Inhibition of NF-kappaB sensitizes A431 cells to epidermal growth factor-induced apoptosis,whereas its activation by ectopic expression of RelA confers resistance

Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically induces apoptosis in cells that overexpress its receptor. We demonstrate for the first time that the EGF-induced apoptosis is accelerated if NF-kappaB is inactivated. To inactivate NF-kappaB, human epidermoid carcinoma cells (A431) that overexpress EGF receptor were stably transfected with an IkappaB-alpha double mutant construct. Under the NF-kappaB-inactivated condition, A431 cells were more sensitive to EGF with decreased cell viability and increased externalization of phosphatidylserine on the cell surface, DNA fragmentation, and activation of caspases (3 and 8 but not 9), typical features of apoptosis. These results were further supported by the potentiation of the growth inhibitory effects of EGF by chemical inhibitors of NF-kappaB (curcumin and sodium salicylate) and the protective role of RelA evidenced by the resistance of A431-RelA cells (stably transfected with RelA) to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431 cells induced DNA binding activity of NF-kappaB (p50 and RelA) and the expression of c-IAP1, a downstream target of NF-kappaB. A431-RelA cells exhibited spontaneous phosphorylation of Akt (a downstream target of phosphatidylinositol 3-kinase and regulator of NF-kappaB) and EGF treatment stimulated it further. Blocking this basal Akt phosphorylation with LY294002, an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor an anti-apoptotic role for NF-kappaB in the regulation of EGF-induced apoptosis.     

Properties of primary mouse myoblasts expanded in culture

We found that the convergently epidermal growth factor (EGF)-induced signal and the collagen-induced signal activate mitogen-activated protein kinase (MAPK), which induces migration. We examined the signaling mechanisms of EGF-induced cell migration on collagen using the A431 carcinoma cell. EGF (10 ng/ml) induced migration on collagen, but inhibited proliferation. Using a MAPK cascade inhibitor, PD98059, it was shown that EGF-induced migration on collagen was mediated by MAPK whereas EGF-induced migration on fibronectin and vitronectin was not. PD98059 also showed that activation of MAPK induced by EGF enhanced the adhesiveness of A431 cells to collagen. By Western blotting analysis, the kinetics of MAPK phosphorylation induced by EGF and collagen was examined separately, and convergently. First of all, EGF without collagen caused transient MAPK phosphorylation. Collagen without EGF caused MAPK to be immediately and transiently dephosphorylated, and rephosphorylated followed by sustained hyperphosphorylation. EGF together with collagen caused an immediate, and sustained, hyperphosphorylation. These facts suggest that the transient MAPK dephosphorylation induced by collagen is required for migration in order to maintain an appropriate level of sustained phosphorylation. Furthermore, we found that adhesion of A431 cells to collagen was blocked by the anti-beta1 integrin antibody or by the mixed antibodies composed of anti-alpha1, -alpha2, and -alpha3 antibodies, indicating that collagen-induced MAPK phosphorylation was mediated through alpha1beta1, alpha2beta1, and alpha3beta1 integrins.     

CREB activity is required for epidermal growth factor‐induced mouse cumulus expansion

The release of a fertilizable oocyte from the ovary is dependent upon the expansion of the cumulus cells. The expansion requires cooperation between epidermal growth factor (EGF) family peptide‐activated mitogen‐activated protein kinase (MAPK)3/1 and oocyte paracrine factor‐activated‐Sma‐ and Mad‐related protein (SMAD)2/3 signaling in cumulus cells. However, the mechanism underlying (MAPK)3/1 signaling is unclear. In the present study, the EGF‐activation of EGF receptor (EGFR) induced cyclic adenosine 3′,5′‐monophosphate (cAMP) response element‐binding protein (CREB) phosphorylation in cumulus cells, and the interruption of CREB functional complex formation by naphthol AS‐E phosphate (KG‐501) completely blocked the EGF‐stimulated expansion‐related gene expression. EGF‐stimulated phosphorylation of CREB was completely inhibited by MAPK3/1 inhibitor U0126, suggesting that EGF‐activated MAPK3/1 results in the activation of CREB for cumulus expansion. Also, the role of EGF‐stimulated calcium signaling was studied. Calcium‐elevating reagents ionomycin and sphingosine‐1‐phosphate mimicked, but calcium chelators bis‐(o'aminophenoxy)‐ethane‐N,N,N,N‐tetraacetic acid, tetra(acetoxymethyl)‐ester, and 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate abolished the activity of EGF on CREB phosphorylation, cumulus expansion, and expansion‐related gene expression. Furthermore, EGF‐induced cumulus expansion was inhibited by calmodulin (CaM)‐dependent protein kinase II (CaMKII) inhibitors, KN‐93 and autocamtide‐2‐related inhibitory peptide. However, the inhibition of SMAD2/3 activity by removal of oocyte from cumulus–oocyte complexes did not affect the EGF‐induced CREB phosphorylation, indicating that EGF‐activated CREB is independent of oocyte‐activated SMAD2/3 signaling. Therefore, EGF‐induced CREB activity by MAPK3/1 and Ca²⁺/CaMKII signaling pathways promotes the expansion‐related gene expression and consequent cumulus expansion.