title: Reduction of c-Src activity by substituted 5,7-diphenyl-pyrrolo[2,3-d]-pyrimidines induces osteoclast apoptosis in vivo and in vitro. Involvement of ERK1/2 pathway.
abstract: We employed potent and selective c-Src inhibitors to investigate the functional and molecular consequences of inhibited c-Src tyrosine kinase activity in osteoclasts. These pyrrolopyrimidine derivatives reduced osteoclast numbers and induced osteoclast disruption in vivo. In vitro, they inhibited resorption pit formation and osteoclastogenesis, impaired adhesion ability and actin ring organization, and induced programmed cell death in mature osteoclasts. The cell death receptor Fas and p53 were insensitive to c-Src modulation. The expression of the cyclin-dependent kinase (CDK)-inhibitor p21WAF1/CIP1 was markedly reduced, but neither Bcl-2 nor Bcl-xL or Bax were modulated by c-Src inhibition. Caspase-9, and to a lesser extent caspase-3, but not caspase-8, were transiently cleaved (activated) by treatment with the c-Src inhibitors. c-Src inhibition stabilized p38 mitogen-activated protein kinase (MAPK), whereas the c-Jun N-terminal kinase (JNK) pathway did not appear to be modulated by our compounds. Most interestingly, transient extracellular signal regulated kinase (ERK1/2) dephosphorylation followed by sustained remarkable rephosphorylation overwhelming control levels was observed in response to c-Src inhibition. Blockade of ERK1/2 rephosphorylation by PD98059 reduced osteoclast nuclear disruption, suggesting the involvement of this pathway in apoptosis. Collectively, these data demonstrate that small pyrrolopyrimidine derivatives impair osteoclast function and induce cell damage suggestive of apoptosis in vivo and in vitro, with mechanisms presumably involving selective sustained ERK1/2 phosphorylation.
authors.affiliation: Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Recchia
authors.forename: Nadia
authors.initials: N
authors.lastname: Rucci
authors.forename: Alessia
authors.initials: A
authors.lastname: Funari
authors.forename: Silvia
authors.initials: S
authors.lastname: Migliaccio
authors.forename: Anna
authors.initials: A
authors.lastname: Taranta
authors.forename: Maurizio
authors.initials: M
authors.lastname: Longo
authors.forename: Michaela
authors.initials: M
authors.lastname: Kneissel
authors.forename: Mira
authors.initials: M
authors.lastname: Susa
authors.forename: Doriano
authors.initials: D
authors.lastname: Fabbro
authors.forename: Anna
authors.initials: A
authors.lastname: Teti
mesh_terms: D000199:Actins / Q000378:metabolism; D018262:Adenocarcinoma, Clear Cell; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001862:Bone Resorption / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000199:Actins; C472714:CGP 76030; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D011758:Pyrroles; D016392:Proto-Oncogene Proteins pp60(c-src); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020169:Caspases; C119957:CGP 77675