1 Abstract Liver fibrosis is a condition described by extensive accumulation of scar tissue in the liver. With further progression, it leads to cirrhosis or even to hepatocellular carcinoma. Liver fibrosis accompanies every chronic liver disease and its prevalence in adult European population is estimated to be around 4%. During my dissertation work, I studied the function of three members of Metzincin family of metalloproteinases - ADAM17, ADAM10 and MMP-19, in liver fibrosis and liver regeneration using mouse genetic models. ADAM17 and ADAM10 are important regulators of signalling pathways which are involved in immune response as well as differentiation. Both proteases are able to cleave ectodomains of their substrates from cell membrane, affecting bioavailability of ligands and functionality of receptors. Several of their substrates are involved in liver pathologies. MMP-19 on the other hand, is a metalloprotease mainly involved in extracellular matrix cleavage, important process in fibrosis development, as well as resolution of fibrosis. Our results demonstrate that ablation of ADAM10 results in increased susceptibility to liver fibrosis in mice, both spontaneous and toxin induced. ADAM10 deficiency affected biliary epithelium, as we detected higher markers of biliary damage in serum of ADAM10 deficient...

Eine Defizienz des LTBP4-Gens ruft schwerwiegende Erkrankungen wie das human autosomal-rezessive Cutis laxa Syndrom Typ 1C (ARCL1C) hervor. Die Interaktion zwischen LTBP4 und TGF-β ist hierbei von pathogenetisch relevanter Bedeutung. Durch fehlende Synthese bzw. Sekretion des TGF β (bedingt durch die LTBP4-Defizienz) kann es zu einer verminderten TGF-β-Aktivität im Lungenparenchym kommen, woraus insgesamt eine fehlerhafte Produktion elastischer Fasern resultiert. Phänotypisch zeigen Patienten Multimorbidität und besonders schwere Defekte in der Lungenentwicklung. Emphyseme infolge fehlerhafter Alveolarisierung und Septierung führen zu früher Mortalität der Patienten innerhalb der ersten Wochen bis Monate. Als Tiermodell wurde die Ltbp4-/- Mauslinie generiert, die nahezu den gleichen Phänotyp aufweist, den auch Patienten mit ARCL1C zeigen. Ziel der Untersuchungen war es, den Einfluss der Pneumozyten Typ I und II auf die gestörte Alveolarisierung, Septierung sowie Angiogenese der Lunge von Ltbp4-/- Mäusen im Vergleich zu WT Mäusen zu überprüfen. Zusätzlich sollte erforscht werden, ob das Sekretom von murinen Ltbp4-/- Lungenfibroblasten (MLF) in vitro die Homöostase der Pneumozyten im Vergleich zu WT MLF hemmt bzw. deren Expression und Sekretion pro- und anti-angiogenetischer Faktoren beeinflusst. In dieser Studie konnte nachgewiesen werden, dass die Defizienz des Ltbp4-Gens postnatal zu schwerwiegenden Lungenmissbildungen und gestörter Alveologenese der Maus führt, wobei sowohl die Pneumozyten Typ I als auch die Pneumozyten Typ II nachweislich in reduzierter Anzahl in Ltbp4-/- Mäusen vorhanden waren. Weiter wurde bestätigt, dass die Pneumozyten Typ II verantwortlich für eine erhöhte Tgf-β-Aktivität sind und somit präfibrotische Prozesse im Lungenparenchym von Ltbp4-/- Mäusen begünstigt werden. Die Ergebnisse verdeutlichen, dass die Defizienz des Ltbp4 Gens erheblichen Einfluss auf das Sekretom der MLF hat. Da das humane ARCL1C Syndrom ebenfalls ein dysfunktionales LTBP4-Gen aufweist, konnten durch dieses Projekt weitere Erkenntnisse der Pathogenese einer fehlerhaften Alveolarisierung in der Lungenentwicklung