Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

Abstract

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.

Inhibition of SMAD2 expression prevents murine palatal fusion

“…The palatal tissues were collected at E13ϩ24h, and the MEE midline region was dissected to extract the proteins Yamamoto et al, 2003). To demonstrate the consistent reduction of SMAD2/p-SMAD2 level, three independent specimens from each group were processed together and run in the same gel.…”

“…This result provides evidence that siRNA inhibition of SMAD2 resulted in the maintenance of MEE cell proliferation. Persistent MEE cell proliferation has been linked to mechanisms that result in a failure of the normal program for palatal fusion Yamamoto et al, 2003;.…”

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

“…EGF and TIMPs (tissue inhibitors of matrix metalloproteinases) may be intrauterine epigenetic modifiers for MEE cell differentiation, because they are contained in amniotic fluid (Sundell et al, 1980;Murphy et al, 1981;Bunning et al, 1984;Kelly, et al, 1997) and can inhibit MEE cell disappearance in the fusing palate in vitro (Dixon and Ferguson, 1992;Blavier et al, 2001;Yamamoto et al, 2003). However, since amniotic fluid could prevent MEE cell differentiation only when the MEE was exposed directly to the fluid, palatal shelf contact and midline seam formation are prerequisite for MEE cell differentiation as well as palatal fusion to occur in utero (Fig.…”

Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

“…ERK1/2 can be activated by various means, all of which result in the activation of various transcriptional factors and ser/thr kinases that contribute to cellular proliferation, differentiation, cell cycle regulation, and cell survival (Chen et al, 1992;Gonzalez et al, 1993;Lenormand et al, 1993). It is evident that MAPK cascades are involved in growth factor-regulated cell proliferation during morphogenesis of quail secondary palate, where EGF-stimulated proliferation of primary cell cultures was accompanied by proliferation and activation of 42-kDa MAPK (Hehn et al, 1998;Yamamoto et al, 2003).…”