CRE Promoter Research Articles

Exploring novel A2AAR antagonists: Design, synthesis, and evaluation of 2,6,9-trisubstituted purine derivatives as promising antifibrotic agents

A series of 2,6,9-trisubstituted purine derivatives were designed and synthesized with diverse chemical moieties. Through a comprehensive biological evaluation, we identified 4-(6-(methylamino)-2-(phenylethynyl)-9H-purin-9-yl)phenol (6a) as a promising A2AAR antagonist with potent antifibrotic properties. Compound 6a demonstrated significant efficacy in inhibiting CRE promoter activity and in reducing the expression of fibrogenic marker proteins and downstream effectors of A2AAR activation, surpassing the A2AAR antagonist ZM241385 and initial screening hits, 9-benzyl-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5a) and 9-((benzyloxy)methyl)-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5j). Further validation revealed that compound 6a effectively inhibited fibrogenic marker proteins induced by A2AAR overexpression or TGF-β1 treatment in hepatic stellate cells, alongside reducing PKA and CREB phosphorylation. These findings suggest that compound 6a exerts its antifibrotic action by modulating the cAMP/PKA/CREB pathway through A2AAR inhibition. Overall, our study provides valuable insights for the development of novel therapeutics that target hepatic fibrosis through A2AAR antagonism.

P-621 Rekovelle® vs Gonal-F®: in vitro characterization of intracellular signals induced by the two FSH preparations

Abstract Study question Do Rekovelle® and Gonal-F® modulate preparation-specific signalling? Summary answer Gonal-F® and Rekovelle® induce preparation-specific pattern of intracellular signalling. What is known already Rekovelle® and Gonal-F® are two commercial preparations of recombinant FSH used in assisted reproduction. These preparations differ in originator cells (PER.C6® cell line of human fetal retinal origin for Rekovlle®; modified Chinese Hamster Ovary cell line for Gonal-F®), likely reflecting glycosylation profiles impacting their mode of action. Study design, size, duration 3-year in vitro functional comparison of Rekovelle® and Gonal-F®, using the transfected HEK293 cell line expressing FSH receptor (FSHR). Participants/materials, setting, methods FSHR-expressing HEK293 cells were treated with increasing concentrations of GonalF® and Rekovelle® (pM-µM) to evaluate receptor-receptor interaction and trafficking, intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ increase by bioimaging techniques, as well as phospho-protein by Western blotting, and genomic effects by CRE-luciferase reporter gene activity. Main results and the role of chance Both the preparations induced similar, dose-dependent increase of receptor trafficking through intracellular organelles. These data were obtained by evaluation of the interaction between FSHR and endosomal markers RAB-GTPases (Rab) 5 and 7. Rekovelle® induced more pronounced steroifogenic signals than Gonal-F®, detected as FSHR-FSHR interactions at the cell membrane, cAMP accumulation, cAMP-responsive elements binding protein (CREB) phosphorylation and CRE promoter activity. Interestingly, while cell treatment by Gonal-F® mediated complete dose-response of cAMP, consisting in the continuous increase of the second messenger together with the increase of FSH concentration, Rekovelle® had biphasic action where cAMP increased from 0 to 100 nM although decreased at 1 µM Rekovelle® dose. Moreover, Rekovelle® was 10-times more potent than Gonal-F® in activating intracellular Ca2+ activation. The two preparations induced similar pattern of extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation. In conclusion, Rekovelle® has higher potency than Gonal-F® in activating cAMP- and Ca2+-dependent intracellular events, potentially resulting in preparation-specific steroidogenic pattern. Limitations, reasons for caution Experiments were performed in the transfected HEK293 cell line in vitro and should be confirmed by clinical observations. Wider implications of the findings We described different intracellular events mediated by Gonal-F® and Rekovelle® in vitro, likely originated by different glycan profiles and possibly impacting the ovarian response during assisted reproduction. Trial registration number Not Applicable

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness.

