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Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway
A Khwaja1, K Lehmann, B M Marte
1Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Abstract:
Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.
Insights
Hepatocyte growth factor/scatter factor (HGF/SF) triggers cell scattering and tubule formation via phosphoinositide 3-OH kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways. PI 3-kinase is key for scattering, while MAPK plays a supporting role.
Area of Science:
- Cell biology
- Molecular signaling
- Epithelial cell dynamics
Background:
- Hepatocyte growth factor/scatter factor (HGF/SF) is a key regulator of epithelial cell morphogenesis.
- Madin-Darby canine kidney (MDCK) cells are a widely used model for studying epithelial cell behavior.
- HGF/SF induces scattering and tubulogenesis, but the underlying signaling pathways are complex.
Purpose of the Study:
- To elucidate the specific roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-OH kinase (PI 3-kinase) pathways in HGF/SF-induced MDCK cell scattering and tubulogenesis.
- To identify downstream effectors involved in these morphological changes.
Main Methods:
- MDCK cells were treated with HGF/SF and specific pathway inhibitors (PD98059 for MAPK, LY294002 for PI 3-kinase).
- Constitutively active forms of signaling molecules (PI 3-kinase, Ras, R-Ras, Raf, PKB/Akt, Rac) were expressed.
- Cell scattering in monolayer and tubule formation in collagen gels were analyzed morphologically.
Main Results:
- HGF/SF induced cell scattering and tubulogenesis through prolonged activation of ERK2 (MAPK) and PKB/Akt (PI 3-kinase pathway).
- Inhibition of either MAPK or PI 3-kinase pathways blocked HGF/SF-induced scattering, with distinct morphological outcomes.
- PI 3-kinase activation, but not MAPK activation alone, was sufficient for scattering; however, basal MAPK activity was required.
- PI 3-kinase and R-Ras induced tubulogenesis, while Ras and Raf caused disorganized growth; MAPK inhibition also blocked tubulogenesis.
Conclusions:
- Multiple signaling pathways downstream of Met and Ras are involved in HGF/SF-induced morphological effects.
- PI 3-kinase pathway is the primary driver of cell scattering, utilizing effectors other than PKB/Akt or Rac, and requires basal MAPK function.
- PI 3-kinase activity is essential for tubulogenesis, whereas both complete inhibition and excessive activation of MAPK oppose this process.