Tenofovir Modulates Semaphorin 4D Signaling and Regulates Bone Homeostasis, Which Can Be Counteracted by Dipyridamole and Adenosine A2A Receptor

Semaphorin 4D (Sema4D) is a neurotrophin that is secreted by osteoclasts and binds to its receptor PlexinB1 on osteoblasts to inhibit their differentiation and function. Adenosine A2A activation inhibits osteoclast Sema4D-mediated secretion, diminishes inflammatory osteolysis and prevents bone loss following tenofovir (one of the most used antivirals in HIV). Therefore, tenofovir might activate Sema4D signaling to alter bone turnover. Female C57Bl/6/A2AKO mice were ovariectomized and treated with saline (control), tenofovir 75 mg/Kg/day, dipyridamole 25 mg/Kg/day or a combination for 5 weeks and long bones were prepared for histology. Primary murine-induced osteoclast/osteoblast were challenged with tenofovir/dipyridamole 1 μM each, and the expression of Sema4D/PlexinB1, RhoA/ROCK/IGF1R was studied by RT-PCR, Western blot and immunostaining. In vivo tenofovir showed an increased expression of Sema4D when compared to control mice, and dipyridamole reverted the expression in an A2A-dependent manner. In vitro, tenofovir increases Sema4D expression and secretion in osteoclast precursors, and pre-treatment with dipyridamole reverted this effect. pRhoA and ROCK1 activation were increased and IRS1/IGF1R expression was diminished by tenofovir in the Vav3/ARHGAP18 mechanism in osteoblast precursors and reverted by dipyridamole in an A2A-dependent manner. This suggests that tenofovir increases bone loss by activation of Sema4D/PlexinB1 signaling, which inhibits osteoblast differentiation. Agents that increase local adenosine concentrations, such as dipyridamole, might prevent bone loss following the inhibition of this pathway.

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Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

The Journal of Cell Biology ◽

10.1083/jcb.201204067 ◽

2012 ◽

Vol 199 (7) ◽

pp. 1145-1158 ◽

Cited By ~ 42

Author(s):

Hyung Joon Kim ◽

Vikram Prasad ◽

Seok-Won Hyung ◽

Zang Hee Lee ◽

Sang-Won Lee ◽

...

Keyword(s):

Plasma Membrane ◽

Bone Mass ◽

Osteoclast Differentiation ◽

Premenopausal Women ◽

Fine Tuning ◽

Bone Homeostasis ◽

Regulatory Loop ◽

And Function

The precise regulation of Ca2+ dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand–induced Ca2+ oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca2+ efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca2+ signaling in osteoclasts.

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Ethnopharmacological basis for antispasmodic, antidiarrheal and antiemetic activities of Ceratonia siliqua pods

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v12i4.33218 ◽

2017 ◽

Vol 12 (4) ◽

pp. 384

Author(s):

Irfan Hamid ◽

Khalid Hussain Janbaz

Keyword(s):

Crude Extract ◽

Video Clip ◽

Dependent Manner ◽

Ceratonia Siliqua ◽

Spasmolytic Activity ◽

Rabbit Jejunum ◽

Pre Treatment ◽

Dose Dependent

The study was conducted to provide the ethnopharmacological bases of the crude extract of seed pods of Ceratonia siliqua in the gastrointestinal spasm, diarrhea and emesis. In segregated rabbit jejunum, it showed dose-dependent (0.01-10 mg/mL) relaxation of spontaneous as well as carbachol (1 µM)-induced contraction. Pre-treatment of segregated rat ileum with C. siliqua, significantly (p<0.0001) suppressed the carbachol (1 µM)-induced contraction similar to atropine (1 µM). These results indicated that C. siliqua possesses spasmolytic activity through possible blockage of muscarinic receptor in jejunum preparations. Furthermore, the crude extract inhibited the castor oil-induced diarrhea, charcoal meal propulsion in mice and copper sulfate-induced retches in chicks in a dose-dependent manner (100, 200, 300 mg/kg). These in vitro and in vivo results indicate that C. siliqua possesses the spasmolytic and antidiarrheal activities mediated possibly through blockage of muscarinic receptors. Thus, this study provides a rationale for its folkloric use.Video Clip of Methodology:12 min 42 sec <a href="https://www.youtube.com/v/BQGWdIZqpsY">Full Screen</a> <a href="https://www.youtube.com/watch?v=BQGWdIZqpsY">Alternate</a>