Myelination allows fast and synchronized nerve influxes and is provided by Schwann cells (SCs) in the peripheral nervous system. Glucocorticoid hormones are major regulators of stress, metabolism and immunity affecting all tissues. They act by binding to two receptors, the low-affinity glucocorticoid receptor (GR) and the high-affinity mineralocorticoid receptor (MR). Little is known about the effect of glucocorticoid hormones on the PNS, and this study focuses on deciphering the role of MR in peripheral myelination. In this work, the presence of a functional MR in SCs is demonstrated and the expression of MR protein in mouse sciatic nerve SC is evidenced. Besides, knockout of MR in SC (SCMRKO using Cre-lox system with DesertHedgeHog (Dhh) Cre promoter) was undertaken in mice. SCMRKO was not associated with alterations of performance in motor behavioral tests on 2- to 6-month-old male mice compared to their controls. No obvious modifications of myelin gene expression or MR signaling gene expression were observed in the SCMRKO sciatic nerves. Nevertheless, Gr transcript and GR protein amounts were significantly increased in SCMRKO nerves compared to controls, suggesting a possible compensatory effect. Besides, an increase in myelin sheath thickness was noted for axons with perimeters larger than 15 µm in SCMRKO illustrated by a significant 4.5% reduction in g-ratio (axon perimeter/myelin sheath perimeter). Thus, we defined MR as a new player in peripheral system myelination and in SC homeostasis.

Olfactory receptor OR52N2 for PGE2 in mediation of guppy courtship behaviors

Prostaglandins (PGs) are a type of physiologically active unsaturated fatty acids. As an important sex pheromone, PGs play a vital role in regulating the reproductive behaviors of species by mediating nerve and endocrine responses. In this study, guppy (Poecilia reticulate) was used as the model specie to detect the function of PGE2 in inducing the onset of courtship behaviors. Our results showed that adding PGE2 into the water environment could activate the courtship behavior of male guppy, indicating that the peripheral olfactory system mediated the PGE2 function. Thereafter, the open reading frame (ORF) of olfactory receptor or52n2 was cloned, which was 936 bp in length, coding 311 amino acids. As a typical G protein-coupled receptor, OR52N2 had a conservative seven α-helix transmembrane domains. To confirm the regulatory relationship between OR52N2 and PGE2, dual-luciferase reporter assay was employed to verify the activation of downstream CREB signaling pathways. Results showed that PGE2 significantly enhanced CRE promoter activity in or52n2 ORF transient transfected HEK-293 T cells. Finally, localization of or52n2 mRNA were observed in ciliated receptor cells of the olfactory epithelium using in situ hybridization. Our results provide a novel insight into sex pheromone signaling transduction in reproductive behavior.

Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse.

The development of tools to manipulate the mouse genome, including knockout and transgenic technology, has revolutionized our ability to explore gene function in mammals. Moreover, for genes that are expressed in multiple tissues or at multiple stages of development, the use of tissue-specific expression of the Cre recombinase allows gene function to be perturbed in specific cell types and/or at specific times. However, it is well known that putative tissue-specific promoters often drive unanticipated 'off-target' expression. In our efforts to explore the biology of the male reproductive tract, we unexpectedly found that expression of Cre in the central nervous system resulted in recombination in the epididymis, a tissue where sperm mature for ~1-2 weeks following the completion of testicular development. Remarkably, we not only observed reporter expression in the epididymis when Cre expression was driven from neuron-specific transgenes, but also when Cre expression in the brain was induced from an AAV vector carrying a Cre expression construct. A surprisingly wide range of Cre drivers - including six different neuronal promoters as well as the adipose-specific Adipoq Cre promoter - exhibited off-target recombination in the epididymis, with a subset of drivers also exhibiting unexpected activity in other tissues such as the reproductive accessory glands. Using a combination of parabiosis and serum transfer experiments, we find evidence supporting the hypothesis that Cre may be trafficked from its cell of origin to the epididymis through the circulatory system. Together, our findings should motivate caution when interpreting conditional alleles, and suggest the exciting possibility of inter-tissue RNA or protein trafficking in modulation of reproductive biology.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Multipotent Progenitor Niche Factors

