Date of Award
Doctor of Philosophy
Judith A Cole
Charles A Lessman
Intermediate filament proteins (IF) are cytoskeletal proteins that are primary determinants of the mechanical strength of tissues. IF proteins also modulate signaling pathways involved in cell death, survival, and growth. These proteins are expressed in a tissue specific manner and their regulation is strictly controlled in embryonic development and pathological conditions. Synemin is an IF protein that, unlike other IF proteins, is expressed in a broad range of tissues rather than in specific cell types. Synemin is expressed in all muscle types, glial cells of the retina and optic nerve, hepatic stellate cells, some mature neurons of the peripheral nervous system, and in astrocyte progenitors. The expression of synemin in astrocyte precursor cells is developmentally regulated as these cells stop expressing synemin after they mature into astrocytes. However, synemin is re-expressed in mature astrocytes during brain injury and when astrocytes become malignant (astrocytomas). Surprisingly, information about the factors and/or signaling pathways regulating synemin expression in development and disease is lacking.Therefore, one of our goals was to determine which factors regulate synemin protein levels in different tumor cells. To this end, we treated U373-MG astrocytoma, HeLa cervical carcinoma, and N2a neuroblastoma cells with factors that regulate expression of IF proteins other than synemin in tumor cells, such as transforming growth factor-α (TGF-α), all-trans retinoic acid (ATRA), and γ-secretase inhibitor-1 (GSI-1). Following each treatment, the protein levels of the two major synemin isoforms (α and ß) were compared between control and treated cells by densitometry of Western blots. Our results demonstrated that the levels of synemin are regulated by these agents in a cell-type dependent manner. TGF-α upregulated synemin in astrocytoma cells, but not in carcinoma or neuroblastoma cells. This suggests that synemin is expressed during the malignant transformation of astrocytoma due to regulation by factors such as TGF-α. On the other hand, ATRA and GSI-1, decrease synemin levels in carcinoma and neuroblastoma cells, but not in astrocytoma cells. Since mature, normal epithelial cells and peripheral neurons express little synemin, these results suggest that synemin is expressed in carcinoma and neuroblastoma due to the absence of differentiation factors in the tumor environment.With regards to the role of synemin in tumors, our laboratory had shown that synemin positively regulates the proliferation of astrocytoma cells by antagonizing protein phosphatase 2A (PP2A). This prevents PP2A from inactivating Akt, a kinase that promotes astrocytoma proliferation. These previous studies also showed that synemin and PP2A co-immunoprecipitate. However, these experiments did not reveal in which cellular compartments these interactions took place. To answer that question and to examine in-situ the interaction between synemin and PP2A, we used the newly developed proximity ligation assay (PLA). Initial experiments showed that PLA is a very sensitive technique. Therefore, we initiated experiments to optimize the conditions of the assay, and in particular, titrating the dilutions of the different antibodies, as well as developed guidelines for the use of proper controls. Using these optimized PLA conditions, we determined that synemin and PP2A interaction was localized mainly in the nuclear region in astrocytoma cells. PLA also revealed that when synemin is expressed, there is very little interaction between PP2A and Akt in astrocytoma cells. However, when synemin is downregulated by shRNA, PP2A is distributed in the cytoplasm of these cells. Also, there is increased interaction between PP2A and Akt in synemin silenced astrocytoma cells. Therefore, these results suggest that synemin binding to PP2A plays a role in determining the cellular distribution of PP2A and in sequestering PP2A from Akt. This explains that in synemin silenced astrocytoma cells, Akt remains phosphorylated and active, enabling the astrocytoma cells to enter the cell cycle and proliferate. We also used PLA to demonstrate that PP2A interaction with synemin involves amino acids 700-1028 in the C-terminus of synemin, a region that has not previously been found to bind with synemin interacting partners. These PLA data were validated by proliferation rescue experiments.Therefore, based on our results we conclude that synemin functions as a scaffolding protein in astrocytoma cells. By binding to PP2A, synemin spatially regulates its activity to control astrocytoma proliferation by regulating Akt phosphorylation and activity. Our work also demonstrates the utility of the PLA technique to study interactions between synemin and its binding partners in-situ. These studies establish PLA as a valuable tool in studying interactions between IF proteins other cellular proteins.
dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Paul, Madhumita, "Intermediate Filament Protein Synemin: Regulation by Exogenous Factors and Role in Astrocytoma Cell Proliferation through Interaction with Protein Phosphatase 2A" (2016). Electronic Theses and Dissertations. 1514.