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Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

Journal of Cell Science ◽

10.1242/jcs.109.6.1479 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1479-1495 ◽

Cited By ~ 4

Author(s):

L.A. Temesvari ◽

J.M. Rodriguez-Paris ◽

J.M. Bush ◽

L. Zhang ◽

J.A. Cardelli

Keyword(s):

Morphological Changes ◽

Specific Inhibitor ◽

Fluid Phase ◽

Contractile Vacuole ◽

Dependent Manner ◽

Concanamycin A ◽

Latex Beads ◽

And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

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Notch Signaling-Dependant Expulsion of Parasites through Mast Cell-Mediated Immunity.

Blood ◽

10.1182/blood.v104.11.1473.1473 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1473-1473

Author(s):

Mamiko Sakata-Yanagimoto ◽

Etsuko Yamaguchi-Nakagami ◽

Toru Sakai ◽

Keiki Kumano ◽

Atsushi Kunisato ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Notch Signaling ◽

Cell Generation ◽

Pre Treatment ◽

And Function ◽

Tgf Β1

Abstract [Background] Notch signaling is known to be important in hematopoiesis, but very little information is available about its significance in mast cells. Here we provide direct evidence that notch signaling is critical for both development and function of mast cells in vitro and in vivo. [Methods] A Lin− fraction of mouse bone marrow cells was cultured on immobilized Delta1 in the presence of SCF and IL-3, and emerging Lin−FcεRI+c-Kit+ mast cells were characterized. Next, production of mouse mast cell protease-1 (mMCP-1), which is specific for nematode infection through locally expressed TGF-β1 in vivo, by bone marrow-derived mast cells (BMMC) was analyzed after the stimulation with Delta1 in the presence of TGF-β1. Finally, mice were infected with Strongyloides venezuelensis after pre-treatment with Delta1, and expulsion of the worms was examined. [Results] Lin−FcεRI+c-Kit+ mast cells developed remarkably earlier if stimulated with Delta1 (at one week, 15% vs. 3%). DAPT, a γ-secretase inhibitor, blocked the Delta1 effect, while it did not affect the regular time-course mast cell generation by SCF and IL-3. SB431542, a selective inhibitor of TGF-β1 signaling, also blocked early mast cell generation by Delta1. Delta1 augmented mMCP-1 expression and secretion from BMMC by 50 fold. Both DAPT and SB431542 showed a dose-dependent inhibition of Delta1 effect on mMCP-1 expression and secretion. Pre-treatment of the hosts with Delta1 promoted the expulsion of S. venezuelensis, (left/inoculated ratios of worms, 3% vs. 40%) while Delta1 had no effect in the mast cell-deficient W/Wv mice. [Discussion] Our observations reveal that notch signaling regulates both development and function of mast cells in vitro in conjunction with TGF-β1 signaling. In vivo, it is also likely that Delta1 facilitates the functional maturation of intestinal mast cells to eradicate parasites. More precise mechanism of Delta1 action on mast cells in vivo is under a study.

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β2-Adrenergic Receptor Signaling in Osteoblasts Contributes to the Catabolic Effect of Glucocorticoids on Bone

Endocrinology ◽

10.1210/en.2010-0881 ◽

2011 ◽

Vol 152 (4) ◽

pp. 1412-1422 ◽

Cited By ~ 35

Author(s):

Yun Ma ◽

Jeffry S. Nyman ◽

Huan Tao ◽

Heather H. Moss ◽

Xiangli Yang ◽

...