gewonnen werden. Von großer Bedeutung ist auch, dass dieses Mausmodell den Phänotyp pädiatrischer Lungenerkrankungen aufweist, sodass diese Erkenntnisse zum Verständnis der Pathogenese der prä- und neonatalen Entwicklung pädiatrischer Lungenerkankungen beitragen.:Inhaltsverzeichnis 1. Einleitung 1 2. Literaturübersicht 3 2.1 Die Lunge 3 2.2 Morphologie der Lunge 4 2.3 Alveolarepithel 4 2.4 Die Blut-Gas-Schranke 6 2.5 Embryologie der Lunge 6 2.6 Anatomie der Lunge von Maus und Mensch im Vergleich 9 2.7 LTBP – latent TGF-β bindende Proteine 10 2.8 Isoformen des LTBPs 13 2.9 Ltbp4 in der Lunge 15 2.10 Angiogenese 15 2.11 Autosomal-rezessives Cutis laxa Syndrom Typ 1C 17 3. Tiere, Material und Methoden 19 3.1 Tiere 19 3.2 Material 20 3.2.1 Geräte 20 3.2.2 Chemikalien und Verbrauchsmaterialien 22 3.2.3 Kits und Assays 25 3.2.4 Puffer und Lösungen 26 3.2.5 Gele 28 3.2.6 Antikörper 28 3.2.7 Primer 29 3.3 Zelllinien 30 3.4 Methoden 31 3.5 Organentnahme der Versuchstiere 31 3.6 Histologie 33 3.7 Zellkultur 34 3.7.1 Isolierung primärer muriner Lungenfibroblasten 35 3.7.2 MLE 12-Zellkultivierung 36 3.7.3 Tube Formation Assay 36 3.7.4 TGF-β Minc Lung Assay 37 3.7.5 Proliferations Assay 39 3.7.6 Viabilitäts Assay 40 3.7.7 Caspase-3 Assay 40 3.7.8 Fluoreszenz Activating Cell Sorting (FACS) 41 3.8 Molekularbiologische Methoden 44 3.8.1 Genotypisierung 44 3.8.2 Polymerase-Ketten-Reaktion 45 3.8.3 Western Blot 47 3.8.4 RNA-Isolierung mit konditioniertem Medium MLF stimulierter MLE 12-Zellen 50 3.8.5 cDNA-Synthese und Aktin-Kontroll-PCR 51 3.8.6 Real time quantitative PCR 53 3.9 Statistische Auswertung 55 4. Ergebnisse 56 4.1 Lungen von Ltbp4-/- Mäusen weisen eine reduzierte Anzahl von Pneumozyten Typ II auf 56 4.2 Lungen von Ltbp4-/- Mäusen weisen eine reduzierte Anzahl von Pneumozyten Typ I auf 59 4.3 Aktivierung von Tgf-β nach Stimulation mit konditioniertem Medium von Ltbp4-/- MLF in MLE 12-Zellen 61 4.4 Konditioniertes Medium von Ltbp4-/- MLF vermindert die Proliferation von MLE 12-Zellen und erhöht die Viabilität 63 4.5 Die Apoptoserate (Casp3/7) der MLE 12-Zellen nach Stimulation mit konditio-niertem Medium von Ltbp4-/- MLF wird nicht beeinflusst 64 4.6 mRNA Expression von Sp-C und Sp-B in MLE 12-Zellen nach Stimulation mit konditioniertem Medium von Ltbp4-/- MLF 64 4.7 MLE 12-Zellen weisen eine erhöhte mRNA Expression von Ltbp1 und eine unveränderte mRNA Expression von Ltbp4 nach Stimulation mit konditioniertem Medium von Ltbp4-/- MLF auf 65 4.8 Die angiogene Stimulation von Endothelzellen und die mRNA Expression angiogener Faktoren von MLE 12-Zellen, die mit konditioniertem Medium von Ltbp4-/- MLF vergleichend zum WT MLF stimuliert wurden, wird stark beeinflusst 66 5. Diskussion 69 6. Zusammenfassung 80 7. Summary 82 8. Literaturverzeichnis 84 9. Anhang 91

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20080号 / 医博第4173号 / 新制||医||1018(附属図書館) / 33196 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fibrosis is a disease where the normally transitory wound healing response enters a chronic state. Bleomycin and Adenovector models of pulmonary fibrosis have implicated TGF-β1 in this disease. Concern regarding a synergistic combination of TGF-β1 with an adaptive immune response within the Adenovector model prompted its use within mice devoid of T Lymphocytes, Balb/c SCIDs. The lack of an adaptive immune response within these mice did not affect the severity of fibrogenesis, as compared to Balb/c data in a hydroxyproline