Mediator cyclin-dependent kinases upregulate transcription of inflammatory genes in cooperation with NF-κB and C/EBPß on stimulation of Toll-like receptor 9.

In eukaryotes, the Mediator complex has important roles in regulation of transcription by RNA polymerase II. Mediator is a large complex with more than 20 subunits that form head, middle, tail and CDK/cyclin modules. Among them, CDK8 and/or CDK19 (CDK8/19), and their counterpart cyclin C, form the CDK/cyclin module together with Mediator subunits MED12 and MED13. Despite evidences of both activation and repression, the precise functional roles of CDK8/19 in transcription are still elusive. Our previous results indicate that CDK8/19 recruits epigenetic regulators to repress immunoresponse genes. Here, this study focused on Toll-like receptors (TLRs), which exert innate immune responses through recognition of pathogen-associated molecular patterns and examined the functional roles of CDK8/19. As a result, CDK8/19 regulated transcription of inflammatory genes on stimulation of TLR9 in myeloma-derived RPMI8226 cells, which led to expression of inflammation-associated genes such as IL8, IL10, PTX3 and CCL2. Mediator subunits CDK8/19 and MED1, inflammation-related transcriptional activator NF-κB and C/EBPß, and general transcription factors TFIIE and TFIIB colocalized at the promoter regions of these genes under this condition. Our results show that CDK8/19 positively regulates inflammatory gene transcription in cooperation with NF-κB and C/EBPß on stimulation of TLR9.

[Viewing sepsis and autoimmune disease in relation with infection and NETs-formation].

Neutrophil has been widely recognized as body's first line of defence against pathogens. NETosis was first reported in 2004 as a programmed cell death of neutrophil and distinguished from apoptosis and necrosis. This phenomenon has been already observed in both basic and clinical research. NETosis is induced by various stimulants such as PMA, IL-8, DAMPs/PAMPs, bacteria, and antigen-antibody complex including self-antibody such as ANCA. It is known that there are two types of NETosis following bacterial infections. Although both of them have the ability to capture and kill bacteria, they also damage the host tissues. The inhibition of the NETs-related enzymes prevents the NETs formation at that time. The production of O2- from respiratory burst of neutrophils triggers NETs formation. In the first type of NETosis, neutrophils are completely collapsed, while in the second type, they maintain the morphology and the ability of phagocytosis. However, bacteria can escape from NETs by degrading NETs with their secreting nucleases. Thus the animal models of infection, using these bacteria, oftentimes suffer from severe infectious diseases. Human CGD (Chronic Granulomatosis Disease) patients who do not have Nox2 are immunocompromised, and highly susceptible to infection due to the defect of NETs formation. On the other hand, SLE patients are unable to break down the NETs as their serum inhibits the DNase1 activity, which results in autoantibody generation against NETs as well as self-DNA. It is getting clear that there is a relationship between inflammatory diseases, including infectious diseases, Sepsis and autoimmune diseases, and NETs. Therefore, it is important to re-evaluate the inflammatory disorders from NETs' perspective, and to incorporate the emerging concepts for better understanding the mechanisms involved.

Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11/hippostasin.

The human kallikrein (KLK) family consists of 15 genes located on human chromosome 19q13.4. KLK11/hippostasinis a member of the kallikrein family and is expressed in various tissues. Two types of KLK11 isoforms, isoform 1 and isoform 2, have been predicted from cDNA sequences. Isoform 1 has been isolated from human hippocampus, whereas isoform 2 has been isolated from prostate. However, the regulation and characteristics of these isoforms are unknown. We identified the first three exons (1a, 1b, and 1c) by determining their transcription initiation sites. Exon 1b contained the initiation codon of isoform 2, and noncoding exons 1a and 1c contributed to isoform 1 mRNA. The dual luciferase promoter assay revealed three promoter regions, corresponding to the first exon of each isoform. Reverse transcription and PCR showed that exon 1a was expressed in the hippocampus, thalamus, and non-central nervous system (CNS) tissues, whereas exon 1b was detected only in non-CNS tissues. Exon 1c was observed in both CNS and non-CNS tissues, except for salivary glands. In vitro mutagenesis revealed that the initiation codon for isoform 2 in exon 1b was functional. Isoform 2 had additional hydrophilic amino acids at the amino terminal and was secreted from the neuroblastoma cell line Neuro2a. Isoform 1 fused with green fluorescent protein (GFP) was distributed to cellular processes, whereas isoform 2-GFP was retained in the Golgi apparatus. We suggest that not only alternative splicing but also tissue-specific use of multiple promoters regulate the expression and intracellular trafficking of KLK11/hippostasin isoforms.

Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma.