Keyword(s):

Bone Loss ◽

Bone Cells ◽

Sympathetic Nerves ◽

Bone Diseases ◽

Autonomic Nerves ◽

High Dose ◽

Bone Homeostasis ◽

Adrenergic Stimulation

Abstract The sympathetic nervous system is a physiological regulator of bone homeostasis. Autonomic nerves are indeed present in bone, bone cells express the β2-adrenergic receptors (β2AR), and pharmacological or genetic disruption of sympathetic outflow to bone induces bone gain in rodents. These recent findings implied that conditions that affect β2AR signaling in osteoblasts and/or sympathetic drive to bone may contribute to bone diseases. In this study, we show that dexamethasone stimulates the expression of the β2AR in differentiated primary calvarial osteoblasts, as measured by an increase in Adrβ2 mRNA and β2AR protein level after short-term dexamethasone treatment. Isoproterenol-induced cAMP accumulation and the expression of the β2AR target gene Rankl were also significantly increased after dexamethasone pretreatment, indicating that dexamethasone promotes the responsiveness of differentiated osteoblasts to adrenergic stimulation. These in vitro results led to the hypothesis that glucocorticoid-induced bone loss, provoked by increased endogenous or high-dose exogenous glucocorticoids given for the treatment of inflammatory diseases, might, at least in part, be mediated by increased sensitivity of bone-forming cells to the tonic inhibitory effect of sympathetic nerves on bone formation or their stimulatory effect on bone resorption. Supporting this hypothesis, both pharmacological and genetic β2AR blockade in mice significantly reduced the bone catabolic effect of high-dose prednisolone in vivo. This study emphasizes the importance of sympathetic nerves in the regulation of bone homeostasis and indicates that this neuroskeletal signaling axis can be modulated by hormones or drugs and contribute to enhance pathological bone loss.

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Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.01075-15 ◽

2016 ◽

Vol 36 (19) ◽

pp. 2451-2463 ◽

Cited By ~ 13

Author(s):

Takashi Iezaki ◽

Kazuya Fukasawa ◽

Gyujin Park ◽

Tetsuhiro Horie ◽

Takashi Kanayama ◽

...

Keyword(s):

Bone Loss ◽

Osteoclast Differentiation ◽

Nuclear Factor Κb ◽

Bone Diseases ◽

Nuclear Factor ◽

Activator Protein 1 ◽

Bone Homeostasis ◽

Activated T Cells

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) through activator protein 1. Global deletion of murineIfrd1increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion ofIfrd1in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impairedin vitroinIfrd1-deleted bone marrow macrophages (BMMs).Ifrd1deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-κB-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-κB/NFATc1 axis plays a pivotal role in bone remodelingin vivoand represents a therapeutic target for bone diseases.

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Repositioning Azelnidipine as a Dual Inhibitor Targeting CD47/SIRPα and TIGIT/PVR Pathways for Cancer Immuno-Therapy

Biomolecules ◽

10.3390/biom11050706 ◽

2021 ◽

Vol 11 (5) ◽

pp. 706

Author(s):

Xiuman Zhou ◽

Ling Jiao ◽

Yuzhen Qian ◽

Qingyu Dong ◽

Yixuan Sun ◽

...

Keyword(s):

