GLUTAMINE SUPPLEMENTATION STIMULATES RANKL-INDUCED OSTEOCLAST GENERATION AND DIMINISHES APOPTOSIS IN RAW 264.7 MACROPHAGES

SCOTTO FLORENCIA; SANTOS VANESSA; SOUSA CAROLINA; CASTRO MS; GROTZ, ESTEFANIA; PARRADO CECILIA; REY-ROLDÁN, ESTELA; GENTILE, MARÍA TERESA; MENEZES FREIRE, SONGELI; NEY FREIRE, ANDRÉ; CANELLADA, ANDREA

Buzios

Congreso; XXXIX Congress of The Brazilian Society of Immunology; 2014

Sociedad Brasilera de Inmunología

GLUTAMINE SUPPLEMENTATION STIMULATES RANKL-INDUCEDOSTEOCLAST GENERATION AND DIMINISHES APOPTOSIS IN RAW 264.7MACROPHAGES.FLORENCIA SCOTTO1*, VANESSA SANTOS2*, CAROLINA SOUSA2*, MARISACASTRO1, ESTEFANÍA GROTZ1, CECILIA PARRADO1, ESTELA REY1, TERESAGENTILE1, SONGELI MENEZES FREIRE2#, ANDRÉ NEY FREIRE2, ANDREACANELLADA1#. * participated equally in this work. # participated equally in this work1-Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires;Instituto de Estudios de la Inmunidad Humoral Prof. RA Margni, CONICET-UBA, Argentina.2- Universidade Federal da Bahia-Brasil.INTRODUCTION: Osteoclast (OC) is a giant multinucleate cell that resorbs calcified matrixduring bone formation and remodeling. Molecular mechanisms underlying metabolic adaptationto the energetic demands of osteoclastic bone resorption remain poorly understood. Murinemacrophage RAW 264.7 cells have been widely used as an in vitro model of OC differentiation.Cytokines produced by activated immune cells, as RANKL, TNF-a, IL-17, induce OCdifferentiation. Glutamine (Gln) is a nonessential amino acid with antioxidant capacity thatmaintains normal immunological function under stress or pathological conditions. It wasdemonstrated that the RANKL-mediated osteoclastogenesis transiently generate reactive oxygenspecies that can trigger apoptosis. OBJECTIVE: To evaluate the effect of Gln on the OCdifferentiation of RAW cells in vitro. METHODS: RAW cells were cultured during 5 dayswith RANKL (0 or 50 nM) and Gln (0-2-4-7 mM), or in the presence or absence of supernatantsof Balb/c mouse spleen cells that had been previously cultured during 24 h with Gln (2-4-7mM) and concanavalin A (ConA, 0 or 5ug/mL). OCs were measured by Tartrate Resistant AcidPhosphatase (TRAP) expression and matrix metaloprotease activity (MMP), determined bycytochemistry and gel zymography respectively. Apoptosis was evaluated in RAW cells byanalysis of DNA integrity in agarose gels, and by tunel assay. RESULTS: We found that in theabsence of Gln in the culture media, OCs generation did not occur in response to RANKL. Inthe presence of Gln (2mM), RANKL increased 2 fold the osteoclast differentiation compared tocontrol unstimulated cells (p