E11 Cells Research Articles

Transcriptomic profiling of the immune response to Siniperca chuatsi rhabdovirus (SCRV) in E11 cells

Transcriptomic profiling of the immune response to Siniperca chuatsi rhabdovirus (SCRV) in E11 cells

Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes.In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

Protein tyrosine phosphatase 1B is a regulator of alpha-actinin4 in the glomerular podocyte

Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.

Inhibition of nervous necrosis virus replication by Shewanella putrefaciens Pdp11 extract

One of the most relevant infectious diseases affecting farmed fish is the viral encephalopathy and retinopathy, caused by the nervous necrosis virus (NNV). The two commercial vaccines available against this virus have been designed to protect European seabass against the infection with a particular NNV species, RGNNV. Therefore, the development of strategies to protect different fish species against different viruses is a key issue for the aquaculture industry. In this regard, there are numerous studies focused on the use of probiotics, although their antiviral activity has been poorly investigated. Shewanella putrefaciens Pdp11, SpPdp11, is a probiotic with proven positive effects on gilthead seabream and Senegalese sole, protecting those species against several bacterial pathogens; however, its antiviral activity remains to be investigated. The current study is a step forward in the use of probiotics against viral infections, evaluating the anti-RGNNV activity of sonicated-SpPdp11 extracts both, in vitro and in vivo. According to our results, SpPdp11 extracts compromised RGNNV multiplication in E11 cells, affecting viral assembly and/or exit rather than genome replication. Furthermore, this antiviral activity may be produced by the capacity of the SpPdp11-extract to induce an immune response in treated cells. This anti-RGNNV activity has been confirmed in vivo, since 82% of fish fed with the SpPdp11-supplemented diet survived an experimental-RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%. These results suggest that SpPdp11-supplemented feeding can be a promising prophylactic tool against RGNNV and encourage further research on other fish species and viral pathogens.

Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2.

Podocyte damage and renal inflammation are the main features and pathogenesis of diabetic nephropathy (DN). Inhibition of lysophosphatidic acid (LPA) receptor 1 (LPAR1) suppresses glomerular inflammation and improves DN. Herein, we investigated LPA-induced podocyte damage and its underlying mechanisms in DN. We investigated the effects of AM095, a specific LPAR1 inhibitor, on podocytes from streptozotocin (STZ)-induced diabetic mice. E11 cells were treated with LPA in the presence or absence of AM095, and the expression of NLRP3 inflammasome factors and pyroptosis were measured. A chromatin immunoprecipitation assay and Western blotting were performed to elucidate underlying molecular mechanisms. Gene knockdown by transfecting small interfering RNA was used to determine the role of the transcription factor Egr1 (early growth response protein 1) and histone methyltransferase EzH2 (Enhancer of Zeste Homolog 2) in LPA-induced podocyte injury. AM095 administration inhibited podocyte loss, NLRP3 inflammasome factor expression, and cell death in STZ-induced diabetic mice. In E11 cells, LPA increased NLRP3 inflammasome activation and pyroptosis via LPAR1. Egr1 mediated NLRP3 inflammasome activation and pyroptosis in LPA-treated E11 cells. LPA decreased H3K27me3 enrichment at the Egr1 promoter in E11 cells by downregulating EzH2 expression. EzH2 knockdown further increased LPA-induced Egr1 expression. In podocytes from STZ-induced diabetic mice, AM095 suppressed Egr1 expression increase and EzH2/H3K27me3 expression reduction. Collectively, these results demonstrate that LPA induces NLRP3 inflammasome activation by downregulating EzH2/H3K27me3 and upregulating Egr1 expression, resulting in podocyte damage and pyroptosis, which may be a potential mechanism of DN progression.

Biocontrol Capabilities of Bacillus subtilis E11 against Aspergillus flavus In Vitro and for Dried Red Chili (Capsicum annuum L.).