assay. TGF-β1 is a pluripotent cytokine with key roles in wound healing, immune regulation, and development, making it a dangerous molecule to therapeutically modulate directly. Future strategies will likely focus on downstream fibrotic molecules uninvolved in immune regulation, such as CTGF. While CTGF has been associated with fibrosis and is likely activated by TGF-β1, no conclusive evidence is available within an animal model. TGF-β1 stimulates cells by binding its receptor and signaling through the Smad signal transduction pathway. Smad3 knockout mice were used to examine the regulation of CTGF by TGF-β1, and study its role in pulmonary fibrosis. We show that these mice produce dramatically less CTGF in response to TGF-β1 than littermates expressing Smad3, and they show protection against TGF-β1 induced pulmonary fibrosis, using the Adenovector system. TGF-β1 can alter lung development, and is thought to be a causative agent in Bronchopulmonary Dysplasia, a disease affecting immature lungs. Utilizing the Adenovector system, we developed a neonatal rat model of BPD that closely resembles the human disease, providing researchers with a system to study the disease course. TGF-β1 is part of a family of growth factors, of which TGF-β3 is also a member. What role TGF-β3 plays in pulmonary fibrosis has not been evaluated. To allow future in vivo studies on the effect of TGF-β3 on lung morphology, we constructed a replication deficient Adenovector expressing constitutively active TGF-β3. / Thesis / Master of Science (MSc)

Activator Protein-1(AP-1) family plays a central role in the transcriptional regulation of many genes that are associated with cell proliferation, migration, metastasis, and survival. Transforming growth factor beta (TGF-β) is a multi-functional regulatory cytokine that regulates many aspects of cellular function, including cellular proliferation, migration, and survival. This study investigated the role of FOS proteins in TGF-β signaling in prostate cancer cell proliferation, migration, and invasion. DU145 and PC3 prostate cancer cells were exposed to TGF-β1 at varying time and dosage, RT-PCR, western blot and immunofluorescence analyses were used to determine TGF-β1 effect on FOS mRNA and protein expression levels as well as FosB sub-cellular localization. Transient silencing of FOS protein was used to determine their role in cell proliferation, migration and invasion. Our data showed that FOS mRNA and proteins were differentially expressed in human prostate epithelial (RWPE-1) and prostate cancer cell lines (LNCaP, DU145, and PC3). TGF-β1 induced the expression of FosB at both the mRNA and protein levels in DU145 and PC3 cells, whereas cFos and Fra1 were unaffected and Fra2 protein expression increased in PC3 cell only. Immunofluorescence analysis showed an increase in the accumulation of FosB protein in the nucleus of PC3 cells after treatment with exogenous TGF-β1. Selective knockdown of endogenous FosB by specific siRNA did not have any effect on cell proliferation in PC3 and DU145 cells. However, basal and TGF-β1-and EGF- induced cell migration was significantly reduced in DU145 and PC3 cells lacking endogenous FosB. TGF-β1- and EGF-induced cell invasion were also significantly decreased after FosB knockdown in PC3 cells. Transient silencing of Fra2 resulted in decrease in cell proliferation in PC3 cells whereas transient silencing of cFos resulted in an increase in cell number in PC3 cells. And lastly, TGF-β1 reduced FosB: cJun dimerization; cJun knockdown increased cell migration in PC3 cells and its over expression decreased cell migration in DU145 cells. Our data suggest that FosB is required for migration and invasion in prostate cancer cells. We also conclude that TGF-β1 effect on prostate cancer cell migration and invasion may be mediated through the induction of FosB.