The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA expression and human diseases, such as cancer. The contribution of miRNAs to TGF-β-mediated cellular responses remains an open question. Thus, I became interested if miRNAs offered an additional layer of regulation in TGF-β signaling through which this cytokine exerts its pro-metastasis function. To address this inquiry, in the first part of this dissertation I investigated whether miRNAs influenced the ability of TGF-β to induce cellular responses directly involved with carcinoma metastasis, such as epithelial-mesenchymal transition (EMT). Here, I identified two miRNAs, miR-21 and miR-31, that are upregulated during EMT in LIM 1863 organoids, a colon carcinoma model of EMT driven by TGF-β. We performed in vitro studies to characterize the function of miR-21 and miR-31 and found that these two miRNAs positively impact the induction of EMT, migration and invasion by TGF-β. Furthermore, we uncovered TIAM1 (T lymphoma and metastasis gene 1) as a novel target of both miR-21 and miR-31 and show that downregulation of TIAM1 is critical for the pro-migration and pro-invasion activities of miR-21 and miR-31. Together these findings reveal miR-21 and miR-31 as downstream effectors of TGF-β signaling by facilitating EMT, migration and invasion of colon carcinoma cells. How TGF-β regulates miR-21 and miR-31 became important questions and thus the focus of the second part of this thesis. Interestingly, I found that TGF-β and TNF-α synergize to increase miR-21 and miR-31 levels in LIM 1863 organoids and that the synthesis of new factors induced by TGF-β/TNF-α are required for this upregulation. Moreover, I report that regulation of miR-21 by TGF-β/TNF-α occurs at multiple levels of biogenesis. More specifically data provided here show that Smad4 binds to the promoter of miR-21 to upregulate its expression thereby specifying miR-21 as a typical TGF-β target gene. This mechanism is different from one recently observed in smooth muscle cells in which TGF-β did not stimulate miR-21 transcription, but interestingly, Smad4 enhanced the Drosha-mediated processing of the miR-21 precursor. These two mechanisms suggest that TGF-β regulation of miR-21 is contextual and highlight the complexity of TGF-β signaling. As a whole, my findings establish important roles for miR-21 and miR-31 in TGF-β-mediated cellular responses that facilitate the pro-metastasis utility of TGF-β in colon cancer. Also, I describe a novel mechanism by which TGF-β/TNF-α signaling elevates the level of miR-21 and miR-31. Future studies that identify additional targets of miR-21 and miR-31 may offer further insight into the molecular mechanisms underlying cellular regulation by TGF-β. This information will be vital for the design of therapeutic interventions for colon cancer patients.

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The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

Transforming growth factor beta 3 (TGF3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGF3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGF3 in an in vitro model. TGF3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGF3 were not detected in the rinsing fluid of the TGF3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGF3 to assess its bioactivity. Both scaffold-associated TGF3 and TGF3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGF3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaold-associated TGF3 is preserved, thus TGF3 application via absorption in decellularized tendon scaffolds is a feasible approach.

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<p><strong> </strong>Fibrotic cataracts such as anterior subcapsular cataract (ASC) are induced by transforming growth factor beta (TGFβ). The mechanism which governs TGFβ-mediated ASC has not been elucidated. What is known is that TGFβ initiates the conversion of lens epithelial cells (LECs) to myofibroblast-like cells, through a process known as epithelial to mesenchymal transition (EMT). TGFβ-induced EMT leading to ASC has been associated with the upregulation of two matrix metalloproteinases (MMPs), MMP2 and MMP9. However, roles for either of these MMPs have yet to be established in ASC. To determine the involvement of MMP2 and MMP9 I used synthetic inhibitors in conjunction with an established <em>ex vivo </em>rat lens model initiated by TGFβ. The results demonstrated that co-culturing rat lenses with TGFβ and the matrix metalloproteinase inhibitor (MMPI), GM6001 or an MMPI specific for MMP2/9 suppressed ASC. Additionally, studies conducted on the conditioned media from these treatments revealed that TGFβ induces the cleavage of E-cadherin ectodomain which is suppressed by coculturing with either MMPI. To further delineate a role for MMP9 <em>in vivo</em>, ASC formation was examined in two models of lens specific TGFβ overexpression in the absence of functional MMP9. Adenoviral delivery of TGFβ to the anterior chamber of the eye in the absence of functional MMP9 resulted in complete suppression of ASC. Similarly, lens specific TGFβ overexpression in the absence of MMP9 suppressed ASC in 75% of mouse lenses. Additional studies determined that connective tissue growth factor is able to mediate ASC, albeit to a lesser degree than TGFβ.</p> / Doctor of Philosophy (PhD)