Signal transducer and activator of transcription 5 (STAT5a and STAT5b isoforms) are latent transcription factors that are intrinsically critical for normal hematopoiesis but are also expressed in bone marrow stromal cells. However, despite known STAT5a (prolactin) and STAT5b (growth hormone) functions in breast and liver tissue respectively, hematopoiesis-supporting stromal functions have not been explored. Here, floxed STAT5ab double knockout (KO) was generated with a variety of hematopoietic and stromal Cre-expressing transgenic mouse strains to test whether STAT5a or STAT5b play a role in extrinsic regulation of hematopoiesis. Pan-hematopoietic deletion with Vav1-Cre was the positive control for loss of multipotent hematopoietic function but surprisingly Vav1-Cre also deleted STAT5ab in CD45neg cells, dysregulated stromal niche factor mRNA expression, and altered MSC osteo/chondro-lineage differentiation priming. Single cell transcriptome analysis of bone marrow from wild-type or Vav1-Cre KO mice long-term repopulating hematopoietic stem cells (LT-HSC) showed upregulated oxidative phosphorylation (P<10-10) and protein translation (P<10-39) genes in LT-HSCs and throughout myeloid-primed clusters, while in contrast common lymphoid progenitor-like cells were completely eliminated. Similarly, like Vav1+ cells and based on scRNAseq analysis, Nes+ cells were also detected in both CD45neg and CD45+ cell clusters. Deletion of STAT5ab with Nes-Cre reduced the number of LT-HSCs (30% reduction compared to Nes-Cre control mice, P=0.02, N=4) and caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45+ and CD45neg bone marrow cell populations, additional stroma-specific Cre strains (LepR, Osx, Ocn, Prrx1, Nes-CreERT2) as well as endothelial-specific (Cdh5) strains were generated lacking STAT5ab. Viable mice were assessed for peripheral blood hematology, competitive repopulation, as well as hematopoietic stem/progenitor subsets (LT-HSC, MPP1, MPP2, MPP3, MPP4, KLS) using well-accepted flow cytometry antibody panels. Deletion of STAT5 in stromal cells with LepR-Cre significantly reduced MPP4 (35% reduction, P=0.025, N=4). Overall, extrinsic stromal (but not endothelial) cells were deficient in supporting multipotent and lineage-committed hematopoietic progenitors. However, peripheral blood hematology was rescued through homeostatic production. Gene regulation profiles resembled that of cytokine-starved MPPs. Functional support for niche-supporting activity was also assessed using STAT5ab KO MSCs in vitro. With Lepr-Cre mediated STAT5ab deletion, bone marrow niche factor mRNAs were downregulated (Cxcl12, Angpt1, Igfbp4, Igf1, Il34) and support of multipotent hematopoietic cells was significantly impaired. ELISA assay showed that SDF-1a was reduced 3-fold (P<0.001, N=5) and IGF-1 was reduced 2-fold (P<0.01, N=5) compared to wild type MSCs. Inversely, mRNAs for Kitl and Grem1 were increased in the same stromal cells, consistent with loss of multipotency and mixed HSC vs. MPP stromal support activities. Furthermore, novel scRNAseq analysis on simultaneous bone marrow hematopoietic and stromal cells compared wild-type and KO mice. Differentially expressed gene sets with Maximum Mean Discrepancy (MMD) co-expression revealed a key central role for coordinated STAT5ab/Cish balance as a biomarker of the cellular progenitor activity state (metabolic, protein translation, survival) associated with multipotency, comparing KO to wild-type cells. Importantly, Cish was strongly co-expressed in cells primed for lineage-specific differentiation (MSC - osteo/chondrogenic-primed; HSC - myeloid-primed) with STAT5ab co-expressed genes being associated with multipotent potential. Therefore, novel STAT5ab/Cish-mediated gene regulation programs operate within the bone marrow microenvironment to mediate cytokine production and responsiveness. This work highlights the importance of examining heterotypic cell types within bone marrow to elucidate functional cross-talk underlying hematopoietic homeostasis and defines conserved STAT5ab-mediated gene signatures as potential biomarkers to test in human hematopoietic and immune cell disorders.

