Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2’s role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
Fungus; Coccomyces proteae; Aurapex penicillata; CMG2; Phenolic
The purpose of this study was to compare the effects of Nordic walking with conventional walking and band-based resistance exercise on functional fitness, static balance and dynamic balance in older adults. Volunteers (n = 65) were divided into four groups: Nordic walking (NW), conventional walking (CW), resistance (RES), and control. Each group performed activity 50-70 min·day−1 (warm-up 10-15 min, main exercise 30-40, and cool down 10-15 min), 3 days·week−1 (NW and CW) or 2 day·week−1 (RES) for 12 wks. Upper-body strength improved (p < 0. 05) in the RES (22.3%) and the NW (11.6%) groups compared to the CW and control groups. Cardio- respiratory fitness improved more in the NW (10.9%) and CW (10.6%) groups compared to the RES and control groups. Upper- and lower-body flexibility also improved in all exercise groups compared to the control group. There were no improvements in balance measures in any group. While all modes of exercise improved various components of fitness, Nordic walking provided the best well-rounded benefits by improving upper-body strength, cardiovascular endurance, and flexibility. Therefore, Nordic walking is recommended as an effective and efficient mode of concurrent exercise to improve overall functional fitness in older adults.
Nordic walking, conventional walking, and resistance training are beneficial for older adults.
Nordic walking and conventional walking both improve cardio-respiratory fitness while resistance training does not.
Nordic walking provides additional benefits in upper-body muscular strength compared to conventional walking.
Nordic walking is an effective and efficient mode of exercise to improve overall fitness in older adults.
Walking; resistance exercise; concurrent exercise; aging; functional fitness
As the older adult population increases in size, the number of older adults participating in sport activities will also likely increase proportionally with a concomitant increase in musculoskeletal injuries. Age-associated functional declines in muscle strength and the sensory systems, in addition to several other issues, contribute to reductions in balance that may increase fall risk There are a variety of ways to evaluate balance and fall-risk, and each older adult should be regularly screened in order to evaluate any changes in the ability to maintain postural stability. Balance training is a useful intervention in rehabilitation of postural stability impairments as well as in training programs for performance enhancement. One scientifically-based approach is Sensorimotor Training (SMT) which can be characterized as a progressive balance training program using labile surfaces to provide adequate and safe challenges to the older athlete's balance. SMT addresses both static and dynamic components of balance as well as the multitude of systems that control balance in order to train effective strategies and elicit automatic postural responses in order to enhance postural stability. The authors believe that SMT should become part of the regular training regimen for the aging athlete. For the sport and orthopedic healthcare professional, an understanding of the physiologic changes that occur with age, the means by which balance can be assessed, and how SMT programs can be developed and implemented is crucial in addressing the growing number of older athletes that they will see.
Level of Evidence:
Aging; balance assessment; balance training; postural stability
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The article entitled “Monosodium glutamate (MSG) intake is associated with the prevalence of metabolic syndrome in a rural Thai population”, concluded that higher amounts of individual’s MSG consumption are associated with the risk of having the metabolic syndrome and being overweight independent of other major determinants. However, this epidemiological study is the only study indicating such a relationship between MSG intake and the prevalence of metabolic syndrome and there is no direct supporting evidence for a causal relationship between MSG intake and prevalence of metabolic syndrome. This study does not indicate that MSG causes metabolic syndrome. Furthermore, there are several questionable points concerning study methods. Further carefully designed studies taking into account all glutamate sources are necessary to demonstrate the relationship between overweight, metabolic syndrome, MSG intake and umami sensitivity.
Monosodium glutamate; Intake; Overweight; Metabolic syndrome
Determine the reliability of two different modified (MOD1 and MOD2) testing methods compared to a standard method (ST) for testing trunk flexion and extension endurance.
Twenty‐eight healthy individuals (age 26.4 ± 3.2 years, height 1.75 ± m, weight 71.8 ± 10.3 kg, body mass index 23.6 ± 3.4 m/kg2).
Trunk endurance time was measured in seconds for flexion and extension under the three different stabilization conditions. The MOD1 testing procedure utilized a female clinician (70.3 kg) and MOD2 utilized a male clinician (90.7 kg) to provide stabilization as opposed to the ST method of belt stabilization.
No significant differences occurred between flexion and extension times. Intraclass correlations (ICCs3,1) for the different testing conditions ranged from .79 to .95 (p <.000) and are found in Table 3. Concurrent validity using the ST flexion times as the gold standard coefficients were .95 for MOD1 and .90 for MOD2. For ST extension, coefficients were .91 and .80, for MOD1 and MOD2 respectively (p <.01).
