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One-third of postmenopausal women experience at least one osteoporotic bone fracture in their lifetime that occurs spontaneously or from low-impact events. However, osteoporosis-associated jaw bone fractures are extremely rare. It was also observed that jaw bone marrow stem cells (BMSCs) have a higher capacity to form mineralized tissues than limb BMSCs. At present, the underlying causes and mechanisms of variations between jaw bone and limb bone during postmenopause are largely unknown. Thus, the objective of the current study was to examine the site-specific effects of estrogen deficiency using comprehensive analysis of bone quantity and quality, and its association with characterization of cellular components of bone. Nine rats (female, 6 months old) for each bilateral sham and ovariectomy (OVX) surgery were obtained and maintained for 2 months after surgery. A hemi-mandible and a femur from each rat were characterized for parameters of volume, mineral density, cortical and trabecular morphology, and static and dynamic mechanical analysis. Another set of 5 rats (female, 9 months old) was obtained for assays of BMSCs. Following cytometry to identify BMSCs, bioassays for proliferation, and osteogenic, adipogenic, chondrogenic differentiation, and cell mitochondrial stress tests were performed. In addition, mRNA expression of BMSCs was analyzed. OVX decreased bone quantity and quality (mineral content, morphology, and energy dissipation) of femur while those of mandible were not influenced. Cellular assays demonstrated that mandible BMSCs showed greater differentiation than femur BMSCs. Gene ontology pathway analysis indicated that the mandibular BMSCs showed most significant differential expression of genes in the regulatory pathways of osteoblast differentiation, SMAD signaling, cartilage development, and glucose transmembrane transporter activity. These findings suggested that active mandibular BMSCs maintain bone formation and mineralization by balancing the rapid bone resorption caused by estrogen deficiency. These characteristics likely help reduce the risk of osteoporotic fracture in postmenopausal jawbone.

The objective of this study was to examine relationships among a variety of bone characteristics, including volumetric, mineral density, geometric, dynamic mechanical analysis, and static fracture mechanical properties. As MYO9B is an unconventional myosin in bone cells responsible for normal skeletal growth, bone characteristics of wild-type (WT), heterozygous (HET), and MYO9B knockout (KO) mice groups were compared as an animal model to express different bone quantity and quality. Forty-five sex-matched 12-week-old mice were used in this study. After euthanization, femurs were isolated and scanned using microcomputed tomography (micro-CT) to assess bone volumetric, tissue mineral density (TMD), and geometric parameters. Then, a non-destructive dynamic mechanical analysis (DMA) was performed by applying oscillatory bending displacement on the femur. Finally, the same femur was subject to static fracture testing. KO group had significantly lower length, bone mineral density (BMD), bone mass and volume, dynamic and static stiffness, and strength than WT and HET groups (p 0.019). On the other hand, TMD parameters of KO group were comparable with those of WT group. HET group showed volumetric, geometric, and mechanical properties similar to WT group, but had lower TMD (p 0.014). Non-destructive micro-CT and DMA parameters had significant positive correlations with strength (p 0.015) without combined effect of groups and sex on the correlations (p 0.077). This comprehensive characterization provides a better understanding of interactive behavior between the tissue- and organ-level of the same femur. The current findings elucidate that MYO9B is responsible for controlling bone volume to determine the growth rate and fracture risk of bone.

