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Background As elegant structures designed for neural communication, synapses are the building bricks of our mental functions. Recently, many studies have pointed out that synaptic proteinassociated mutations may lead to dysfunctions of social cognition. Dlgap2, which encodes one of the main components of scaffold proteins in postsynaptic density (PSD), has been addressed as a candidate gene in autism spectrum disorders. To elucidate the disturbance of synaptic balance arising from Dlgap2 loss-of-function in vivo, we thus generated Dlgap2-/-mice to investigate their phenotypes of synaptic function and social behaviors. Methods The creation of Dlgap2-/-mice was facilitated by the recombineering-based method, Cre-loxP system and serial backcross. Reversal learning in a water T-maze was used to determine repetitive behaviors. The three-chamber approach task, resident-intruder test and tube task were performed to characterize the social behaviors of mutant mice. Cortical synaptosomal fraction, Golgi-Cox staining, whole-cell patch electrophysiology and transmission electron microscopy were all applied to investigate the function and structure of synapses in the orbitofrontal cortex (OFC) of Dlgap2-/-mice. Results Dlgap2-/-mice displayed exacerbated aggressive behaviors in the resident-intruder task, and elevated social dominance in the tube test. In addition, Dlgap2-/-mice exhibited a clear reduction of receptors and scaffold proteins in cortical synapses. Dlgap2-/-mice also demonstrated lower spine density, decreased peak amplitude of miniature excitatory postsynaptic current and ultra-structural deficits of PSD in the OFC. Conclusions Our findings clearly demonstrate that Dlgap2 plays a vital role in social behaviors and proper synaptic functions of the OFC. Moreover, these results may provide valuable insights into the neuropathology of autism. [ABSTRACT FROM AUTHOR]

Hypoxia affects the tumor microenvironment and is considered important to metastasis progression and therapy resistance. Thus far, the majority of global analyses of tumor hypoxia responses have been limited to just a single omics level. Combining multiple omics data can broaden our understanding of tumor hypoxia. Here, we investigate the temporal change of the metabolite composition with gene expression data from literature to provide a more comprehensive insight into the system level in response to hypoxia. Nuclear magnetic resonance spectroscopy was used to perform metabolomic profiling on the MDA-MB-231 breast cancer cell line under hypoxic conditions. Multivariate statistical analysis revealed that the metabolic difference between hypoxia and normoxia was similar over 24 h, but became distinct over 48 h. Time dependent microarray data from the same cell line in the literature displayed different gene expressions under hypoxic and normoxic conditions mostly at 12 h or earlier. The direct metabolomic profiles show a large overlap with theoretical metabolic profiles deduced from previous transcriptomic studies. Consistent pathways are glycolysis/gluconeogenesis, pyruvate, purine and arginine and proline metabolism. Ten metabolic pathways revealed by metabolomics were not covered by the downstream of the known transcriptomic profiles, suggesting new metabolic phenotypes. These results confirm previous transcriptomics understanding and expand the knowledge from existing models on correlation and co-regulation between transcriptomic and metabolomics profiles, which demonstrates the power of integrated omics analysis. [ABSTRACT FROM AUTHOR]

The rhizome of Davallia formosana is commonly used to treat bone disease including bone fracture, arthritis, and osteoporosis in Chinese herbal medicine. Here, we report the effects of WL1101, the ethanol extracts of fresh rhizomes of Davallia formosana on ovariectomy-induced osteoporosis. In addition, excess activated bone-resorbing osteoclasts play crucial roles in inflammation-induced bone loss diseases, including rheumatoid arthritis and osteoporosis. In this study, we examined the effects of WL1101 on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. Treatment with WL1101 significantly inhibited RANKL-stimulated osteoclastogenesis. Two isolated active compounds, ((-)-epicatechin) or WL14 (4-hydroxy-3-aminobenzoic acid) could also inhibit RANKL-induced osteoclastogenesis. WL1101 suppressed the RANKL-induced nuclear factor-kB (NF- kB) activation and nuclear translocation, which is the key process during osteoclastogenesis, by inhibiting the activation of IkB kinase (IKK) and I/cBα. In animal model, oral administration of WL1101 (50 or 200 mg/kg/day) effectively decreased the excess bone resorption and significantly antagonized the trabecular bone loss in ovariectomized rats. Our results demonstrate that the ethanol extracts of fresh rhizomes of Davallia formosana inhibit osteoclast differentiation via the inhibition of NF-kB activation and effectively ameliorate ovariectomy-induced osteoporosis. WL1101 may thus have therapeutic potential for the treatment of diseases associated with excessive osteoclastic activity [ABSTRACT FROM AUTHOR]

