Repeated cocaine administration alters the expression of genes in corticolimbic circuitry after a 3-week withdrawal: a DNA macroarray study

Addiction to psychostimulants elicits behavioral and biochemical changes that are assumed to be mediated by alterations of gene expression in the brain. The changes in gene expression after 3 weeks of withdrawal from chronic cocaine treatment were evaluated in the nucleus accumbens core and shell, dorsal prefrontal cortex and caudate using a complementary DNA (cDNA) array. The level of mRNA encoded by several genes was identified as being up- or down-regulated in repeated cocaine versus saline subjects. The results from the cDNA array were subsequently confirmed at the protein level with immunoblotting. Of particular interest, parallel up-regulation in protein and mRNA was found for the adenosine A1 receptor in the accumbens core, neuroglycan C in the accumbens shell, and the GluR5 glutamate receptor subtype in dorsal prefrontal cortex. However, there was an increase in TrkB protein in the nucleus accumbens core of cocaine-treated rats without a corresponding alteration in mRNA. These changes of gene expression in corticolimbic circuitry may contribute to the psychostimulant-induced behavioral changes associated with addiction.     

Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.     

Directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast

The extracellular domain of epidermal growth factor receptor (EGFR-ECD) has been engineered through directed evolution and yeast surface display using conformationally-specific monoclonal antibodies (mAbs) as screening probes for proper folding and functional expression in Saccharomyces cerevisiae. An EGFR mutant with four amino acid changes exhibited binding to the conformationally-specific mAbs and human epidermal growth factor, and showed increased soluble secretion efficiency compared with wild-type EGFR. Full-length EGFR containing the mutant EGFR-ECD was functional, as assayed by EGF-dependent autophosphorylation and intracellular MAPK signaling in mammalian cells, and was expressed and localized at the plasma membrane in yeast. This approach should enable engineering of other complex mammalian receptor glycoproteins in yeast for genetic, structural, and biophysical studies.     

Effects of cis and trans regulatory variations on the expression divergence of heat shock response genes between yeast strains

Recently, two genome-wide association studies in Asia identified gene polymorphisms known as rs4488809, rs9816619 in TP63 and rs2131877, rs952481 in C3orf21. It has been proposed that these polymorphisms are susceptibility loci for non-small cell lung cancer (NSCLC) development among Japanese and Korean populations. We ask whether susceptibility to NSCLC is limited to the Chinese population or whether the environment also affects genetic polymorphisms. We conducted a matched case-control study to explore this question. Results show that polymorphism of TP63 was not associated with NSCLC development, whereas variant genotypes of C3orf21 were nominally associated with a reduced risk of lung adenocarcinoma (OR=0.619, 95% CI=0.390-0.976). These results strongly suggest that environmental agents interact with human genetic polymorphism independent of ethnic background. In addition, the C3orf21 gene may be a potential susceptibility marker for lung adenocarcinoma independent of ethnic background and environmental agents.     

Synergistic effects of pravastatin and pioglitazone in renal tubular epithelial cells induced by transforming growth factor-beta1

Recent studies suggest that treatment with PPAR-gamma agonists and statins have beneficial effects on renal disease. However, the combined effects of PPAR-gamma agonists and statins in human renal epithelial cells are unknown. Our present study revealed that there were synergistic effects of pravastatin and pioglitazone in the expression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1) and collagen 1 in human renal proximal tubular epithelial cells induced by transforming growth factor-beta 1 (TGF-beta1). The beneficial effects of combined therapy against renal tubular epithelial cell injury are attributed, at least in part, to the inhibition of transdifferentiation, extracellular matrix deposition and cytokine production.     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

Requirement of the enzymatic and signaling activities of plasmin for phorbol-ester-induced scattering of colon cancer cells