T Cell ◽

Cancer Immunotherapy ◽

Cd8 T Cell ◽

Dependent Manner ◽

Dual Inhibitor ◽

Macrophage Phagocytosis ◽

Hypertensive Drug ◽

And Function

Strategies boosting both innate and adaptive immunity have great application prospects in cancer immunotherapy. Antibodies dual blocking the innate checkpoint CD47 and adaptive checkpoint PD-L1 or TIGIT could achieve durable anti-tumor effects. However, a small molecule dual blockade of CD47/SIRPα and TIGIT/PVR pathways has not been investigated. Here, an elevated expression of CD47 and PVR was observed in tumor tissues and cell lines analyzed with the GEO datasets and by flow cytometry, respectively. Compounds approved by the FDA were screened with the software MOE by docking to the potential binding pockets of SIRPα and PVR identified with the corresponding structural analysis. The candidate compounds were screened by blocking and MST binding assays. Azelnidipine was found to dual block CD47/SIRPα and TIGIT/PVR pathways by co-targeting SIRPα and PVR. In vitro, azelnidipine could enhance the macrophage phagocytosis when co-cultured with tumor cells. In vivo, azelnidipine alone or combined with irradiation could significantly inhibit the growth of MC38 tumors. Azelnidipine also significantly inhibits the growth of CT26 tumors, by enhancing the infiltration and function of CD8+ T cell in tumor and systematic immune response in the tumor-draining lymph node and spleen in a CD8+ T cell dependent manner. Our research suggests that the anti-hypertensive drug azelnidipine could be repositioned for cancer immunotherapy.

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Mitochondrial Phosphoenolpyruvate Carboxykinase (PCK2) Maintains the Function of Bone Marrow Mesenchymal Stromal/stem Cells to Rescue Osteoporotic Phenotype Partly Through Autophagy Dependent Manner

10.21203/rs.3.rs-115769/v1 ◽

2020 ◽

Author(s):

Zheng Li ◽

Xuenan Liu ◽

Xuejiao Liu ◽

Yangge Du ◽

Yuan Zhu ◽

...

Keyword(s):

Stem Cells ◽

Bone Loss ◽

Phosphoenolpyruvate Carboxykinase ◽

Adipogenic Differentiation ◽

Osteoporosis Treatment ◽

Dependent Manner ◽

Aged Mice ◽

Stromal Stem Cells

Abstract BackgroundMitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a rate-limiting enzyme that plays critical roles in multiple physiological processes. We unveiled the important role of PCK2 on the regulation of osteogenesis by mesenchymal stromal/stem cells (MSCs) in our previous work. Here we further investigated the roles of PCK2 on regulating adipogenesis of MSCs and its therapeutic effect on osteoporosis. MethodsWe investigated PCK2 function in adipogenic differentiation of MSCs in vitro through loss-and-gain-of-function experiments. This was followed by heterotopic adipose formation assay in nude mice. In addition, ovariectomized (OVX) and aged mice were used as osteoporotic models to test the effect of PCK2 on osteoporosis. The bone formation and adipocyte accumulation were assessed by micro-CT and histological analysis. The multipotent capacity of control and osteoporotic BMMSCs were evaluated by quantitative real time-polymerase chain reaction (qRT-PCR) and western blot analysis. ResultsPCK2 expression levels were signiﬁcantly decreased in BMMSCs from OVX and aged mice. Furthermore, PCK2 could inhibit adipogenesis of BMMSCs and thus resisting lipid droplet formation and attenuating bone loss in osteoporotic mice. Mechanistically, we detected that autophagy level was decreased in BMMSCs of osteoporotic mice, while overexpression of PCK2 in vivo could rescued the autophagy activity. We further indicated that PCK2 could reverse osteopenia phenotype and adipose formation in OVX and aged mice partially via autophagy.ConclusionsCollectively, we suggested that PCK2 could attenuate bone loss and adipocyte accumulation of BMMSCs in osteoporotic mice through autophagy dependent manner. Our study indicated that PCK2 could be a brand and effective therapeutic target for osteoporosis treatment.

