Kallikreins Research Articles

Validation of a Novel Biomarker Panel for the Detection of Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy. Our integrated -omics approach to ovarian cancer biomarker discovery has identified kallikrein 6 (KLK6) and folate-receptor 1 (FOLR1) as promising candidates but these markers require further validation. KLK6, FOLR1, CA125, and HE4 were investigated in three independent serum cohorts with a total of 20 healthy controls, 150 benign controls, and 216 ovarian cancer patients. The serum biomarker levels were determined by ELISA or automated immunoassay. All biomarkers demonstrated elevations in the sera of ovarian cancer patients compared with controls (P < 0.01). Overall, CA125 and HE4 displayed the strongest ability (AUC 0.80 and 0.82, respectively) to identify ovarian cancer patients and the addition of HE4 to CA125 improved the sensitivity from 36% to 67% at a set specificity of 95%. In addition, the combination of HE4 and FOLR1 was a strong predictor of ovarian cancer diagnosis, displaying comparable sensitivity (65%) to the best-performing CA125-based models (67%) at a set specificity of 95%. The markers identified through our integrated -omics approach performed similarly to the clinically approved markers CA125 and HE4. Furthermore, HE4 represents a powerful diagnostic marker for ovarian cancer and should be used more routinely in a clinical setting. The implications of our study are 2-fold: (i) we have demonstrated the strengths of HE4 alone and in combination with CA125, lending credence to increasing its usage in the clinic; and (ii) we have demonstrated the clinical utility of our integrated -omics approach to identifying novel serum markers with comparable performance to clinical markers. Cancer Epidemiol Biomarkers Prev; 25(9); 1333-40. ©2016 AACR.

Kallikrein-related peptidases in pancreatic cancer: expression and significance

Pancreatic cancer is a one of the most malignant digestive cancer. Because the lack of effective methods for early diagnosis, most patients have been ineligible for surgical resection when diagnosed. Kallikrein family is a group of serine proteases, because of its ability to decompose the extracellular matrix proteins, it may be closely related to the invasion and metastasis of various cancers. And some members of kallikrein family may become cancer diagnostic biomarkers. This paper reviews all the recent articles about kallikrein family study in pancreatic cancer. Key words: Tissue kallikreins; Pancreatic neoplasms; Biological marker; Drug resistance

An integrated cell line-based discovery strategy identified follistatin and kallikrein 6 as serum biomarker candidates of breast carcinoma

Secreted proteins constitute a relevant source of putative cancer biomarkers. Here, we compared the secretome of a series of four genetically-related breast cancer cell lines as a model of aggressiveness using quantitative mass spectrometry. 537 proteins (59.5% of the total identified proteins) predicted to be released or shed from cells were identified. Using a scoring system based on i) iTRAQ value, ii) breast cancer tissue mRNA expression levels, and iii) immunohistochemical staining (public database), a short list of 10 candidate proteins was selected. Using specific ELISA assays, the expression level of the top five proteins was measured in a verification set of 56 patients. The four significantly differentially expressed proteins were then validated in a second independent set of 353 patients. Finally, follistatin (FST) and kallikrein 6 (KLK6) in serum were significantly higher (p-value < 0.0001) in invasive breast cancer patients compared with non-cancerous controls. Using specific cut-off values, FST distinguished breast cancer samples from healthy controls with a sensitivity of 65% and an accuracy of 68%, whereas KLK6 achieved a sensitivity of 55% and an accuracy of 61%. Therefore, we concluded that FST and KLK6 may have significance in breast cancer detection. Biological significanceDiscovery of new serum biomarkers that exhibit increased sensitivity and specificity compared to current biomarkers appears to be an essential field of research in cancer. Most biological markers show insufficient diagnostic sensitivity for early breast cancer detection and, for the majority of them, their concentrations are elevated only in metastatic forms of the disease. It is therefore essential to identify clinically reliable biomarkers and develop effective approaches for cancer diagnosis. One promising approach in this field is the study of secreted proteins through proteomic analysis of in vitro progression breast cancer models. Here we have shown that FST and KLK6 are elevated in breast cancer patient serum compared to healthy controls. We expect that our discovery strategy will help to identify cancer-specific and body-fluid-accessible biomarkers.

