptor (AR) may play an important role in the development and progression of prostate cancer. To find new, useful compounds that effectively may attenuate the function of AR in prostate cancer cells, the authors investigated the effect of gum mastic, a natural resin, on AR activity. An androgen-responsive prostate cancer cell line LNCaP was used as a model for this study. Gene transfer, reverse transcriptase-polymerase chain reaction analysis, electrophoretic mobility shift assay, and Western blot analysis were used to test the effect of gum mastic on the expression and function of the AR. To demonstrate the inhibitory effect of gum mastic on the function of the AR, the expression of androgen-regulated genes, including prostate-specific antigen (PSA), human kallikrein 2 (hK2), and NKX3.1 were measured. In addition, transient transfection assays with the PSA promoter and the AR promoter also were used to test the effects of mastic. The results showed that gum mastic inhibited the expression of the AR at the transcriptional level, resulting in the down-regulation of both AR messenger RNA and protein levels. Therefore, the function of the AR was inhibited, as reflected by the reduced expression of NKX3.1 and PSA and by androgen-stimulated growth. Because gum mastic exhibited a strong in vitro potency to attenuate the expression and function of the AR, further investigation will be required to determine whether this naturally occurring substance has in vivo potency to inhibit prostate cancer development.