Inhibition of Osteoclastogenesis by Mechanically Loaded Osteocytes: Involvement of MEPE

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes

SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2−/− calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter

Single nucleotide polymorphisms in bone turnover-related genes in Koreans: ethnic differences in linkage disequilibrium and haplotype

<p>Abstract</p> <p>Background</p> <p>Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to their function in bone formation and resorption during bone remodeling.</p> <p>Methods</p> <p>We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity depicted.</p> <p>Results</p> <p>We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When compared SNPs in the Korean population with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly differentiated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five sub-populations were substantially different from one another.</p> <p>Conclusion</p> <p>Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) > 0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies.</p

Tartrate resistant acid phosphatase (TRAP) positive cells in rheumatoid synovium may induce the destruction of articular cartilage

Objective: To examine the role of tartrate resistant acid phosphatase (TRAP) positive mononuclear and multinucleated cells in the destruction of articular cartilage in patients with rheumatoid arthritis (RA). Methods: The presence of TRAP positive cells in the synovial tissue of patients with RA was examined by enzyme histochemistry and immunohistochemistry. Expression of mRNAs for matrix metalloproteinases (MMPs) was assessed by the reverse transcriptase-polymerase chain reaction (RT-PCR) and northern blot analysis. Production of MMPs by mononuclear and multinucleated TRAP positive cells was examined by immunocytochemistry, enzyme linked immunosorbent assay (ELISA) of conditioned medium, and immunohistochemistry of human RA synovial tissue. In addition, a cartilage degradation assay was performed by incubation of (35)S prelabelled cartilage discs with TRAP positive cells. Results: TRAP positive mononuclear cells and multinucleated cells were found in proliferating synovial tissue adjacent to the bone-cartilage interface in patients with RA. Expression of MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-12 (macrophage metalloelastase), and MMP-14 (MT1-MMP) mRNA was detected in TRAP positive mononuclear and multinucleated cells by both RT-PCR and northern blot analysis. Immunocytochemistry for these MMPs showed that MMP-2 and MMP-9 were produced by both TRAP positive mononuclear and multinucleated cells, whereas MMP-12 and MMP-14 were produced by TRAP positive multinucleated cells. MMP-2 and MMP-9 were detected in the conditioned medium of TRAP positive mononuclear cells. TRAP positive mononuclear cells also induced the release of (35)S from prelabelled cartilage discs. Conclusion: This study suggests that TRAP positive mononuclear and multinucleated cells located in the synovium at the cartilage-synovial interface produce MMP-2 and MMP-9, and may have an important role in articular cartilage destruction in patients with RA