Synthesis, biological evaluation and molecular modeling of pseudo-peptides based statine as inhibitors for human tissue kallikrein 5

Human kallikrein 5 (KLK5) is a potential target for the treatment of skin inflammation and cancer. A new series of statine based peptidomimetic compounds were designed and synthesized through simple and efficient reactions. Some KLK5 inhibitors (2a–c compounds) were identified with nanomolar affinity showing Ki values of 0.12-0.13 μM. Our molecular modeling studies suggest that the inhibitors binding at the KLK5 through H-bond interactions with key residues (mainly His108, Gln242, Gly243, Ser245, and Ser260), disrupting the correlated motions mainly among the Ile67-Tyr127, Glu128-Val187, and Gly237-Ser293 subdomains, which seems to be crucial for KLK5 activity. Therefore, we believe that these findings will significantly facilitate our understanding of the conformational dynamics in the course of KLK5 inhibition and, consequently, the development of more potent molecules as alternative for cancer treatment.

Synthesis, antiproliferative activities, and computational evaluation of novel isocoumarin and 3,4-dihydroisocoumarin derivatives

A series of novel isocoumarin derivatives were synthesized using Castro–Stephens cross-coupling. Moreover, novel 3,4-dihydroisocoumarin derivatives were obtained by catalytic hydrogenation of the corresponding isocoumarin precursors. The antiproliferative activity of all compounds was evaluated in vitro in different tumor cells. Furthermore, docking calculations were performed for the kallikrein 5 (KLK5) active site to predict the possible mechanism of action of this series of compounds. Theoretical findings indicate that the 3,4-dihydroisocoumarin derivative 10a forms hydrogen bonds with Ser190 and Gln192 residues of KLK5. This derivative was the most active compound in the series with potent antiproliferative activity and high selectivity index (SI > 378.79) against breast cancer cells (MCF-7, GI50 = 0.66 μg mL−1). This compound represents a promising matrix for developing new antiproliferative agents.

Effects of Qingpeng ointment on skin barrier function in volunteers with xerosis and atopic dermatitis-like mouse models

Objective To estimate effects of Qingpeng ointment on skin barrier function, and to explore their potential mechanisms. Methods A total of 12 female volunteers with xerosis on the extensor surfaces of legs were enrolled into this study. All the volunteers were topically treated with Qingpeng ointment on the right leg (Qingpeng side) and its vehicle on the left leg (vehicle side) twice daily for 7 consecutive days. Skin barrier function-associated indexes including transepidermal water loss (TEWL) and the water content of the stratum corneum were measured before the treatment, after 3-and 7-day treatment separately. Thirty-six BALB/c female mice were randomly and equally divided into 6 groups: a blank control group receiving no induction or treatment, a model group induced by 2, 4-dinitrofluorobenzene (DNFB) on the back for 5 consecutive weeks, 3 Qingpeng groups and a vehicle group induced by DNFB on the back for 5 consecutive weeks followed by topical treatment with different concentrations (50%, 75%, 100%) of Qingpeng ointment or its vehicle twice a day for 2 consecutive weeks. At the end of treatment, skin appearance of mice was observed with naked eyes, skinfold thickness and TEWL were measured. Then, all the mice were sacrificed, and skin tissue specimens were resected from the back of mice followed by histopathologic examination and immunohistochemical staining for the detection of filaggrin (FLG), involucrin (IVL) and kallikrein 7 (KLK7) expressions in the epidermis. Results After start of treatment, both the Qingpeng side and vehicle side showed significantly decreased TEWL on day 3 (t = 2.651, 2.996, respectively, both P 0.05). In addition, there were no marked differences in mouse skin appearance, skinfold thickness or histopathologic manifestations between the vehicle groupand model group, while the manifestations of dermatitis were attenuated to different extents in the three Qingpeng groups compared with the model group. Both TEWL and KLK7 expression levels were significantly decreased in the vehicle group and three Qingpeng groups compared with the model group (all P 0.05). Conclusion The vehicle of Qingpeng ointment can improve skin barrier function. Key words: Dermatitis, atopic; Models, animal; Mice; Skin; Volunteer; Qingpeng ointment; Skin barrier

The role of human kallikrein 6, clusterin and adiponectin as potential blood biomarkers of dementia

