Prevention of bone loss after ovariectomy in mice with preferential overexpression of the transcription factor paired box-5 in osteoblasts.

We have recently shown that the transcription factor Paired box-5 (Pax5) promotes bone formation in vivo and osteoblastogenesis in vitro. Here, we demonstrated the involvement of Pax5 in bone remodeling after ovariectomy (OVX). A significant increase was seen in vertebrae bone volume in transgenic mice preferentially overexpressing Pax5 in osteoblasts by using the mouse α1(I)Collagen promoter, whereas OVX significantly reduced vertebrae bone volume in wild-type (WT) mice without significantly affecting that in Pax5 transgenic mice. Preferential osteoblastic Pax5 overexpression invariably led to significant increases in osteoblastic and osteoclastic parameters in mice with sham operation. However, OVX significantly increased osteoclastic parameters in WT mice, without additionally increasing osteoblastic and osteoclastic parameters in Pax5 transgenic mice. These results suggest that osteoblastic Pax5 would play a role in OVX-induced bone loss through a mechanism relevant to the promotion of both osteoblastic bone formation and osteoclastic bone resorption in vivo.

The transcription factor paired box-5 promotes osteoblastogenesis through direct induction of Osterix and Osteocalcin.

Although skeletal abnormalities are seen in mice deficient of particular paired box (Pax) family proteins, little attention has been paid to their role in osteoblastogenesis so far. Here, we investigated the possible involvement of several Pax family members in mechanisms underlying the regulation of differentiation and maturation of osteoblasts. Among different Pax family members tested, Pax5 was not markedly expressed in murine calvarial osteoblasts before culture, but progressively expressed by osteoblasts under differentiation toward maturation. Immunoreactive Pax5 was highly detectable in primary cultured mature osteoblasts on immunoblotting and in osteoblastic cells attached to cancellous bone in mouse tibial sections on immunohistochemistry, respectively. Knockdown by small interfering RNA (siRNA) of endogenous Pax5 led to significant inhibition of the expression of Osteocalcin, and Osterix through deterioration of gene transactivation, in addition to a1(I)Collagen expression and alkaline phosphatase (ALP) staining, without affecting runt-related transcription factor-2 (Runx2) expression and cell viability in osteoblastic MC3T3-E1 cells. The introduction of Pax5 enhanced promoter activities of Osteocalcin and Osterix in a manner dependent on the paired domain in MC3T3-E1 cells. Putative Pax5 binding sites were identified in the 5'-flanking regions of mouse Osteocalcin and Osterix, whereas chromatin immunoprecipitation assay revealed the direct binding of Pax5 to particular regions of Osteocalcin and Osterix promoters in MC3T3-E1 cells. Overexpression of Pax5 significantly increased Osteocalcin, Osterix, and a1(I)Collagen expression, ALP activity, and Ca(2+) accumulation, without affecting Runx2 expression, in MC3T3-E1 cells. In vertebrae of transgenic mice predominantly expressing Pax5 in osteoblasts, a significant increase was seen in the ratio of bone volume over tissue volume and the bone formation rate. These findings suggest that Pax5 could positively regulate osteoblastic differentiation toward maturation in vitro, in addition to promoting bone formation and remodeling in vivo, as one of the transcription factors essential for controlling osteoblastogenesis independently of Runx2.

The natural polyamines spermidine and spermine prevent bone loss through preferential disruption of osteoclastic activation in ovariectomized mice.

BACKGROUND AND PURPOSE: Although naturally occurring polyamines are indispensable for a variety of cellular events in eukaryotic cells, little attention has been paid to their physiological and pathological significance in bone remodelling to date. In this study, we evaluated the pharmacological properties of several natural polyamines on the functionality and integrity of bone in both in vitro and in vivo experiments. EXPERIMENTAL APPROACH: Mice were subjected to ovariectomy (OVX) and subsequent oral supplementation with either spermidine or spermine for determination of the bone volume together with different parameters regarding bone formation and resorption by histomorphometric analyses in vivo. Pre-osteoclasts were cultured with receptor activator of NF-κB ligand (RANKL), with or without spermidine and spermine to determine cellular maturation by tartrate-resistant acid phosphatase (TRAP) staining and cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction in vitro. KEY RESULTS: Spermidine or spermine, given in drinking water for 28 days, significantly prevented the increased osteoclast surface/bone surface ratio and the reduced bone volume following OVX in mice. Either spermidine or spermine significantly inhibited the increased number of multinucleated TRAP-positive cells in osteoclasts cultured with RANKL in a concentration-dependent manner without affecting cell survival. CONCLUSIONS AND IMPLICATIONS: The natural polyamines spermidine and spermine prevented OVX-induced bone loss through the disruption of differentiation and maturation of osteoclasts, rather than affecting osteoblasts. The supplementation with these natural polyamines could be beneficial for the prophylaxis as well as therapy of metabolic bone diseases such as post-menopausal osteoporosis.