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KISS1R Signals Independently of G?q/11 and Triggers LH Secretion via the ?-Arrestin Pathway in the Male Mouse.
Endocrinology
PUBLISHED: 08-22-2014
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Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a G?q/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the G?q/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of G?q/11 via ?-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the G?q/11- and ?-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either ?-arrestin-1 or ?-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that ?-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that G?q/11-uncoupled KISS1R mutants, like L148S, will display G?q/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from G?q/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking ?-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is ?-arrestin-dependent. Overall, we conclude that KISS1R signals via G?q/11 and ?-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of G?q/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism.
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Leptin-responsive GABAergic neurons regulate fertility through pathways that result in reduced kisspeptinergic tone.
J. Neurosci.
PUBLISHED: 04-25-2014
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The adipocyte-derived hormone leptin plays a critical role in the central transmission of energy balance to modulate reproductive function. However, the neurocircuitry underlying this interaction remains elusive, in part due to incomplete knowledge of first-order leptin-responsive neurons. To address this gap, we explored the contribution of predominantly inhibitory (GABAergic) neurons versus excitatory (glutamatergic) neurons in the female mouse by selective ablation of the leptin receptor in each neuronal population: Vgat-Cre;Lepr(lox/lox) and Vglut2-Cre;Lepr(lox/lox) mice, respectively. Female Vgat-Cre;Lepr(lox/lox) but not Vglut2-Cre;Lepr(lox/lox) mice were obese. Vgat-Cre;Lepr(lox/lox) mice had delayed or absent vaginal opening, persistent diestrus, and atrophic reproductive tracts with absent corpora lutea. In contrast, Vglut2-Cre;Lepr(lox/lox) females exhibited reproductive maturation and function comparable to Lepr(lox/lox) control mice. Intracerebroventricular administration of kisspeptin-10 to Vgat-Cre;Lepr(lox/lox) female mice elicited robust gonadotropin responses, suggesting normal gonadotropin-releasing hormone neuronal and gonadotrope function. However, adult ovariectomized Vgat-Cre;Lepr(lox/lox) mice displayed significantly reduced levels of Kiss1 (but not Tac2) mRNA in the arcuate nucleus, and a reduced compensatory luteinizing hormone increase compared with control animals. Estradiol replacement after ovariectomy inhibited gonadotropin release to a similar extent in both groups. These animals also exhibited a compromised positive feedback response to sex steroids, as shown by significantly lower Kiss1 mRNA levels in the AVPV, compared with Lepr(lox/lox) mice. We conclude that leptin-responsive GABAergic neurons, but not glutamatergic neurons, act as metabolic sensors to regulate fertility, at least in part through modulatory effects on kisspeptin neurons.
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Central precocious puberty that appears to be sporadic caused by paternally inherited mutations in the imprinted gene makorin ring finger 3.
J. Clin. Endocrinol. Metab.
PUBLISHED: 03-14-2014
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Loss-of-function mutations in makorin ring finger 3 (MKRN3), an imprinted gene located on the long arm of chromosome 15, have been recognized recently as a cause of familial central precocious puberty (CPP) in humans. MKRN3 has a potential inhibitory effect on GnRH secretion.
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TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.
FASEB J.
PUBLISHED: 12-27-2013
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Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.-Noel, S. D., Abreu, A. P., Xu, S., Muyide, T., Gianetti, E., Tusset, C., Carroll, J., Latronico, A. C., Seminara, S. B., Carroll, R. S., Kaiser, U. B. TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.
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Dynamic kisspeptin receptor trafficking modulates kisspeptin-mediated calcium signaling.
Mol. Endocrinol.
