Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.
OBJECTIVE
The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling.


METHODS
DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells.


RESULTS
Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration.


CONCLUSIONS
Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.
Department of Physiology, University of Tübingen, Tübingen, Germany.
Wenting
W
Yang
Meerim K
MK
Nurbaeva
Evi
E
Schmid
Antonella
A
Russo
Ahmad
A
Almilaji
Kalina
K
Szteyn
Jing
J
Yan
Caterina
C
Faggio
Ekaterina
E
Shumilina
Florian
F
Lang
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D054421:Chemokine CCL21 / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D020868:Gene Silencing / Q000187:drug effects; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D047028:Macrocyclic Compounds / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D010080:Oxazoles / Q000494:pharmacology; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000172:deficiency / Q000378:metabolism*
D054421:Chemokine CCL21; D016207:Cytokines; C494042:Ets1 protein, mouse; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008070:Lipopolysaccharides; D047028:Macrocyclic Compounds; D010080:Oxazoles; D050784:Proto-Oncogene Protein c-ets-1; C511704:xestospongin C; C494922:Akt2 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium