Epidermal Growth Factor-Induced Proliferation of Chicken Primordial Germ Cells: Involvement of Calcium/Protein Kinase C and NFKB

Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10–100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(betaaminoethyl ether)-N,N'-tetraacetic acid (EGTA; an intracellular Ca²⁺ chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H₇, or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H₇, and SN50, respectively. On the other hand, inhibition of EGFR, Ca²⁺/PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGFstimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca²⁺/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.