Beta-Catenin Downregulation Contributes to Epidermal Growth Factor-induced Migration and Invasion of MDAMB231 Cells

We previously demonstrated that epidermal growth factor (EGF) enhances cell migration and invasion of breast cancer cells in a SMAD ubiquitination regulatory factor 1 (SMURF1)-dependent manner and that SMURF1 induces degradation of β-catenin in C2C12 cells. However, the relationship between EGF-induced SMURF1 and β-catenin expression in breast cancer cells remains unclear. So, we investigated if EGF and SMURF1 regulate β-catenin expression in MDAMB231 human breast cancer cells. When MDAMB231 cells were incubated with EGF for 24, 48, and 72 hours, EGF significantly increased expression levels of SMURF1 mRNA and protein while suppressing expression levels of β-catenin mRNA and protein. Overexpression of SMURF1 downregulated β-catenin mRNA and protein, whereas knockdown of SMURF1 increased β-catenin expression and blocked EGF-induced β-catenin downregulation. Knockdown of β-catenin enhanced cell migration and invasion of MDAMB231 cells, while β-catenin overexpression suppressed EGF-induced cell migration and invasion. Furthermore, knockdown of β-catenin enhanced vimentin expression and decreased cytokeratin expression, whereas β-catenin overexpression decreased vimentin expression and increased cytokeratin expression. These results suggest that EGF downregulates β-catenin in a SMURF1-dependent manner and that β-catenin downregulation contributes to EGF-induced cell migration and invasion in MDAMB breast cancer cells.

TNFalpha Increases the Expression of beta2 Adrenergic Receptors in Osteoblasts

Tumor necrosis factor alpha (TNFalpha) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that beta2 adrenergic receptor (beta2AR) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether TNFalpha modulates betaAR expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by TNFalpha. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of betaAR, beta2 and beta3AR were found in our analysis to be upregulated by TNFalpha. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in TNFalpha-primed C2C12 cells, indicating that TNFalpha enhances beta2AR signaling in osteoblasts. TNFalpha was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a beta2AR antagonist, attenuated this TNFalpha suppression of osteogenic differentiation. TNFalpha increased the expression of receptor activator of NF-kappaB ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that TNFalpha increases beta2AR expression in osteoblasts and that a blockade of beta2AR attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by TNFalpha. These findings imply that a crosstalk between TNFalpha and beta2AR signaling pathways might occur in osteoblasts to modulate their function.