PUBLISHED: 12-02-2013
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Kisspeptin receptor (KISS1R) signaling plays a critical role in the regulation of reproduction. We investigated the role of kisspeptin-stimulated KISS1R internalization, recycling, and degradation in the modulation of KISS1R signaling. Kisspeptin stimulation of Chinese hamster ovary or GT1-7 cells expressing KISS1R resulted in a biphasic increase in intracellular Ca(2+) ([Ca(2+)]i), with a rapid acute increase followed by a more sustained second phase. In contrast, stimulation of the TRH receptor, another Gq/11-coupled receptor, resulted in a much smaller second-phase [Ca(2+)]i response. The KISS1R-mediated second-phase [Ca(2+)]i response was abolished by removal of kisspeptin from cell culture medium. Notably, the second-phase [Ca(2+)]i response was also inhibited by dynasore, brefeldin A, and phenylarsine oxide, which inhibit receptor internalization and recycling, suggesting that KISS1R trafficking contributes to the sustained [Ca(2+)]i response. We further demonstrated that KISS1R undergoes dynamic ligand-dependent and -independent recycling. We next investigated the fate of the internalized kisspeptin-KISS1R complex. Most internalized kisspeptin was released extracellularly in degraded form within 1 hour, suggesting rapid processing of the internalized kisspeptin-KISS1R complex. Using a biotinylation assay, we demonstrated that degradation of cell surface KISS1R was much slower than that of the internalized ligand, suggesting dissociated processing of the internalized kisspeptin-KISS1R complex. Taken together, our results suggest that the sustained calcium response to kisspeptin is dependent on the continued presence of extracellular ligand and is the result of dynamic KISS1R trafficking.
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Central precocious puberty caused by mutations in the imprinted gene MKRN3.
N. Engl. J. Med.
PUBLISHED: 06-05-2013
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The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified.
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GnRH pulse frequency-dependent stimulation of FSH? transcription is mediated via activation of PKA and CREB.
Mol. Endocrinol.
PUBLISHED: 02-07-2013
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Expression of pituitary FSH and LH, under the control of pulsatile GnRH, is essential for fertility. cAMP response element-binding protein (CREB) has been implicated in the regulation of FSH? gene expression, but the molecular mechanisms by which pulsatile GnRH regulates CREB activation remain poorly understood. We hypothesized that CREB is activated by a distinct signaling pathway in response to pulsatile GnRH in a frequency-dependent manner to dictate the FSH? transcriptional response. GnRH stimulation of CREB phosphorylation (pCREB) in the gonadotrope-derived L?T2 cell line was attenuated by a protein kinase A (PKA) inhibitor, H89. A dominant negative PKA (DNPKA) reduced GnRH-stimulated pCREB and markedly decreased GnRH stimulation of FSH? mRNA and FSH?LUC activity, but had little effect on LH?LUC activity, indicating relative specificity of this pathway. In perifusion studies, FSH? mRNA levels and FSH?LUC activities were increased by pulsatile GnRH, with significantly greater increases at low compared with high pulse frequencies. DNPKA markedly reduced these GnRH-stimulated FSH? responses at both low and high pulse frequencies. Correlating with FSH? activation, both PKA activity and levels of pCREB were increased to a greater extent by low compared with high GnRH pulse frequencies, and the induction of pCREB was also attenuated by overexpression of DNPKA at both low and high pulse frequencies. Taken together, these data indicate that a PKA-mediated signaling pathway mediates GnRH activation of CREB at low-pulse frequencies, playing a significant role in the decoding of the hypothalamic GnRH signal to result in frequency-dependent FSH? activation.
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Imaging of human mesenchymal stromal cells: homing to human brain tumors.
J. Neurooncol.
PUBLISHED: 05-06-2011
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Human mesenchymal stromal cells (hMSC) can be used as a drug delivery vehicle for the treatment of GBM. However, tracking the migration and distribution of these transplanted cells is necessary to interpret therapeutic efficacy. We compared three labeling techniques for their ability to track the migration of transplanted hMSC in an orthotopic mouse xenograft model. hMSC were labeled with three different imaging tags (fluorescence, luciferase or ferumoxide) for imaging by fluorescence, bioluminescence or magnetic resonance imaging (MRI), respectively. hMSC were labeled for all imaging modalities without the use of transfection agents. The labeling efficacy of the tags was confirmed, followed by in vitro and in vivo migration assays to track hMSC migration towards U87 glioma cells. Our results confirmed that the labeled hMSC retained their migratory ability in vitro, similar to unlabeled hMSC. In addition, labeled hMSC migrated towards the U87 tumor site, demonstrating their retention of tumor tropism. hMSC tumor tropism was confirmed by all three imaging modalities; however, MRI provides both real time assessment and the high resolution needed for clinical studies. Our findings suggest that ferumoxide labeling of hMSC is feasible, does not alter their migratory ability and allows detection by MRI. Non invasive tracking of transplanted therapeutic hMSC in the brain will allow further development of human cell based therapies.