Human kallikrein 11 (hK11) is a putative serine protease of the human kallikrein gene family. Currently, no methods are available for measuring hK11 in biological fluids and tissues. Our aim was to develop immunological reagents and assays for measuring hK11 and examine if the concentration of this kallikrein is altered in disease states. We produced recombinant hK11 protein in a baculovirus system and used it to develop monoclonal and polyclonal antibodies against hK11. We then developed an immunofluorometric procedure for measuring hK11 in biological fluids and tissue extracts with high sensitivity and specificity. We further quantified hK11 in various biological fluids and in serum of patients with various cancers. The hK11 immunofluorometric assay is highly sensitive (detection limit, 0.1 microg/l) and specific (no detectable cross-reactivity for other homologous kallikreins). We established the tissue expression pattern of hK11 at the protein level and found the highest levels in the prostate, followed by stomach, trachea, skin, and colon. We have immunohistochemically localized hK11 in epithelial cells of various organs. We further detected hK11 in amniotic fluid, milk of lactating women, cerebrospinal fluid, follicular fluid, and breast cancer cytosols. However, highest levels were seen in prostatic tissue extracts and seminal plasma. hK11 in seminal plasma and prostatic extracts is present at approximately 300-fold lower levels than prostate-specific antigen and at approximately the same levels as hK2. hK11 expression in breast cancer cell lines is up-regulated by estradiol. Elevated serum levels of hK11 were found in 70% of women with ovarian cancer and in 60% of men with prostate cancer. This is the first reported immunological assay for hK11. Analysis of this biomarker in serum may aid in the diagnosis and monitoring of ovarian and prostatic carcinoma.

A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells.

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.

Decreased cerebrospinal fluid levels of neurosin (KLK6), an aging-related protease, as a possible new risk factor for Alzheimer's disease.

Neurosin is a kallikrein-like serine protease expressed preferentially in the human brain. It is localized in senile plaques and neurofibrillary tangles in the brains of individuals with Alzheimer's disease (AD) and in Lewy bodies in patients with Parkinson's disease. Neurosin is present in the cerebrospinal fluid (CSF) as a proenzyme and does not show any enzymatic activity. We have developed a sandwich ELISA system using monoclonal and polyclonal antibodies against human neurosin and have measured neurosin levels in the CSF from AD and non-CNS disease patients. Both male and female patients with peripheral neuropathy showed statistically positive correlations between CSF neurosin concentrations and age (males, n = 52, r = 0.482, p < 0.005; females, n = 43, r = 0.365, p < 0.005). In contrast, such positive correlation was not observed in the CSF from patients with AD. Further, some such patients showed extremely low levels of CSF neurosin. Our results suggest that neurosin is an aging-related protease and that a decreased CSF concentration of neurosin may be a risk factor for developing AD.

Molecular cloning and expression of a variant form of hippostasin/KLK11 in prostate.

BACKGROUND: Hippostasin is a kallikrein-like serine protease, which has two alternatively spliced isoforms, brain-type and prostate-type. We previously reported alternative expression of hippostasin in prostate cancer cell lines. METHODS: We studied the expression of a variant-form hippostasin (isoform 3) mRNA by RT-PCR. Localization of the isoform 3 protein was examined by immunohistochemistry. The enzymatic activity of the recombinant protein was measured with synthetic substrates. RESULTS: A novel isoform of hippostasin contains 25 additional amino acids in the catalytic triad of brain-type hippostasin. Its mRNA was expressed in normal prostate tissue, BPH, and prostate cancer cell lines. The protein was localized in the prostate secretory epithelium. The enzyme activity was similar to that of brain-type hippostasin, which has kallikrein-like activity. CONCLUSIONS: In this study, we have identified a third isoform of hippostasin, which was designated variant-form (isoform 3). Hippostasin isoform 3 may play a role in the prostate, including reproductive and/or tumorigenic functions.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5.

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression.

Spinesin/TMPRSS5, a novel transmembrane serine protease, cloned from human spinal cord.

A cDNA encoding a novel serine protease, which we designated spinesin, has been cloned from human spinal cord. The longest open reading frame was 457 amino acids. A homology search revealed that the human spinesin gene was located at chromosome 11q23 and contained 13 exons, the gene structure being similar to that of TMPRSS3 whose gene is also located on 11q23. Spinesin has a simple type II transmembrane structure, consisting of, from the N terminus, a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger receptor-like domain, and a serine protease domain. Unlike TMPRSS3, it carries no low density lipoprotein receptor domain in the stem region. The extracellular region carries five N-glycosylation sites. The sequence of the protease domain carried the essential triad His, Asp, and Ser and showed some similarity to that of TMPRSS2, hepsin, HAT, MT-SP1, TMPRSS3, and corin, sharing 45.5, 41.9, 41.3, 40.3, 39.1, and 38.5% identity, respectively. The putative mature protease domain preceded by H(6)DDDDK was produced in Escherichia coli, purified, and successfully activated by immobilized enterokinase. Its optimal pH was about 10. It cleaved synthetic substrates for trypsin, which is inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride but not by antipain or leupeptin. Northern blot analysis against mRNA from human tissues including liver, lung, placenta, and heart demonstrated a specific expression of spinesin mRNA in the brain. Immunohistochemically, spinesin was predominantly expressed in neurons, in their axons, and at the synapses of motoneurons in the spinal cord. In addition, some oligodendrocytes were clearly stained. These results indicate that spinesin is transported to the synapses through the axons after its synthesis in the cytoplasm and may play important roles at the synapses. Further analyses are required to clarify its roles at the synapses and in oligodendrocytes.