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Streptozotocin induces G2 arrest in skeletal muscle myoblasts and impairs muscle growth in vivo

AJP Cell Physiology ◽

10.1152/ajpcell.00338.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. C1033-C1040 ◽

Cited By ~ 32

Author(s):

Adam P. W. Johnston ◽

Jonathan E. Campbell ◽

Jeremy G. Found ◽

Michael C. Riddell ◽

Thomas J. Hawke

Keyword(s):

Skeletal Muscle ◽

Muscle Growth ◽

Phase Cell ◽

Saline Control ◽

Dependent Manner ◽

Β Cell ◽

Cross Sectional ◽

M Phase

Streptozotocin (STZ) is used extensively to induce pancreatic β-cell death and ultimately diabetes mellitus in animal models. However, the direct effects of STZ on muscle are largely unknown. To delineate the effects of STZ from the effects of hypoinsulinemia/hyperglycemia, we injected young rats with 1) saline (control), 2) STZ (120 mg/kg) or 3) STZ and insulin (STZ-INS; to maintain euglycemia). STZ rats demonstrated significantly elevated blood glucose throughout the 48-h protocol, while control and STZ-INS rats were euglycemic. Body mass increased in control (13 ± 4 g), decreased by 19 ± 2 g in STZ and remained unchanged in STZ-INS rats (−0.3 ± 2 g). Cross-sectional areas of gastrocnemius muscle fibers were smaller in STZ vs. control (1,480 ± 149 vs. 1,870 ± 40 μm2, respectively; P < 0.05) and insulin treatment did not rescue this defect (STZ-INS: 1,476 ± 143 μm2). Western blot analysis revealed a detectable increase in ubiquitinated proteins in the STZ skeletal muscles compared with control and STZ-INS. To further define the effects of STZ on skeletal muscle, independent of hyperglycemia, myoblasts were exposed to varying doses of STZ (0.25–3.0mg/ml) in vitro. Both acute and chronic exposures of STZ significantly impaired proliferative capacity in a dose-dependent manner. Within STZ-treated myoblasts, increased reactive oxygen species was associated with significant G2/M phase cell-cycle arrest. Taken together, our findings show that the effects of STZ are not β-cell specific and reveal that STZ should not be used for studies examining diabetic myopathy.

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The Progesterone Receptor Coactivator Hic-5 Is Involved in the Pathophysiology of Endometriosis

Endocrinology ◽

10.1210/en.2009-0008 ◽

2009 ◽

Vol 150 (8) ◽

pp. 3863-3870 ◽

Cited By ~ 56

Author(s):

Lusine Aghajanova ◽

Michael C. Velarde ◽

Linda C. Giudice

Keyword(s):

Progesterone Receptor ◽

Reproductive Age ◽

Dependent Manner ◽

Women Of Reproductive Age ◽

Stromal Fibroblasts ◽

Cycle Dependent ◽

Regulated Gene Expression ◽

And Function

Endometriosis is an estrogen-dependent disorder primarily associated with pelvic pain and infertility in up to 10% of women of reproductive age. Recent studies suggest that resistance to progesterone action may contribute to the development and pathophysiology of this disorder. In this study we examined the in vivo and in vitro expression and function of one progesterone receptor (PR) coactivator, Hic-5, in human endometrium and endometrial stromal fibroblasts (hESFs) from 29 women with and 30 (control) women without endometriosis. Hic-5 was highly expressed in stromal, but not epithelial, cells in women without endometriosis, in a cycle-dependent manner. In contrast, Hic-5 expression was not regulated during the menstrual cycle in hESFs from women with endometriosis and was significantly reduced in hESFs from women with vs. without disease. Hic-5 mRNA expression throughout the cycle in endometrium from control women, but not those with endometriosis, correlated with expression of PR. Hic-5 mRNA in hESFs was significantly up-regulated in control but not endometriosis hESFs after treatment in vitro with 8-bromoadenosine-cAMP for 96 h but only modestly after 14 d of progesterone treatment. Hic-5 silencing did not influence cAMP-regulated gene expression but affected genes regulated solely by progesterone (e.g. DKK1 and calcitonin). Together the data suggest that the proposed progesterone resistance in endometrium from women with endometriosis derives, in part, from impaired expression of the PR coactivator, Hic-5, in endometrial tissue and cultured endometrial stromal fibroblasts.

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