PAMM: A Novel Antioxidant Protein That Regulates Osteoclast Differentiation And Mediates Estrogen Bone Protective Effect

Objectives:Bone homeostasis is influenced by decreases in the defense against oxidative stress as well as increases in the production of reactive oxygen species (ROS). At the cellular level, defects in bone remodeling caused by oxidative stress are associated with decreased osteoblast and osteoclast numbers and decreased bone formation rate as well as increased osteoblast and osteocyte apoptosis. Aging and loss of sex steroids also result in increased oxidative stress as reflected by an increase in the levels of reactive oxygen species, and decreased activity of glutathione reductase. ROS also play a central role in estrogen deficiency-induced boneloss. The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts.We identified and characterized a novel member of the Peroxiredoxin (PRX)-like 2 family that we called PAMM:Peroxiredoxin Activated in M-CSF stimulated Monocytes, a redox regulatory protein that modulates osteoclast differentiation in vitro.In this study, the objectives are:(1) to characterize the molecule and the expression of PAMM; (2) to determine the role of PAMM in octeoclastes differentiation; (3) to identify PAMM as an antioxidant; (4) to examine the role of PAMM in ovariectomy-induced bone loss and estrogen replace treatment.Methods:(1) Microarray hybridization, a genome-wide expression screening was conducted to identify genes upregulated during RANKL-induced osteoclast differentiation. Identificated the sequence, structure, and orthologues of pamm; Analysised PAMM expression in multiple mouse tissue by Western Blotting; Studied pamm expression further during macrophage activation and osteoclast differentiation; Investigated the expression of PAMM in bone marrow monocyte activation in vivo, induced by M-CSF in bone sections from Csf-1-null toothless (tl/tt) rats.(2) Generated stable over express PAMM-RAW264.7 cell lines named PAMM#1 and PAMM#2; Compared PAMM#1 and PAMM#2 cells with control vector-transfected RAW264.7 cells under osteoclasts differentiation induced by RANKL; Determined the activation of NF-κB and c-Jun in the stables PAMM cell lines stimulated with RANKL; Mutated C85 to G and C88 to G and checked the effect on the octeoclastes differentiation.(3) MTT analysised the proliferation of PAMM stable cell line and the control cells with H2O2 stimulation; Checked the change of GSH/GSSG ratio in the PAMM cell line and the control cells induced by RANKL, and the GSH/GSSG in C85G, C88G, and C85G/C88G transfected HEK293 cells as well.(4) Examined the expression of PAMM and Akt activation in ovariectomy (OVX) mice with or without estrogen replace treatment; Determined the PAMM expression in M-CSF induced human CD14+PBMC with E2 or E2/ICI182780; Stimulated human CD14+ PBMCs with M-CSF and determined PAMM abundance and phosphorylated Akt in the presence of Wortmannin or Rapamycin to determine if M-CSF stimulation of PAMM expression required Akt activation.(5) Data are presented as means plus or minus SDs from at least three independ entexperiments. Statistical significance was determined by using one-way analysis of variance (ANOVA) followed by the Student t test (*P<0.05;**P<0.01). Statistical analysis was performed using SPSS for Windows 22.0.Results:(1) Identified a gene, which we named pamm [Peroxiredoxin (PRX)-like 2 Activated in M-CSF stimulated monocytes] by a genome-wide microarray screening in RAW 264.7 cells as well as in normal BMM stimulated with RANKL and with M-CSF/RANKL, respectively to identify genes involved in RANKL-induced osteoclast formation. Unstimulated cells were used as controls. Pamm mRNA is upregulated in stimulated RAW264.7 cells and BMM (fold changes relative to unstimulated cells:1.41 and 1.48, respectively). Pamm is a member of the peroxiredoxin (PRX)-like 2 family. Pamm nucleotide-sequence analysis predicts the existence of a 229-amino acid protein. PAMM contains a putative CXXC motif (C in positions 85 and 88), which suggests that PAMM has redox activity. PAMM was found in mouse kidney, liver, skin, and brain, and expressed in M-CSF-activated macrophages in vitro and in vivo. PAMM is a secreted protein and expressed by stimulation with M-CSF.(2) Constitutive expression of PAMM virtually abolished osteoclast formation, as judged by the formation of osteoclast-like TRAP-positive multinuclear cells, and inhibited NF-κB and c-Jun activation as well. It suggested that PAMM overexpression blocks RANKL induced osteoclast differentiation by inhibiting NF-kB2 and c-Jun activation. But, Mutations in the CXXC motif and the addition of H2O2 abolish PAMM ability to inhibit osteoclast formation.(3) PAMM expression increased cellular GSH/GSSG ratio and protected cells from oxidative stress. Cell viability decreased in a dose dependent manner as a result of oxidative stress induced by H2O2 treatment of control RAW 264.7 cells, but increased in PAMM#1 cells, as a result of PAMM antioxidant activity. Moreover, the GSH/GSSG ratios were decreased in C85G and C88G mutations transfected HEK293 cells. It indicated both C residues are required for PAMM activity. And, oxidative stress induced PAMM and Nrf2 mRNA expression.(4) Ovariectomy induces bone loss and oxidative stress. The significant increased PAMM protein expression and Akt activation were detected in the OVX/Vehicle and OVX/E2 groups compared to the Sham/Vehicle group (P<0.05). We also found that M-CSF stimulation of PAMM expression requires Akt phosphorylation, estrogen stimulates M-CSF induced PAMM Expression in preosteoclasts, and both Wortmannin and Rapamycin blocked M-CSF induced PAMM ex-pression via inhibited phosphorylation of Akt.Conclusions:(1) We described the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin-like 2 protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. PAMM is a secreted protein contained 229 amino acids. The expression of PAMM is detected in osteoclast precursor stimulated by M-CSF, and downrelugated by RANKL stimulation. PAMM also express in mouse bone, brain, liver and kidney.(2) PAMM expression completely abolished RANKL-induced p100 NF-kB and c-Jun activation, as well as osteoclast formation. CXXC motif is the essential part to suppress osteoclasts differentiation. PAMM expression may affect bone resorption in vivo and help to maintain bone mass.(3) Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro.(4) PAMM expression is inducible by oxidativestress in vitro and in vivo.The increased PAMM abundance and phosphorylated Akt in OVX mice treated with estrogen. M-CSF-induced PAMM expression is mediated by Akt phosphorylation. Estrogen-induced PAMM expression is also mediated by phosphorylation of Akt. These findings point to PAMM as a potential candidate for Akt-mediated protection against oxidative stress. The PAMM secreted from adipocytes maybe also involved in estrogen deficiency osteoporosis.(5) Our findings provide evidence of a novel mechanism linking cellular redox status and bone homeostasis and may suggest a new approaches to the prevention and/or treatment of human bone diseases by targeting molecules, like PAMM, that modulate cellular redox status in bone cells.