ObjectivesProgressive degenerative syndromes which affect brain, altering memory, behavior, cognition and emotion, are commonly defined as dementia. It was suggested that serum human kallikrein 6 (KLK6), clusterin (CLU) and adiponectin (ADPN) in combination with inflammation markers, neuroimaging and neuropsychological testing could assist in discriminating dementia patients from control individuals. Our aim was therefore to compare serum concentrations of KLK6, CLU and ADPN and inflammatory marker, interleukin-6 (IL-6), in patients suffering from Alzheimer's disease (AD), patients with vascular dementia (VAD), cognitively healthy participants (CHP) and those with mild cognitive impairment (MCI). Design and methodsSerum samples were collected from AD, VAD and MCI patients admitted to the University Department of Neurology (Zagreb, Croatia) for regular follow-up. All patients underwent standard neuroimaging procedures including brain CT, neurosonological assessment with intima-media thickness (IMT) and breath holding index (BHI) calculations. Cognitive abilities were tested using standard Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Concentrations of KLK6, CLU, ADPN and IL-6 were determined in all serum samples. ResultsWe have recruited a total of 235 participants, divided in 4 groups: AD (N=70), VAD (N=67), MCI (N=48) and CHP (N=50). Serum concentrations of KLK6 (P=0.137), CLU (P=0.178) and ADPN (P=0.268) did not differ between AD, VAD, MCI and cognitively healthy control group of participants, whereas IL-6 was significantly higher in VAD patients than in AD, MCI and CHP individuals (P=0.014). There was no association between investigated biomarkers and clinical patient parameters. ConclusionsSerum concentrations of KLK6, CLU and ADPN do not differ between AD, VAD and controls with and without mild cognitive impairment. Higher IL-6 levels in VAD group point to the inflammatory component in the development of vascular dementia. Investigated biomarkers are not associated with neuroimaging findings and neuropsychological patient data.

A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

The Co-Expression of Kallikrein 5 and Kallikrein 7 Associates with Poor Survival in Non-HPV Oral Squamous-Cell Carcinoma

Objective: Oral squamous-cell carcinoma (OSCC) still has a poor prognosis. Lymph node metastasis (LNM) is a major determinant of treatment decisions and prognosis. Serine protease inhibitor Kazal-type 5 (SPINK5) is the inhibitor of kallikrein 5 (KLK5) and KLK7. SPINK5, KLK5 and KLK7 are three of the genes of a recently validated LNM-predicting gene expression profile in OSCC. This study evaluates their clinicopathological role and value as biomarkers in OSCC. Methods: Eighty-three patients with primary OSCC, treated surgically between 1996 and 2000, were included. Gene expression data were acquired from a previously reported study. Human papillomavirus (HPV) status was determined by an algorithm for HPV-16. Protein expression for KLK5, KLK7 and SPINK5 was semi-quantitatively determined in all 83 tumours by immunohistochemistry. All expression data were correlated with clinicopathological parameters. Results: Concurrent loss of KLK5 and KLK7 correlates with worse disease-specific and overall survival (DSS and OS). Multivariate analysis proved that co-expression is an independent prognostic factor for DSS (p = 0.029) and OS (p = 0.001). Conclusion: This report demonstrates that concurrent loss of KLK5 and KLK7 associates with a poor clinical outcome in OSCC and could therefore serve as prognostic marker in this disease.

SPINK5 expression in keratinocytes

The balance between serine proteases, kallikreins (KLKs), and their inhibitor, Kazal type‐5 (SPINK5), is important to maintain steady desquamation. To explore regulation of SPINK5 expression in skin, its promoters (nucleotide ‐676/‐532 and ‐318/‐146 from the major transcription start site) were subjected to luciferase assay because these two sides displayed high luciferase intensity in NHEK human keratinocyte. Electrophoretic mobility shift assay (EMSA) appeared specific nuclear protein binding to these two sides with almost the same size shift. However, in DNA foot printing, ‐676/‐532 and ‐318/‐146 had no specific sequence protection. Thus, at this moment, we could not confirm the major regulator of SPNK5 gene expression in skin. We, at least, acknowledge that the exploration of the relationship between KLKs and SPINK5 will give crucial information related to skin differentiation/proliferation. (partially supported by Grant‐in‐Aid C‐264607944).

Cigarette smoke mediates epigenetic repression of miR-217 during esophageal adenocarcinogenesis.

Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative RT-PCR (qRT-PCR) techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/ primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA cross-link immunoprecipitation, qRT-PCR and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 (NF-1) within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression, and down-regulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and up-regulation of KLK7 in IEE and EACC. Over-expression of miR-217 significantly decreased, whereas over-expression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via up-regulation of KLK7, and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.

Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.

Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population.

