Neuregulin-1 regulates cell adhesion via an ErbB2/phosphoinositide-3 kinase/Akt-dependent pathway: potential implications for schizophrenia and cancer

Background

Neuregulin-1 (NRG1) is a putative schizophrenia susceptibility gene involved extensively in central nervous system development as well as cancer invasion and metastasis. Using a B lymphoblast cell model, we previously demonstrated impairment in NRG1α-mediated migration in cells derived from patients with schizophrenia as well as effects of risk alleles in NRG1 and catechol-O-methyltransferase (COMT), a second gene implicated both in schizophrenia susceptibility and in cancer.

Methodology/Principal Findings

Here, we examine cell adhesion, an essential component process of cell motility, using an integrin-mediated cell adhesion assay based on an interaction between ICAM-1 and the CD11a/CD18 integrin heterodimer expressed on lymphoblasts. In our assay, NRG1α induces lymphoblasts to assume varying levels of adhesion characterized by time-dependent fluctuations in the firmness of attachment. The maximum range of variation in adhesion over sixty minutes correlates strongly with NRG1α-induced migration (r² = 0.61). NRG1α-induced adhesion variation is blocked by erbB2, PI3K, and Akt inhibitors, but not by PLC, ROCK, MLCK, or MEK inhibitors, implicating the erbB2/PI3K/Akt1 signaling pathway in NRG1-stimulated, integrin-mediated cell adhesion. In cell lines from 20 patients with schizophrenia and 20 normal controls, cells from patients show a significant deficiency in the range of NRG1α-induced adhesion (p = 0.0002). In contrast, the response of patient-derived cells to phorbol myristate acetate is unimpaired. The COMT Val108/158Met genotype demonstrates a strong trend towards predicting the range of the NRG1α-induced adhesion response with risk homozygotes having decreased variation in cell adhesion even in normal subjects (p = 0.063).

Conclusion/Significance

Our findings suggest that a mechanism of the NRG1 genetic association with schizophrenia may involve the molecular biology of cell adhesion.     

Subchronic peripheral neuregulin-1 increases ventral hippocampal neurogenesis and induces antidepressant-like effects

Background

Adult hippocampal neurogenesis has been implicated in the mechanism of antidepressant action, and neurotrophic factors can mediate the neurogenic changes underlying these effects. The neurotrophic factor neuregulin-1 (NRG1) is involved in many aspects of brain development, from cell fate determination to neuronal maturation. However, nothing is known about the influence of NRG1 on neurodevelopmental processes occurring in the mature hippocampus.

Methods

Adult male mice were given subcutaneous NRG1 or saline to assess dentate gyrus proliferation and neurogenesis, as well as cell fate determination. Mice also underwent behavioral testing. Expression of ErbB3 and ErbB4 NRG1 receptors in newborn dentate gyrus cells was assessed at various time points between birth and maturity. The phenotype of ErbB-expressing progenitor cells was also characterized with cell type-specific markers.

Results

The current study shows that subchronic peripheral NRG1β administration selectively increased cell proliferation (by 71%) and neurogenesis (by 50%) in the caudal dentate gyrus within the ventral hippocampus. This pro-proliferative effect did not alter neuronal fate, and may have been mediated by ErbB3 receptors, which were expressed by newborn dentate gyrus cells from cell division to maturity and colocalized with SOX2 in the subgranular zone. Furthermore, four weeks after cessation of subchronic treatment, animals displayed robust antidepressant-like behavior in the absence of changes in locomotor activity, whereas acute treatment did not produce antidepressant effects.

Conclusions

These results show that neuregulin-1β has pro-proliferative, neurogenic and antidepressant properties, further highlight the importance of peripheral neurotrophic factors in neurogenesis and mood, and support the role of hippocampal neurogenesis in mediating antidepressant effects.     

The induction of microRNA targeting IRS-1 is involved in the development of insulin resistance under conditions of mitochondrial dysfunction in hepatocytes

Background

Mitochondrial dysfunction induces insulin resistance in myocytes via a reduction of insulin receptor substrate-1 (IRS-1) expression. However, the effect of mitochondrial dysfunction on insulin sensitivity is not understood well in hepatocytes. Although research has implicated the translational repression of target genes by endogenous non-coding microRNAs (miRNA) in the pathogenesis of various diseases, the identity and role of the miRNAs that are involved in the development of insulin resistance also remain largely unknown.

Methodology

To determine whether mitochondrial dysfunction induced by genetic or metabolic inhibition causes insulin resistance in hepatocytes, we analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in SK-Hep1 hepatocytes. We used qRT-PCR to measure cellular levels of selected miRNAs that are thought to target IRS-1 3′ untranslated regions (3′UTR). Using overexpression of miR-126, we determined whether IRS-1-targeting miRNA causes insulin resistance in hepatocytes.

