Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

9 1 Review of the literature. 11 1.1 Extracellular matrix (ECM). 11 1.1.1 Functions of the ECM. 11 1.1.1.1 ECM regulates cell signaling pathways through adhesion receptors. 11 1.1.1.2 ECM determines the mechano-physical properties of tissues. 15 1.1.1.3 ECM serves as a repository for signaling molecules. 16 1.1.2 Structural organization of the ECM. 18 1.1.2.1 Interstitial ECM. 19 1.1.2.1.1 Collagen matrix. 19 1.1.2.1.2 Elastin matrix. 20 1.1.2.1.3 Fibronectin matrix. 21 1.1.2.2 Basement membrane. 22 1.1.3 Structural remodeling of the ECM. 23 1.2 Transforming growth factor β (TGF-β). 24 1.2.1 Biological functions of TGF-β. 24 1.2.2 Synthesis and secretion as a large latent complex. 26 1.2. 3 Activation of latent TGF-β. 27 1.2. 4 TGF-β signaling. 28 1.2. 4.1 Classical Smad pathway. 29 1.2. 4.2 MAPK pathway. 30 1.3 Latent TGF-β binding proteins. 31 1.3.1 Structural characteristics of LTBPs. 31 1.3.2 Biological roles and disease associations. 32 1.3.3 Interactions with other ECM constituents. 34 1.4 Transcriptional regulation of gene expression. 35 1.4.1 Initiation of transcription. 35 1.4.2 Regulatory elements within a promoter sequence. 36 1.4.3 Transcriptional regulators. 37 1.4.3.1 Smad transcription factors. 37 2 Aims of the study. 39 3 Materials and methods. 40 3.1 Cell lines and reagents. 40 3.2 Construction of expression vectors. 42 3.3 Production and purification of recombinant LTBP-4 (II). 43 3.4 DNA analysis. 44 3.4.1 Transfection and promoter activity assay (I, III). 44 3.5 RNA analysis. 44 3.5. 1 RNA isolation and Northern hybridization (I, III). 44 3.5. 2 RLM-RACE analyses of transcription initiation sites (I, III). 44 3.5. 3 Quantitative PCR (III). 45 3.6 Protein analysis. 45 3.6. 1 Collection of cell conditioned media (II, III). 45 3.6. 2 Isolation of ECM (II). 45 3.6. 3 Immunoprecipitation (III). 46 3.6. 4 SDS-PAGE and immunoblotting (II, III). 46 3.6. 5 Immunofluorescence analysis (II, III). 46 3.6. 6 Heparin binding assays (II). 47 3.6. 7 ELISA binding assays (II). 47 3.6. 8 TGF-β activity assays (II). 47 3.7 Cell adhesion assays (II). 48 4 Results and discussion. 49 4.1 Characterization of human LTBP-3 promoter (I). 49 4.1.1 Identification of potential regulatory elements within the LTBP-3 promoter by sequence analysis. 49 4.1.2 Functional analysis of LTBP-3 promoter in cultured cells. 50 4.1.3 Smad and Erk pathways mediate the TGF-β induced transcriptional activation of LTBP-3 promoter. 51 4.2 Identification of ECM binding domains of LTBP-4S (II). 52 4.2.1 LTBP-4S has several heparin binding regions. 52 4.2.2 The C-terminal domain of LTBP-4 supports fibroblast adhesion and partially employs HSPGs. 53 4.2. 3 The N-terminal FN binding sites are critical for the ECM targeting of LTBP-4S. 54 4.2. 4 Disturbed LTBP-mediated sequestration of TGF-β exposes growth factor to increased activation. 55 4.3 Functional characterization of N-terminal variants of LTBP-4 (III). 56 4.3.1 Tissue distribution of LTBP-4 variants refers to specific roles in human tissues. 57 4.3.2 Two independent promoters control the expression of LTBP-4S and -4L. 57 4.3.3 LTBP-4S and -4L have significant differences in their secretion, processing and ECM targeting in cultured cells. 59 5 Perspective. 62 6 Acknowledgements. 64 7 References. 66