These methods proved to be a reliable substitute for previously accepted ST testing methods in normal college‐aged individuals. These modified testing procedures can be implemented in athletic training rooms and weight rooms lacking appropriate tables for the ST testing.
Level of Evidence:
Core; stabilization; trunk endurance
The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12–/– OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12–/–, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 gene knockout (Itgb3–/–) OCs, but its effects were substantially blunted in P2ry12–/– OCs. In vivo, P2ry12–/– mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.
Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.
To determine the effects of a supervised strength training program on body composition and physical capacity of older women using three different devices: weight machines, elastic bands, and aquatic devices that increase drag forces (ADIDF). Four groups were formed: control group, weight machine group (WMG), elastic band group (EBG) and a group that used ADIDF (ADIDFG). Body composition and physical capacity were assessed before and after the intervention period. The ADIDFG showed improvements in fat mass (FM), fat-free mass of the left arm (FFM-LA) and right arm (FFM-RA), knee push-up test (KPT), squat test (ST) and crunch test (CT) (p <0.05). Individuals in the EBG and WMG also improved their FM, fat free mass (FFM), FFM-LA, FFM-RA, KPU, ST and CT. ADIDF training improves body composition and physical capacity of postmenopausal women as does performing land-based training programs.
aquatic training; weight machines; elastic bands; fitness
Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.
Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.
Fluorescent; bisphosphonate; imaging; conjugates
Originally identified as axonal guidance cues, semaphorins are expressed throughout many different tissues and regulate numerous non-neuronal processes. We demonstrate that most class III semaphorins are expressed in mouse osteoblasts and are differentially regulated by cell growth and differentiation: Sema3d expression is increased and Sema3e expression decreased during proliferation in culture, while expression of Sema3a is unaffected by cell density but increases in cultures of mineralizing osteoblasts. Expression of Sema3a, -3e, and -3d is also differentially regulated by osteogenic stimuli; inhibition of GSK3β decreased expression of Sema3a and -3e, while 1,25-(OH)2D3 increased expression of Sema3e. Parathyroid hormone had no effect on expression of Sema3a, -3b, or -3d. Osteoblasts, macrophages, and osteoclasts express the Sema3e receptor PlexinD1, suggesting an autocrine and paracrine role for Sema3e. No effects of recombinant Sema3e on osteoblast proliferation, differentiation, or mineralization were observed; but Sema3e did inhibit the migration of osteoblasts in a wound-healing assay. The formation of multinucleated, tartrate-resistant acid phosphatase–positive osteoclasts was decreased by 81% in cultures of mouse bone marrow macrophages incubated with 200 ng/mL Sema3e. Correspondingly, decreased expression of osteoclast markers (Itgb3, Acp5, Cd51, Nfatc1, CalcR, and Ctsk) was observed by qPCR in macrophage cultures differentiated in the presence of Sema3e. Our results demonstrate that class III semaphorins are expressed by osteoblasts and differentially regulated by differentiation, mineralization, and osteogenic stimuli. Sema3e is a novel inhibitor of osteoclast formation in vitro and may play a role in maintaining local bone homeostasis, potentially acting as a coupling factor between osteoclasts and osteoblasts.
Macrophage; Plexin; Mineralization; Vitamin D3; Migration
Falls are a serious health risk for older adults. But for those living in rural and frontier areas of the USA, the risks are higher because of limited access to health care providers and resources. This study employed a community-based participatory research approach to develop a fall prevention toolkit to be used by residents of rural and frontier areas without the assistance of health care providers. Qualitative data were gathered from both key informant interviews and focus groups with a broad range of participants. Data analysis revealed that to be effective and accepted, the toolkit should be not only evidence based but also practical, low-cost, self-explanatory, and usable without the assistance of a health care provider. Materials must be engaging, visually interesting, empowering, sensitive to reading level, and appropriate for low-vision users. These findings should be useful to other researchers developing education and awareness materials for older adults in rural areas.
MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir.
We employed a proteomics technique called “stable isotope labelling by amino acids in cell culture” (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation.
To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs.
Osteoclasts are multinucleated cells that are responsible for resorption of bone, and increased activity of these cells is associated with several common bone diseases, including postmenopausal osteoporosis. Upon adhesion to bone, osteoclasts become polarized and reorganise their cytoskeleton and membrane to form unique domains including the sealing zone (SZ), which is a dense ring of F-actin-rich podosomes delimiting the ruffled border (RB), where protons and proteases are secreted to demineralise and degrade the bone matrix, respectively. These processes are dependent on the activity of small GTPases. Rho GTPases are well known to control the organization of F-actin and adhesion structures of different cell types, affecting subsequently their migration. In osteoclasts, RhoA, Rac, Cdc42, RhoU and also Arf6 regulate podosome assembly and their organization into the SZ. By contrast, the formation of the RB involves vesicular trafficking pathways that are regulated by the Rab family of GTPases, in particular lysosomal Rab7. Finally, osteoclast survival is dependent on the activity of Ras GTPases. The correct function of almost all these GTPases is absolutely dependent on post-translational prenylation, which enables them to localize to specific target membranes. Bisphosphonate drugs, which are widely used in the treatment of bone diseases such as osteoporosis, act by preventing the prenylation of small GTPases, resulting in the loss of the SZ and RB and therefore inhibition of osteoclast activity, as well as inducing osteoclast apoptosis. In this review we summarize current understanding of the role of specific prenylated small GTPases in osteoclast polarization, function and survival.
