Serine Protease Inhibitor Kazal Type Research Articles

Molecular characterization, expression and functional analysis of two Kazal-type serine protease inhibitors from Venerupis philippinarum

Kazal-type serine protease inhibitors (KSPIs) act as negative regulators in immune signaling pathway by controlling the extent of serine protease (SP) activities. In this study, the full-length cDNA of two KSPIs (designed as VpKSPI-1 and VpKSPI-2) were identified from Venerupis philippinarum by rapid amplification of cDNA ends (RACE) approaches. The open reading frame (ORF) of VpKSPI-1 and VpKSPI-2 was of 552 bp and 402 bp, encoding a polypeptide of 183 and 133 amino acids, respectively. The transcripts of VpKSPI-1 and VpKSPI-2 were ubiquitously expressed in all tissues tested with the highest expression level in hepatopancreas. After Vibrio anguillarum challenge, the relative mRNA expression of VpKSPI-1 and VpKSPI-2 in hepatopancreas was both up-regulated within 96 h. The recombinant VpKSPI-1 (rVpKSPI-1) displayed weak activities towards chymotrypsin, moderate inhibitory activity to trypsin, while rVpKSPI-2 showed significant inhibitory activities against chymotrypsin and trypsin. When the molar ratio of rVpKSPI-2 to chymotrypsin and trypsin reached 1:4 and 1:2, the protease activities could be almost entirely inhibited. All these results suggested that both VpKSPI-1 and VpKSPI-2 perhaps play a vital role in the innate immunity of V. philippinarum.

Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways.

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G2 /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.

High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector Aedes aegypti.

Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 Å resolution crystal structure of AaTI is reported from extremely tightly packed crystals (∼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.

Salivary gland transcripts of the kissing bug, Panstrongylus chinai, a vector of Chagas disease

The saliva of hematophagous arthropods injected during blood feeding contains potent pharmacologically active components to counteract the host hemostatic and inflammatory systems. In the present study, dominant salivary gland transcripts of Panstrongylus chinai, a vector of Chagas disease, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library. This analysis showed that 56.5% of the isolated transcripts coded for putative secreted proteins, of which 73.7% coded for proteins belonging to the lipocalin family. The most abundant transcript of lipocalin family proteins was a homologue of pallidipin 2, an inhibitor of collagen-induced platelet aggregation of Triatoma pallidipennis. In addition, homologues of triafestin, an inhibitor of the kallikrein-kinin system of T. infestans, were identified as the dominant transcript. Other salivary transcripts encoding lipocalin family proteins had homology to triplatin (an inhibitor of platelet aggregation) and others with unknown function. Other than lipocalin family proteins, homologues of a Kazal-type serine protease inhibitor (putative anticoagulant), a hemolysin-like protein (unknown function), inositol polyphosphate 5-related protein (a regulator of membrane phosphoinositide), antigen 5-related protein (unknown function) and apyrase (platelet aggregation inhibitor) were identified.

Abstract 2972: SPINK1, a soluble factor released by the therapy-damaged tumor microenvironment, promotes prostate cancer resistance

Abstract Cancer evolution is driven by not only the genetic and/or epigenetic alterations of cancer cells, but also diverse factors that are derived from the surrounding tumor microenvironment (TME). Upon chemotherapy or radiation, stromal cells in the TME become senescent and develop a senescence-associated secretory phenotype (SASP) that is characterized by secretion of a large number of cytokines, chemokines, growth factors, and proteases. In most cases, the SASP is functionally regulated by the DNA damage secretory program (DDSP) and pathophysiologically responsible in vivo for disease exacerbation. We recently disclosed significant upregulation of the serine protease inhibitor Kazal type I (SPINK1) in primary normal human prostate fibroblasts after exposure to DNA damaging agents. SPINK1 plays critical roles in cancer cell proliferation, survival and motility in multiple human malignancies, but its influence as a soluble TME-associated factor on cancer progression remains unknown. In this study, we performed a series of molecular and cellular studies to define the biological roles of SPINK1 in an activated TME. Promoter analysis indicated that SPINK1 expression in the fibroblasts is regulated by multiple transcriptional factors including the NF-κB complex in response to genotoxic stress. Fibroblast-derived SPINK1 can significantly enhance the aggressiveness of prostate cancer cells including accelerated proliferation, increased migration, elevated invasion and more importantly, enhanced chemoresistance. SPINK1 triggers a typical epithelial-mesenchymal transition (EMT) in cancer cells, a process mediated by EGFR/PI3K/Akt and MAPK/Erk signaling pathways. Consistent with the in vitro data, our in vivo studies suggested that SPINK1 expression in the prostate TME substantially promoted cancer survival and disease progression. Clinical investigation revealed increased expression of SPINK1 in the stroma of multiple organ types including the prostate, lung and breast of cancer patients after chemotherapy, implying the systemic induction of SPINK1 by anticancer agents. Overall, our study demonstrates that SPINK1 is a soluble biomarker of therapeutically damaged TME and represents a potentially exploitable molecular target in human prostate cancer clinics. Note: This abstract was not presented at the meeting. Citation Format: Fei Chen, Da Fu, Eric Lam, Yu Sun. SPINK1, a soluble factor released by the therapy-damaged tumor microenvironment, promotes prostate cancer resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2017-2972

