京都大学 / 新制・課程博士 / 博士(医学) / 甲第23065号 / 医博第4692号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 平井 豊博, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multispecific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.

Phosphateses (EC 3.1.3.x) are a group of enzymes that hydrolyze phosphoesters. That way they affect the energetic metabolism of a cell and its regulation. Phosphatases that dephosphorylate proteins are an integral part of signaling pathways, stress responses and they modulate enzymatic activity. This thesis deals with the study of phosphatases obtained from tobacco leaves (Nicotiana tabacum, L.). Solution of enzymes was prepared by extraction in both acidic and alkaline buffers. Through the use of the chromogenic substrate pNP-phosphate it was determined that there is a higher phosphatase activity in the glycosylated fraction than in the fraction that did not bind to Con A Sepharose. The research of the ions effect on the phosphatase activity has determined that Mg2+ and Ca2+ show positive effect on the phosphatase activity while the effect of Co2+ and Mn2+ is inhibitory. The Zn2+ ions have shown no effect whatsoever. The glycosylated phosphatases also dephosphorylated low-weight-molecular substrates phosphoserine, ATP and glucose-6-phosphate. The research of protein phosphatase activity discovered the affinity to the substrate phosvitin, although neither to casein nor its tryptic cleaves. Detailed experiments have shown that the pH optima for all the substrates lie from pH 5 to 6. Glycosylated phosphatases...

Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance. Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors. Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors. Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

Pim (Provirus Integration site for Moloney murine leukemia virus) kinases are a family of three serine/threonine kinases involved in cell cycle, survival and metabolism. These kinases were first identified in malignant cells and are most often associated with their role in cancer. Their role in immunity and lymphocytes is less well known. To date, it has been shown that Pim 1 and/or Pim 2 are important for T lymphocyte survival and activation when the Akt signaling pathway is inhibited by rapamycin. In addition, our laboratory has shown that Pim 2 is critical for BLyS-mediated naive B lymphocyte survival in the presence of rapamycin. This thesis extends the role(s) for Pim 1 and/or 2 to include functions during B cell activation and the generation of immune responses. We found that during in vitro activation of purified resting splenic B cells from wild type mice with a variety of activators that use multiple signaling pathways, including the BCR, TLR and CD40 receptors, both Pim 1 and 2 kinases were induced by 48 hours post-activation, suggesting that they could play a role in B cell activation and differentiation to antibody secreting or memory B cells. Immunization of Pim 1-/-2-/- knockout mice with T cell dependent antigens showed impairment in antibody and antibody secreting cell generation as well as lack of germinal center formation clearly demonstrating an involvement of Pim 1 and/or 2 in the immune response. FACS examination of B cell populations from naive Pim 1-/-2-/- knockout mice revealed normal levels of splenic marginal zone and follicular B cells and T cells, however, decreased numbers of all peritoneal B cell populations and decreased B cells in Peyer's Patches was seen. An examination of serum antibody found in naive Pim 1-/-2-/- knockout mice showed decreased levels of natural antibody, which is likely due to loss of the peritoneal B1 cells but does not explain the significantly decreased TD immune response. To determine whether the defect was B cell intrinsic or a more complex interaction between B and T cells, we determined whether Pim 1-/-2-/- mice would respond to T cell independent, TI-1 and TI-2, antigens. Antibody production and antibody secreting cell formation were also significantly decreased in these mice supporting our notion of a B cell intrinsic defect. To further examine the B cell response problem, we attempted to establish chimeric mice using either bone marrow derived cells or fetal liver cells from WT or Pim 1-/-2-/- donors so that the B cells were derived from Pim 1-/-2-/- mice and the T cells would be WT. Unfortunately, we were not able to consistently engraft and develop mature Pim 1-/-2-/- B cells, which indicate that there is a stem cell defect in these knockout mice that requires further investigation. Because one of the major failures in activated Pim 1-/-2-/- B cells is the generation of antibody secreting cells, an analysis of the expression of transcription factors IRF-4 and BLIMP-1, known to play a role in this process was carried out. Although IRF-4 induction was not affected by the loss of Pim 1 and 2, the number of cells able to increase BLIMP-1 expression was significantly decreased, revealing a partial block in the generation of ASCs. Taken together the data presented in this thesis reveals a new and critical role for Pim 1 and 2 kinases in the humoral immune response.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation.

Obesity has been on the rise over the last 30 years, reaching worldwide epidemic proportions. Obesity has been linked to multiple metabolic disorders and co-morbidities such as Type 2 Diabetes Mellitus (T2DM), cardiovascular disease, non-alcoholic steatohepatitis and various cancers. Furthermore, obesity is associated with a chronic state of low-grade inflammation in adipose tissue (AT), and it is thought that insulin resistance (IR) and T2DM is associated with the inflammatory state of AT. Endothelial cells (ECs) mediate the migration of immune cells into underlying tissues during times of inflammation, including obesity- and cardiovascular disease-associated inflammation. Cytokines and chemoattractants released from inflamed tissues promote EC activation. Upon activation, ECs increase the expression of leukocyte adhesion molecules (LCAMs) including intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), E-selectin (E-sel) and P-selectin (P-sel). Increased expression of these LCAMs and increased infiltration of inflammatory cells such as macrophages, have been linked to IR, diabetes and atherosclerosis in obese individuals. Preliminary data from our lab suggests that lipolysis induced by the β-adrenergic receptor agonist CL 316,243 causes an increase in endothelial LCAM gene expression. In addition, histological analyses show increased content of immune cells within AT after the ECs become activated. Here, we demonstrate that CL 316,243-induced lipolysis causes infiltration of neutrophils in wild type (WT) but not E-sel knockout (KO) mice. Following EC activation, there was also a marked increase in cytokine gene expression including IL-1β, MCP-1, and TNF-α in an E-sel-dependent manner. In contrast, fasting-induced lipolysis was associated with increased macrophage infiltration into AT in the absence of EC activation in an E-sel-independent manner. We also examined the role of mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potential contributor to endothelial activation and atherosclerosis. Here we demonstrate that deletion of MAP4K4 in ECs in vitro diminishes TNF-α-induced EC activation. Additionally, MAP4K4 depletion in primary ECs derived from lungs of mice expressing MAP4K4 shRNA decreases EC activation. Finally, endothelial specific depletion or loss of MAP4K4 reduced atherosclerotic plaque formation in vivo. Taken together, these results highlight the importance of the endothelium in modulating obesity-associated comorbidities. Furthermore, these data implicate endothelial MAP4K4 as a novel regulator of EC activation and consequently AT inflammation and atherosclerosis.