In vitro evaluation of the biological effect of SOFAT on osteoblasts.
Osteoclastogenesis is regulated by osteoblasts especially through the production of receptor activator of nuclear factor kappa-B ligand (RANKL). Immune cells present in inflamed tissues markedly increase this process by upregulating RANKL directly or by secreting proinflammatory cytokines, which stimulate RANKL expression by osteoblasts. A novel T-cell-secreted cytokine, termed secreted osteoclastogenic factor of activated T cells (SOFAT) was recently described. To better understand how SOFAT affects bone metabolism, we investigated its effect on osteoblastic cells. We demonstrate here that SOFAT did not influence MC3T3 cells viability and proliferation, evaluated by trypan blue exclusion and MTT tests, respectively. SOFAT stimulated the secretion of IL-6, IL-10 and GM-CSF in MC3T3 cells, as shown by the analysis of an inflammatory cytokines ELISA array. The upregulation of the corresponding genes was checked by qPCR. Both RANKL mRNA and protein levels did not significantly change in the presence of SOFAT, evaluated by qPCR and western blotting, respectively. In addition, analysis of a PCR array for IL6/STAT3 pathway demonstrated that SOFAT induced the expression of BCL2, IL1B, IL10, IL22, IL2RA, IL4, IL6, TNFSF10 and PIAS3, while IL2, IL21, CD4, CSF3R and TNF were repressed. Our results confirm that the SOFAT mechanism of action is RANKL-independent and indicate that, by co-opting osteoblasts to increase the production of osteoclastogenic cytokines, SOFAT may exacerbate inflammation and support osteoclast formation and bone destruction.
Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil. Electronic address: napimogamh@yahoo.com.
Marcelo Henrique
MH
Napimoga
Laboratory of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Ana Paula Dias
AP
Demasi
Periodontal Medicine Research Group, Department of Periodontology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Christian Rado
CR
Jarry
Periodontal Medicine Research Group, Department of Periodontology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Mauricio Cardoso
MC
Ortega
Laboratory of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Vera Cavalcanti
VC
de Araújo
Laboratory of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil.
Elizabeth Ferreira
EF
Martinez
D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000276:immunology; D050828:Protein Inhibitors of Activated STAT / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000378:metabolism*; D016184:Receptors, Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism
C517047:CSF3R protein, human; D016207:Cytokines; D053645:Interleukin-2 Receptor alpha Subunit; D018832:Molecular Chaperones; C484745:PIAS3 protein, human; D050828:Protein Inhibitors of Activated STAT; D019253:Proto-Oncogene Proteins c-bcl-2; D053245:RANK Ligand; D016184:Receptors, Colony-Stimulating Factor; C546006:THNSL2 protein, human; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human
Cytokines; Inflammation; Osteoblast; SOFAT