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Etsuko Abe

Publications and source records attributed to Etsuko Abe.

17 recordsLinked to original sources
PubMed2006

Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts.

Here, we demonstrate that the Ca(2+)/calmodulin-sensitive phosphatase calcineurin is a necessary downstream mediator for osteoclast differentiation. Using quantitative PCR, we detected the calcineurin isoforms Aalpha, Abeta, Agamma (catalytic), and B1 (regulatory) in osteoclast precursor RAW-C3 cells. We found that, although the expression of these isoforms remained relatively unchanged during osteoclast differentiation, there was a profound increase in the expression of their primary substrate for calcineurin, nuclear factor of activated T cells (NFAT)c1. For gain-of-function studies, we incubated osteoclast precursors for 10 min with a calcineurin fusion protein (TAT-calcineurin Aalpha); this resulted in its receptorless influx into >90% of the precursor cells. A marked increase in the expression of the osteoclast differentiation markers tartrate-resistant acid phosphatase (TRAP) and integrin beta(3) followed. In addition, the expression of NFATc1, as well as the alternative substrate for calcineurin, IkappaBalpha, was significantly enhanced. Likewise, transfection with constitutively active NFAT resulted in an increased expression of both TRAP and integrin beta(3). In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATc1, TRAP, and integrin beta(3) by TAT-calcineurin Aalpha. The expression of these markers was also inhibited by calcineurin Aalpha U1 small nuclear RNA, which significantly reduced calcineurin Aalpha mRNA and protein expression. Consistent with these observations, we observed a reduction in osteoclastogenesis in calcineurin Aalpha(-/-) cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-of-function experiments establish that calcineurin Aalpha is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism.

Acid Phosphatase↗
PubMed2006

TNFalpha mediates the skeletal effects of thyroid-stimulating hormone.

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNFalpha, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR-/- and TSHR+/- mice is abrogated in compound TSHR-/-/TNFalpha-/- and TSHR+/-/TNFalpha+/- mice, respectively. In parallel studies, we find that TSH directly inhibits TNFalpha production, reduces the number of TNFalpha-producing osteoclast precursors, and attenuates the induction of TNFalpha expression by IL-1, TNFalpha, and receptor activator of NF-kappaB ligand. TSH also suppresses osteoclast formation in murine macrophages and RAW-C3 cells. The suppression is more profound in cells that overexpress the TSHR than those transfected with empty vector. The overexpression of ligand-independent, constitutively active TSHR abrogates osteoclast formation even under basal conditions and in the absence of TSH. Finally, IL-1/TNFalpha and receptor activator of NF-kappaB ligand fail to stimulate AP-1 and NF-kappaB binding to DNA in cells transfected with TSHR or constitutively active TSHR. The results suggest that TNFalpha is the critical cytokine mediating the downstream antiresorptive effects of TSH on the skeleton.

Animals↗
PubMed2006

Function of BMPs and BMP antagonists in adult bone.

The expression and function of BMPs and BMPs in bone tissues have been studied for a long time because of their remarkable activities. However, their biological functions in normal bone remodeling in adults were not fully understood until recently. Advanced technologies using gene manipulation were used to study their roles in adulthood. In addition, findings of new BMP antagonists and the effect of Wnt-canonical pathways on bone features also provided new insights in bone studies.

Animals↗
PubMed2006

TSH and bone loss.

We have recently challenged the view that the bone loss associated with hyperthyroidism is solely due to elevated thyroid hormone levels. We find that thyroid-stimulating hormone (TSH), derived from the anterior pituitary gland, inhibits bone resorption by the osteoclast. Mice haploinsufficient in the TSH receptor show reduced bone density and evidence of enhanced bone resorption in the face of normal thyroid function. In humans, TSH inhibits markers of bone resorption with a single administration, and low TSH levels correlate with increased fracture risk. The evidence that low TSH levels predispose to osteoporosis in hyperthyroidism is discussed in view of the emerging role of pituitary hormones in bone biology.

Animals↗
PubMed2005

Calcineurin regulates bone formation by the osteoblast.

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.

Animals↗
PubMed2005

Bisphosphonates induce inflammation and rupture of atherosclerotic plaques in apolipoprotein-E null mice.

The apolipoprotein E knockout (Apo-E-/-) mouse is a well-known model of atherosclerosis. Bisphosphonates, through their affinity to hydroxyapatite, are known to reduce arterial calcification in several animal models. Thus, we examined the effect of two therapeutically used oral bisphosphonates, alendronate and risedronate, on plaque formation in the Apo-E-/- mouse. The drugs were administered by gavage to 16-week-old Apo-E-/- mice for 8 weeks. At 8 weeks, there was no difference in bone mineral density (BMD) of the alendronate- and risedronate-treated mice at any site. A time-dependent increase in BMD was demonstrated in Apo-E-/- mice with risedronate (p<0.01). Histological evaluation revealed that both bisphosphonates caused atherosclerotic plaque rupture. Five of 17 mice had severe inflammation with or without plaque rupture, while seven mice showed inflammation, but without plaque rupture. Neither caspase 3 nor metalloproteinases 2 and 9 were increased in ruptured plaques on immunocytochemistry. Quantitative measurements of arterial caliber remained unaffected. Our finding of plaque inflammation and rupture in bisphosphonate-treated Apo-E-/- mice may provide the first animal model for studies aimed at characterizing mechanisms of plaque rupture in animal models.