Serine peptidase inhibitor Kazal type 1 (SPINK1) as novel downstream effector of the cadherin-17/β-catenin axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide. Previously, we reported that cadherin-17 (CDH17) and its related CDH17/β-catenin axis may be responsible for inducing HCC in a subset of patients exhibiting CDH17 over-expression. Here we aimed at obtaining a better understanding of the CDH17-related HCC biology and to obtain further indications for the design of targeted therapies in CDH17 over-expressing HCC patients. We found that SPINK1 acts as a downstream effector of the CDH17/β-catenin axis in HCC. In addition, we found that SPINK1 expression exhibited a positive correlation with CDH17 expression in human HCCs and was over-expressed in up to 70% of the tumors. We identified SPINK1 as a downstream effector of the CDH17/β-catenin axis using a spectrum of in vitro assays, including gene expression modulation and inhibitor assays, bioinformatics analyses and luciferase reporter assays. These in vitro results were validated in primary human HCCs, including the observation that alteration in β-catenin expression (a core component of the CDH17/β-catenin axis) in tumors affects SPINK1 serum levels in HCC patients. Similar to CDH17, SPINK1 expression in HCC cells was found to be associated with specific tumor-related properties via activating the c-Raf/MEK/ERK pathway. Our current data substantiate our knowledge on the role of CDH17 in the biology of HCC and suggest that components of the CDH17/β-catenin axis may serve as therapeutic targets in CDH17 over-expressing HCC patients.

Serine protease inhibitor Kazal type 1 (SPINK1) downregulates E‐cadherin and induces EMT of hepatoma cells to promote hepatocellular carcinoma metastasis via the MEK/ERK signaling pathway

To investigate serine protease inhibitor Kazal type 1 (SPINK1) expression and its influence on the prognosis of human hepatocellular carcinoma (HCC) and to explore the underlying molecular mechanisms involved. Altogether 80 patients with HCC who underwent curative resection were followed up for a median of 58.6 months. SPINK1 expression was detected in the primary HCC samples by immunohistochemistry. Its role in tumor invasion and metastasis was evaluated in vitro by gene silencing using a small interfering RNA-mediated approach, recombinant SPINK1 and U0126 (an inhibitor of MEK/ERK). The proteins in the MEK/ERK signaling pathway were detected by Western blot. Patients with high SPINK1 expression showed poor overall survival (P = 0.0001) and recurrence-free survival (P = 0.001) compared with those with low SPINK1 expression. The suppression of SPINK1 resulted in reduced cell migration and invasion. SPINK1 overexpression was significantly associated with increased cell migration and invasion in vitro. Furthermore, SPINK1 promoted cancer cells motility and epithelial-mesenchymal transition (EMT) via the mitogen-activated protein kinase kinase (MAPK) and extracellular regulated kinase (ERK) pathway, resulting in increased vimentin expression and decreased E-cadherin expression. SPINK1 may be an oncogene that induces EMT via the MEK/ERK pathway and is a potential target for HCC therapy.

The association between SPINK1 and clinical outcomes in patients with prostate cancer: a systematic review and meta-analysis.