Heme-oxygenase-1 (HO-1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO-1 inhibits osteoclastogenesis. However, the effect of HO-1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO-1 and HO-1 inducer hemin to study the effects of HO-1 in primary cultured osteoblasts. The results showed that induction of HO-1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO-1, bilirubin, carbon monoxide, and iron, are involved in the inhibitory action of HO-1. HO-1 can be induced by H2O2, lipopolysaccharide and inflammatory cytokines such as TNF-α and IL-1β in osteoblasts and also in STZ-induced diabetic mice. In addition, endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) markedly increased both mRNA and protein levels of HO-1 in osteoblasts via PI3K-Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d-PGJ2. Our results indicate that upregulation of HO-1 inhibits the maturation of osteoblasts and HO-1 may be involved in oxidative- or inflammation-induced bone loss. J. Cell. Physiol. 222: 757–768, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-13 (MMP-13) is involved in the degradation of extracellular matrix in many kinds of tissues. Here we found that hypoxia increased MMP-13 protein and mRNA levels in primary rat astrocyte cultures. Hypoxia stimulation also increased the secretion of MMP-13 from astrocytes, as shown by zymographic analysis. In addition, exposure to hypoxia up-regulated the expression of c-Fos and c-Jun time-dependently. Hypoxia-induced MMP-13 overexpression was antagonized by transfection with antisense oligodeoxynucleotides (AS-ODN) of c-Fos or c-Jun. Furthermore, hypoxic-conditioned medium (Hx-CM) collected from astrocytes exposed to hypoxia increased paracellular permeability of adult rat brain endothelial cells (ARBECs). Administration of MMP-13 neutralizing antibody antagonized Hx-CM-induced paracellular permeability of ARBECs. Furthermore, pre-transfection of astrocytes with AS-ODN of c-Fos, c-Jun or MMP-13-shRNA significantly decreased hyperpermeability of ARBECs induced by Hx-CM. The arrangement of tight junction protein (TJP) zonular occludens-1 (ZO-1) of ARBECs disorganized in response to Hx-CM. Administration of Hx-CM to ARBECs also resulted in the production of proteolytic fragments of ZO-1, which was antagonized by transfection of MMP-13-shRNA in primary astrocytes. Administration of MMP-13 recombinant protein to ARBECs led to the disorganization and fragmentation of ZO-1 protein and also increased paracellular permeability. These results suggest that hypoxia-induced MMP-13 expression in astrocytes is regulated by c-Fos and c-Jun. MMP-13 is an important factor leading to the disorganization of ZO-1 and hyperpermeablility of blood–brain barrier in response to hypoxia. J. Cell. Physiol. 220: 163–173, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The article examines how osteoblast-derived TGF-ß1 stimulates IL-8 release through AP-1 and NF-kB in human cancer cells. The scientists' purpose for this research paper was to examine whether osteoblast-derived TGF-ß1 is associated with osteolytic bone diseases. Chemokine interleukin (IL)-8 mRNA levels were measured using RT-PCR analysis.

The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory synaptic transmission on DG. Evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (eEPSCampa and eEPSCnmda ) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60–120 g). Exogenously applied AMPA and NMDA-induced currents and AMPA receptor-mediated miniature EPSC (mEPSCampa ) were recorded in the presence of specific antagonists. LTG inhibited both eEPSCampa and eEPSCnmda , and had no effect on exogenously applied NMDA-induced current indicating LTG inhibited glutamate release. Previous studies demonstrated that alteration in glutamate concentration in synaptic cleft causes parallel changes of eEPSCampa and eEPSCnmda . Our results showed that LTG inhibited eEPSCampa significantly more than eEPSCnmda (p < 0.05), suggesting that LTG may also have blocked the postsynaptic AMPA receptor. The hypothesis is further supported by the facts that; (1) LTG (30–100 μM) inhibited direct exogenously applied AMPA-induced currents (to 90%), (2) LTG significantly reduced the amplitude, but not the frequency of mEPSCampa and asynchronous (EPSC), and (3) LTG-induced reduction of eEPSCampa was not associated with a modification of the paired-pulse ratio. To sum up, LTG exerts a postsynaptic inhibitory mechanism on the AMPA receptor. Our results demonstrate that LTG suppress es postsynaptic AMPA receptors and reduces glutamate release in granule cells of DG. The postsynaptic effect can be one of the underlying mechanisms of LTG's anticonvulsant action. [ABSTRACT FROM AUTHOR]

