The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF

We have previously reported that epidermal growth factor (EGF) stimulates b ranching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kas himata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF rec eptor (EGER) is localized principally, if not exclusively, on the epithelia l components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Ma thews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651- 1657). The EGFR is a receptor tyrosine kinase, and after binding of its lig and, it triggers several intracellular signaling cascades, among them the o ne activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimula te branching morphogenesis in the fetal mouse SMG. SMG rudiments were colle cted as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); plac ed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induce d the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to t he phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots show ed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/ 2 in response to EGF. Immunohistochemical staining for phosphotyrosine incr eased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2 by itself inhibited branching mo rphogenesis and, furthermore, decreased the stimulatory effect of EGF on br anching. Western blots confirmed that this inhibitor blocked phosphorylatio n of ERK-1/2 in fetal SMGs exposed to EGF. These results show that componen ts of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this casc ade perturbs branching morphogenesis. However, EGF did not cause phosphoryl ation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SM G. (C) 2000 Academic Press.