Regensburg, Univ., Diss., 2003. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (181-196). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Prostate cancer micrometastasis allows cancer cells to vacate their original tumor sites and migrate to distant parts of the body via the bloodstream, lymphatic system, or by direct extension. Cells synthesize and secrete matrix metalloproteinases (MMPs) that degrade proteins of the surrounding extracellular matrix (ECM); thus allowing them to escape into the lymphatic or circulatory systems to invade other tissues. Transforming growth factor β (TGF-β) induces the migration and invasion of cancer cells and the expression of matrix metalloproteinases (MMPs), specifically MMP-2, and -9 in several malignancies. In this study, we examined the role of MMP-7, a known activator of MMP-2 and MMP-9, in TGF-β signaling in cell proliferation, migration, and invasion in prostate cancer cells. Basal expression levels of MMP7 mRNA, protein, and secreted protein were determined using RT-PCR, western blot analysis, and ELISA, respectively. Our data show that MMP7 mRNA and proteins were differentially expressed in several cell line models representing different stages of prostate cancer. TGF-β1 induces MMP-7 gene expression and protein levels 24 and 48 hours after treatment in PC3 cells. Our data also show that TGF-β induces cell migration and invasion in PC3 and E006AA cells; however, the selective knockdown of MMP7 expression using siRNA resulted in a significant decrease in control and TGFβ-induced cell migration and invasion in both PC3 and E006AA cells. MMP-7 knockdown also caused significant reduction in cell proliferation in PC3 cells. Our data suggest that MMP7 is essential for cell migration and invasion in prostate cancer cells indicating that it may be required for TGFβ-induced cancer metastases.

A organização de fibras de colágeno no leito de uma ferida é componente importante da cicatrização e contração da ferida, determinando em última instância a qualidade final da cicatriz. Neste estudo realizamos a implantação de modelo de biotecnologia constituído de géis de colágeno povoados por fibroblastos humanos, que foi utilizado como instrumento para a melhor compreensão dos fenômenos ainda pouco elucidados, envolvidos na contração de feridas. Utilizando fibroblastos procedentes de pele normal ou quelóides, observou-se maior contração dos géis povoados por fibroblastos oriundos de quelóide. O modelo implementado foi considerado eficiente para a avaliação da presença de moduladores da fase de remodelação da cicatriz, tais como o Fator de Crescimento Transformador Beta (TGF beta). A comparação entre a curva de contração gerada por fibroblastos oriudos de pele normal sob o efeito do TGF beta e a contração gerada por fibroblastos de quelóides, demonstra que as mesmas apresentam comportamento igual do ponto de vista estatístico. O modelo proposto demonstrou ser adequado para a melhor compreensão dos mecanismos responsáveis pela contração de feridas, bem como possui potencial na avaliação de novas drogas capazes de modular este fenômeno / An important component of tissue healing and wound contraction is the re-arrangement of ground collagen fibers, which can ultimately influence the final quality of scars. In this study we used a biotechnology experimental model with contracting collagen gels seeded with human fibroblasts in order to better understand the phenomena involved in wound contraction. We compared the contraction of the collagen gels using fibroblasts from normal skin and from keloids, and we observed that the collagen gels seeded with keloid fibroblasts suffered a bigger contraction. The model was considered efficient to test growth factors with the potential to modulate the remodeling phase of the scar, for example, the Transforming Growth Factor Beta (TGF beta). The analysis of the changing macroscopic gel area comparing the contraction generated by the normal fibroblasts after the treatment with TGF beta with the contraction in the gels with keloid\'s fibroblasts showed that they have the same behavior. The experimental model proved to be an useful tool to better understand the wound contraction and to test new drugs to modulate this phenomenon.

In the late stages of cancer, tumors acquire the ability to spread throughout the body and invade distant tissues in a process called metastasis. Studies have shown that metastasis is responsible for 90% of all cancer-related deaths, making this an important field of study. In breast cancers, 30% of patients overexpress the HER2 oncoprotein, causing a more invasive and metastatic disease. Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called EMT (epithelial-to-mesenchymal transition), where epithelial cells transition into a migratory phenotype through cell-cell junction breakdown. SLK is a Ste20-like kinase that has been linked to many processes, including cell migration and signaling downstream of the HER2 receptor complex. Here we show that the cellular migration and invasion of TGFβ-treated normal mammary epithelial cells is significantly impaired in the absence of SLK. Additionally, immunofluorescence analyses demonstrate that SLK knockdown conditions decrease a cell’s ability to progress through EMT due to the visible staining of epithelial markers. We find that SLK-depleted cultures express significantly lower levels of Snail1,and fibronectin mRNA levels following TGF-β treatment. Surprisingly, our data demonstrates that SLK kinase activity is not activated downstream of TGF-β stimulation, and that a kinase-dead SLK rescues Snail1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced epithelial-to-mesenchymal transition in a kinase activity-independent manner.

Idiopathic pulmonary fibrosis is a chronic and fatal disease that causes the stiffening of lung tissue and gradual lung function decline. Currently, there are no effectives therapies for this disease. Fibrotic lungs are characterized by accumulation of smooth muscle α actin- (SMA) expressing myofibroblasts and excessive deposition of a collagen rich extracellular matrix. The differentiation of lung fibroblasts into myofibroblasts is stimulated by numerous growth factors, including transforming growth factor β (TGFβ), and potentiated by a stiff mechanical environment. Our laboratory has identified a secreted matrix protein, aortic carboxypeptidase-like protein (ACLP), which is upregulated in idiopathic pulmonary fibrosis. Additionally, ACLP knockout mice are protected from experimentally induced fibrosis. This led to the hypothesis that ACLP promotes the fibroblast to myofibroblast transition, and the goal of this research was to characterize the mechanism of ACLP action. ACLP expression preceded SMA and collagen type I expression in rapidly differentiating primary mouse lung myofibroblasts. In gain of function studies, recombinant ACLP induced SMA and collagen I expression in both primary differentiating myofibroblasts as well as IMR90 human lung fibroblasts. ACLP knockdown by siRNA slowed myofibroblast differentiation and partially reverted fully differentiated myofibroblasts into fibroblasts. Because of the similarities among ACLP targets and TGFβ targets, it was hypothesized that ACLP stimulates TGFβ signaling. In lung fibroblasts, ACLP induced Smad3 phosphorylation and nuclear translocation, a feature of TGFβ signaling. The effects of ACLP on myofibroblast differentiation were dependent on TGFβ receptor (TβR) kinase activity and ACLP interacted directly with T&betaR II to promote myofibroblast differentiation. A recombinant TβR II Fc chimera was used to inhibit ACLP-induced SMA expression, but this reagent had no effect on ACLP-induced collagen type I expression, which suggests a differential regulation of SMA and collagen by ACLP. Additionally, ACLP modulated changes in differentiation between cells grown on softer versus stiffer matrices. Using recombinant fragments of the ACLP protein, the N-terminal thrombospondin repeat domain was found to be necessary and sufficient to promote myofibroblast differentiation. Taken together, these studies identified a novel mechanism of ACLP action in fibroblasts and may lead to new therapeutic strategies to treat fibrotic disease.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23380号 / 医博第4749号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.

No description available.