It has been shown that ultrasound (US) stimulation accelerates fracture healing, bone maturation, and remodeling in the animal models and in clinical studies. One of the major factor involves in remodeling process is matrix metalloproteinases (MMPs) such as MMP-13 that has been shown to degrade the native interstitial collagens in several tissues. Here we found that US stimulation increased the secretion of MMP-13 in cultured rat osteoblasts, as shown by zymographic analysis. US stimulation also increased the mRNA level of MMP-13, c-Fos, and c-Jun. Cycloheximide (an inhibitor of protein translocation) and actinomycin D (an inhibitor of gene transcription) did not inhibit the MMP-13, c-Fos, and c-Jun mRNA expression, suggesting that such expression does not require de novo protein synthesis and not change their stabilities. p38 inhibitor, SB203580 or JNK inhibitor, SP600125 but not ERK inhibitor, PD98059 attenuated the US-induced MMP-13, c-Fos, and c-Jun expression; these results were further substantiated by transfecting with the dominant negative mutants of p38 or JNK. The binding of c-Fos and c-Jun to the AP-1 element on the MMP-13 promoter and the enhancement of AP-1 luciferase activity was enhanced by US stimulation. Taken together, our results provide evidence that US stimulation increases MMP-13 expression through p38 and JNK signaling pathway to regulate bone remodeling. J. Cell. Physiol. 215: 356–365, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. bFGF increased Fn expression, which was attenuated by phosphatidylinositol phospholipase inhibitor (U73122), protein kinase C inhibitor (GF109203X), Src inhibitor (PP2), NF-κB inhibitor (PDTC), IκBα phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (TPCK). bFGF-induced increase of Fn-luciferase activity was antagonized by cells transfected with Fn construct without NF-κB regulatory site. Stimulation of osteoblasts with bFGF activated IκB kinase α/β (IKK α/β) and increased IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-protein complex and κB-luciferase activity. bFGF-mediated an increase of IKKα/β activity and DNA-binding activity was inhibited by U73122, GF109203X, or PP2. The binding of p65 to the NF-κB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the Fn promoter was enhanced by bFGF. Overexpression of constitutively active FGF receptor 2 (FGFR2) increased Fn-luciferase activity, which was inhibited by co-transfection with dominant negative (DN) mutants of PLCγ2, PKCα, c-Src, IKKα, or IKKβ. Our results suggest that bFGF increased Fn expression in rat osteoblasts via the FGFR2/PLCγ2/PKCα/c-Src/NF-κB signaling pathway. J. Cell. Physiol. 211: 45–55, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. We generated viable and fertile CRMP-1 knock-out (CRMP-1-/-) mice with no evidence of gross abnormality in the major organs. CRMP-1-/- mice exhibited intense microtubule-associated protein 2 (MAP2) staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 (growth-associated protein-43) and PSD95 (postsynaptic density-95) (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knock-out mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1-/- mice showed a normal synapsin I labeling pattern in CA1 and normal paired-pulse facilitation. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory. [ABSTRACT FROM AUTHOR]

1 Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX-2 expression were investigated in RAW264.7 macrophages. 2 Capsaicin and resiniferatoxin (RTX) can inhibit LPS- and IFN-γ-mediated NO production, and iNOS protein and m RNA expression with similar 1C[sub50] values of around 10 μM. 3 Capsaicin also transcriptionally inhibited LPS-and PMA-induced COX-2 expression and PGE[sub2] production. However, this effect exhibited a higher potency (1C[sub50] 0.2μM). and RTX failed to elicit such responses at 10μM. 4 Interestingly, we found that capsazepine, a competitive TRPVI antagonist, did not prevent the inhibition elicited by Capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX-2/PGE[sub2] induction with an IC[sub50] value of 3μM. RT-PCR and immunoblotting analysis excluded the expression of TRPVl in RAW264.7 macrophages. 5 The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS-elicited NF-[subk]B and AP-I activation and IFN-Y-elicited STAT1 activation. The reporter assay of AIM activity also supported this action. 6 The kinase assay indicated that ERK,JNK, and IKK activation by LPS were inhibited by vanilloids. 7 In conclusionvanilloids can modulate the expression of inflammatory iNOS and COX-2 genes in macrophages through interference with upstream signalling events of LPS and IFN-γ. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions. [ABSTRACT FROM AUTHOR]

Lung caner cells have a striking tendency to metastasize to bone. The chemokine stromal cell-derived factor-1 (SDF-1) is constitutively secreted by osteoblasts and bone marrow stromal cells and plays a key role for homing of hematopoietic cells to the bone marrow. Reverse transcriptase–polymerase chain reaction and flow cytometry studies demonstrated SDF-1 receptor (CXCR4) messenger RNA (mRNA) and surface expression of CXCR4 in lung cancer cell lines, especially higher in those with high invasiveness (A549) as compared with lower level in H928 cells and H1299 cells. SDF-1, osteoblast-conditioned medium (OBCM) and stromal cell-conditioned medium all induced the invasiveness of lung cancer cells. Matrix metalloproteinase (MMP)-9 small interfering RNA inhibited the SDF-1α- or OBCM-induced MMP-9 expression and thereby significantly inhibited the SDF-1α- or OBCM-induced cell invasion. Furthermore, mitogen-activated protein kinase kinase inhibitor PD98059 suppressed SDF-1α-induced MMP-9 mRNA expression. Transient transfection with dominant-negative extracellular signal-regulated kinase (ERK) mutant also showed that the ERK-signaling pathway was involved in SDF-1α-induced MMP-9 expression. Moreover, nuclear factor-κB (NF-κB) decoy oligodeoxynucleotide suppressed the MMP-9 promoter activity enhanced by SDF-1α. Both mitogen-activated protein kinase kinase inhibitor and ERK mutant also antagonized SDF-1α-induced NF-κB-driven luciferase promoter activity. Taken together, our results indicated that bone marrow-derived-SDF-1α enhances the invasiveness of lung cancer cells by increasing MMP-9 expression through the CXCR4/ERK/NF-κB signal transduction pathway. [ABSTRACT FROM AUTHOR]

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE2 enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immimofluorescence staining and enzyme-linked immunosorbent assay. PGE2 also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated up-regulation of Fn. At the mechanistic level, Ca2+ chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phosphalipase C inhibitor (U73122), or Sre inhibitor (PP2) attenuated the PGE2-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE2 Furthermore, treatment with antisense oligonucleotides of various PKC isoform& including ɑ, β, ∈ and δ, demonstrated that ɑ isozyme plays an important role in the enhancement action of PGE2 on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE2 and 17-phenyl trinor PGE2 (EP1/EP3 agonist) increased the surface expression and mRNA level of ɑ5 or β1 integrins. Fn promoter activity was enhanced by PGE2 and 17-phenyl trinor PGE2 in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCɑ or c-Src inhibited the potentiating action of PGE2 on Fn pro-rooter activity. Local administration of PGE2 or 17-phenyl trinor PGE2 into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and ɑ5β1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE2 increased Fn and promoted bone formation in rat osteoblasts via the EP1/phospholipase C/PKCɑ/c-Src signaling pathway. [ABSTRACT FROM AUTHOR]

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and clinical studies. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US stimulation increased NO formation and the protein level of inducible nitric-oxide synthase (iNOS). US-mediated iNOS expression was attenuated by anti-integrin αvβ3 or β1 antibodies but not anti-integrin αvβ3 or β3 antibodies or focal adhesion kinase mutant. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) also inhibited the potentiating action of US. US stimulation increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, US stimulation also increased the stability and activity of HIF-l protein. The binding of HIF-la to the HRE elements on the iNOS promoter was enhanced by US stimulation. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for US-induced HIP-la activation and subsequent iNOS up-regulation. Taken together, our results provide evidence that US stimulation up-regulates iNOS expression in osteoblasts by an HIF-lα-dependent mechanism involving the activation of ILK/Akt and mTOR pathways via integrin receptor. [ABSTRACT FROM AUTHOR]

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 µg/ml, 10 µl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases. [ABSTRACT FROM AUTHOR]