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-17T14:43:42Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ImunoreatividadeTgfCaspase.pdf: 1588662 bytes, checksum: 11bae3a9256acce980d29907ea3977c8 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-31T15:08:31Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ImunoreatividadeTgfCaspase.pdf: 1588662 bytes, checksum: 11bae3a9256acce980d29907ea3977c8 (MD5) / Made available in DSpace on 2017-10-31T15:08:31Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ImunoreatividadeTgfCaspase.pdf: 1588662 bytes, checksum: 11bae3a9256acce980d29907ea3977c8 (MD5) Previous issue date: 2007-11-30 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hanseníase é uma doença infecto-contagiosa crônica que acompanha a humanidade há muitos anos. O Brasil ocupa o segundo lugar em números de casos, sendo que o estado do Pará é o que apresenta o maior número de casos absolutos. Este estudo teve como objetivo avaliar a imunorreatividade para TGF-β e caspase-3 nas formas clínicas virchowiana e tuberculóide do mal de hansen procurando correlacionar o padrão de imunomarcação com o controle tecidual da resposta imune do hospedeiro ao bacilo, através de um estudo de caso controle com 30 pacientes, sendo quinze apresentando na forma virchowiana e quinze com o tipo tuberculóide. Os pacientes seguiram o protocolo de diagnóstico de hanseníase segundo critérios do Ministério da Saúde do Brasil. Observou-se que a forma virchowiana da hanseníase apresentou uma correlação estatisticamente significativa (p=0,4630) entre o TGF-β a caspase-3, evidenciando que tanto a citocina quanto o imunomarcador da apoptose aumentam gradativamente e simultaneamente nesta forma polar e que indiretamente aponta para um papel do TGF-β no controle da resposta imunológica in situ à infecção pelo Mycobacterium leprae. / Leprosy is a chronic infect-contagious disease that accompanies the humanity for many years. Brazil occupies the second place in numbers of cases, and the state of Para is that presents the largest number of absolute cases. This study had as objective to evaluate the immunorreactivity for TGF-β and caspase-3 in the lepromatous and tuberculoid clinical forms of the leprosy evil trying to correlate the immunomarking pattern with the tissue control of the immune response of the host to the bacillus, through a retrospective study of paraffin enclosed tissue of 30 patients, being fifteen presenting in the lepromatous form and fifteen with the tuberculoid type of disease. The patients followed the protocol of leprosy diagnosis according to criteria of Health Ministry of Brazil and histopathologic features. It was observed that the lepromatous form of the leprosy presented a significant statistically correlation (p=0,4630) among TGF-β to caspase-3, evidencing that as the cytokine as the imunomarking of the apoptosis increase inch by inch and simultaneously in this polar form and that indirectly appear to a role of TGF-β in the control of the immunologic response in its place to the infection for the Mycobacterium leprae.