Abstract 2009: Role of kallikrein 6 secretion in metastatic colon cancer

Abstract Growing evidence indicates that serine proteases kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikrein 6 (KLK6) is a member of the family of 15 highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Overexpression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and cancer. Activating K-RAS mutations are common in colon cancer progression and contribute to many cancerous phenotypes including increased proliferation, evasion of apoptosis, and increased migration and invasion. We have shown that acquisition of K-RAS mutations in colon cancer cells leads to overexpression of membrane protein caveolin 1 and results in increase of KLK6 expression and secretion. The aim of this study was to elucidate the role of KLK6 expression in metastatic CRC in relation to the status of K-RAS oncogene. Analysis of KLK6 expression in CRC patients by immunohistochemistry shows the statistically significant increase in the staining score for KLK6 protein in the microscopically normal tissue 10 cm away from tumors, which carry mutant K-RAS (G12A mutation) (p=0.05 for crypts staining and p=0.0445 for the surface area). These data suggest a specific role of KLK6 in the invasive phenotype associated with the presence of K-RAS mutation. To investigate the significance of KLK6 expression in metastatic CRC, we developed HCT116 human colon cancer isogenic cell lines (K-RAS G13D mutation) with the genetic knock down of KLK6 expression (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with the minimal effect on the cell growth and viability in cell culture. SCID mice injected with shKLK6 cells exhibited a statistically significant increase in the survival rates (p=0.005), decrease in the incidence of distant metastases and the shift in the location of the metastatic foci closer to the cells injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6 clone compared to HCT116 control clone (p&amp;lt;0.04). A comprehensive array-based profiling and paired analysis of mRNA and miRNA expression in HCT116 control and KLK6 knockout cells revealed a group of specific miRNAs- mRNA target interactions involved in KLK6-mediated metastasis formation during CRC. The data reported here underscore the significance of KLK6 overexpression for distant metastasis formation in CRC and present the opportunity for therapeutic interventions. Citation Format: Bethany A. Skovan, Ritu Pandey, Raymond B. Nagle, Haiyan Cui, Julie A. Buckmeier, Robert S. Krouse, Patricia A. Thompson, Natalia A. Ignatenko. Role of kallikrein 6 secretion in metastatic colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2014-2009

Expression of kallikrein-related peptidase 7 is decreased in prostate cancer

Recent evidence suggests that the human kallikrein 7 (KLK7) is differentially regulated in a variety of tumors. The aim of this study was to determine the expression of kallikrein-related peptidase 7 and KLK7 in our large collection of prostate samples. Between August 2000 and December 2012, 116 patients with histologically confirmed prostate cancer (PCa) and 92 with benign prostate hyperplasia (BPH) were recruited into the study. Using immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot, kallikrein-related peptidase 7 expression in BPH and PCa tissues was determined at the mRNA and protein levels. The relationships between kallikrein-related peptidase 7 mRNA expression and clinicopathological features were analyzed. A total of 64 of 92 (69.57%) benign cases showed positive staining for KLK7 and 23 of 116 (19.83%) malignant cases showed positive, the difference of KLK7 expression between PCa and BPH was statistically significant (P < 0.001). The expression level of kallikrein-related peptidase 7 mRNA was significantly decreased in PCa tissues compared with that in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 mRNA exhibited different expression patterns in terms of localization depending on pathological category of PCa. Similarly, our western immunoblot analyses demonstrated that the protein expression levels of KLK7 was lower in PCa than in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 and KLK7 expression are down-regulated in PCa and lower expression of kallikrein-related peptidase 7 closely correlates with higher Gleason score and higher prostate-specific antigen level.

Expression of Human Tissue Kallikreins (KLKs) in Polymorphous Low Grade Adenocarcinoma (PLGA)

Expression of Human Tissue Kallikreins (KLKs) in Polymorphous Low Grade Adenocarcinoma (PLGA)

LEKTI, A Physiological Inhibitor of Multiple Serine Proteinases, Blocks Migration and Invasion of Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive expression in normal oral mucosa and lost or down regulated expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 lines. We then determined the consequences of LEKTI re-expression on migration and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 clones show striking morphological changes and enhanced adhesionon type I, III, IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent manner. Microarray analysis identified 186 genes which are differentially regulated in both OSC19 LEKTI clones. MMP-14, KLK5, and ADAM8 are down regulated while MMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT-PCR and Western blot results confirmed microarray results for MMP-14 and MMP-3in OSC19 LEKTI clones. In addition we discover that MMP-9 protein expression and pro-MMP-9 activity are severely reduced in LEKTI expressing clones as shown by WB and zymogram. Together, this work provides mechanistic insights into how loss of LEKTI protein expression promotes an invasive phenotype in HNSCC tumors.

