Abstract
Neuronal precursor cells are differentiated into functional neurons during embryonic development. Various processes regulate neuronal differentiation, including signal transduction, gene expression, and cell adhesion. PC12 cell line is a well-established model for studying neuronal differentiation. After nerve growth factor (NGF) treatment, activation of the receptor tropomyosin-related kinase A (TrkA) induces downstream signaling such as mitogen-activated protein kinases (MAPK) cascade, phosphatidylinositol 3 kinase (PI3K)-Akt, phospholipase C gamma (PLCγ) and ultimately leads to neurite outgrowth in PC12 cells. Our previous results have shown that SH2B1β-overexpressing PC12 cells (PC12-SH2B1β cells) enhance neurite outgrowth compared with control PC12 cells (PC12-GFP cells). On the other hand, cells overexpressing the dominant negative form of SH2B1β [PC12-SH2B1β(R555E) cells] significantly reduce NGF-induced neuronal differentiation. Nevertheless, the effect of SH2B1β on NGF-induced neurite outgrowth is solely on MAPK, PI3K-Akt and PLCγ signaling because PC12-SH2B1β(R555E) cells do not inhibit NGF initiated signaling pathways. Interestingly, we have found that PC12-SH2B1β(R555E) cells show much more aggregated phenotype than PC12-SH2B1β and PC12-GFP cells. This result reveals the possibility that SH2B1β may regulate NGF-induced neuronal differentiation by modulating cell adhesion. In line with this finding, our preliminary results suggest a subset of NGF responsive genes including N-cadherin, which participates in cell-cell adhesion, is differentially regulated by SH2B1β and SH2B1β(R555E). This thesis investigated the possible mechanisms by which SH2B1β and SH2B1β(R555E) would regulate NGF-induced neurite outgrowth. The data in this thesis suggest that overexpressing SH2B1β□reduces the expression of N-cadherin and its binding to β-catenin. Moreover, overexpressing SH2B1β□also□increases the phosphorylation of β-catenin on Y654, and disrupts the association between N-cadherin and β-catenin. Furthermore, in response to NGF, SH2B1β□stabilizes the interaction between N-cadherin and β-catenin likely through modulating cyclin-dependent kinase 5 (CDK5) and Src kinase and thus downregulating the level of pβ-catenin (Y654). In contrast, in the absence of NGF, overexpressing SH2B1β(R555E) increases the expression of N-cadherin and its binding to β-catenin through inhibiting the phosphorylation of β-catenin on Y654. Taken together, the data in this thesis suggest that overexpressing SH2B1β downregulates the N-cadherin-β-catenin complex in the absence of NGF to allow faster neurite initiation, but increases the complex formation in response to NGF likely to facilitate cell-cell communication during neuronal differentiation. On the contrary, overexpressing SH2B1β(R555E) increases the interaction between N-cadherin and β-catenin and ultimately blocks neurite outgrowth.