Pituitary Adenylyl Cyclase-activating Polypeptide 38 Reduces Astroglial Proliferation by Inhibiting the GTPase RhoA

Abstract

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G 1 phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G 1 phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) 1 and vasoactive intestinal polypeptide (VIP) are neuropeptides of the secretin-glucagon family (1). Whereas both peptides stimulate the G protein-coupled receptors VPAC1 and VPAC2 at subnanomolar concentrations, only PACAP38 stimulates PAC1 receptors with a potency in the same range (2, 3). During the embryonic and postnatal period, the neuropeptides and the receptors are expressed in cells of the rodent neocortex and its growth zones. There is strong evidence for the involvement of these peptides in neural cell proliferation, phenotypic determination, differentiation, and survival (4 -12).Activation of PAC1 receptors by PACAP38 can change the proliferation of neural cells. However, inhibitory as well as stimulatory effects mediated by protein kinase A (PKA) have been observed in cultured neocortical or cerebellar neurons and spinal cord glial cells (3,4,(12)(13)(14)(15)(16). These contradictory effects may be related to the diversity in receptor isoforms and their signal transduction pathways. The 7 isoforms reported differ in their third intracellular loop and can couple to adenylyl cyclase as well as phospholipases C and D (12, 17).In the cellular proliferation cycle, cyclins and cyclin-dependent kinases (CDKs) determine the progression through the G 1 phase into the S phase. Thus, the association of cyclin D with CDK4/6 and of cyclin E with CDK2 is important for G 1 progression and G 1 /S transition, respectively. The complexes induce the phosphorylation of the retinoblastoma gene product that allows the transcription of E2F-regulated genes (18). Upon activation, extracellular signal-related kinases 1 and 2 (ERK1 and ERK2) increase the activity of cyclin D1. However, only the sustained activation of ERKs and cyclin D1 leads to G 1 progression (19,20). In primary cells, such a ...

Reelin Signals through Apolipoprotein E Receptor 2 and Cdc42 to Increase Growth Cone Motility and Filopodia Formation

“…However, when Rho-kinase was inhibited with Y27632 (10μM), Reelin was also capable to increase the number of axonal branches (Figure 5B); Y27632 alone did not influence the number of axonal branches compared to controls. The neuropeptides vasoactive intestinal peptide (VIP) and pituitary-adenylyl-cyclase-activating polypeptide-38 (PACAP38), which have an established role in neuronal development (Dickson and Finlayson, 2009; Waschek, 2002; Vaudry et al, 2009) can activate Cdc42 and Rac1, but also decrease RhoA and Rho-kinase activity in neural cells (Meyer et al, 2005; Henle et al, 2006). As VIP-mediated Rho-kinase inhibition induces the elongation of dendrites and axons by stabilizing microtubules (Leemhuis et al, 2007) we used VIP alone and in combination with Reelin.…”

Reelin modulates cytoskeletal organization by regulating Rho GTPases

“…We could demonstrate that Reelin functionally interacts with the peptidergic VIP/PACAP38-receptor system to increase axonal branch formation. 24 As VIP-mediated Rho-kinase inhibition 35,36 induces the elongation of dendrites and axons by stabilizing microtubules, 37 these results demonstrate that Reelin and a neuropeptidergic system can cooperate to promote neuronal development by inducing axonal branching. In addition, these findings pinpoint the influence of the tubulin cytoskeleton in mediating Reelin's effect on neuromorphogenesis, which is also targeted by other effectors of the Reelin-Dab1 signaling cascade, including the Tau kinase GSK3beta, 12 Lis1, 38 and Map1b.…”

Small GTP‐binding protein RhoB is expressed in glial müller cells in the vertebrate retina

“…Activation and/or inactivation of Rho proteins in other glial cell types such as astrocytes, Schwann, and Bergmann cells have demonstrated that small GTPases may be important factors for polarity, motility, maintenance of morphology, and proliferation (Manning et al, 1998;Ramakers and Moolenaar, 1998;Etienne-Manneville and Hall, 2001;Kaartinen et al, 2001;Sepp and Auld, 2003;Guasch et al, 2003;Meyer et al, 2005). However, little is known about the expression and role of Rho GTPases, particularly RhoB, and the regulation of cellular morphology in vertebrate retina Mü ller cell.…”