Dynamic reorganization of the astrocyte actin cytoskeleton elicited by cAMP and PACAP: a role for phosphatidylInositol 3-kinase inhibition.
Cyclic AMP (cAMP)-raising agents induce astrocytes grown in vitro to adopt a stellate morphology resembling their in vivo appearance, through the depolymerization of actomyosin stress fibres. The signalling pathways responsible for cAMP-induced astrocyte stellation have thus far remained largely elusive. We showed in this study that the neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) mimicked the effect of forskolin, a direct activator of adenylate cyclase, on the actin cytoskeleton of primary rat astrocytes. The depolymerization of stress fibres induced by PACAP or forskolin was prevented by the expression of a constitutively active mutant of RhoA, but not by a protein kinase A (PKA) blocker, indicating that cAMP-raising agents act upstream of RhoA, in a PKA-independent manner. In addition, PACAP and forskolin inhibited basal Akt phosphorylation, and basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3-kinase (PI 3-K) activities. Incubation with a PI 3-K blocker resulted in the depolymerization of stress fibres. This effect was blocked by the expression of a constitutively active mutant of RhoA, indicating that PI 3-K inhibition acted upstream of RhoA. Together, these data demonstrate for the first time that depolymerization of stress fibres, and the resulting astrocyte stellation, induced by stimulation of cAMP production involves the inhibition of the PI 3-K-RhoA pathway.
Perez
Virgili
V
UPR 2580 CNRS, Laboratoire de Génomique Fonctionnelle,141, rue de la cardonille, 34094 Montpellier Cedex 05, France. Laurent.Journot@ccipe.cnrs.fr
Bouschet
Tristan
T
Fernandez
Céline
C
Bockaert
Joël
J
Journot
Laurent
L
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D002867:Chromones / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009479:Neuropeptides / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014162:Transfection / Q000379:methods; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics
D000199:Actins; D002867:Chromones; D005576:Colforsin; D000242:Cyclic AMP; D005904:Glial Fibrillary Acidic Protein; D049452:Green Fluorescent Proteins; D007211:Indoles; D009025:Morpholines; D009414:Nerve Growth Factors; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020742:rhoA GTP-Binding Protein; C495403:Adcyap1 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C007293:DAPI; C494920:Akt1 protein, rat