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KISS1R intracellular trafficking and degradation: effect of the Arg386Pro disease-associated mutation.
Endocrinology
PUBLISHED: 02-01-2011
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The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R trafficking--instead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increase on mutant receptor recycled back to the plasma membrane.
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Genomic profiling reveals alternative genetic pathways of meningioma malignant progression dependent on the underlying NF2 status.
Clin. Cancer Res.
PUBLISHED: 08-03-2010
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Meningiomas are the most common central nervous system tumors in the population of age 35 and older. WHO defines three grades predictive of the risk of recurrence. Clinical data supporting histologic malignant progression of meningiomas are sparse and underlying molecular mechanisms are not clearly depicted.
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Novel local drug delivery system using thermoreversible gel in combination with polymeric microspheres or liposomes.
Anticancer Res.
PUBLISHED: 06-10-2010
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The purpose of our study was to evaluate the application of thermoreversible gelation polymer (TGP) as a local drug delivery system for malignant glioma.
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Inhibition of thromboxane synthase activity improves glioblastoma response to alkylation chemotherapy.
Transl Oncol
PUBLISHED: 02-19-2010
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Thromboxane synthase (TXSA), an enzyme of the arachidonic acid metabolism, is upregulated in human glial tumors and is involved in glioma progression. Here, we analyzed the in vitro and in vivo effects of pharmacological inhibition of TXSA activity on human glioblastoma cells. Furegrelate, a specific inhibitor of TXSA, significantly inhibited tumor growth in an orthotopic glioblastoma model by inducing proapoptotic, antiproliferative, and antiangiogenic effects. Inhibition of TXSA induced a proapoptotic disposition of glioma cells and increased the sensitivity to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea, significantly prolonging the survival time of intracerebral glioma-bearing mice. Our data demonstrate that the targeted inhibition of TXSA activity improves the efficiency of conventional alkylation chemotherapy in vivo. Our study supports the role of TXSA activity for the progression of malignant glioma and the potential utility of its therapeutic modulation for glioma treatment.
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Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.
Exp. Neurol.
PUBLISHED: 02-16-2010
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Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs promotes the migration and proliferation of embedded cells, which is an important factor for designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF (vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural tissue. We examined the morphological changes of the printed C17.2 cells embedded in the collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%) after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within 1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their morphology. The cells printed in this range also migrated toward the fibrin gel, with the total migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of the GF in the collagen scaffold. The presented method can be gainfully used in the development of three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.
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Reproductive hormone-dependent and -independent contributions to developmental changes in kisspeptin in GnRH-deficient hypogonadal mice.
PLoS ONE
PUBLISHED: 01-30-2010
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Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.
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SNAI2/Slug promotes growth and invasion in human gliomas.
BMC Cancer
PUBLISHED: 01-12-2010
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Numerous factors that contribute to malignant glioma invasion have been identified, but the upstream genes coordinating this process are poorly known.
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Frequency-dependent regulation of follicle-stimulating hormone beta by pulsatile gonadotropin-releasing hormone is mediated by functional antagonism of bZIP transcription factors.
Mol. Cell. Biol.
PUBLISHED: 12-14-2009
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Oscillatory synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), under the control of pulsatile hypothalamic gonadotropin-releasing hormone (GnRH), is essential for normal reproductive development and fertility. The molecular mechanisms by which various patterns of pulsatile GnRH regulate gonadotrope responsiveness remain poorly understood. In contrast to the alpha and LH beta subunit genes, FSH beta subunit transcription is preferentially stimulated at low rather than high frequencies of pulsatile GnRH. In this study, mutation of a cyclic AMP response element (CRE) within the FSH beta promoter resulted in the loss of preferential GnRH stimulation at low pulse frequencies. We hypothesized that high GnRH pulse frequencies might stimulate a transcriptional repressor(s) to attenuate the action of CRE binding protein (CREB) and show that inducible cAMP early repressor (ICER) fulfills such a role. ICER was not detected under basal conditions, but pulsatile GnRH stimulated ICER to a greater extent at high than at low pulse frequencies. ICER binds to the FSH beta CRE site to reduce CREB occupation and abrogates both maximal GnRH stimulation and GnRH pulse frequency-dependent effects on FSH beta transcription. These data suggest that ICER production antagonizes the stimulatory action of CREB to attenuate FSH beta transcription at high GnRH pulse frequencies, thereby playing a critical role in regulating cyclic reproductive function.
