Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway

Abstract

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3β (GSK-3β) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGFinduced phosphorylation of Akt and GSK-3β, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3β, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3β pathway.

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

“…Silencing of ␤-arrestin 1 and ␤-arrestin 2 expression was assessed by immunoblotting using anti-␤-arrestin 1 (1: 2000; EMD Millipore, Billerica, MA) and anti-␤-arrestin 2 (1:1000; Novus Biologicals, Littleton, CO) antibodies, respectively. Hippocampal cultures were prepared as described previously (30). In brief, dissociated hippocampal neurons from embryonic day 18 rats were plated on 15-mm-diameter coverslips coated with poly-D-lysine (Sigma-Aldrich) and laminin (ATCC, Manassas, VA) in Neurobasal medium supplemented with B-27, 25 M glutamate, 500 M glutamine (Invitrogen), and 2-mercaptoethanol at a density of ϳ150/mm 2 and maintained at 37°C in a humidified 5% CO 2 incubator.…”

Allosteric Modulator ORG27569 Induces CB1 Cannabinoid Receptor High Affinity Agonist Binding State, Receptor Internalization, and Gi Protein-independent ERK1/2 Kinase Activation

“…24 h post-transfection, membranes of transfected cells expressing the wild-type or mutant receptors were prepared as described previously (28). Hippocampal cultures were prepared as described previously (29). In brief, dissociated hippocampal neurons from embryonic day 18 rats were plated on 15-mm-diameter coverslips coated with poly-D-lysine (Sigma-Aldrich) and laminin (ATCC, Manassas, VA) in Neurobasal medium supplemented with B-27, 25 M glutamate, 500 M glutamine (Invitrogen), and 2-mercaptoethanol at a density of ϳ150/mm 2 and maintained at 37°C in a humidified 5% CO 2 incubator.…”

c-Met and NF-κB–Dependent Overexpression of Wnt7a and -7b and Pax2 Promotes Cystogenesis in Polycystic Kidney Disease

“…14,24,25 In turn, the NF-kB family of transcription factors is a known mediator acting downstream of Akt. [26][27][28] Luciferase reporter activity expressed from an NF-kBresponsive promoter was increased over fivefold in Pkd1 2/2 cells compared with Pkd WT cells and was sensitive to SU11274 (Figure 6A).…”