osteoclast; bone; Rab; Rho; Rac; Ras; Arf; bisphosphonate; prenylation
Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function. The gunmetal (gm/gm) mouse bears a mutation in the catalytic subunit of RGGT, causing a loss of 75% of the activity of this enzyme and hence hypoprenylation of several Rabs in melanocytes, platelets and cytotoxic T cells. We have now found that prenylation of several Rab proteins is also defective in gm/gm osteoclasts. Moreover, while osteoclast formation and cytoskeletal polarization occurs normally, gm/gm osteoclasts exhibit a substantial reduction in resorptive activity in vitro compared with osteoclasts from +/gm mice, which do not have a prenylation defect. Surprisingly, rather than the osteosclerosis that would be expected to result from defective osteoclast function in vivo, gm/gm mice exhibited a slightly lower bone mass than +/gm mice, indicating that defects in other cell types, such as osteoblasts, in which hypoprenylation of Rabs was also detected, may contribute to the phenotype. However, gm/gm mice were partially protected from ovariectomy-induced bone loss, suggesting that levels of Rab prenylation in gm/gm osteoclasts may be sufficient to maintain normal physiological levels of activity, but not pathological levels of bone resorption in vivo.
osteoclast; bone resorption; bone; Rab; small GTPase; prenylation; gunmetal
The anthrax toxin receptors tumor endothelial marker-8 (TEM-8) and capillary morphogenesis gene-2 (CMG-2) are responsible for allowing entry of anthrax toxin into host cells. However, these receptors were first discovered due to their enhanced expression on endothelial cells undergoing blood vessel growth or angiogenesis in in vitro or in vivo model systems. Targeting and inhibiting angiogenesis is an important strategy for current anti-cancer therapies and treatment of retinal diseases. Structures, tissue expression, and interactions of the TEM-8 and CMG-2 proteins have been documented, and functional roles for these receptors in angiogenesis have recently emerged. TEM-8 appears to regulate endothelial cell migration and tubule formation whereas a role for CMG-2 in endothelial proliferation has been documented. TEM-8 and CMG-2 bind differentially to extracellular matrix proteins including collagen I, collagen IV and laminin and these properties may be responsible for their apparent roles in regulating endothelial cell behavior during angiogenesis. TEM-8-binding moieties have also been suggested to be useful in selectively targeting anti-angiogenic and anti-tumorigenic therapies to tumor endothelium. Additionally, studies of modified forms of lethal toxin (LeTx) have demonstrated that targeted inhibition of MAPKs within tumor vessels may represent an efficacious anti-angiogenic strategy.
Endothelial; angiogenesis; anthrax; intracellular signaling; extracellular matrix
Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647–labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo. © 2010 American Society for Bone and Mineral Research
bisphosphonates; fluorescent conjugates; cellular uptake; osteocytes; monocytes
Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src-/- mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.
Apomine, a 1,1-bisphosphonate-ester with antitumor activity, has previously been reported to strongly down-regulate 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the mevalonate pathway responsible for the prenylation of proteins. Here, we show that although apomine down-regulated HMG-CoA reductase protein levels in myeloma cells, it did not inhibit protein prenylation, and apomine-induced apoptosis could not be prevented by mevalonate, indicating that apomine cytotoxicity is independent from its effects on HMG-CoA reductase. Instead, apomine cytotoxicity was prevented by the addition of phosphatidylcholine, which is similar to the previously reported ability of phosphatidylcholine to overcome the cytotoxicity of farnesol, whereas phosphatidylcholine had no effect on down-regulation of HMG-CoA reductase by apomine. These findings raised the possibility that apomine, independent from its own cytotoxic effects, could enhance the antitumor effects of the competitive HMG-CoA reductase inhibitor lovastatin via down-regulating HMG-CoA reductase. Indeed, treatment with apomine in combination with lovastatin resulted in synergistic decreases in viable cell number and induction of apoptosis. At the concentrations used, apomine down-regulated HMG-CoA reductase protein levels without being cytotoxic. Accumulation of unprenylated Rap1A by lovastatin was enhanced in the presence of apomine. Furthermore, synergy was completely prevented by mevalonate, and apomine did not synergize with desoxolovastatin, which does not inhibit HMG-CoA reductase. We conclude that the synergistic drug interaction results from an enhancement by apomine of the effects of lovastatin, mediated by down-regulation of HMG-CoA reductase by apomine. Thus, these findings demonstrate a novel strategy for enhancing the antitumor effects of lovastatin.