Alendronate↗
PubMed2003

TSH is a negative regulator of skeletal remodeling.

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.

Animals↗
PubMed2003

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ "sensor," particularly during periods of active motility and secretion.

ADP-ribosyl Cyclase↗
PubMed2003

Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice.

We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense beta-gal histo- or cytostaining in adult noggin+/- mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.

Aging↗
PubMed2003

Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics.

Over the past decade, advances in molecular tools, stem cell differentiation, osteoclast and osteoblast signaling mechanisms, and genetically manipulated mice models have resulted in major breakthroughs in understanding osteoclast biology. This review focuses on key advances in our understanding of molecular mechanisms underlying the formation, function, and survival of osteoclasts. These include key signals mediating osteoclast differentiation, including PU.1, RANK, CSF-1/c-fms, and src, and key specializations of the osteoclast including HCl secretion driven by H+-ATPase and the secretion of collagenolytic enzymes including cathepsin K and matrix metalloproteinases (MMPs). These pathways and highly expressed proteins provide targets for specific therapies to modify bone degradation. The main outstanding issues, basic and translational, will be considered in relation to the osteoclast as a target for antiresorptive therapies.

Animals↗
PubMed2003

The structure of medical malpractice decision-making in Japan.

The article summarises the problems in the medical malpractice litigation systems in the United Kingdom and Japan, demonstrating the similarities and identifying the length of time between initiating an action and its decision and other factors responsible for lengthy litigation. Based on analysis of decisions of medical malpractice cases between 1986 and 1998 in Japan, the functioning of the Japanese medical malpractice litigation system is discussed. Lengthy litigation is shown to be correlated with outcome and implies that the Japanese medical dispute resolution mechanism favours those who can endure lengthy litigation, namely the defendants, who are physicians or hospitals. In view of the similarities between the two systems, it is likely that the same bias--that the wealthier party in the litigation is more likely to win the case--also occurs in medical malpractice litigation in the United Kingdom and Australia.

Compensation and Redress↗
PubMed2002

Transduction of TAT fusion proteins into osteoclasts and osteoblasts.

It has been difficult to transduce primary cultures of bone cells with proteins of interest. Here, we report the development and validation of a new technology for transduction of osteoblasts and osteoclasts with peptides and moderately sized proteins. Fusion proteins between TAT, an 11 amino acid Arg-rich sequence derived from the HIV protein, and either hemagglutinin or calcineurin Aalpha were synthesized and purified. Exposure of osteoblasts and osteoclasts in primary culture to either TAT-HA or TAT-calcineurin Aalpha resulted in a rapid (within 10 min) intracellular movement of the fusion protein evident on co-immunostaining. Almost 99% of cells were transduced and the fusion protein was retained in approximately 50% of the cells for up to 5 days. TAT did not abolish the functionality of calcineurin Aalpha; the fusion protein stimulated osteoblast differentiation and inhibited osteoclastic resorption. We expect that our studies will provide a firm basis for the future development of TAT fusion proteins for critical molecules involved in bone cell differentiation and function.

Animals↗
PubMed2002

Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed2002

A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl cyclase, a putative intracellular NAD+ sensor.

CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+-->cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km approximately 5.13 microM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Delta(-49)-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr-Delta(-49)-CD38-EGFP or Delta(-49)-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.

3T3 Cells↗
PubMed2002

Ca(2+) influx through the osteoclastic plasma membrane ryanodine receptor.

We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca(2+) influx channel and as a divalent cation (Ca(2+)) sensor. Cytosolic Ca(2+) measurements revealed Ca(2+) influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca(2+) change was, as expected, not seen in Ca(2+)-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (approximately 25 mV) ryanodine triggered extracellular Ca(2+) influx that was blocked by Ni(2+). In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective approximately 60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential (E(rev)) approximately 0 mV]. In the presence of a Ba(2+) gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to approximately 120 pS and E(rev) shifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca(2+) channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab(34). To demonstrate that extracellular Ca(2+) sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca(2+) signals triggered by either Ni(2+) (5 mM) or Cd(2+) (50 microM). Finally, intracellular application of antiserum Ab(34) potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca(2+) influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates Ca(2+) influx but also functions as a sensor for extracellular divalent cations.

Animals↗