Evidence of the prognostic role of serine peptidase inhibitor Kazal type 1 (SPINK1) in prostate cancer (PCa) is controversial. The aim of this study was, therefore, to evaluate the association between SPINK1 and clinical outcomes in PCa. Searches were made of PubMed, Medline, Embase, and the China Biology Medicine disc (CBMdisc) up to January 2017. The Newcastle–Ottawa Scale was used to assess the risk of bias of included studies. RevMan software was used to perform meta-analysis, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method was employed for assessing the quality of the evidence. Ten studies with 17,161 patients were included in the analysis. Random-effect models were adopted for all outcomes with significant heterogeneities. In patients treated with radical prostatectomy, SPINK1 was associated with biochemical recurrence (BCR) (hazard ratio [HR] =1.41, 95% confidence interval [CI]: 1.01–1.97; P=0.04), but not PCa-specific mortality (HR =0.93, 95% CI: 0.33–2.57; P=0.88), and overall survival (OS) (HR =0.89, 95% CI: 0.58–1.35; P=0.57). In metastatic PCa, SPINK1 was significantly associated with castration-resistant PCa-free survival (HR =3.87, 95% CI: 1.87–8.00; P=0.0003) and OS (HR =2.59, 95% CI: 1.16–5.78; P=0.02). However, the quality of the evidence was very low for all study outcome measures. In conclusion, although SPINK1 was not a predictor of PCa mortality or OS among patients who underwent radical prostatectomy, it may have prognostic value in metastatic PCa.

The skin barrier function gene SPINK5 is associated withchallenge-proven IgE-mediated food allergy in infants.

A defective skin barrier is hypothesized to be an important route of sensitization to dietary antigens and may lead to food allergy in some children.Missense mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) skin barrier gene have previously been associated with allergic conditions. To determine whether genetic variants in and around SPINK5 are associated with IgE-mediated food allergy. We genotyped 71 "tag" single nucleotide polymorphisms (tag-SNPs) within a region spanning ~263kb including SPINK5 (~61kb) in n=722 (n=367 food-allergic, n=199 food-sensitized-tolerant and n=156 non-food-allergic controls) 12-month-old infants (discovery sample) phenotyped for food allergy with the gold standard oral food challenge. Transepidermal water loss (TEWL) measures were collected at 12months from a subset (n=150) of these individuals. SNPs were tested for association with food allergy using the Cochran-Mantel-Haenszel test adjusting for ancestry strata. Association analyses were replicated in an independent sample group derived from four paediatric cohorts, total n=533 (n=203 food-allergic, n=330 non-food-allergic), mean age 2.5years, with food allergy defined by either clinical history of reactivity, 95% positive predictive value (PPV) or challenge, corrected for ancestry by principal components. SPINK5 variant rs9325071 (A⟶G) was associated with challenge-proven food allergy in the discovery sample (P=.001, OR=2.95, CI=1.49-5.83). This association was further supported by replication (P=.007, OR=1.58, CI=1.13-2.20) and by meta-analysis (P=.0004, OR=1.65). Variant rs9325071 is associated with decreased SPINK5 gene expression in the skin in publicly available genotype-tissue expression data, and we generated preliminary evidence for association of this SNP with elevated TEWL also. We report, for the first time, association between SPINK5 variant rs9325071 and challenge-proven IgE-mediated food allergy.

PRSS1 and SPINK1 Mutations in Alcoholic and Idiopathic Chronic Pancreatitis.

Several recent studies have reported associations between gene mutations and chronic pancreatitis (CP); however, little is known about their association with risk of CP in the Chinese Han population. The aim of this study was to describe mutations in the cationic trypsinogen (PRSS1) and serine protease inhibitor Kazal type 1 (SPINK1) genes in patients with alcoholic chronic pancreatitis (ACP) and idiopathic chronic pancreatitis (ICP) and to investigate their influence on the clinical course of the disease. One hundred patients (24 with ACP, 76 with ICP) and 100 healthy volunteers (control group) were enrolled in the study. PRSS1 (R122H) mutations were detected in one (1.3%) patient with ICP and SPINK1 (N34S) mutations were present in one (4.1%) patient with ACP. PRSS1 and SPINK1 mutations were not detected in the control populations. There were no statistically significant differences between the CP patients and the control group. Those preliminary data suggest low prevalence of SPINK1 and PRSS1 mutations in the Chinese population, generally, as well as in CP patients, indicating that these mutations do not contribute to the development of CP.

SPINK6 Promotes Metastasis of Nasopharyngeal Carcinoma via Binding and Activation of Epithelial Growth Factor Receptor.

