Enhancement of erythropoietin-stimulated cell proliferation by Anandamide correlates with increased activation of the mitogen-activated protein kinases ERK1 and ERK2.
BACKGROUND
Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells.


METHODS
32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies.


RESULTS
Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated.


CONCLUSIONS
These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.
Valk
P
P
Institute of Hematology, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands.
Verbakel
S
S
von Lindern
M
M
Löwenberg
B
B
Delwel
R
R
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001095:Arachidonic Acids / Q000494:pharmacology*; D001892:Camphanes / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D016895:Culture Media, Serum-Free; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004357:Drug Synergism; D063388:Endocannabinoids; D004789:Enzyme Activation; D004921:Erythropoietin / Q000494:pharmacology*; D016762:Genes, fos; D051379:Mice; D008894:Milk Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D001095:Arachidonic Acids; D001892:Camphanes; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D063388:Endocannabinoids; D008894:Milk Proteins; D053284:Polyunsaturated Alkamides; D016760:Proto-Oncogene Proteins c-fos; D011720:Pyrazoles; D012333:RNA, Messenger; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; C110630:SR 144528; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C078814:anandamide