EGF‐stimulated signaling by means of PI3K, PLCγ1, and PKC isozymes regulates branching morphogenesis of the fetal mouse submandibular gland

Abstract

Branching morphogenesis of fetal mouse submandibular glands (SMGs) partly depends on the epidermal growth factor (EGF) receptor that triggers at least three intracellular signaling pathways involving (1) the mitogen-activated protein kinases ERK-1/2, (2) phospholipase C␥1 (PLC␥1), and (3) phosphatidylinositol-3-kinase (PI3K). PLC␥1 directly activates protein kinase C (PKC) isozymes; PI3K stimulates protein kinase B (PKB, also known as Akt), which ultimately activates PKCs and other proteins. We reported that the pattern of phosphorylation of ERK-1/2 in response to EGF in SMGs varies with fetal age and that blockade of EGF-stimulated ERK-1/2 signaling partially inhibits branching (Kashimata et al. [2000] Dev. Biol. 220:183-196). Here, we report on components of the PLC␥1, PI3K, and PKC families of signaling molecules in fetal SMGs from the 13th day of gestation to postnatal ages. Western blotting revealed that (1) PLC␥1 is present from E13 to E18 but drops off precipitously to negligible levels on the day of birth and thereafter, and (2) PI3K, PKB(Akt), and several PKC isozymes are expressed from E13 onward through adult life. Both PLC␥1 and PI3K are phosphorylated in response to EGF. Inhibition of PI3K by LY294002 inhibited EGF-stimulated branching, but inhibition of PLC␥1 by U73122 had no effect. Western blotting showed that the concentrations of 8 PKC isozymes vary with age in the fetal and postnatal SMG. However, general inhibition of PKCs by Calphostin C or specific inhibition of PKC␣ or of PKC⑀ by Gö 6976 or Ro-32-0432, respectively, increased EGF-stimulated branching. Calphostin C also increased EGF-stimulated phosphorylation of ERK-1/2. These findings indicate that signaling from the EGF receptor in the fetal mouse SMG varies with development and triggers stimulatory effects by means of ERK-1/2 and PI3K but inhibitory effects by means of PKC isozymes.

P2Y2 nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells

“…It is well-established that MAPKs, including JNK and ERK1/2, regulate cell proliferation, migration, and differentiation (15,18,21,36,63,72,86,102,120), processes important for salivary gland morphogenesis (60,70) and regeneration of a wide variety of tissues (19,24,50,53,54,56,65,71,80,100,105,107,111,122,124). The P2Y 2 R-mediated activation of ERK1/2 has been reported in HSG cells (94), corneal epithelial cells (13), and human coronary artery endothelial cells (HCAEC) (29).…”

Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium

“…LPA is reported to cross-talk with EGF signaling in activating Ras-MAP kinase, which results in the initiation of DNA synthesis (Moolenaar et al, 2004); thus, it seems likely that the same mechanism works in the branching morphogenesis of submandibular epithelium. However, branching morphogenesis is a complex event that includes cell contraction and migration as well as cell proliferation, and LPA and EGF signaling may work cooperatively in other cascades, such as the activation of phospholipase C␥1 and phosphatidylinositol-3-kinase, which may be involved in submandibular branching morphogenesis (Koyama et al, 2003;Larsen et al, 2003;Anliker and Chun, 2004;Moolenaar et al, 2004). An additional consideration is that LPA stimulates cell binding to fibronectin (Zhang et al, 1994).…”

“…The salivary epithelium thereby was proved to perform branching morphogenesis when embedded in a laminin-rich basementmembrane-like matrix and stimulated by epidermal growth factor (EGF; Nogawa and Takahashi, 1991). Since then, many studies have reported the involvement of EGF family ligands (transforming growth factor ␣, heparin-binding EGF, and neuregulin) and laminins in the branching morphogenesis of the salivary gland (Kadoya et al, 1995;Kashimata and Gresik, 1997;Hosokawa et al, 1999;Kashimata et al, 2000;Umeda et al, 2001;Koyama et al, 2003;Miyazaki et al, 2004;Kadoya and Yamashina, 2005). During the development of the mesenchyme-free culture of salivary epithelium, we introduced a serum-supplemented medium but paid little attention to the serum, because we had typically added serum as a source of nutrients for in vitro organ cultures.…”

Salivary Gland Branching Morphogenesis — Recent Progress and Future Opportunities

“…A recent study has attempted to dissect the signaling components that contribute to the epithelial phenotypes induced by EGF, FGF7, and FGF10 (Koyama et al , 2008). Previous studies had shown that a number of signaling pathways are important for SMG branching morphogenesis, including mitogen-activated protein kinases (ERK-1/2), phospholipase Cγ1 (PLCγ1), and phosphatidylinositol-3-kinase (PI3K) (Kashimata et al , 2000; Koyama et al , 2003; Larsen et al , 2003; Steinberg et al , 2005). Comparisons of the ability of these three growth factors to stimulate each of these pathways, and importantly the ability of specific inhibitors to block their induced epithelial phenotypes, suggest that ERK-1/2 (stimulated by EGF and FGF7) is important for bud formation, whereas PLCγ1 (stimulated by FGF7 and FGF10) may be important for bud/duct elongation.…”