Nasopharyngeal carcinoma has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. The underlying molecular mechanisms of nasopharyngeal carcinoma metastasis are not fully understood. Here, we report the identification of serine protease inhibitor Kazal-type 6 (SPINK6) as a functional regulator of nasopharyngeal carcinoma metastasis via EGFR signaling. SPINK6 mRNA was upregulated in tumor and highly metastatic nasopharyngeal carcinoma cells. Immunohistochemical staining of 534 nasopharyngeal carcinomas revealed elevated SPINK6 expression as an independent unfavorable prognostic factor for overall, disease-free, and distant metastasis-free survival. Ectopic SPINK6 expression promoted in vitro migration and invasion as well as in vivo lymph node metastasis and liver metastasis of nasopharyngeal carcinoma cells, whereas silencing SPINK6 exhibited opposing effects. SPINK6 enhanced epithelial-mesenchymal transition by activating EGFR and the downstream AKT pathway. Inhibition of EGFR with a neutralizing antibody or erlotinib reversed SPINK6-induced nasopharyngeal carcinoma cell migration and invasion. Erlotinib also inhibited SPINK6-induced metastasis in vivo Notably, SPINK6 bound to the EGFR extracellular domain independent of serine protease-inhibitory activity. Overall, our results identified a novel EGFR-activating mechanism in which SPINK6 has a critical role in promoting nasopharyngeal carcinoma metastasis, with possible implications as a prognostic indicator in nasopharyngeal carcinoma patients. Cancer Res; 77(2); 579-89. ©2016 AACR.

High Prevalence of Serine Protease Inhibitor Kazal Type 1 Gene Variations Detected by Whole Gene Sequencing in Patients with Fibrocalculous Pancreatic Diabetes.

Aim of Study:The aim is to study the prevalence and pattern of serine protease inhibitor Kazal type 1 (SPINK1) gene variations in patients with fibrocalculous pancreatic diabetes (FCPD) using whole gene sequencing.Materials and Methods:A total of 56 consecutive patients of FCPD were recruited for the study. Diagnosis of FCPD was based on the presence of diabetes mellitus in patients having chronic pancreatitis with radiological evidence of ductal calcifications, in the absence of other known causes for pancreatitis. Ethylenediaminetetraacetic acid samples were collected from all patients, and complete gene sequencing was performed for SPINK1 gene using Sanger technique.Results:Overall 35 patients (62.5%) were detected to have genetic alterations in SPINK1 gene. N34S polymorphism was seen in 23 participants (41.07%) out of which 3 were homozygous. N34S was seen to be in linkage disequilibrium with IVS1 − 37T>C (18/23) and IVS3-69insAAAA (19/23) polymorphisms. Seven patients (12.5%) had a 272 C>T 3'UTR polymorphism while one patient (1.8%) had a P55S polymorphism. Two patients (3.5%) had an IVS3 + 2T>C mutation which has been shown to be associated with loss of function of SPINK protein. Overall 48.2% of FCPD patients had genetic variations that were significant compared to the control population. There was no difference in anthropometric and biochemical parameters between those with or without SPINK1 gene variations.Conclusions:Variations in SPINK1 gene are frequently observed in FCPD. N34S polymorphism was the most common variation followed by intronic variations. Two patients had the pathogenic intronic IVS3 + 2T>C mutation. Whole gene sequencing of the SPINK1 gene enabled detection of an additional 7.1% of patients with significant SPINK1 gene variations as compared to targeted screening for the N34S variation.

Serine protease inhibitor kazal-type 6 inhibits tumorigenesis of human hepatocellular carcinoma cells via its extracellular action.

Hepatocellular carcinoma (HCC) causes significant medical burdens worldwide. Diagnosis, especially in the early stages, is still challenging. Therapeutic options are limited and often ineffective. Although several risk factors have been known important for development of HCC, the molecular basis of the process is rather complex and has not been fully understood. We have found that a subpopulation of HCC cells which are resistant to oncolytic parvovirus H1 superinfection highly express serine protease inhibitor Kazal-type 6 (SPINK6). This protein is specifically reduced in all HCC cell lines and tissues we analyzed. When upregulated, SPINK6 could suppress the malignant phenotypes of the HCC cells in several in vitro models. The putative tumor suppression role of SPINK6 is, however, independent of its protease inhibitory activity. To suppress the malignancy of HCC cells, SPINK6 has to be secreted to trigger signals which regulate an intracellular signaling molecule, ERK1/2, as well as a series of downstream factors involved in cell cycle progression, apoptosis and migration. Our study supports that SPINK6 is an important tumor suppressor in liver, and further investigations may help develop more effective diagnostic and therapeutic approaches.