Regulation of serine protease inhibitor Kazal type-5 (SPINK5) gene expression in the keratinocytes.

ObjectivesSerine protease inhibitor Kazal type-5 (SPINK5) plays a crucial role in deciding the timing of desquamation of the skin. Its gene expression is limited at the very surface of the stratum granulosum (SG), whereas expression of kallikreins (KLKs) encoding proteases is usually found throughout the stratum spinosum and SG.MethodsTo explore the difference in expression regulation of these proteases/inhibitors, the function of SPINK5 promoter was examined using luciferase assay.ResultsLuciferase assay targeting the SPINK5 promoters (nucleotide −676/−532 and −318/−146 from the major transcription start site) showed high intensity in NHEK human keratinocyte. These two sites had neither common cis-elements nor GATA3 element but electrophoretic mobility shift assay showed similar retardation bands. Moreover, DNA footprinting did not display specific protected bands. Thus, we could not identify cis-element(s) that controlled these elements. Differentiation induced by high Ca2+ medium failed to alter their luciferase activities. Transfection of GATA3 expressing vector significantly but slightly increased them and that of vector expressing its dominant negative form decreased.ConclusionsAlthough GATA3 is reportedly important for inhibition of proliferation and induction of differentiation of keratinocytes, its effect on SPINK5 expression was indirect and GATA3 alone was insufficient for final differentiation of keratinocytes where full SPINK5 expression was observed.

Current drug therapies for rosacea: a chronic vascular and inflammatory skin disease.

Rosacea is a chronic skin disorder that presents with abnormal vascular and inflammatory conditions. Clinical manifestations include flushing, facial erythema, inflammatory papules and pustules, telangiectasias, edema, and watery or irritated eyes. To discuss the evolving pathophysiology of rosacea, factors involved in promoting the chronic vascular and inflammatory abnormalities seen in rosacea, and the available drug therapies for the condition. Chronic inflammation and vascular changes are believed to be underlying factors in the pathophysiology of rosacea. Aberrant cathelicidin expression, elevated kallikrein 5 (KLK5) proteolytic activity, and altered toll-like receptor 2 (TLR2) expression have been reported in rosacea skin leading to the production of proinflammatory cytokines. Until recently, drug therapies only targeted the inflammatory lesions (papules and pustules) and transient erythema associated with these inflammatory lesions of rosacea. Brimonidine tartrate gel 0.5% was recently approved for the treatment of persistent (nontransient) facial erythema of rosacea, acting primarily on the cutaneous vascular component of the disease. Rosacea is a chronic vascular and inflammatory skin disease. Understanding the role of factors that trigger the onset of rosacea symptoms and exacerbate the condition is crucial in treating this skin disease.

Putative kallikrein substrates and their (patho)biological functions.

Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3-13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates.

Identification of Human Tissue Kallikrein 6 as a Potential Marker of Laryngeal Cancer Based on the Relevant Secretory/Releasing Protein Database

Objective. This study was aimed to create a large-scale laryngeal cancer relevant secretory/releasing protein database and further discover candidate biomarkers. Methods. Primary tissue cultures were established using tumor tissues and matched normal mucosal tissues collected from four laryngeal cancer patients. Serum-free conditioned medium (CM) samples were collected. These samples were then sequentially processed by SDS-PAGE separation, trypsin digestion, and LC-MS/MS analysis. The candidates in the database were validated by ELISA using plasma samples from laryngeal cancer patients, benign patients, and healthy individuals. Results. Combining MS data from the tumor tissues and normal tissues, 982 proteins were identified in total; extracellular proteins and cell surface proteins accounted for 15.0% and 4.3%, respectively. According to stringent criteria, 49 proteins were selected as candidates worthy of further validation. Of these, human tissue kallikrein 6 (KLK6) was verified. The level of KLK6 was significantly increased in the plasma samples from the cancer cohort compared to the benign and healthy cohorts and moreover showed a slight decrease in the postoperative plasma samples in comparison to the preoperative plasma samples. Conclusions. This laryngeal cancer-derived protein database provides a promising repository of candidate blood biomarkers for laryngeal cancer. The diagnostic potential of KLK6 deserves further investigation.