RESUMO
An enriched environment is associated with hippocampal plasticity, including improved cognitive performance and increased neurogenesis. Here, we show that hippocampal expression of vascular endothelial growth factor (VEGF) is increased by both an enriched environment and performance in a spatial maze. Hippocampal gene transfer of VEGF in adult rats resulted in approximately 2 times more neurogenesis associated with improved cognition. In contrast, overexpression of placental growth factor, which signals through Flt1 but not kinase insert domain protein receptors (KDRs), had negative effects on neurogenesis and inhibited learning, although it similarly increased endothelial cell proliferation. Expression of a dominant-negative mutant KDR inhibited basal neurogenesis and impaired learning. Coexpression of mutant KDR antagonized VEGF-enhanced neurogenesis and learning without inhibiting endothelial cell proliferation. Furthermore, inhibition of VEGF expression by RNA interference completely blocked the environmental induction of neurogenesis. These data support a model in which VEGF, acting through KDR, mediates the effect of the environment on neurogenesis and cognition.
Assuntos
Hipocampo/fisiologia , Aprendizagem , Memória , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Divisão Celular , Endotélio Vascular/fisiologia , Hipocampo/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Fator de Crescimento Placentário , Proteínas da Gravidez/fisiologia , RatosRESUMO
In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.
Assuntos
Moléculas de Adesão Celular/fisiologia , Extensões da Superfície Celular/patologia , Neoplasias do Colo/patologia , Proteínas dos Microfilamentos/fisiologia , Fosfoproteínas/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Colágeno Tipo IV/metabolismo , Guanilato Ciclase/fisiologia , Humanos , Invasividade Neoplásica , Fosforilação , Transdução de SinaisRESUMO
Glucagon-like peptide-1 (GLP-1) is a gut peptide that, together with its receptor, GLP-1R, is expressed in the brain. Here we show that intracerebroventricular (i.c.v.) GLP-1 and [Ser(2)]exendin(1-9) (HSEGTFTSD; homologous to a conserved domain in the glucagon/GLP-1 family) enhance associative and spatial learning through GLP-1R. [Ser(2)]exendin(1-9), but not GLP-1, is also active when administered peripherally. GLP-1R-deficient mice have a phenotype characterized by a learning deficit that is restored after hippocampal Glp1r gene transfer. In addition, rats overexpressing GLP-1R in the hippocampus show improved learning and memory. GLP-1R-deficient mice also have enhanced seizure severity and neuronal injury after kainate administration, with an intermediate phenotype in heterozygotes and phenotypic correction after Glp1r gene transfer in hippocampal somatic cells. Systemic administration of [Ser(2)]exendin(1-9) in wild-type animals prevents kainate-induced apoptosis of hippocampal neurons. Brain GLP-1R represents a promising new target for both cognitive-enhancing and neuroprotective agents.
Assuntos
Glucagon/farmacologia , Hipocampo/fisiologia , Aprendizagem/fisiologia , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Administração Intranasal , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Expressão Gênica , Técnicas de Transferência de Genes , Glucagon/administração & dosagem , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Ácido Caínico/efeitos adversos , Aprendizagem/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/administração & dosagem , Precursores de Proteínas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/genética , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
AIMS: Vasodilator-stimulated phosphoprotein (VASP) controls actin dynamics associated with the malignant phenotype of colorectal tumors. Oncogenic VASP function, in turn, is finely regulated by cyclic nucleotide-dependent phosphorylation of serine (Ser) residues 157 and 239, whose differential expression determines cell survival behavior in colon cancer. However, the role of differential VASP Ser phosphorylation in colorectal carcinogenesis remains unclear. MAIN METHODS: Specific VASP phosphomutant constructs were employed to selectively silence Ser157 or Ser239 phosphorylation in human colon carcinoma cells. Cyclic nucleotide-dependent manipulation of VASP Ser phosphorylation was performed with 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP) or 8-chlorophenylthio 3',5'-cyclic guanosine monophosphate (8-CPT-cGMP). Tumorigenic and locomotory phenotypes were examined in vitro with clonogenic and wound healing assays, respectively. Finally, tumor formation and growth were investigated in vivo employing two distinct xenograft models of colorectal cancer. KEY FINDINGS: Disruption of VASP Ser157 phosphorylation weakened the clonogenic and migratory abilities of human colon cancer cells, effects mimicked by 8-CPT-cGMP-dependent regulation of VASP Ser239. In contrast, inhibition of VASP Ser239 phosphorylation enhanced cell clonogenicity and migration and was phenocopied by 8-Br-cAMP-dependent regulation of VASP Ser157. Importantly, cancer cells bearing the phosphomutant construct targeting VASP Ser157 decreased, while those with the phosphomutation at Ser239 improved their abilities to establish productive tumor colonies and grow in the peritoneal cavity or subcutaneous tissues of nude mice. SIGNIFICANCE: Together, present observations suggest differential VASP Ser phosphorylation is a relevant, targetable molecular event underlying tumor formation and progression in colon cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Fosforilação/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.
Assuntos
Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Enterotoxinas/farmacologia , Receptores de Detecção de Cálcio/fisiologia , Apoptose , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Neoplasias Colorretais/patologia , Humanos , Precondicionamento Isquêmico , Necrose , Receptores de Detecção de Cálcio/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion. Phosphorylation of VASP serine (Ser) residues 157 and 239 regulate VASP function, directing subcellular localization and inhibiting actin polymerization, respectively. METHODS: The expression levels of VASP protein, pSer157-VASP, and pSer239-VASP were determined by immunohistochemistry in tumors and matched normal adjacent tissue from 141 CRC patients, divided into 2 cohorts, and the association of VASP biomarker expression with clinicopathologic features and disease recurrence was examined. RESULTS: We report that changes in VASP expression and phosphorylation were significantly associated with tumor invasion and disease recurrence. Furthermore, we disclose a novel 2-tiered methodology to maximize VASP positive and negative predictive value performance for prognostication. CONCLUSION: VASP biomarkers may serve as prognostic biomarkers in CRC and should be evaluated in a larger clinical study.
RESUMO
Sex differences have been observed in the antinociceptive effects of opioids in rodents and rhesus monkeys. Sex differences in the affinity of opioid ligands for opioid receptors may contribute to these findings. To test this hypothesis, the relative affinity of the competitive opioid antagonist quadazocine for mu and kappa opioid receptors was determined in rhesus monkeys using in vivo pA(2) analysis. The antinociceptive effects of the mu opioid agonist fentanyl and the kappa opioid agonist U50,488 were determined alone and after pretreatment with quadazocine in 4 females and 4 males using a warm-water tail-withdrawal assay of thermal nociception. The relative potency of quadazocine antagonism of fentanyl and U50,488 in females and males was used to assess sex differences in the relative affinity of quadazocine for mu and kappa receptors. Fentanyl was equipotent in female and male monkeys, and quadazocine was equipotent as an antagonist of fentanyl in females and males. In contrast, U50,488 was significantly less potent in females, and quadazocine was less potent as an antagonist of U50,488 in females. These findings suggest that opioid ligands have similar affinity for mu receptors but lower affinity for at least some kappa receptors in female than in male rhesus monkeys.
RESUMO
Matrix metalloproteinase-9 (MMP-9) produced by colorectal cancer cells is a critical determinant of metastatic disease progression and an attractive target for antimetastatic strategies to reduce colon cancer mortality. Cellular signaling by cyclic GMP (cGMP) regulates MMP-9 dynamics in various cell systems, and the bacterial enterotoxin receptor guanylyl cyclase C (GCC), the principle source of cGMP in colonocytes, which is overexpressed in colorectal cancers, inhibits tumor initiation and progression in the intestine. Here, we show that ligand-dependent GCC signaling through cGMP induces functional remodeling of cancer cell MMP-9 reflected by a compartmental redistribution of this gelatinase, in which intracellular retention resulted in reciprocal extracellular depletion. Functional remodeling of MMP-9 by GCC signaling reduced the ability of colon cancer cells to degrade matrix components, organize the actin cytoskeleton to form locomotory organelles and spread, and hematogenously seed distant organs. Of significance, GCC effects on cancer cell MMP-9 prevented establishment of metastatic colonies by colorectal cancer cells in the mouse peritoneum in vivo. Because endogenous hormones for GCC are uniformly deficient in intestinal tumors, reactivation of dormant GCC signaling with exogenous administration of GCC agonists may represent a specific intervention to target MMP-9 functions in colon cancer cells. The notion that GCC-mediated regulation of cancer cell MMP-9 disrupts metastasis, in turn, underscores the unexplored utility of GCC hormone replacement therapy in the chemoprevention of colorectal cancer progression.
Assuntos
Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Guanilato Ciclase/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Receptores de Peptídeos/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular/fisiologia , GMP Cíclico/metabolismo , Células Epiteliais/enzimologia , Humanos , Inibidores de Metaloproteinases de Matriz , Metástase Neoplásica , Neoplasias Peritoneais/enzimologia , Neoplasias Peritoneais/secundário , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Transdução de SinaisRESUMO
Colorectal cancer mortality largely reflects metastasis, the spread of the disease to distant organs. Matrix metalloproteinase 9 (MMP-9) is a key regulator of metastasis and a target for anticancer strategies in colon cancer. Here, the overexpression of MMP-9 in pure tumor epithelial, but nor stromal, cell populations was associated with metastatic progression of colorectal cancer, as defined by reverse transcriptase-polymerase chain reaction (qRT-PCR) and confirmed by immunostaining. Thus, cancer cell MMP-9 represents a novel, selective prognostic and predictive factor that may be exploited for more effective disease stage stratification and therapeutic regimen selection in patients with colorectal cancer.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Metaloproteinase 9 da Matriz/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Compartimento Celular , Separação Celular , Neoplasias Colorretais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/patologia , Masculino , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , PrognósticoRESUMO
The motor abnormalities of Parkinson's disease (PD) are caused by alterations in basal ganglia network activity, including disinhibition of the subthalamic nucleus (STN), and excessive activity of the major output nuclei. Using adeno-associated viral vector-mediated somatic cell gene transfer, we expressed glutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of the neurotransmitter GABA, in excitatory glutamatergic neurons of the STN in rats. The transduced neurons, when driven by electrical stimulation, produced mixed inhibitory responses associated with GABA release. This phenotypic shift resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype. This strategy suggests that there is plasticity between excitatory and inhibitory neurotransmission in the mammalian brain that could be exploited for therapeutic benefit.