| Abstract |
Transforming growth factor β (TGFβ) is a pleiotropic cytokine which regulates several biological processes such as cell proliferation, cell cycle regulation, apoptosis, differentiation and other developmental processes. TGFβ is a member of a superfamily of cytokines encoded by 29 different genes in humans and signals via receptor serine/threonine kinases, termed type I and type II transmembrane receptors, that form complexes with the ligand on the cell surface resulting in activation of the dormant kinase activity of the type I receptor. This receptor then phosphorylates and activates members of the Smad family of tumour suppressors which act as transcriptional activators. The cellular response to TGFβ depends on its negative or positive cross talk with other signaling pathways which infuences the transcriptional activity of Smad proteins. The purpose of this Thesis, was to a) study the mechanisms of positive and negative cross talk of TGFβ with other signaling pathways and b) characterise regions of the Smad3 protein which are required for the transcriptional activation of gene expression. The first part of the Thesis was focused on the mechanisms of the TGFβ signaling suppression by the Latent Membrane Protein 1 (LMP1) of the Epstein-Barr virus. LMP1 is responsible for the tumourigenic action of Epstein-Barr on various cell types. Our studies revealed that LMP1 suppresses TGFβ signaling through activation of the transcriptional factor NF-kB, which either antagonizes Smad3 for the interaction with common co-activators such as p300, or/and interacts directly with Smad3 preventing its transcriptional activity in the nucleus. We exhibited physical, in vitro interactions between the 122-90 region of Smad3 and the RHD (Rel Homology Domain)of the p65 and p50 subunits of NF-kB. In the second part of the Thesis, we examined functional interactions detween Smad3 and the orphan nuclear receptor HNF-4 (Hepatocyte Nuclear Factor-4), that result in TGFβ-mediated transcriptional activation of specific hepatic genes, such as those of apolipoproteins. Smad3/4 proteins enhance HNF-4 trancriptional activity through ditect physical interactions with the AF-1 region (1-25 aa) of HNF-4. Mutagenesis of the hydrophobic aminoacids of the AF-1 region inhibited the Smad cooperative activity. Interestingly, not only HNF-4 but also Smad3/4 proteins are co-activated by members of the p160 family of coactivators (TIF2, SRC-1, RAC3) and by PGC1. In the third part of the Thesis, we performed a detailed functional analysis of the transcriptional active regions of Smad3 protein, which revealed that besides MH2 domain, a second region located in aa 200-248, that includes part of the linker and the first 18 aa of the MH2 domain, is essential for the transcriptional activity of Smads and for their interactions with other proteins. By using a recently discovered method of protein interactions based on biotinylation in vivo, we showed that the internal deletion of the 230-248 region and point mutation of the 238 aminoacid to alanine (E238A) which in Smad4 is related with tumourigenesis, inhibited the transcriptional capacity of Smad3 and its ability to homo- and hetero-polymerise with Smad4. Oppositively, internal deletion of the 200-230 region of the linker did not affect the polymerizing ability of Smad3 but prenented its interaction with the coactivator p/CAF. The above mentioned results indicate the importance of the anti-proliferative action of the TGFβ cytocine (through activation of the inhibitors of cell cecle) which is the target of the viral protein LMP1 in epithelial cells. Also, the results underline that TGFβ signaling is implicated in the expression of specific hepatic genes and conscequently in biolofical processes such as metabolism, lipid homoeostasis, reduction of cholesterol, through synergistic interactions of Smads with HNF-4. Cross talk of TGFβ signaling with hormone nuclear receptors (through functional interactions with their coactivators) indicates that TGFβ is involved in processes such as the function of the human reproductive system, or/and glycogen homoeostasis. The detailed mapping of regions that are responsible for the synergistic cooperativity between Smads and HNF-4 will be a useful tool in cases that TGFβ signaling is upregulated (fibrosis, hepatocyte tumourigenesis), or in cases where HNF-4 is malfunctioning (MODY I). Elucidation of the transcriptionaly active regions of Smad3 protein which are crucial for its interactions with other proteins could be important for construction of molecules that either enhance TGFβ signaling (when Smad proteins are mutated in cancers of the lung or gastrointentinal cancers), or suppress TGFβ signaling (when its function is upregulated in fibrosis and metastasis). These results are expected to provide new insights into the mechanisms of TGFβ signaling regulation which will further facilitate to devise new strategies for gene therapy of severe disorders such as fibrosis, atherosclerosis and cancer.
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