<p>Thyroid growth and function is partly regulated by growth factors binding to receptors on the cell surface. In the present thesis, the transforming growth factor-β (TGF-β) superfamily members have been studied for their role in regulation of growth and differentiation of both normal and neoplastic thyroid epithelial cells.</p><p>TGF-β1 is a negative regulator of thyrocyte growth and function. However, the importance of other TGF-β superfamily members has not been fully investigated. TGF-β1, activin A, bone morphogenetic protein (BMP)-7 and their receptors were found to be expressed in porcine thyrocytes. In addition to TGF-β1, activin A was also found to be a negative regulator of thyroid growth and function, and both stimulated phosphorylation and nuclear translocation of Smad proteins. Furthermore, TGF-β1 and epidermal growth factor (EGF) demonstrated a synergistic negative effect on thyrocyte differentiation. Simultaneous addition of the two factors resulted in a loss of the transepithelial resistance and expression of the epithelial marker E-cadherin. This was followed by a transient expression of N-cadherin.</p><p>Despite the extremely malignant character of anaplastic thyroid carcinoma (ATC) tumor cells, established cell lines are still responsive to TGF-β1. A majority of the cell lines were also found to be growth inhibited by BMP-7. BMP-7 induced cell cycle arrest of the ATC cell line HTh 74 in a dose- and cell density-dependent manner. This was associated with upregulation of p21^CIP1 and p27^KIP1, decreased cyclin-dependent kinase (Cdk) activity and hypophosphorylation of the retinoblastoma protein (pRb). TGF-β1, and to some extent also BMP-7, induced the expression of N-cadherin and matrix metalloproteinase (MMP)-2 and -9. Stimulation of HTh 74 cells with TGF-β1 increased the migration through a reconstituted basement membrane indicating an increased invasive phenotype of the cells.</p><p>Taken together, these data show that TGF-β superfamily members not only affect growth and function of normal thyroid follicle cells but may also, in combination with EGF, play a role in cell dedifferentiation. This study additionally suggests that the TGF-β superfamily members may be important for the invasive properties of ATC cells.</p>

<p>Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. </p><p>In order to study the BMP role for catecholaminergic neurons <i>in vivo</i>, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. </p><p>Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.</p><p>The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.</p><p>Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. </p><p>These results demonstrate a positive role of BMPs for MDN fate<i> in vivo</i>. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.</p>

<p>The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.</p><p>The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, <i>Id2</i> and <i>Id3</i>. Interestingly, we found that TGF-β-induced repression of <i>Ids</i> is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of <i>Ids</i> and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.</p><p>Furthermore, we identified a serine/threonine kinase, <i>SNF1LK</i>, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling. </p>

Thyroid growth and function is partly regulated by growth factors binding to receptors on the cell surface. In the present thesis, the transforming growth factor-β (TGF-β) superfamily members have been studied for their role in regulation of growth and differentiation of both normal and neoplastic thyroid epithelial cells. TGF-β1 is a negative regulator of thyrocyte growth and function. However, the importance of other TGF-β superfamily members has not been fully investigated. TGF-β1, activin A, bone morphogenetic protein (BMP)-7 and their receptors were found to be expressed in porcine thyrocytes. In addition to TGF-β1, activin A was also found to be a negative regulator of thyroid growth and function, and both stimulated phosphorylation and nuclear translocation of Smad proteins. Furthermore, TGF-β1 and epidermal growth factor (EGF) demonstrated a synergistic negative effect on thyrocyte differentiation. Simultaneous addition of the two factors resulted in a loss of the transepithelial resistance and expression of the epithelial marker E-cadherin. This was followed by a transient expression of N-cadherin. Despite the extremely malignant character of anaplastic thyroid carcinoma (ATC) tumor cells, established cell lines are still responsive to TGF-β1. A majority of the cell lines were also found to be growth inhibited by BMP-7. BMP-7 induced cell cycle arrest of the ATC cell line HTh 74 in a dose- and cell density-dependent manner. This was associated with upregulation of p21CIP1 and p27KIP1, decreased cyclin-dependent kinase (Cdk) activity and hypophosphorylation of the retinoblastoma protein (pRb). TGF-β1, and to some extent also BMP-7, induced the expression of N-cadherin and matrix metalloproteinase (MMP)-2 and -9. Stimulation of HTh 74 cells with TGF-β1 increased the migration through a reconstituted basement membrane indicating an increased invasive phenotype of the cells. Taken together, these data show that TGF-β superfamily members not only affect growth and function of normal thyroid follicle cells but may also, in combination with EGF, play a role in cell dedifferentiation. This study additionally suggests that the TGF-β superfamily members may be important for the invasive properties of ATC cells.