Serine protease inhibitor Kazal-type 2 is expressed in the male reproductive tract of carp with a possible role in antimicrobial protection

The presence of the low-molecular-mass serine protease inhibitor Kazal-type (Spink) is a characteristic feature of vertebrate semen. Its main function is control of the serine protease in the acrosome, acrosin. Here we showed for the first time that Spink is present in the seminal plasma of carp, which have anacrosomal spermatozoa. Using a three-step isolation procedure that consisted in gel filtration and RP-HPLC and re-RP-HPLC, we isolated this inhibitor and identified it as serine protease inhibitor Kazal-type 2 (Spink2), a reproductive-derived member of the Spink family. The cDNA sequence of this inhibitor obtained from carp testis encoded 77 amino acids, including a 17 amino acids signal peptide; this sequence was distinct from fish Kazal-type inhibitors. The mRNA expression analysis showed that Spink2 is expressed predominantly in carp testis and spermatic duct. Immunohistochemical analysis demonstrated its localization in testis in Sertoli, Leydig and germ cells at all developmental stages (with the exception of spermatozoa) and in the epithelium of the spermatic duct. Aside from strong inhibition of trypsin, this inhibitor acts strongly against subtilisin and possesses bacteriostatic activities against Lactobacillus subtilis, Escherichia coli and Aeromonas hydrophila. The localization of Spink2 in carp reproductive tract suggests an important function in spermatogenesis and in maintenance of the microenvironment in which sperm maturation occurs and sperm are stored. Our results suggest that Spink2 from carp seminal plasma may play a role in antibacterial semen defense, protecting semen against unwanted proteolysis within the reproductive tract.

Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome.

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed “X-SPINK1“. Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/− mice rescued perinatal lethality, but the resulting Spink3−/−;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3−/−;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.

Genetic Analysis of Japanese Children With Acute Recurrent and Chronic Pancreatitis.

Causes of acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP) are sometimes difficult to determine in children. In such patients, genetic analysis may prove helpful. The present study analyzed mutations of cationic trypsinogen (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC), and carboxypeptidase A1 (CPA1) and investigated the clinical features of children with these mutations. Genetic analyses of mutations in these 4 genes were conducted in 128 patients with ARP or CP. Characteristics of the patients showing mutations were investigated using medical records. Fifty of the 128 (39.1%) subjects had at least 1 mutation (median age at onset, 7.6 years). Abdominal pain was the presenting symptom of pancreatitis in 48 of the 50 patients (96%). Fifteen of those 50 patients (30.0%) had a family history of pancreatitis. Gene mutations were present in PRSS1 in 26 patients, SPINK1 in 23, CTRC in 3, and CPA1 in 5. In the 31 patients with mutations in SPINK1, CTRC, or CPA1, 16 (51.6%) had homozygous or heterozygous mutations with other mutations. Three patients underwent surgery and another 4 patients underwent endoscopy to manage ARP or CP. Although 3 of the 7 patients complained of mild abdominal pain, none of those 7 patients experienced any obvious episode of ARP after treatment. In pediatric patients with idiopathic ARP and CP, genetic analysis is useful for identifying the cause of pancreatitis. Early endoscopic or surgical treatment prevents ARP by extending the interval between episodes of pancreatitis in this population.

Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?

Insulin-like peptides (ILPs) have regulatory roles in reproduction, development and metabolism in invertebrates. The mode of ILP actions has not been well studied in invertebrates in regard to the role of binding partners, i.e., ILP binding protein (ILPBP). In this study, the full-length cDNA of Callinectes sapidus ILPBP (Cas-ILPBP, 960 bp) has been isolated using RACE cloning, having short 5′ and 3′ UTRs of 30 and 162 bp, respectively. The predicted precursor of Cas-ILPBP (255 aa) contains, in order a signal peptide (23 aa), an insulin-like growth factor (IGF) binding (IB) domain (79 aa), a kazal-type serine protease inhibitor (KI) domain (36 aa) and an immunoglobulin (Ig) domain (101 aa). Phylogenetic analysis shows that Cas-ILPBP is grouped with the ILPBPs of other crustacean species, and it shares the closest relationship with the ILPBP from another crab species, Scylla paramamosain. Transcripts of Cas-ILPBP are found in all examined tissues, with the highest levels in the nervous tissues (eyestalk ganglia, brain and thoracic ganglia complex) and followed by midgut, the pericardial organ, abdominal muscle and the heart. As Cas-ILPBP contains a putative Ig domain, it is hypothesized that this protein may be involved in immunity, particularly in the adult females infected with a reo-like virus (CsRV1). The expression levels of Cas-ILPBP are examined in several tissues (hemocytes, midgut, eyestalk ganglia) from the animals carrying varying levels of CsRV1 at 17 and 23 °C water temperatures. Cas-ILPBP levels in the midgut are most significantly affected by high levels of CsRV1 infection. Reduction in Cas-ILPBP levels in the midguts is noted from the animals infected with high levels of CsRV1 that show reduced or stop feeding activity, indicating that it may play an important role in midgut functions such as digestion and nutrient absorption.

