Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.

The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex. In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2IDR). Biophysical analyses show that MBD2IDR is an intrinsically disordered region (IDR). Despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical area of contact requiring two contiguous amino acid residues, Arg286 and Leu287. Mutation of these critical residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene Prostasin in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function. In Chapter 3, we have discussed a novel mechanism for MBD2-mediated silencing of the fetal γ-globin gene. Through microarray expression analyses in adult erythroid cells of MBD2-/- mice, we identified ZBTB32 and miR-210 as downstream targets of MBD2. Over-expression of ZBTB32 and miR-210 in adult erythroid cells causes increased expression of the silenced fetal γ-globin gene. Thus, our results indicate that MBD2 may regulate γ-globin gene expression indirectly though ZBTB32 and miR-210 in adult erythroid cells.