The First Cambridge Conference on Advances in Treating Metastatic Bone Cancer, a symposium held in Cambridge, Massachusetts, October 28 to 29, 2005, was convened to discuss recent advances and research related to the natural history of bone metastases and skeletal complications, bone cancer biology, treatment of myeloma and other solid tumors, and treatment-induced bone loss. The conference format combined brief presentations with extended periods of discussion. The conclusions reached during the 2-day meeting are summarized in this article and presented in more detail in the individual articles and accompanying discussion sessions that comprise the conference proceedings.
Bisphosphonates are the most widely used class of drug for inhibiting osteoclast-mediated bone loss, but their effectiveness at preventing joint destruction in rheumatoid arthritis has generally been disappointing. We examined whether the ability of bisphosphonates to induce osteoclast apoptosis and inhibit bone resorption in vitro is influenced by the cytokine receptor activator of nuclear factor-kappa B ligand (RANKL), an important mediator of inflammation-induced bone loss.
Rabbit osteoclasts were treated with the bisphosphonates clodronate or alendronate for up to 48 hours in the absence or presence of RANKL. Changes in cell morphology and induction of apoptosis were examined by scanning electron microscopy, whilst resorptive activity was determined by measuring the area of resorption cavities. Changes in the level of anti-apoptotic proteins, including Mcl-1, Bcl-2, and Bcl-x>L, were determined in rabbit osteoclasts and in cytokine-starved mouse osteoclasts by Western blotting.
RANKL significantly attenuated the ability of both clodronate and alendronate to induce osteoclast apoptosis and inhibit bone resorption. Treatment of rabbit osteoclasts with RANKL was associated with an increase in the anti-apoptotic protein Mcl-1 but not Bcl-2. A role for Mcl-1 in osteoclast survival was suggested using osteoclasts generated from mouse bone marrow macrophages in the presence of RANKL + macrophage colony-stimulating factor (M-CSF) since cytokine deprivation of mouse osteoclasts caused a rapid loss of Mcl-1 (but not Bcl-2 or Bcl-xL), which preceded the biochemical and morphological changes associated with apoptosis. Loss of Mcl-1 from mouse osteoclasts could be prevented by factors known to promote osteoclast survival (RANKL, M-CSF, tumour necrosis factor-alpha [TNF-α], or lipopolysaccharide [LPS]).
RANKL protects osteoclasts from the apoptosis-inducing and anti-resorptive effects of bisphosphonates in vitro. The ability of RANKL (and other pro-inflammatory factors such as TNF-α and LPS) to increase the level of Mcl-1 in osteoclasts may explain the lack of effectiveness of some bisphosphonates in preventing inflammation-induced bone loss.
Nitrogen-containing bisphosphonates indirectly activate Vγ9Vδ2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vγ9Vδ2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid-pulsed monocytes triggered activation of γδ T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vγ9Vδ2 T cell activation.
bisphosphonates; alkylamines; monocytes; gamma delta T cells; isopentenyl diphosphate
Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational
geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab
GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction
by bisphosphonate analogs in which one phosphonate group is replaced by a
carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were
3-PEHPC, which was previously reported, and
acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by
both IC50 and Ki.(+)-3-IPEHPC behaves as a
mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an
uncompetitive inhibitor with respect to Rab substrates. We propose that
phosphonocarboxylates prevent only the second GG transfer onto Rabs based on
the following evidence. First, geranylgeranylation of Rab proteins ending with
a single cysteine motif such as CAAX, is not affected by the
inhibitors, either in vitro or in vivo. Second, the addition
of an -AAX sequence onto Rab-CC proteins protects the substrate from
inhibition by the inhibitors. Third, we demonstrate directly that in the
presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by
only a single GG addition. The presence of (+)-3-IPEHPC resulted in a
preference for the Rab N-terminal cysteine to be modified first, suggesting an
order of cysteine geranylgeranylation in RGGT catalysis. Our results further
suggest that the inhibitor binds to a site distinct from the GGPP-binding site
on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine
binding site adjacent to the active site, which is necessary to align the
mono-GG-Rab for the second GG addition. These inhibitors may represent a novel
therapeutic approach in Rab-mediated diseases.
In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-α. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC-class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.
Lymphocytic Choriomeningitis Virus (LCMV); Toll-like receptors; CNS glial cells
This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.