Context matters - Daxx and Atrx are not robust tumor suppressors in the murine endocrine pancreas.

ABSTRACTGenome sequencing has revealed the importance of epigenetic regulators in tumorigenesis. The genes encoding the chromatin remodeling complex DAXX:ATRX are frequently mutated in pancreatic neuroendocrine tumors; however, the underlying mechanisms of how mutations contribute to tumorigenesis are only partially understood, in part because of the lack of relevant preclinical models. Here, we used genetically engineered mouse models combined with environmental stress to evaluate the tumor suppressor functions of Daxx and Atrx in the mouse pancreas. Daxx or Atrx loss, alone or in combination with Men1 loss, did not drive or accelerate pancreatic neuroendocrine tumorigenesis. Moreover, Daxx loss did not cooperate with environmental stresses (ionizing radiation or pancreatitis) or with the loss of other tumor suppressors (Pten or p53) to promote pancreatic neuroendocrine tumorigenesis. However, owing to promiscuity of the Cre promoter used, hepatocellular carcinomas and osteosarcomas were observed in some instances. Overall, our findings suggest that Daxx and Atrx are not robust tumor suppressors in the endocrine pancreas of mice and indicate that the context of a human genome is essential for tumorigenesis. This article has an associated First Person interview with the first author of the paper.

Kindlin-2 deficiency induces fatal intestinal obstruction in mice.

Rationale: Smooth muscle-motility disorders are mainly characterized by impaired contractility and functional intestinal obstruction. Some of these cases are caused by genetic mutations of smooth muscle genes ACTA2, ACTG2, MYH11, MYLK and LMOD1. Still the etiology is complex and multifactorial and the underlying pathology is poorly understood. Integrin interaction protein Kindlin-2 is widely expressed in striated and smooth muscle cells (SMC). However, the function of Kindlin-2 in the smooth muscle remains elusive.Methods: We generated two mouse models using different cre promoter transgenic mice, Kindlin-2fl/fl SM22α-cre+ (cKO mice) and Kindlin-2fl/fl; MYH-cre+ (iKO mice). Embryos and adult tissues were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay. We investigated ultrastructure changes of mouse smooth muscle using transmission electron microscopy (TEM) and measured smooth muscle contractile force in mounting aortic and intestinal rings using the multiwire myograph system (DMT 620M). In addition, cell traction force microscopy (CTFM) was applied to observe the functional change of primary SMC after Kindlin-2 depletion by RNAi.Results: Depletion of Kindlin-2 encoding gene Fermt2 in embryonic smooth muscles leads to apoptosis, downregulates the key components of SMC, impairs smooth muscle development, and finally causes embryonic death at E14.5. Tamoxifen-induced Kindlin-2-specific knockout in adult mouse smooth muscle showed decreased blood pressure, intestinal hypoperistalsis, and eventually died of intestinal obstruction. Kindlin-2 depletion also leads to downregulated Myh11, α-SMA, and CNN, shortened myofilament, broken myofibrils, and impaired contractility of the smooth muscles in iKO mice. Mechanistically, loss of Kindlin-2 decreases Ca2+ influx in primary vascular smooth muscle cells (PVSMC) by downregulating the expression of calcium-binding protein S100A14 and STIM1.Conclusion: We demonstrated that Kindlin-2 is essential for maintaining the normal structure and function of smooth muscles. Loss of Kindlin-2 impairs smooth muscle formation during embryonic development by inducing apoptosis and jeopardizes the contraction of adult smooth muscle by blocking Ca2+ influx that leads to intestinal obstruction. Mice with Kindlin-2 depletion in adult smooth muscle could be a potent animal model of intestinal obstruction for disease research, drug treatment and prognosis.

