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Beclin-1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non-autophagic manner. Although studies have examined the non-autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin-1 is unclear. Here, we examined the role of Beclin-1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin-1 upon RANKL stimulation in a p38- and NF-kappa B-dependent manner. During OC differentiation, Beclin-1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin-1 in RANKL-primed BMMs led to a significant reduction in RANKL-dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin-1 inhibited RANKL-mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin-1 knockdown, indicating that Beclin-1 mediates RANKL-induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin-1 plays a non-autophagic role in RANKL-induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1. J. Cell. Physiol. 229: 1963–1971, 2014. © 2014 Wiley Periodicals, Inc.

Pentraxin-3 (PTX3), also known as tumor necrosis factor-stimulated gene 14 (TSG-14), is produced by immune and vascular cells in response to pro-inflammatory signals and is therefore a multipotent inflammatory mediator. The present study showed that during human osteoblast (OB) differentiation, precursor OBs (pOBs), but not mature OB, highly expressed PTX3. TNFα treatment elevated the PTX3 expression of pOBs. When mice were injected with lipopolysaccharide, which induces an inflammatory osteolytic condition characterized by trabecular bone destruction and high osteoclastogenesis, their bone marrow cells expressed elevated levels of PTX3 protein. Exogenous PTX3 did not directly affect osteoclast (OC) or OB differentiation. However, when pOBs and precursor OCs were co-cultured, exogenous PTX3 significantly increased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (i.e., OC cells) by increasing the pOB mRNA expression and protein secretion of RANK ligand (RANKL). This was accompanied with increased Runt-related transcription factor 2 (Runx2) expression in the pOBs. Knock-down of endogenous PTX3 with small-interfering RNA did not change the osteogenic potential of pOBs but suppressed their production of RANKL and reduced osteoclastogenesis. Finally, TNFα treatment of the co-culture elevated PTX3 expression by the pOBs and increased OC formation. This effect was suppressed by PTX3 knock-down by decreasing RANKL expression. Thus, the PTX3-driven increase in the osteoclastogenic potential of pOBs appears to be mediated by the effect of PTX3 on pOB RANKL production. These findings suggest that PTX3 is an inflammatory mediator that contributes to the deteriorating osteolytic condition of inflamed bone. J. Cell. Physiol. 229: 1744-1752, 2014. © 2014 Wiley Periodicals, Inc.

Pentraxin-3 (PTX3), also known as tumor necrosis factor-stimulated gene 14 (TSG-14), is produced by immune and vascular cells in response to pro-inflammatory signals and is therefore a multipotent inflammatory mediator. The present study showed that during human osteoblast (OB) differentiation, precursor OBs (pOBs), but not mature OB, highly expressed PTX3. TNFα treatment elevated the PTX3 expression of pOBs. When mice were injected with lipopolysaccharide, which induces an inflammatory osteolytic condition characterized by trabecular bone destruction and high osteoclastogenesis, their bone marrow cells expressed elevated levels of PTX3 protein. Exogenous PTX3 did not directly affect osteoclast (OC) or OB differentiation. However, when pOBs and precursor OCs were co-cultured, exogenous PTX3 significantly increased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (i.e., OC cells) by increasing the pOB mRNA expression and protein secretion of RANK ligand (RANKL). This was accompanied with increased Runt-related transcription factor 2 (Runx2) expression in the pOBs. Knock-down of endogenous PTX3 with small-interfering RNA did not change the osteogenic potential of pOBs but suppressed their production of RANKL and reduced osteoclastogenesis. Finally, TNFα treatment of the co-culture elevated PTX3 expression by the pOBs and increased OC formation. This effect was suppressed by PTX3 knock-down by decreasing RANKL expression. Thus, the PTX3-driven increase in the osteoclastogenic potential of pOBs appears to be mediated by the effect of PTX3 on pOB RANKL production. These findings suggest that PTX3 is an inflammatory mediator that contributes to the deteriorating osteolytic condition of inflamed bone. J. Cell. Physiol. 229: 1744-1752, 2014. © 2014 Wiley Periodicals, Inc.

Beclin-1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non-autophagic manner. Although studies have examined the non-autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin-1 is unclear. Here, we examined the role of Beclin-1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin-1 upon RANKL stimulation in a p38- and NF-kappa B-dependent manner. During OC differentiation, Beclin-1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin-1 in RANKL-primed BMMs led to a significant reduction in RANKL-dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin-1 inhibited RANKL-mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin-1 knockdown, indicating that Beclin-1 mediates RANKL-induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin-1 plays a non-autophagic role in RANKL-induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1.