Elsevier

Cellular Signalling

Volume 20, Issue 5, May 2008, Pages 825-835
Cellular Signalling

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

https://doi.org/10.1016/j.cellsig.2007.12.013Get rights and content

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 HGF-induced 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.

Introduction

Growth factors and their associated receptor protein tyrosine kinases can regulate a variety of neuronal functions such as dendrite formation, neurite extension, and migration [1], [2], [3]. The importance of hepatocyte growth factor (HGF) and c-Met in development and maintenance of neural circuits is just beginning to be recognized. HGF promotes neurite outgrowth, stimulates dendrite growth in mature neurons, and guides axons to targets [4], [5], [6], [7], [8], [9], [10]. In addition, c-Met is clustered at excitatory synapses and can stimulate expression of synaptic proteins and promote their clustering at synapses [11].

The downstream signaling pathways by which HGF and c-Met can enhance dendritic development have not been studied. Several downstream signaling proteins activated by growth factor receptors, such as Akt, calcium/calmodulin-dependent protein kinase II (CaMKII), p44/42 mitogen activated protein kinase (MAPK), and glycogen synthase kinase-3β (GSK-3β), are crucial regulators of dendritic growth and development [12], [13], [14], [15], [16]. Recently Akt has been implicated in signaling downstream of c-Met [17], [18]. Among the downstream targets of Akt, GSK-3β, which is inactivated upon phosphorylation by Akt, can regulate microtubule dynamics by phosphorylating downstream targets, including microtubule-associated protein 2 (MAP2) [19], [20]. MAP2 binds to and stabilizes microtubules and thus can regulate microtubule-dependent changes in axons and dendrites [21]. Phosphorylation of MAP2 by GSK-3β inhibits its association with microtubules and decreases microtubule stability [22], [23].

Although the functions of HGF and c-Met in regulating neurite growth and dendritic morphogenesis in neurons suggest crucial roles for these molecules during development, little is known about the intracellular pathways triggered by HGF and c-Met during dendrite maturation in hippocampal neurons. We show that HGF treatment promotes dendrite elongation, induces signaling through the Akt/GSK-3β pathway, and decreases phosphorylation of MAP2. These effects were reversed by pre-treatment with the selective c-Met inhibitor, PHA-665752 [24], [25] or by genetic suppression of c-Met expression with use of short hairpin RNAs (shRNAs). Furthermore, inhibitors of Akt blocked the ability of HGF to promote dendrite elongation, but inhibitors of GSK-3β enhanced this effect. Together, these data indicate that signaling by HGF and c-Met via Akt is important in modulating dendrite maturation during the early stages of hippocampal neuron differentiation.

Section snippets

Animals

All studies were conducted with a protocol approved by the University of Connecticut Animal Care and Use Committee in compliance with National Institutes of Health guidelines for the care and use of experimental animals.

Primary dissociated hippocampal neuron cultures

Cultures of dissociated hippocampal neurons from embryonic day 18 rats were prepared as previously described [11], [26]. Briefly, hippocampal neurons were dissociated with trypsin and plated on 15 mm diameter coverslips coated with poly-DL-lysine (Sigma) and laminin (ATCC) in

HGF and c-Met expressions are developmentally regulated during neuron differentiation in vitro

We investigated the temporal relationship between HGF expression and c-Met expression and activation during the early stages of hippocampal neuron differentiation in vitro. HGF expression at 1 DIV was low but detectable by immunoblot. The expression of HGF increased about 5-fold by day 3 and 8-fold by day 7 compared to 1 DIV (Fig. 1A). We examined the expression of the HGF receptor, c-Met, in the same samples. c-Met, which migrates as a 140 and 170 kDa doublet [30], was already apparent at 1

Discussion

Growth factors and their associated receptor tyrosine kinases (RTKs) play important roles in various neuronal functions such as neuronal polarization, neurite extension, and dendrite maturation during central nervous system (CNS) development [1], [2], [3], [32]. Although many signals have been identified that modulate dendrite growth and maturation, relatively few have been identified that regulate the initial stages of dendrite elongation [34]. HGF and c-Met are expressed by specific classes

Acknowledgement

PHA-665752 was kindly provided by Pfizer. This work was supported by a National Institutes of Mental Health grant (MH069778) to RSW.

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