Gγ7-specific prothymosin alpha deletion causes stress- and age-dependent motor dysfunction and anxiety

We previously showed that prothymosin alpha (ProTα) improves cerebral ischemia-induced motor dysfunction. Our recent study also demonstrated that heterozygous ProTα deletion exhibited an enhanced anxiety-like behavior in mice. However, it remains elusive which brain regions or cells are related to these phenotypes. Here we generated conditional Gγ7-specific ProTα knockout mice using G protein γ7 subunit gene (Gng7)-cre promoter to see the brain robustness roles of ProTα in the striatum and hippocampus. The younger conditional ProTα (Gng7) knockout mice at the age of 10 weeks showed no significant phenotypes in motor dysfunction in the Rotarod test and locomotor activity in the open-field test, whereas significant motor dysfunction was obtained by 15 min transient middle cerebral artery occlusion (tMCAO)-induced cerebral ischemia. The aged conditional ProTα (Gng7) knockout mice at the age of 20 weeks showed hypolocomotor activity with less center time in the open-field test and impaired motor coordination in the Rotarod test without ischemia. Thus, this study suggests that ProTα has important roles in the maintenance of motor coordination and anxiety-like behavior.

Evidence for the Direct Effect of the NPFF Peptide on the Expression of Feeding-Related Factors in Spotted Sea Bass (Lateolabrax maculatus).

Neuropeptide FF (NPFF) is a family member of RF-amide peptides, which are suggested to be involved in the control of vertebrate feeding behavior. However, little is known about the effect of the NPFF peptide on feeding-related processes in basal vertebrates. In this study, four full-length cDNAs, npff, npffr1, npffr2-1, and npffr2-2, were cloned from spotted sea bass and characterized. The conserved NPFF peptide is biologically active because it functionally interacts with different receptors expressed in cultured eukaryotic cells to enhance CRE promoter activity. Tissue distribution analysis showed that the highest npff mRNA expression occurred in the telencephalon, hypothalamus, medulla, gonad and muscle, but the npffrs mRNAs were mainly distributed within the central nervous system (CNS). In situ hybridization (ISH) detected npff-expressing cells in several specific regions ranging across the telencephalon and midbrain to the hypothalamus. Incubation of the spotted sea bass conserved NPFF peptide significantly increased the expression of orexin (orx) and neuropeptide Y (npy) mRNA and decreased the expression of leptin (lep), somatostatin (ss), and cholecystokinin (cck) mRNA in brain cells. Similarly, the conserved NPFF peptide also heightened the expression of gastrin (gas), ghrelin (ghrl), and motilin (mtl) mRNA and significantly reduced the expression of cck mRNA in the intestine and stomach. Taken together, these data suggest that the NPFF peptide may play a stimulating role in regulating feeding-related processes in spotted sea bass.

Stat3 in osteocytes mediates osteogenic response to loading

Signal transducer and activator of transcription 3 (Stat3) is a member of the Stat family of proteins involved in signaling in many different cell types, including osteocytes. Osteocytes are considered major mechanosensing cells in bone due to their intricate dendritic networks able to sense changes in physical force and to orchestrate the response of osteoclasts and osteoblasts. We examined the role of Stat3 in osteocytes by generating mice lacking Stat3 in these cells using the Dmp-1(8kb)-Cre promoter (Stat3cKO mice). Compared to age-matched littermate controls, Stat3cKO mice of either sex (18 weeks old) exhibit reduced bone formation indices, decreased osteoblasts and increased osteoclasts, and altered material properties, without detectable changes in bone mineral density (BMD) or content of either trabecular or cortical bone. In addition, Stat3cKO mice of either sex show significantly decreased load-induced bone formation. Furthermore, pharmacologic inhibition of Stat3 in osteocytes in vitro with WP1066 blocked the increase in cytosolic calcium induced by ATP, a mediator of the cellular responses to sheer stress. WP1066 also increased reactive oxygen species (ROS) production in cultured MLO-Y4 osteocytes. These data demonstrate that Stat3 is a critical mediator of mechanical signals received by osteocytes and suggest that osteocytic Stat3 is a potential therapeutic target to stimulate bone anabolism.

