Chemoattractant action and molecular signaling pathways of Kit ligand on mouse primordial germ cells.
Using a Transwell chamber as migration assay for mouse primordial germ cells (PGCs), we show here that these cells posses directional migration in the absence of somatic cell and defined matrix support and in response to a Kit ligand (KL) gradient or medium conditioned by Aorta/Gonad/Mesonephros and gonadal ridges. Other putative PGC chemoattractants such as SDF1 and TGFbeta did not exert any attractive action on PGCs. The chemoattractant activity of KL and conditioned medium was also evidenced by their ability to stimulate actin reorganization in PGCs. In the aim to identify downstream signaling pathways governing KL chemoattraction on PGCs, we demonstrated that in such cells KL rapidly (5 min) increased autophosphorylation of its receptor c-Kit and caused phosphorylation of the serine-threonine kinase AKT through the action of PI3K. 740Y-P peptide, a direct activator of PI3 kinase, stimulated PGC migration at levels similar to those elicited by KL. LY294002 (a specific inhibitor of PI3K) abolished KL-dependent PGC migration or the chemoattractant activity of the conditioned medium and inhibited AKT phosphorylation; Src kinase inhibitors PP2 and SU6656, caused significant reduction of the KL-dependent PGC migration and AKT phosphorylation, while U0126, a selective inhibitor of the MEK/ERK protein kinase cascade, reduced PGC migration and AKT phosphorylation at lesser extent. SU6656 completely abolished the chemoattractant activity of the conditioned medium. Finally, SB202190 (a p38 inhibitor) and rapamycin (mTOR inhibitor) did not affect PGC migration. In addition, to demonstrate that somatic cells are not essential for PGC motility and directional migration, we evidenced a novel role for KL as PGC chemoattractant and for PI3K/AKT and Src kinase, as players involved in the activation of the PGC migratory machinery and likely important for their directional movement towards the gonadal ridges.
Farini
Donatella
D
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00173 Rome, Italy.
La Sala
Gina
G
Tedesco
Marianna
M
De Felici
Massimo
M
D000818:Animals; D002448:Cell Adhesion; D002465:Cell Movement; D002630:Chemotactic Factors / Q000378:metabolism*; D002633:Chemotaxis; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D002630:Chemotactic Factors; D010455:Peptides; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D019061:src-Family Kinases