Colon cancer progression is associated with the activation of protein kinase C (PKC), the downregulation of functional E-cadherin and an increased expression of the serine protease urokinase (u-PA) and its receptor (u-PAR). HT29-M6 intestinal epithelial cells represent an in vitro model to study colon cancer progression. These cells are induced to scatter and to invade by phorbol esters. Using proteolytic and cell signaling inhibitors, we show that HT29-M6 cells require plasminogen for the acquisition of the scattering response to PMA. Our results indicate that, prior to inducing a state of competency for plasminogen-dependent scattering, PMA triggers an ordered succession of events where upregulation of the activity of u-PA precedes proteolysis of u-PAR and active degradation of the extracellular matrix (ECM). These events poise HT29-M6 cells to a scatter-competent state that allows the subsequent localized proteolytic activation of plasminogen to plasmin, required for the execution of scattering. Finally, we show that, in addition to its enzymatic activity directed at the degradation of ECM, plasmin generates an intracellular signal resulting in the phosphorylation of ERK 1/2. For a full motogenic activity, plasmin requires this signal since the use of a MEK inhibitor (PD98059) specifically blocks the plasmin-dependent phase of cell scattering. Our observations suggest that plasmin exerts a dual role in PMA-induced scattering of HT29-M6 cells, one directed extracellularly to promote proteolysis of the ECM and one directed to generate intracellular signaling.     

Effects and mechanisms of basic fibroblast growth factor on the proliferation and regenerative profiles of cryopreserved dental pulp stem cells

ObjectivesVarious factors could interfere the biological performance of DPSCs during post‐thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3‐month cryopreservation as well as the underlying mechanisms.Materials and methodsDPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146⁺ DPSCs (P3) were identified by CCK‐8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK‐8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre‐treated DPSCs was cultivated in bFGF‐free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation.ResultsIt is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up‐regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre‐stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised.ConclusionsThis study illustrated a safe and feasible cell culture technique to rapidly amplify post‐thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.     

Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.     

Antagonistic functional duality of cancer genes

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer “gene-chameleons”, which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP⁺-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically “the drivers” of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and “paradoxical” effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.     

Neurotensin downregulates the pro-inflammatory properties of skin dendritic cells and increases epidermal growth factor expression

In the last decades some reports reveal the neuropeptide neurotensin (NT) as an immune mediator in the Central Nervous System and in the gastrointestinal tract, however its effects on skin immunity were not identified. The present study investigates the effect of NT on signal transduction and on pro/anti-inflammatory function of skin dendritic cells. Furthermore, we investigated how neurotensin can modulate the inflammatory responses triggered by LPS in skin dendritic cells. We observed that fetal-skin dendritic cells (FSDCs) constitutively express NTR1 and NTR3 (neurotensin receptors) and that LPS treatment induces neurotensin expression. In addition, NT downregulated the activation of the inflammatory signaling pathways NF-κB and JNK, as well as, the expression of the cytokines IL-6, TNF-α, IL-10 and the vascular endothelial growth factor (VEGF), while the survival pathway ERK and epidermal growth factor (EGF) were upregulated. Simultaneous dendritic cells exposure to LPS and NT induced a similar cytokine profile to that one induced by NT alone. However, cells pre-treated with NT and then incubated with LPS, completely changed their cytokine profile, upregulating the cytokines tested, without changes on growth factor expression. Overall, our results could open new perspectives in the design of new therapies for skin diseases, like diabetic wound healing, where neuropeptide exposure seems to be beneficial.     

Polymorphism analysis in genes of the somatotropic axis in Nellore cattle selected for growth

The somatotropic axis consists of genes that are involved in muscular development. These genes are potential regions of study to identify possible QTL for economically important traits in beef cattle. The aim of this study was to verify the existence of GH1, POU1F1, and GHR polymorphisms in Nellore cattle to verify the influence of selection in these mutations and to analyse the association between molecular markers and body weight at different ages, yearling hip height, carcass fat thickness and loin eye area. Six hundred forty-five animals from the Centro APTA Bovinos de Corte, were genotyped by PCR–RFLP techniques. The association analyses were performed with general mixed models taking into consideration the effect of one marker, and other model taking into consideration interactions between two molecular markers. Only the molecular markers rs81109601 on GH1 and rs109136815 on GHR were polymorphic; however, they were not found to be under selection. The association of the GHR rs109136815 marker and loin eye area was observed (p < 0.05), as well as the effect of interaction between the markers and the female body weight at 550 days of age (p < 0.04). The interaction effect should be considered in situations where the interactivity between two genes is known.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.