Structural and functional analysis of two novel somatostatin receptors identified from topmouth culter (Erythroculter ilishaeformis)

In the present study, we cloned and characterized two somatostatin (SS) receptors (SSTRs) from topmouth culter (Erythroculter ilishaeformis) designated as EISSTR6 and EISSTR7. Analysis of EISSTR6 and EISSTR7 signature motifs, 3D structures, and homology with the known members of the SSTR family indicated that the novel receptors had high similarity to the SSTRs of other vertebrates. EISSTR6 and EISSTR7 mRNA expression was detected in 17 topmouth culter tissues, and the highest level was observed in the pituitary. Luciferase reporter assay revealed that SS14 significantly inhibited forskolin-stimulated pCRE-luc promoter activity in HEK293 cells transiently expressing EISSTR6 and EISSTR7, indicating that the receptors can be activated by SS14. We also identified phosphorylation sites important for the functional activity of EISSTR6 and EISSTR7 by mutating Ser23, 43, 107, 196, 311 and Ser7, 29, 61, 222, 225 residues, respectively, to Ala, which significantly reduced the inhibitory effects of SS14 on the CRE promoter mediated by EISSTR6 and EISSTR7. Furthermore, treatment of juvenile topmouth culters with microcystin-LR or 17β-estradiol significantly affected EISSTR6 and EISSTR7 transcription in the brain, liver and spleen, suggesting that these receptors may be involved in the pathogenic mechanisms induced by endocrine disruptors. Our findings should contribute to the understanding of the structure-function relationship and evolution of the SSTR family.

Identification of 5-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve Memory and Cognition.

Initial work in Drosophila and mice demonstrated that the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a master control gene for memory formation. The relationship between CREB and memory has also been found to be true in other species, including aplysia and rats. It is thus well-established that CREB activation plays a central role in memory enhancement and that CREB is activated during memory formation. On the basis of these findings, a phenotypic high-throughput screening campaign utilizing a CRE-luciferase (CRE-Luci) SK-N-MC cell line was performed to identify compounds that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin. A number of small-molecule hits of unknown mechanisms of action were identified in the screening campaign, including HT-0411. Follow-up studies suggested that the CREB activation by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-B). Further, HT-0411 was shown to improve 24 h memory in rodents in a contextual fear conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel, potent, and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of this and other related analogue series identified a number of potential clinical development candidates; ultimately, compound 8f was identified as a candidate molecule with high selectivity toward MAO-B (29-56 nM) over MAO-A (19% inhibition at a screening concentration of 50 μM), an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic properties in rodents and dogs, and efficacy in multiple rodent memory models.

Neutrophils in cisplatin AKI—mediator or marker?

Inflammation is an important mediator of most forms of acute kidney injury (AKI). Although neutrophils are prominent components of theinflammatory cascade, the precise role of neutrophils in AKI and the mechanisms by which they contribute to AKI remain controversial. In this issue, Deng etal. identify an important cross talk between renal epithelial cells and neutrophils involving the production and action ofleukotriene B4 in mediating cisplatin AKI. We discuss the possible explanations for the discrepant findings that have been reported for neutrophils in cisplatin AKI.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Molecular identification of the Dyn/Kor system and its potential role in the reproductive axis of goldfish

To ascertain the significance of the dynorphin/kappa-opioid receptor (Dyn/Kor) system in fish reproduction, prodynorphin (pdyn) cDNA was cloned from goldfish. Two Dyn peptides (DynA and DynB) are present in the goldfish prodynorphin precursor. Both DynA and DynB are biologically active as they are able to functionally interact with the goldfish Kor expressed in cultured eukaryotic cells to suppress forskolin-induced CRE promoter activity. RT-PCR analysis showed that pdyn is widely expressed in brain regions, with the highest expression in hypothalamus. During ovarian development, hypothalamic pdyn and kor mRNA levels are lower in the early vitellogenic stage. Then the biological effects of Dyn peptides on salmon gonadotropin releasing hormone (sgnrh), luteinizing hormone beta (lhb) and follicle stimulating hormone beta (fshb) mRNA synthesis were further investigated in goldfish. Intraperitoneal injections of DynA and DynB significantly reduced hypothalamic sgnrh and pituitary lhb and fshb mRNA levels in male goldfish, but these two peptides only down-regulated sgnrh and lhb mRNA expression in female goldfish. In vitro studies revealed that DynA also decreased lhb mRNA levels in primary cultures of pituitary cells, indicating that this peptide can exert its actions at the pituitary level. Our findings suggest that the Dyn/Kor system plays a negative role in regulating the reproductive axis in goldfish.