Abstract A42: Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer

Abstract Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) is primarily secreted by the cells of the tumor microenvironment (TME), mainly by inflammatory cells but also by activated fibroblasts. IL-6 stimulates growth and survival signaling in CRC. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. Expression of serine proteases trypsin-1 and -2, the main targets of SPINK1, also correlate with malignancy and metastatic potential. The aim of this study was to assess the paracrine signaling between fibroblast-derived IL-6 and cancer cell-derived SPINK1, trypsin-1 and -2. The following expression analyses were used: quantitative PCR, immunohistochemistry, immunofluorometric assays and Western blotting. The results show that CRC cells Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. Conditioned media from fibroblasts had the same effect, and conversely CRC media increased IL-6 secretion in the fibroblasts, indicating paracrine signaling between these cell types. In CRC tissues cancer cells were positive for SPINK1, while IL-6 was found in fibroblasts surrounding the cancer cells, confirming the in vitro results. In Colo205 cells the baseline levels of trypsin-1 and -2 and the respective genes PRSS1 and PRSS2 were much higher compared to HT-29 cells. In Colo205 cells IL-6 led to concomitant increase in the secretion of trypsin-1 and -2, whereas in HT-29 cells these remained constantly low. Mechanistically, addition of IL-6 led to activation of the canonical Stat3 pathway, as indicated by Stat3 phosphorylation. Stat3 inhibitor reduced both SPINK1 and trypsin-1 and -2 levels, demonstrating that Stat3 is the transcription factor driving their expression. Taken together, our results show a connection between TME-derived inflammatory response and increased SPINK1 and trypsin-1 and -2 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. The study also strengthens the role of tumor microenvironment in tumor progression and emphasizes the importance of studying tumor-stroma crosstalk of proteolytic processing. Citation Format: Kati Räsänen, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Outi Itkonen, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A42.

Purification and Characterization of a Novel Kazal-Type Trypsin Inhibitor from the Leech of Hirudinaria manillensis.

Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of “non-classical” Kazal inhibitors according to its CysI-CysII disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F) XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (Ki) of (8.12 ± 0.18) × 10−9 M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin.

Expressions of isopeptide bonds and corneodesmosin in middle ear cholesteatoma.

Isopeptide bonds form cross-links between constituent proteins in the horny layer of the epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes, which bind corneocytes together. Both play important roles in maintaining epidermal barrier functions. In the present study, we investigated the expressions of isopeptide bonds, CDSN, and related enzymes in middle ear cholesteatoma in comparison with the skin. Prospective case series of patients with middle ear cholesteatoma. Tertiary medical institute. Cholesteatoma and normal postauricular skin were collected from patients with acquired middle ear cholesteatoma during tympanomastoidectomy. Expression of e-(g-glutamyl)lysine isopeptide bonds was examined by immunohistochemistry; Expressions of transglutaminase (TGase)1, TGase2, TGase3, and TGase5 by immunohistochemistry and quantitative RT-PCR (qRT-PCR); expression of CDSN by immunohistochemistry, qRT-PCR, and Western blot; and expressions of tissue kallikrein-related peptidase (KLK)5, KLK7, KLK14, and serine peptidase inhibitor Kazal type 5 (SPINK5) by qRT-PCR. TGase2 was higher (P=0.0046) and TGase5 was lower (P=0.0008) in cholesteatoma than in the postauricular skin. Immunoreactivity for isopeptide bonds was localized in the granular and horny layers, and was not different between the two tissues. Immunoreactivity for CDSN was localized in the granular layer, and was lower in cholesteatoma than in the skin (P=0.0090). Western blot and qRT-PCR confirmed that the expression of CDSN was lower in cholesteatoma than in the skin. Expressions of KLK5, KLK7, KLK14, or SPINK5 were not different between the two tissues. These results indicate that the production of CDSN is likely to be suppressed in cholesteatoma, which would account, at least in part, for the mechanical fragility and increased permeability of the cholesteatoma epithelium.