Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein–induced bone formation

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)–induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2–induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts

Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldBone mineral density (BMD) is a heritable complex trait used in the clinical diagnosis of osteoporosis and the assessment of fracture risk. We performed meta-analysis of five genome-wide association studies of femoral neck and lumbar spine BMD in 19,195 subjects of Northern European descent. We identified 20 BMD loci that reached genome-wide significance (GWS; P < 5 x 10(-8)), of which 13 map to regions not previously associated with this trait: 1p31.3 (GPR177), 2p21 (SPTBN1), 3p22 (CTNNB1), 4q21.1 (MEPE), 5q14 (MEF2C), 7p14 (STARD3NL), 7q21.3 (FLJ42280), 11p11.2 (LRP4, ARHGAP1, F2), 11p14.1 (DCDC5), 11p15 (SOX6), 16q24 (FOXL1), 17q21 (HDAC5) and 17q12 (CRHR1). The meta-analysis also confirmed at GWS level seven known BMD loci on 1p36 (ZBTB40), 6q25 (ESR1), 8q24 (TNFRSF11B), 11q13.4 (LRP5), 12q13 (SP7), 13q14 (TNFSF11) and 18q21 (TNFRSF11A). The many SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism and highlight the complex genetic architecture that underlies osteoporosis and variation in BMD

Prostate cancer bone metastases promote both osteolytic and osteoblastic activity

Advanced prostate cancer is frequently accompanied by the development of metastasis to bone. In the past, prostate cancer bone metastases were characterized as being osteoblastic (i.e., increasing bone density) based on radiographs. However, emerging evidence suggests that development of prostate cancer bone metastases requires osteoclastic activity in addition to osteoblastic activity. The complexities of how prostate tumor cells influence bone remodeling are just beginning to be elucidated. Prostate cancer cells produce a variety of pro-osteoblastic factors that promote bone mineralization. For example, both bone morphogenetic proteins and endothelin-1 have well recognized pro-osteoblastic activities and are produced by prostate cancer cells. In addition to factors that enhance bone mineralization prostate cancer cells produced factors that promote osteoclast activity. Perhaps the most critical pro-osteoclastogenic factor produced by prostate cancer cells is receptor activator of NFΚB ligand (RANKL), which has been shown to be required for the development of osteoclasts. Blocking RANKL results in inhibiting prostate cancer-induced osteoclastogenesis and inhibits development and progression of prostate tumor growth in bone. These findings suggest that targeting osteoclast activity may be of therapeutic benefit. However, it remains to be defined how prostate cancer cells synchronize the combination of osteoclastic and osteoblastic activity. We propose that as the bone microenvironment is changed by the developing cancer, this in turn influences the prostate cancer cells' balance between pro-osteoclastic and pro-osteoblastic activity. Accordingly, the determination of how the prostate cancer cells and bone microenvironment crosstalk are important to elucidate how prostate cancer cells modulate bone remodeling. © 2003 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34904/1/10662_ftp.pd

BMP Regulation of SOST and Osterix Expression During Embryonic Osteogenesis

Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis since loss of function mutation in SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in osteogenic front in embryonic 16.5 day postcoitum (E16.5) embryonic calvariae and this expression persisted in the peripheral area of cranial bone in the later developmental stage (E18.5). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar co-expression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of BMP2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expressing adenovirus into the primary calvarial osteoblastic cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.American Society for Bone and Mineral Research 26th annual meetin