Neuroprotective effect of a novel Chinese herbal decoction on cultured neurons and cerebral ischemic rats.

BackgroundHistorically, traditional Chinese medicine has been widely used to treat stroke. Based on the theory of Chinese medicine and the modern pharmacological knowledge of herbal medicines, we have designed a neuroprotective formula called Post-Stroke Rehabilitation (PSR), comprising seven herbs – Astragalus membranaceus (Fisch.) Bunge, Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Cassia obtusifolia L., Ligusticum chuanxiong Hort., Angelica sinensis (Oliv.) Diels, and Glycyrrhiza uralensis Fisch. We aim to examine the neuroprotective activity of PSR in vitro and in vivo, and to explore the underlying molecular mechanisms, to better understand its therapeutic effect and to further optimize its efficacy.MethodsPSR extract or vehicle was applied to primary rat neurons to examine their survival effects against N-methyl-d-aspartate (NMDA)-elicited excitotoxicity. Whole-cell patch-clamp recording was conducted to examine the NMDA-induced current in the presence of PSR. ERK- and CREB-activation were revealed by western blot analysis. Furthermore, PSR was tested for CRE promoter activation in neurons transfected with a luciferase reporter. The protective effect of PSR was then studied in the rat middle cerebral artery occlusion (MCAO) model. MCAO rats were either treated with PSR extract or vehicle, and their neurobehavioral deficit and cerebral infarct were evaluated. Statistical differences were analyzed by ANOVA or t-test.ResultsPSR prominently reduced the death of cultured neurons caused by NMDA excitotoxicity in a dose-dependent manner, indicating its neuroprotective property. Furthermore, PSR significantly reduced NMDA-evoked current reversibly and activated phosphorylation of ERK and CREB with distinct time courses, with the latter’s kinetics slower. PSR also triggered CRE-promoter activity as revealed by the increased expression of luciferase reporter in transfected neurons. PSR effectively reduced cerebral infarct and deficit in neurological behavior in MCAO rats when PSR decoction was administered starting either 6 days before or 6 h after onset of ischemia.ConclusionsPSR is neuroprotective both in vitro and in vivo – it protects cultured neurons against NMDA excitotoxicity, and effectively reduces ischemic injury and neurobehavioral deficit in MCAO rats in both the pre- and post-treatment regimens. The underlying neuroprotective mechanisms may involve inhibition of NMDA receptor current and activation of ERK and CREB. This study provides important preclinical data necessary for the further development of PSR for stroke treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1417-1) contains supplementary material, which is available to authorized users.

Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1−/− mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1−/− mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4week old Osx:EfnB1−/− mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.

P38α MAPK is required for arsenic-induced cell transformation.

Arsenic exposure has been reported to cause neoplastic transformation through the activation of PcG proteins. In the present study, we show that activation of p38α mitogen-activated protein kinase (MAPK) is required for arsenic-induced neoplastic transformation. Exposure of cells to 0.5 μM arsenic increased CRE and c-Fos promoter activities that were accompanied by increases in p38α MAPK and CREB phosphorylation and expression levels concurrently with AP-1 activation. Introduction of short hairpin (sh) RNA-p38α into BALB/c 3T3 cells markedly suppressed arsenic-induced colony formation compared with wildtype cells. CREB phosphorylation and AP-1 activation were decreased in p38α knockdown cells after arsenic treatment. Arsenic-induced AP-1 activation, measured as c-Fos and CRE promoter activities, and CREB phosphorylation were attenuated by p38 inhibition in BALB/c 3T3 cells. Thus, p38α MAPK activation is required for arsenic-induced neoplastic transformation mediated through CREB phosphorylation and AP-1 activation.