Wang, Jia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 99-115). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an <i>in vitro </i>and <i>in vivo </i>perspective<i>.</i></p><p>The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration. </p><p>Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity.</p><p>Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs. </p>

Transforming growth factor (TGF) β is a tumor suppressor during early tumor development, by inhibiting proliferation and inducing apoptosis. At later stages of cancer, it becomes a tumor promoter, and promotes tumor cell migration and invasion. TGFβ signals via its type II and type I receptors to several downstream signaling pathways. In the present work we have focused on the TRAF6 (tumor necrosis factor receptor-associated factor 6)/ TAK1 (TGFβ activated kinase 1) signaling pathway and the Smad7-dependent activation of p38 in prostate carcinoma cells (PC3U). We found that TGFβ-induced activation of the ubiquitin ligase TRAF6 was needed for cell invasion, by a mechanism that involves activation of the metalloproteinase TNFα converting enzyme (TACE), via protein kinase Cζ (PKCζ). TACE cleaves the TβRI, whereafter the intracellular domain (ICD) translocates to the nucleus, where it binds to the transcriptional co-activator p300 and regulates gene expression, promoting invasion. Interestingly, the translocation of the TβRI ICD was observed in several cancer cell lines and in sections of primary tumors, but not in primary prostate epithelial cells. We also found that Smad7 and adenomatous polyposis coli (APC) are important for TGFβ- and epidermal growth factor (EGF)-induced cell migration in PC3U cells. TGFβ induces the formation of a complex consisting of Smad7, p38, glycogene synthase kinase 3β (GSK-3β), APC and β-catenin, which localizes to the membrane ruffles in the leading edge of migrating cells. The complex links the TβRI to the microtubule system and promotes membrane ruffling and microtubule polarization, which are known to be important for cell migration. In the EGF signaling pathway, Smad7 was found to be important for phosphorylation of the EGF receptor at Tyr1068, for the activation of p38 and JNK, and for induction of membrane ruffles. Smad7 is required for TGFβ-induced activation of p38 and apoptosis. We found that Smad7 forms a complex with p38 and ataxia telangiectasia mutated (ATM), which is important for activation of p53 mediated apoptosis. Many tumor cells including the PC3U cells lack a functional p53, which is one of the reasons to why cancer cells can avoid the tumor suppressor effects of TGFβ.

Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an in vitro and in vivo perspective. The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration. Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity. Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

No description available.

Smad7作為轉化生長因數-β信號通路中的負性調節因子為人所知，異常的Smad7表達通常會引發癌症及組織纖維化等疾病。而目前對於其在骨重建及其相關疾病中的作用尚未有研究。本研究利用Smad7部分敲除小鼠來探索Smad7在骨重建，骨質疏鬆以及間充質幹細胞分化等方面的作用。 / 本研究所用的Smad7部分敲除小鼠模型來源於已有報導過的Smad7ΔE1(KO)小鼠。該小鼠體內Smad7基因組外顯子I的翻譯區被替換，導致部分蛋白失及其功能破壞。研究結果表明，KO小鼠在6、12、24周齡時股骨遠端幹骺端均有不同程度下降的骨小梁數目、厚度，骨礦化率，骨密度，骨體積分數，及其上升的骨小梁間隙和破骨細胞表面。骨髓來源間充質幹細胞的多向分化實驗表明，KO組呈現出抑制性的成骨能力，表現為鈣結節形成減少，鹼性磷酸酶活性下降，早晚期成骨標記基因表達下降。該組亦表現出促進性的成脂能力，有較多及較早的脂滴形成，成脂標記基因表達上升。而對於骨髓來源巨噬細胞的體外破骨誘導實驗表明，KO組有更多且更大的破骨細胞形成，較大的骨吸收面積，以及上升的破骨標記基因表達。卵巢切除小鼠模型的研究表明，術後4、8、16周，KO组的股骨遠端幹骺端对比野生组有更大程度下降的骨形态学参数，以及明顯升高的破骨細胞融合標記蛋白的表達。體外實驗表明KO组有更多且更大的破骨細胞形成，以及更大面積的骨吸收。積雪草酸曾被證實在肝纖維化模型中誘導Smad7 基因的表達，也在本實驗中用以研究對骨質疏鬆疾病的作用。卵巢切除動物模型連續給藥8周後，骨質疏鬆的現象有明顯逆轉，表現為升高的骨形态学参数，及下降的股骨內破骨細胞融合標記蛋白的表達。 / 總結，本研究證實了Smad7在骨骼發育重建及骨疾病的病理機理等方面的研究提供了突破性的見解。部分敲除Smad7可以導致抑制性的成骨能力，促進性的破骨能力，以及損傷性的骨重建，亦會加速骨質疏鬆的進程，并可作為全新的藥物治療靶點，提示Smad7 本身對於骨重建及骨代謝的保護性作用，為代謝性骨疾病的研究及其臨床藥物開發提供了更廣泛的前景。 / Smad7 has been well documented as a negative regulator of TGF-β signaling, and its altered expression often leads to human diseases such as cancer and fibrosis. However, the role of Smad7 in regulating bone remodeling and related diseases remains unclear. We performed both in vivo and in vitro experiments as well as disease model and drug therapy studies using both wild-type (WT) and Smad7ΔE1 (KO) mice to investigate the functional role of Smad7 in bone remodeling, osteoporosis, and MSCs differentiation. / The Smad7ΔE1 mice were generated by replacing part of the exon1 of Smad7 gene as reported, which resulted in truncated protein and partial loss of Smad7 function. Mice were genotyped by PCR. The μ-CT, histological assays and bone histomorphometric assays in metaphysic region of the femurs showed lower trabecular number (TbN), trabecular thickness (TbTh), mineral apposition rate (MAR), higher trabecular separation (TbSp) and Osteoclast Surface (Oc.S/BS & Oc.N/BS) in the KO mice at 6, 12, to 24 weeks old; as well as lower bone mineral density (BMD) and bone volume fraction (BV/TV) at 24 weeks old in the KO mice. The in vitro BM-MSCs multi-lineage differentiation studies showed the suppressed osteogenic potential in the KO group with fewer mineralized nodules, lower ALP activity and expression of Col1A1, Runx2 and OCN; while the adipogenic potential was elevated with more lipid droplets formation and higher expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was also elevated, showing higher osteoclasts activity and larger resorptive areas, as well as elevated expression of TRAP and CTR. Both in vivo and in vitro studies of the osteoporotic models showed that the KO mice had lower BMD, TbTh, and higher TbSp compared to the WT mice at 4, 8, 16 weeks after OVX, similar results of lower BV/TV and TbN were observed at 4 weeks after OVX in the KO mice. The RANKL-induced osteoclastogenesis potential was elevated compared to WT mice, with more and bigger osteoclasts, larger resorptive areas, as well as elevated expression of TRAP and CTR. The osteoclastic cell fusion was also enhanced. Treatment of Asiatic acid (one traditional Chinese medicine that has been proved to induce the expression of Smad7 as reported) in the OVX mice reversed the osteoporotic process with increase BMD, BV/TV, TbN, TbTh, and decreased TbSp compared to the untreated group. The osteoclastic cell fusion was suppressed after AA treatment. / Partial loss of Smad7 function leads to impaired bone remodeling in vivo, reduced osteogenesis and enhanced osteoclastogenesis in vitro, and also accelerates the osteoporotic development and osteoclastic cell fusion. Asiatic acid may be a novel potential drug for prevention of osteoporosis. Our findings provide new evidences for a better understanding of the biological functions of Smad7 in bone remodeling and its therapeutic potential for metabolic bone diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Nan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-153). / Abstracts also in Chinese.

<p>Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth <i>in vivo</i> as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates <i>in vitro</i> were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with ¹⁸F, ¹¹C or ³H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. </p><p>Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. </p><p>Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. </p><p>In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.</p>

Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth in vivo as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates in vitro were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with 18F, 11C or 3H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.