Principal Findings

Mitochondrial dysfunction resulting from genetic (mitochondrial DNA depletion) or metabolic inhibition (Rotenone or Antimycin A) induced insulin resistance in hepatocytes via a reduction in the expression of IRS-1 protein. In addition, we observed a significant up-regulation of several miRNAs presumed to target IRS-1 3′UTR in hepatocytes with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-126 directly targeted to IRS-1 3′UTR. Furthermore, the overexpression of miR-126 in hepatocytes caused a substantial reduction in IRS-1 protein expression, and a consequent impairment in insulin signaling.

Conclusions/Significance

We demonstrated that miR-126 was actively involved in the development of insulin resistance induced by mitochondrial dysfunction. These data provide novel insights into the molecular basis of insulin resistance, and implicate miRNA in the development of metabolic disease.     

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

Introduction

The present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.

Methods

IL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).

Results

Single injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.

Conclusions

These results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.     

Gemcitabine-mediated radiosensitization of human soft tissue sarcoma

Background/Purpose

Local and systemic control of soft tissue sarcoma (STS) remains a clinical challenge, particularly for retroperitoneal, deep truncal, or advanced extremity disease. 2′,2′-Difluoro-2′-deoxycytidine (gemcitabine) is a potent radiosensitizer in many tumor types, but it has not been studied in human STS. The purpose of this study was to determine the radiosensitizing potential of gemcitabine in preclinical models of human STS.

Materials and Methods

The in vitro radiosensitizing activity of gemcitabine was assessed with clonogenic survival assay on three human STS cell lines: SK-LMS-1 (leiomyosarcoma), SW-872 (liposarcoma), and HT-1080 (fibrosarcoma). Cell cycle distribution was determined using dual-channel flow cytometry. The in vivo radiosensitizing activity of gemcitabine was assessed with subcutaneous SK-LMS-1 nude mice xenografts. Tumor-bearing mice were treated with concurrent weekly gemcitabine and fractionated daily radiotherapy (RT) (2 Gy daily) for 3 weeks (a total dose of 30 Gy).

Results

The 50% inhibitory concentration (IC₅₀) of gemcitabine for the human STS cell lines ranged from 10 to 1000 nM. Significant in vitro radiosensitization was demonstrated in all three human STS cell lines using gemcitabine concentrations at and below the IC₅₀. Maximal radiosensitization was associated with accumulation of cells in early S-phase. SK-LMS-1 xenografts displayed significant tumor growth delay with combined gemcitabine and RT compared to either treatment alone. Treatment related toxicity was greatest in the gemcitabine plus RT arm, but remained at an acceptable level.

Conclusions

Gemcitabine is a potent radiosensitizer in preclinical models of human STS. Clinical trials combining gemcitabine and RT in human STS are warranted.     

Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion

Background

Wnt signaling is important in development and can also contribute to the initiation and progression of cancer. The Secreted Frizzled Related Proteins (SFRPs) constitute a family of Wnt modulators, crucial for controlling Wnt signaling. Here we investigate the expression and role of SFRP3 in melanoma.

Methodology/Principal Findings

We show that SFRP3 mRNA is down-regulated in malignant melanoma tumors as compared to normal/benign tissue. Furthermore, we found that SFRP3 expression was lost in the malignant melanoma cell lines, A2058, HTB63 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. Methylated CpG rich areas were detected in the SFRP3 gene in melanoma cell lines and their SFRP3 expression could be restored using the demethylating agent, 5′aza-deoxycytidine. Addition of recombinant SFRP3 to melanoma cells had no effect on viable cell numbers, but decreased cell migration and invasion. Wnt5a signaling has been shown to increase the migration and invasion of malignant melanoma cells, and high expression of Wnt5a in melanoma tumors has been connected to a poor prognosis. We found that recombinant SFRP3 could inhibit Wnt5a signaling, and that it inhibited melanoma cell migration and invasion in a Wnt5a-dependent manner.

Conclusion/Significance

We conclude that SFRP3 functions as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling.     

Tamoxifen-independent recombination in the RIP-CreER mouse

Background

The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Principal Findings

Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains.

Significance

Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.     

Geographically widespread swordfish barcode stock identification: a case study of its application

Background

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species.

Methodology

We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker.

Case Report

This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products.

Conclusions

The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.     

AGO1 and AGO2 act redundantly in miR408-mediated Plantacyanin regulation

Background

In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 5′ end, respectively. Because most plant miRNAs have a 5′U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 5′A. AGO2 also participates in antiviral defense in association with viral siRNAs.

Principal Findings

This study reveals that miR408, which has a 5′A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 5′ nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 5′A-MIR408 and a mutant 5′U-MIR408 gene complement a mir408 mutant.

Conclusions/Significance

These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 5′ end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 5′ nucleotide.     

