Protein phosphatase 2A is involved in the regulation of protein kinase A signaling pathway during in vitro chondrogenesis.
We have evaluated the importance of the Ser/Thr protein phosphorylation and dephosphorylation for chondrogenesis in high-density chicken limb bud mesenchymal cell cultures (HDCs) by using H89, a cell-permeable protein kinase inhibitor, and okadaic acid (OA), a phosphoprotein phosphatase (PP)-specific inhibitor molecule. When 20 nM OA was applied to the HDCs on Days 2 and 3 of culturing, it significantly inhibited protein phosphatase 2A (PP2A), enhanced cartilage formation, and elevated the activity of cAMP-dependent protein kinase (PKA). Application of 20 microM H89 significantly decreased the activity of PKA and blocked the chondrogenesis in HDCs. Furthermore, OA enhanced cartilage formation and elevated the suppressed activity of PKA even in the H89-pretreated HDCs. cGMP-dependent protein kinase was not detected in HDCs, while protein kinase Cmu (PKCmu), which is also inhibited by nanomolar concentrations of H89, was present throughout the culturing period. Neither OA nor H89 influenced the expression of the catalytic subunit of PKA or the cAMP response element binding protein, CREB. However, a significantly elevated amount of Ser-133-phosphorylated-CREB (P-CREB) was detected following addition of OA, while H89 treatment resulted in a decrease of the amount of P-CREB. Our results demonstrate that PP2A plays a role in the regulation of the PKA signaling pathway and that the phosphorylation level of CREB is influenced by the activity of both enzymes during in vitro chondrogenesis.
Zákány
Róza
R
Department of Anatomy, Histology, and Embryology, Medical and Health Science Centre, University of Debrecen, Debrecen, H-4026, Hungary.
Szucs
Kornélia
K
Bakó
Eva
E
Felszeghy
Szabolcs
S
Czifra
Gabriella
G
Bíró
Tamás
T
Módis
László
L
Gergely
Pál
P
D000818:Animals; D002356:Cartilage / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D018878:Limb Buds / Q000196:embryology / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013449:Sulfonamides; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide