Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice.

BACKGROUND AND AIMS: Kinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs). METHODS: Fibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study. RESULTS: B1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFß, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-ß and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway. CONCLUSIONS: B1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

RGD cadherins and α2ß1 integrin in cancer metastasis: A dangerous liaison.

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Patient Selection and Toxicities of PRRT for Metastatic Neuroendocrine Tumors and Research Opportunities.

OPINION STATEMENT: Neuroendocrine tumors (NETs) are a heterogenous group of neoplasms characterized by varied biological hallmarks and behavior, ranging from indolent to aggressive. For many decades, somatostatin analogues and few targeted therapies were available for NETs and these therapies had minimal response rates. However, there have been a number of recent treatment advances. Peptide receptor radionuclide therapy (PRRT) is a novel approach to treatment of NETs and has changed the landscape of treatment for NETs. It is a form of targeted therapy in which a radiolabeled somatostatin analogue delivers radiation specifically to tumor cells expressing the somatostatin receptor.

The potent small molecule integrin antagonist THR-687 is a promising next-generation therapy for retinal vascular disorders.

Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4 ±â€¯2.7 nM), αvß5 (IC50 of 1.3 ±â€¯0.5 nM) and α5ß1 (IC50 of 6.8 ±â€¯3.2 nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258 ±â€¯113 nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236 ±â€¯173 nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45 mg, 2.25 mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.

An Integrated Pan-Cancer Analysis and Structure-Based Virtual Screening of GPR15.

G protein-coupled receptor 15 (GPR15, also known as BOB) is an extensively studied orphan G protein-coupled receptors (GPCRs) involving human immunodeficiency virus (HIV) infection, colonic inflammation, and smoking-related diseases. Recently, GPR15 was deorphanized and its corresponding natural ligand demonstrated an ability to inhibit cancer cell growth. However, no study reported the potential role of GPR15 in a pan-cancer manner. Using large-scale publicly available data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we found that GPR15 expression is significantly lower in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) than in normal tissues. Among 33 cancer types, GPR15 expression was significantly positively correlated with the prognoses of COAD, neck squamous carcinoma (HNSC), and lung adenocarcinoma (LUAD) and significantly negatively correlated with stomach adenocarcinoma (STAD). This study also revealed that commonly upregulated gene sets in the high GPR15 expression group (stratified via median) of COAD, HNSC, LUAD, and STAD are enriched in immune systems, indicating that GPR15 might be considered as a potential target for cancer immunotherapy. Furthermore, we modelled the 3D structure of GPR15 and conducted structure-based virtual screening. The top eight hit compounds were screened and then subjected to molecular dynamics (MD) simulation for stability analysis. Our study provides novel insights into the role of GPR15 in a pan-cancer manner and discovered a potential hit compound for GPR15 antagonists.

In silico estrogen-like activity and in vivo osteoclastogenesis inhibitory effect of Cicer arietinum extract.

Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

Radiolabeled peptides: current and new perspectives.

Radiolabeled peptides have been the subject of research for over 20 years and during that time possibility/variety of peptide receptor imaging and later targeted radiotherapy increased significantly. The targeted receptors belong to the large family of G-protein-coupled receptors or tyrosine kinases receptors partially connected with them. They both regulate large signaling networks, control multiple cell functions and are implicated in many diseases including cancers. The essential feature of peptides used in nuclear medicine involves their ability to binding with high affinity and specify to their receptors overexpressed on tumor cells. Currently most important peptide radiotracers are somatostatin, GLP-1, bombesin, gastrin/cholecystokinin-2, neurokinin type 1, CXCR4, EGF, VEGF and integrins. These tracers are mainly based on nuclides which are radiometals or on 18F and may be used in both SPECT or PET techniques as well as for hybrid imaging. Using of chelators suitable for peptide labeling with diagnostic and therapeutic radionuclids enables the theranostic approach. In this review we will provide a brief overview over currently available radiopharmaceuticals based on different groups of peptides.

Relaxin-3/RXFP3 system regulates alcohol-seeking.

Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Functional rescue of Kallmann syndrome-associated prokineticin receptor 2 (PKR2) mutants deficient in trafficking.

Mutations in the G protein-coupled prokineticin receptor 2 (PKR2) are known to cause Kallmann syndrome and idiopathic hypogonadotropic hypogonadism manifesting with delayed puberty and infertility. Some of the mutant receptors are not routed to the cell surface; instead, they are trapped in the cellular secretory pathway. The cell-permeant agonists/antagonists have been used to rescue some membrane receptors that are not targeted onto the cell membrane. Here, we chose three disease-associated mutations (W178S, G234D, and P290S), which all resulted in retention of PKR2 intracellularly. We show that a small molecule PKR2 antagonist (A457) dramatically increased cell surface expression and rescued the function of P290S PKR2, but had no effect on W178S and G234D PKR2. Furthermore, we also tested chemical chaperone glycerol on the cell surface expression and function of PKR2 mutants. Treatment with 10% glycerol significantly increased the cell surface expression and signaling of P290S and W178S PKR2. These data demonstrate that some Kallmann syndrome-associated, intracellularly retained mutant PKR2 receptors can be functionally rescued, suggesting a potential treatment strategy for patients bearing such mutations.

SiRNA directed against annexin II receptor inhibits angiogenesis via suppressing MMP2 and MMP9 expression.

BACKGROUND/AIMS: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism. METHODS/RESULTS: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9. CONCLUSION: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension.

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.

Loss of LH-induced down-regulation of anti-Müllerian hormone receptor expression may contribute to anovulation in women with polycystic ovary syndrome.

STUDY QUESTION: Are anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR-II) mRNAs similarly regulated by gonadotrophins in lutein granulosa cells (GCs) from control, normo-ovulatory and oligo/anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: AMH mRNA expression was induced by LH only in lutein GC of oligo/anovulatory PCOS women; down-regulation of AMHR-II, induced by LH in control and normo-ovulatory PCOS women, was absent in oligo/anovulatory women. WHAT IS KNOWN ALREADY: It was suggested that AMH could be responsible for the blockade of follicles at the small antral stage in PCOS women. In keeping with this hypothesis, both AMH and AMHR-II are overexpressed in lutein GCs from oligo/anovulatory PCOS women. STUDY DESIGN, SIZE, DURATION: Women undergoing IVF were included in this prospective study, either in the control group (30 women) or in the PCOS group (21 normo-ovulatory and 19 oligo/anovulatory patients) between January 2010 and July 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human lutein GCs were isolated from follicular fluid during IVF protocols. Twenty-four hours after seeding, lutein GCs from each woman were serum starved and cultured for 48 h ± FSH, LH or cAMP. Then AMH and AMHR-II mRNAs were quantified by quantitative RT-PCR and AMH protein concentration was measured in the culture medium by ELISA. Experimental results were analyzed, within each group of women, by the non-parametric Wilcoxon test for paired comparisons between cells cultured in control medium and FSH, LH or cAMP treated cells. Clinical comparisons between the three groups of women were performed on log values using the ANOVA test with Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: FSH up-regulated both AMH expression and secretion by lutein GCs from the three groups of women (P < 0.05). LH had no effect on AMH mRNAs levels in lutein GCs from controls and normo-ovulatory PCOS women, but increased AMH expression in oligo/anovulatory PCOS women (P < 0.05). Interestingly, LH and cAMP treatments reduced AMHR-II expression by lutein GCs from controls and normo-ovulatory PCOS women (P < 0.05), but had no effect on AMHR-II mRNA levels in oligo/anovulatory PCOS women. LIMITATIONS, REASONS FOR CAUTION: The lutein GCs are not the best model to study AMH and AMHR-II regulation by gonadotrophins. Indeed, AMH and AMHR-II are down-regulated in luteinized cells. Furthermore, these cells have been exposed to non-physiological levels of gonadotrophins and hCG. However, AMH and AMHR-II mRNAs are quantifiable by real-time RT-PCR, and the cells are still responsive to FSH and LH. The age of patients is significantly different between control and oligo/anovulatory PCOS women: this may be a bias in the interpretation of results but older women in the control group had a good ovarian reserve. WIDER IMPLICATIONS OF THE FINDINGS: The overexpression of AMH and AMHR-II in oligo/anovulatory PCOS women could be due to increased LH levels and/or inhibition of its repressive action. The fact that this dysregulation is observed in oligo/anovulatory, but not in normo-ovulatory, PCOS women emphasizes the role of LH in the follicular arrest of PCOS women and suggests that this involves the AMH/AMHR-II system. STUDY FUNDING/COMPETING INTEREST(S): The Assistance-Publique Hôpitaux de Paris provided a Contrat d'Interface and the Agence de Biomédecine provided a grant to Nathalie di Clemente. Schering-Plough provided an FARO grant to Alice Pierre. The authors have nothing to disclose.

Endothelial cell surface expression of protein disulfide isomerase activates ß1 and ß3 integrins and facilitates dengue virus infection.

Infection with dengue virus (DENV) causes diseases ranging from mild dengue fever to severe hemorrhage or shock syndrome. DENV infection of endothelial cells may cause cell apoptosis or vascular leakage and result in clinical illness of hemorrhage. However, the endothelial cell molecules involved in DENV infection and the mechanisms governing virus-cell interactions are still uncertain. Since protein disulfide isomerase (PDI) reducing function at the cell surface was shown to be required for entry of certain viruses and bacteria, we explored the role of PDI expressed on endothelial cell surface in DENV infection. Using siRNA to knock down PDI, DENV infection was reduced which could be reversed by treating cells with a reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). DENV-induced PDI surface expression was mediated through the Lys-Asp-Glu-Leu (KDEL) receptor-Src family kinase signal pathway. Furthermore, cell surface PDI colocalized with ß1 and ß3 integrins after DENV infection, and the activation of integrins was blocked by PDI inhibition. Finally, blockade of PDI inhibited DENV entry into endothelial cells. Our findings suggest a novel mechanism whereby surface PDI which causes integrin activation is involved in DENV entry, and DENV infection further increases PDI surface expression at later time points. These findings may have implications for anti-DENV drug design.

Human relaxin-2: historical perspectives and role in cancer biology.

One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A-B heterodimer linked by three disulfide bonds. This mini-review is divided into three broad themes: an overview of all insulin superfamily members (including structural similarities); roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.

A role for protein kinase C-dependent upregulation of adrenomedullin in the development of morphine tolerance in male rats.

Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and is a pronociceptive mediator. This study investigated whether AM plays a role in the development of tolerance to morphine-induced analgesia. Repetitive intrathecal injection of morphine increased the expression of AM-like immunoreactivity (AM-IR) in the spinal dorsal horn and dorsal root ganglion (DRG) neurons. Ganglion explant culture study showed that this upregulation of AM-IR was µ-opioid receptor dependent through the use of another agonist, fentanyl, and a selective antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)). The coadministration of the selective AM receptor antagonist AM(22-52) markedly attenuated the development of morphine tolerance, associated thermal hyperalgesia, and increase in AM-IR. A likely autocrine mechanism is supported by the finding that AM-IR is colocalized with AM receptor components in DRG neurons. Furthermore, opiate-induced increase in AM content was blocked by protein kinase C (PKC) inhibitors, whereas a PKC activator increased AM synthesis and release. A treatment with AM(22-52) also inhibited increases in the expression of CGRP-IR in the spinal cord and DRGs as well as in culture ganglion explants, whereas exposure to CGRP failed to alter AM content. Together, these results reveal that a sustained opiate treatment induces an upregulation of AM through the activation of µ-opioid receptors and the PKC signaling pathway. This phenomenon contributes to the development of tolerance to the antinociceptive effects of opiates at least partially via the upregulation of CGRP. Targeting AM and its receptors should be considered as a novel approach to preserve the analgesic potency of opiates during their chronic use.

Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila.

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.