Hypoxia changes the expression of the epidermal growth factor (EGF) system in human hearts and cultured cardiomyocytes

Background

The epidermal growth factor (EGF) receptors HER2 and HER4 and the ligands HB-EGF and NRG1 are crucial for heart development. The purpose of our study was to investigate the role of the complete EGF system in relation to hypoxia of the heart.

Methodology/Principal Findings

We examined the mRNA expression by real time PCR of the 4 receptors and 12 ligands from the EGF-system in paired normoxic and hypoxic biopsies isolated from human hearts during coronary artery bypass operation. Compared to normoxic biopsies, hypoxic samples showed down-regulation of HER2 (P = 0.0005) and NRG1 (both α (P = 0.02) and β (P = 0.03) isoforms). In contrast, HB-EGF (P = 0.0008), NRG2β (P = 0.01) and EGFR (P = 0.02) were up-regulated. As HER2 is essential for heart development and we find its expression reduced under hypoxia we investigated the effect of HER2 inhibition in hypoxic HL-1 cardiomyocytes by treatment with trastuzumab (20 nM). This resulted in inhibition of cardiomyocyte proliferation, but interestingly only in hypoxic cells. Co-treatment of HL-1 cells with HB-EGF (10 nM) but not with NRG-1 (5 ng/ml) rescued the cardiomyocytes from HER2 inhibition. HL-1 cardiomyocytes exposed to hypoxia revealed nuclear translocation of activated MAPK and the activity of this downstream signaling molecule was decreased by HER2 inhibition (20 nM trastuzumab), and re-established by HB-EGF (10 nM).

Conclusions/Significance

Hypoxia in the human heart alters the expression of the EGF system. Mimicking the HER2 down-regulation seen in the human heart in cultured cardiomyocytes inhibited their proliferation under hypoxic conditions. Interestingly, HB-EGF is induced in the hypoxic human hearts, and rescues hypoxic cardiomyocytes from the effect of HER2 inhibition in the in vitro model. The results have implications for future treatment strategies of patients with ischemic heart disease.     

Tumor suppression by RNA from C/EBPβ 3'UTR through the inhibition of protein kinase Cε activity

Background

Since the end of last century, RNAs from the 3′untranslated region (3′UTR) of several eukaryotic mRNAs have been found to exert tumor suppression activity when introduced into malignant cells independent of their whole mRNAs. In this study, we sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 3′UTR of C/EBPβ mRΝΑ (C/EBPβ 3′UTR RNA) in human hepatocarcinoma cells SMMC-7721.

Methodology/Principal Findings

By using Western blotting, immunocytochemistry, molecular beacon, confocal microscopy, protein kinase inhibitors and in vitro kinase assays, we found that, in the C/EBPβ 3′UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPβ 3′UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPβ 3′UTR RNA also reduced phosphorylation and expression of keratin 18; and that the enzyme responsible for phosphorylating keratin 18 is protein kinase Cε. We then found that the C/EBPβ 3′UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPβ 3′UTR RNA specifically bound with the protein kinase Cε-keratin 18 conjugate.

Conclusion/Significance

Together, these facts suggest that the tumor suppression in SMMC-7721 by C/EBPβ 3′UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPβ 3′UTR RNA and protein kinase Cε. These facts indicate that the 3′UTR of some eukaryotic mRNAs may function as regulators for genes other than their own.     

SLC11A1 (NRAMP1) polymorphisms and tuberculosis susceptibility: updated systematic review and meta-analysis

Background

Natural resistance associated macrophage protein 1 (NRAMP1), encoded by the SLC11A1 gene, has been described to regulate macrophage activation and be associated with infectious and autoimmune diseases. The relation between SLC11A1 polymorphisms and tuberculosis susceptibility has been studied in different populations.

Methods

We systematically reviewed published studies on SLC11A1 polymorphisms and tuberculosis susceptibility until September 15, 2010 and quantitatively summarized associations of the most widely studied polymorphisms using meta-analysis.

Results

In total, 36 eligible articles were included in this review. In Meta-analysis, significant associations were observed between tuberculosis risk and widely studied SLC11A1 polymorphisms with summarized odds ratio of 1.35 (95%CI, 1.17–1.54), 1.25 (95% CI, 1.04–1.50), 1.23 (95% CI, 1.04–1.44), 1.31 (95%CI, 1.08–1.59) for 3′ UTR, D543N, INT4, and 5′ (GT)n, respectively. Heterogeneity between studies was not pronounced, and the associations did not remarkably vary in the stratified analysis with respect to study population and study base.

Conclusions

The association between SLC11A1 polymorphisms and tuberculosis susceptibility observed in our analyses supports the hypothesis that NRAMP1 might play an important role in the host defense to the development of tuberculosis.