Postmenopausal osteoporosis causes severe loss of bone quantity and quality in limb bone but has a lesser effect on jaw bone. Thus, the objective of this study was to examine whether ovariectomy (OVX) and mastication alter the regional variation of jaw bone characteristics. Sprague-Dawley female rats (6 months) were given a bilateral OVX or a sham operation (SHAM) (n=10 for each group). After 2 months post-OVX, the hemi-mandible from each rat was dissected. A micro-computed tomography based mean, standard deviation (SD), the lower and upper 5(th) percentile (Low(5) and High(5)) values of tissue mineral density (TMD) histograms were assessed for whole bone (WB), alveolar bone (AB), cortical bone (CB), and trabecular bone (TB) regions. Morphology of TB and periodontal ligament (PDL) was also obtained. Layers of AB were segmented up to 400 μm from the PDL. Mechanical properties at the tissue level were measured by nanoindentation at the same site by a single loading-unloading cycle of indentation in hydration. The AB and TB regions had significantly lower TMD Mean, Low(5), and High(5) but higher SD than the CB region for both sham and OVX groups (p0.06). Estrogen deficiency induced by OVX did not deteriorate bone characteristics including mineral density, morphology, and nanoindentation parameters in rat mandibles. Masticatory loading had an effect on the TMD parameters at the limited region of AB. These results provide insight into why osteoporosis-associated jaw bone fractures are extremely rare.

Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.

Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and beta-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.

Tropomyosins (Tms) are alpha-helical dimers that bind and stabilize actin microfilaments while regulating their accessibility to other actin-associated proteins. Four genes encode expression of over forty Tms, most of which are expressed in nonmuscle cells. In recent years, it has become clear that individual Tm isoforms may regulate specific actin pools within cells. In this study, we examined how osteoclast function may be regulated by the tropomyosin isoform Tm-4, which we previously showed to be highly localized to podosomes and sealing zones of osteoclasts. RNAi-mediated knockdown of Tm-4, both in RAW264.7- and mouse marrow-derived osteoclasts, resulted in thinning of the actin ring of the sealing zone. Knockdown of Tm-4 also resulted in diminished bone resorptive capacity and altered resorption pit shape. In contrast, osteoclasts overexpressing Tm-4 demonstrated thickened podosomes on glass as well as thickened, aberrant actin structures on bone, and diminished motility and resorptive capacity. These results indicate that Tm-4 plays a role in regulating adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone.

Vacuolar H+-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49–59 of B1 and 55–65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.

The proton-translocating vacuolar ATPase (V-ATPase) acidifies the endocytic network of eukaryotic cells. Although all eukaryotic cell types require low to moderate levels of V-ATPase, some proton-secreting cells express amplified levels for use in specialized membrane domains. To characterize genetic elements required for this heightened expression, we studied transcription and stability of mRNA encoding the V-ATPase c subunit in a low expressing fibroblast cell line (NIH 3T3) and a high expressing macrophage cell line (RAW 264.7). Isolation of the promoter and mapping of the transcriptional start site indicated that the c subunit promoter is TATA-less and initiates transcription at a single site. Promoter activity was regulated through the same transcription factor binding sites in both cell types, which showed no discernible difference in rates of c subunit transcription. In contrast, c subunit transcripts showed markedly greater stability in RAW cells than in 3T3 cells, as did other constitutively expressed V-ATPase subunit transcripts. Only the B and 'a' subunits, which are expressed in multiple isoforms, were not regulated solely by mRNA stability. These results suggest that overall expression levels of the V-ATPase are set primarily by regulation of mRNA stability and that transcriptional mechanisms determine subunit composition in varying cell types.

During monocyte-to-macrophage differentiation, the cellular content of vacuolar H+-ATPase (V-ATPase) increases more than 4-fold. We have shown previously that amplified expression of the B2 subunit of the V-ATPase occurs solely by increased transcription, and that the 5′-untranslated region of the B2 gene, containing multiple consensus binding sites for the transcription factors AP-2 and Sp1, is required for this expression. The present study demonstrates that AP-2 binding sequences are essential for increased transcription from the B2 promoter during monocyte-macrophage differentiation and that AP-2, expressed exogenously in THP-1 and other cells, activates transcription from the B2 promoter. In mobility shift assays, a nuclear factor from THP-1 and U-937 cells was identified that binds to several AP-2 response elements within the B2 promoter, but does not react with AP-2 antibodies, and has a DNA sequence binding affinity profile that differs from AP-2. These findings suggest that a novel AP-2-like transcription factor is responsible for V-ATPase B subunit amplification during monocyte differentiation.

Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.

In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.