Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

Highly improved metabolic stability and pharmacokinetics of indium-111-DOTA-gastrin conjugates for targeting of the gastrin receptor

The development of metabolically stable radiolabeled gastrin analogues with suitable pharmacokinetics is a topic of recent research activity. These imaging vectors are of interest because the gastrin/CCK2 receptor is highly overexpressed in different tumors such as medullary thyroid cancer, neuroendocrine tumors, and SCLC. The drawback of current targeting agents is either their metabolic instability or their high kidney uptake. We present the synthesis and in vitro and in vivo evaluation of 11 (111)In-labeled DOTA-conjugated peptides that differ by their spacer between the peptide and the chelate. We introduced uncharged but hydrophilic spacers such as oligoethyleneglycol, serine, and glutamine. The affinity of all radiopeptides was high with IC(50) values between 0.5 and 4.8 nM. The improvement of human serum stability is 500-fold within this series of compounds. In addition the kidney uptake could be lowered distinctly and the tumor-to-kidney ratio improved almost 60-fold if compared with radiotracers having charged spacers such as glutamic acid.

The Search for Low Energy Conformational Families of Small Peptides: Searching for Active Conformations of Small Peptides in the Absence of a Known Receptor

Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Previous work revealed that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat model systems, interacting with an unknown receptor, while peptides smaller than eight amino acids did not inhibit breast cancer. We have shown that the use of replica exchange molecular dynamics predicts structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. These simulations identified smaller peptide analogs with a conserved turn, a β-turn formed in the larger peptides. These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used with the experimental information that led to the successful completion of this research.

Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Influence of pregnancy on the adipocytokine and peroxisome proliferator-activated receptor pathways in peripheral blood mononuclear cells from healthy donors and rheumatoid arthritis patients

To identify candidate genes that are regulated by human pregnancy and have the potential to modulate rheumatoid arthritis (RA) disease activity.

L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

Identification of key residues in virulent canine distemper virus hemagglutinin that control CD150/SLAM-binding activity

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

Meprinα transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.

Lead discovery, chemistry optimization, and biological evaluation studies of novel biamide derivatives as CB2 receptor inverse agonists and osteoclast inhibitors

N,N'-((4-(Dimethylamino)phenyl)methylene)bis(2-phenylacetamide) was discovered by using 3D pharmacophore database searches and was biologically confirmed as a new class of CB(2) inverse agonists. Subsequently, 52 derivatives were designed and synthesized through lead chemistry optimization by modifying the rings A-C and the core structure in further SAR studies. Five compounds were developed and also confirmed as CB(2) inverse agonists with the highest CB(2) binding affinity (CB(2)K(i) of 22-85 nM, EC(50) of 4-28 nM) and best selectivity (CB(1)/CB(2) of 235- to 909-fold). Furthermore, osteoclastogenesis bioassay indicated that PAM compounds showed great inhibition of osteoclast formation. Especially, compound 26 showed 72% inhibition activity even at the low concentration of 0.1 μM. The cytotoxicity assay suggested that the inhibition of PAM compounds on osteoclastogenesis did not result from its cytotoxicity. Therefore, these PAM derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent.

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PCR.