Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts.
We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-dependent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrates cross-talk between G protein-coupled second messengers and protein tyrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate (PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increments in tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatment of osteoblasts, which raised intracellular cAMP levels and inhibited DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) as well as inhibited PKC-stimulated MAPK and Raf-1 activities. Despite this, PMA activated the intermediate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presence of a additional distal cAMP-dependent inhibitory mechanisms.
Siddhanti
S R
SR
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hartle
J E
JE
Quarles
L D
LD
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008934:Mitogens / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008934:Mitogens; D011518:Proto-Oncogene Proteins; D005576:Colforsin; D019000:Phosphotyrosine; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate