Abstract not available

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Adult neurogenesis is defined as the generation of new nerve cells in the adult central nervous system (CNS). Stimulating neurogenesis may potentially offer a therapeutic approach for neurodegenerative diseases such as Parkinson???s disease. However, it is not clear why neurogenesis does not normally replace neurons lost in these diseases. As a first step to address this problem it is necessary to identify mechanisms that regulate adult neurogenesis in the normal and diseased brain and further, determine if manipulating these mechanisms may offer therapeutic potential. In this thesis, we identify activin A, a member of the transforming growth factor ?? (TGF??) superfamily, as a significant regulator of neurogenesis. We demonstrate that mRNA encoding activin A is expressed after a KA injury, and that inhibition of this activin A profoundly impairs neurogenesis in the hippocampus. Further we demonstrate that activin A impairs gliosis and also has potent anti-inflammatory effects in the injured hippocampus. Finally, we provide evidence that the majority of activin A???s neurogenic effect results from its potent anti-inflammatory actions. Our study draws a clear link between neurogenesis and inflammation in the CNS and is the first to provide evidence that this process is regulated through activin signalling. Since inflammation is now believed to be an important component of many neurological diseases we suggest that therapeutic compounds that enhance activin A signalling may offer a therapeutic approach for treating these diseases by suppressing inflammation and stimulating neurogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Liver fibrosis is the result of different types of chronic liver diseases, such as cholestatic liver disease and nonalcoholic steatohepatitis, among others. Fibrosis, if left unchecked, may progress to the point of cirrhosis – permanently affecting liver function detrimentally and potentially leading to development of hepatocellular carcinoma. Inflammatory response following tissue injury is vital for the initiation of fibrosis; chronic inflammation results in abnormal tissue healing and promotes a pro-fibrogenic response. Activins are cytokines that have been identified as members of the TGFβ superfamily of growth and differentiation factors. Activin A and B, in particular, have been identified as having roles in the pathophysiology of liver disease, but have not been investigated thoroughly. We treated mice with concanavalin A, a potent T-cell mitogen with liver specificity when administered intravenously, and characterized the resulting response to liver injury and how activin A and B are modulated during this acute inflammatory phase. We showed that activin B is highly increased in circulation following inflammation, as well as locally in the liver as well as the spleen. We then neutralized activin A and B via neutralizing antibodies in our concanavalin A-induced liver injury model to determine if inhibition of these ligands may confer protective effects during the acute inflammatory response in liver. Neutralization of either activin A or activin B protected hepatocytes, improved liver function, and significantly reduced circulating cytokines following concanavalin A administration. Finally, we determined whether inhibition of activin A or B might prevent or reverse the development of liver fibrosis after disease has been established. We induced liver fibrosis in mice via the hepatotoxin carbon tetrachloride, and then treated with neutralizing antibodies while still maintaining carbon tetrachloride administration. Neutralization of activin A and B markedly reduced liver fibrosis, protected hepatocytes, and improved liver function. Our findings implicate both activin A and B as major players in the acute inflammatory response to liver injury, as well as during chronic injury and fibrogenesis, and demonstrate the therapeutic potential of targeting these ligands for the treatment of fibrosis in chronic liver diseases.

Thesis (M. Sc.)--York University, 1999. Graduate Programme in Biological Science. / Typescript. Includes bibliographical references (leaves 71-85). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ39215.

DKD is the leading cause of kidney failure in Canada and its patients suffer the highest morbidity and mortality rates of any kidney failure patient group. Current interventions including strict glycemic control only delay DKD. Thus, there is a major need to identify new therapeutic targets. High glucose (HG) is identified as a major pathogenic factor, inducing the release of growth factors leading to kidney fibrosis. Although treatments have been developed to target these factors, their effectiveness is accompanied by adverse effects due to the lack of specificity. Recently, activins have been suggested to have a prominent role in promoting renal fibrosis and developing a specific anti-activin therapy can avoid potential side effects. Although there is evidence supporting an important role for activin A (ActA) in the induction of fibrosis in DKD, whether ActB also contributes is unknown. In this study, we aim to determine the potential contribution of ActB to promoting fibrosis. Our results show that ActA and ActB are upregulated in rodent and human DKD. We show that hyperglycemia leads to the secretion of ActA and ActB by mesangial cells (MC), whereas only ActB is secreted by renal fibroblasts (RF). Similar to HG, treatment with ActA or ActB leads to Smad2/3 activation and upregulation of extracellular matrix proteins, whereas specific inhibition of either ActA or ActB attenuates these effects. We show that ActA and ActB regulate HG-induced activation of MRTF-A/SRF in MC, leading to an activated phenotype characterized by increased α-SMA expression and ECM production. Lastly, we confirm the specificity and functionality of the activin propeptides in vitro, providing evidence for their effectiveness in vivo. This study will help further our knowledge of the role activins in DKD, potentially providing an alternative therapy. / Thesis / Master of Science (MSc) / As the leading cause of end stage renal disease, diabetic kidney disease (DKD) is described as the reduction in renal function due to chronic exposure to diabetes. This thesis is aimed to understand the pathways and mechanisms that contribute to the development and progression of DKD to help identify novel therapeutic options. This project identified activin B (ActB) as a contributor to the disease and gives evidence that blocking the actions of ActB can prevent profibrotic effects in cells, similar to the profibrotic effects seen in DKD. Furthermore, this thesis demonstrates preliminary evidence for the beneficial effects of anti-ActB therapy, providing a potential alternative therapeutic option for DKD patients.

Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

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