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Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts
S R Siddhanti1, J E Hartle, L D Quarles
1Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
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.
Insights
Protein kinase C (PKC) and cAMP signaling pathways interact to regulate osteoblast DNA synthesis. Forskolin (FSK) inhibits PKC-stimulated mitogen-activated protein kinase (MAPK) activity, suggesting complex cross-talk in bone cells.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Osteoblast differentiation and function are regulated by complex intracellular signaling pathways.
- Protein kinase C (PKC) and G protein-coupled second messengers, like cyclic AMP (cAMP), play crucial roles in cellular processes, including DNA synthesis.
Purpose of the Study:
- To investigate the integration of G protein-coupled second messengers and protein tyrosine phosphorylation within the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade in osteoblasts.
- To elucidate the cross-talk between PKC-dependent pathways and cAMP signaling in regulating osteoblast DNA synthesis and MAPK activation.
Main Methods:
- MC3T3-E1 osteoblasts were treated with phorbol 12-myristate 13-acetate (PMA) to activate PKC and forskolin (FSK) to elevate cAMP levels.
- Assessed effects on DNA synthesis, protein tyrosine phosphorylation, and the activities of Raf-1, MKK, and MAPK (ERK1/ERK2).
Main Results:
- PMA stimulated DNA synthesis, tyrosine phosphorylation of ERK1/ERK2, and activation of Raf-1, MKK, and MAPK.
- FSK inhibited DNA synthesis and blocked PMA-stimulated tyrosine phosphorylation of ERK1/ERK2 and activation of Raf-1 and MAPK.
- PMA still activated MKK in the presence of FSK, indicating both Raf-1-dependent and -independent pathways activated by PKC.
Conclusions:
- PKC activates both Raf-1-dependent and -independent pathways in osteoblasts.
- cAMP elevation by FSK exerts inhibitory effects on PKC-stimulated MAPK activation, suggesting distal cAMP-dependent inhibitory mechanisms.