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Targeting rat brainstem glioma using human neural stem cells and human mesenchymal stem cells.
Clin. Cancer Res.
PUBLISHED: 07-28-2009
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Brainstem gliomas are usually inoperable and have a dismal prognosis. Based on the robust tropisms of neural stem cells (NSC) and mesenchymal stem cells (MSC) to brain tumors, we compared the tumor-tropic migratory capacities of these stem cells and evaluated the therapeutic potential of genetically engineered human NSCs encoding cytosine deaminase (CD) and IFNbeta against brainstem gliomas.
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Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy.
Stem Cells
PUBLISHED: 06-23-2009
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Glioblastoma is among the most aggressive and treatment resistant of all human cancers. Conventional therapeutic approaches are unsuccessful because of diffuse infiltrative invasion of glioma tumor cells into normal brain parenchyma. Stem cell-based therapies provide a promising approach for the treatment of malignant gliomas because of their migratory ability to invasive tumor cells. Our therapeutic strategy was to use human bone marrow-derived mesenchymal stromal cells (hMSCs) as a cellular vehicle for the targeted delivery and local production of the biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) at the glioma tumor site. hMSCs were transduced with a lentivirus expressing secretable TRAIL (S-TRAIL) and mCherry (red fluorescent protein). Our results clearly demonstrate the retention of tumor tropic ability of hMSC S-TRAIL cells by in vitro and in vivo migration assays. In vitro assays confirmed the expression, release, and biological activity of S-TRAIL produced by hMSC S-TRAIL cells. For the in vivo assessment of therapeutic efficacy, hMSCs were injected ipsilateral to an established intracranial glioma tumor in a mouse xenograft model. Genetically engineered hMSC S-TRAIL cells were effective in inhibiting intracranial U87 glioma tumor growth (81.6%) in vivo and resulted in significantly longer animal survival. Immunohistochemical studies demonstrated significant, eight fold greater tumor cell apoptosis in the hMSC S-TRAIL-treated group than in controls. Our study demonstrates the therapeutic efficacy of hMSC S-TRAIL cells and confirms that hMSCs can serve as a powerful cell-based delivery vehicle for the site-specific release of therapeutic proteins.
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Expression of a gonadotropin-releasing hormone receptor-simian virus 40 T-antigen transgene has sex-specific effects on the reproductive axis.
Endocrinology
PUBLISHED: 03-12-2009
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The GnRH receptor (GnRHR) responds to pulsatile GnRH signals to coordinate pituitary gonadotropin synthesis and secretion. Previously, a 1.2-kb fragment of the 5-flanking region isolated from the mouse GnRHR gene was shown to target expression to pituitary gonadotropes in vivo. The 1.2-kb gene promoter fused to the simian virus 40 large T antigen (TAg) was used to generate transgenic mice that form gonadotrope-derived pituitary tumors at 4-5 months of age. Transgenic female mice have hypogonadotropic hypogonadism, infantile gonads, and are infertile throughout their life span, whereas males remain reproductively intact until their tumors become large. We hypothesized that the targeted TAg expression causes a sex-specific disruption of the reproductive axis at the level of the pituitary gland. To test this hypothesis, we characterized the pituitary gonadotropin beta-subunit and TAg expression patterns, and measured plasma gonadotropin and gonadal steroid levels in female and male mice before and after pituitary tumor development. TAg expression was observed in transgenic females and males 15 d of age, before tumor development. Interestingly, and in contrast to the transgenic males, pituitary LH beta and FSH beta subunit protein levels, and plasma LH and FSH levels, were reduced in transgenic females. Reproductive organs in transgenic female mice remained underdeveloped but were normal in transgenic males. We conclude that the expression of the TAg transgene driven by the GnRHR gene promoter results in female-specific infertility due to disruption of gonadotropin production and secretion even before tumor development.
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Local delivery of poly lactic-co-glycolic acid microspheres containing imatinib mesylate inhibits intracranial xenograft glioma growth.
Clin. Cancer Res.
PUBLISHED: 02-03-2009
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In an effort to develop new therapeutic strategies to treat malignant gliomas, we have designed poly (lactic-co-glycolic) acid (PLGA) microparticles that deliver imatinib mesylate, a small molecule tyrosine kinase inhibitor. The local continuous release of imatinib mesylate at the tumor site overcomes many obstacles associated with systemic delivery.
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Vascular endothelial growth factor-stimulated cerebral microvascular endothelial cells mediate the recruitment of neural stem cells to the neurovascular niche.
Brain Res.
PUBLISHED: 01-15-2009
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Endogenous and transplanted neural stem cells (NSC) are highly migratory and display a unique tropism for areas of neuro-pathology. However, signals controlling NSC motility in health and disease are still ill-defined. NSC appear to be intimately associated with the cerebral vasculature and angiogenesis is a hallmark of many neurological disorders. This has led us to investigate the influence of quiescent and angiogenically active human endothelial cells on human NSC migration. In vivo we observed frequent perivascular accumulation of human NSC in the proximity of cerebral microvessels upon induction of angiogenesis by cerebral infusion of vascular endothelial growth factor (VEGF) into the murine brain. We analyzed the in vitro effects of conditioned media from human endothelial cells before and after angiogenic stimulation with VEGF on the migration of human NSC in vitro. Non-stimulated endothelial cells induced a moderate chemotactic migration that was significantly enhanced after angiogenic activation by VEGF. In order to identify cytokines that may function as stimulators of NSC chemotaxis, we screened endothelial cell-conditioned media for the expression of 120 different cytokines. We identified PDGF-BB, RANTES, I-TAC, NAP-2, GROalpha, Ang-2, and M-CSF as endothelial cell-released chemoattractants for human NSC in vitro. VEGF-stimulated cerebral microvascular endothelial cells secreted higher levels of Ang-2 and GROalpha, which in part were responsible for the enhanced chemoattraction of NSC. Our findings support the hypothesis that the angiogenically active microvasculature modulates the local guidance of NSC through endothelial cell-derived chemoattractants.
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Increased neurokinin B (Tac2) expression in the mouse arcuate nucleus is an early marker of pubertal onset with differential sensitivity to sex steroid-negative feedback than Kiss1.
Endocrinology
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At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E(2)) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E(2)-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E(2). Low levels of E(2) significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E(2) levels, supporting differential sensitivity to E(2). Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.
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Evidence of the importance of the first intracellular loop of prokineticin receptor 2 in receptor function.
Mol. Endocrinol.
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Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2.
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Numb regulates glioma stem cell fate and growth by altering epidermal growth factor receptor and Skp1-Cullin-F-box ubiquitin ligase activity.
Stem Cells
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Glioblastoma contains a hierarchy of stem-like cancer cells, but how this hierarchy is established is unclear. Here, we show that asymmetric Numb localization specifies glioblastoma stem-like cell (GSC) fate in a manner that does not require Notch inhibition. Numb is asymmetrically localized to CD133-hi GSCs. The predominant Numb isoform, Numb4, decreases Notch and promotes a CD133-hi, radial glial-like phenotype. However, upregulation of a novel Numb isoform, Numb4 delta 7 (Numb4d7), increases Notch and AKT activation while nevertheless maintaining CD133-hi fate specification. Numb knockdown increases Notch and promotes growth while favoring a CD133-lo, glial progenitor-like phenotype. We report the novel finding that Numb4 (but not Numb4d7) promotes SCF(Fbw7) ubiquitin ligase assembly and activation to increase Notch degradation. However, both Numb isoforms decrease epidermal growth factor receptor (EGFR) expression, thereby regulating GSC fate. Small molecule inhibition of EGFR activity phenocopies the effect of Numb on CD133 and Pax6. Clinically, homozygous NUMB deletions and low Numb mRNA expression occur primarily in a subgroup of proneural glioblastomas. Higher Numb expression is found in classical and mesenchymal glioblastomas and correlates with decreased survival. Thus, decreased Numb promotes glioblastoma growth, but the remaining Numb establishes a phenotypically diverse stem-like cell hierarchy that increases tumor aggressiveness and therapeutic resistance.
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Alternative splicing of CHEK2 and codeletion with NF2 promote chromosomal instability in meningioma.
Neoplasia
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Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas, and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnormalities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions, suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion increases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associated development of aggressive biologic behavior in meningiomas.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.