As a condiment with extensive nutritional value, chili is easy to be contaminated by Aspergillus flavus (A. flavus) during field, transportation, and storage. This study aimed to solve the contamination of dried red chili caused by A. flavus by inhibiting the growth of A. flavus and detoxifying aflatoxin B1 (AFB1). In this study, Bacillus subtilis E11 (B. subtilis) screened from 63 candidate antagonistic bacteria exhibited the strongest antifungal ability, which could not only inhibit 64.27% of A. flavus but could also remove 81.34% of AFB1 at 24 h. Notably, scanning electron microscopy (SEM) showed that B. subtilis E11 cells could resist a higher concentration of AFB1, and the fermentation supernatant of B. subtilis E11 could deform the mycelia of A. flavus. After 10 days of coculture with B. subtilis E11 on dried red chili inoculated with A. flavus, the mycelia of A. flavus were almost completely inhibited, and the yield of AFB1 was significantly reduced. Our study first concentrated on the use of B. subtilis as a biocontrol agent for dried red chili, which could not only enrich the resources of microbial strains for controlling A. flavus but also could provide theoretical guidance to prolong the shelf life of dried red chili.

Susceptibility of Vero E6 cells to tilapia lake virus (TiLV) and anti-TiLV activity of type I interferon

Tilapia lake virus (TiLV) is an emerging orthomyxovirus-like pathogen first identified in Israel in 2014. TiLV can cause up to 90% mortality in tilapia and poses a severe threat to the global tilapia industry. There is no cure for TiLV infection and no commercially available vaccine. It was shown that TiLV can infect cell lines from many different fish species, but TiLV infection and replication in mammalian cells have not been documented. Here, we report that TiLV can infect and replicate in Vero E6 cells, thus expanding the systems available for studying TiLV to a well-characterized cell line. In addition, we investigated the effect of mammalian type I interferon on TiLV infection in this cell culture system. Our results show the robust antiviral activity of mammalian type I interferon against TiLV. Importantly, a recombinant fish type I interferon that we produced in mammalian cells also exhibited potent antiviral activity against TiLV in E11 cells. In conclusion, our studies demonstrate the protective effect of type I interferon against TiLV infection, which may point to a mechanism of TiLV pathogenesis and aid in developing vaccines and antiviral therapies against this important fish pathogen.

Cytokine-like activity of European sea bass ISG15 protein on RGNNV-infected E−11 cells

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.

Characterization of Hagfish (Eptatretus burgeri) Variable Lymphocyte Receptor-Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus.

The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (Petromyzon marinus) and hagfish (Eptatretus burgeri). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.

Pterostilbene surpassed resveratrol for anti-inflammatory application: Potency consideration and pharmacokinetics perspective

This study aimed to evaluate the suitability of pterostilbene for anti-inflammatory application. The in vitro anti-inflammatory activities of pterostilbene were assessed using resveratrol, piceatannol and resveratrol trimethyl ether as comparators while its pharmacokinetics was examined in rats. All tested compounds displayed concentration-dependent anti-proliferative effect in E11 human rheumatoid arthritic synovial fibroblasts. They also suppressed LPS-induced NF-κB p65 nuclear translocation, down-regulated the secretions of IL-6, IL-18, VEGF, nitric oxide, MMP-2 and MMP-9 in E11 cells and attenuated E11-driven migration of THP-1 and U937 monocytes. Similarly, they inhibited LPS-induced pro-inflammatory cytokines in THP-1 cells. Interestingly, pterostilbene usually displayed in vitro anti-inflammatory potencies stronger than resveratrol. In vivo studies revealed that pterostilbene was extensively distributed to major drug target organs like liver, kidney, heart, lung as well as brain and repeated oral dosing did not significantly alter its pharmacokinetics. In conclusion, pterostilbene appears to be a promising candidate for further development.

The involvement of heparin in retinal infection by Toxoplasma gondii in a chick model revealed an ontogenetic-dependent pattern

This work aimed to test the influence of heparin on the susceptibility of retinal cells to Toxoplasma gondii infection. Primary cultures of retinas from chick embryos of 8 (E8) or 11 (E11) days and fibroblasts (control) were used. To determine the influence of heparin in T. gondii infection, tachyzoites of the RH strain were treated with heparin before addition in the culture. A monoclonal anti-heparin antibody was used to analyze the heparin distribution on fibroblast and retinal cell surfaces. Our results showed that retinal cells (E8 and E11) had a higher infection rate than fibroblasts (91% and 24% versus 13%, respectively). Pre-treatment of T. gondii with heparin decreased infection of E8 retinal cells when compared with non-treated parasites (45% versus 91%, respectively), but not of E11 cells (35% versus 48%). In accordance, retinal cells presented an intense heparin staining by immunofluorescence assay. In conclusion, retinal cells from chick embryos were more susceptible to infection by T. gondii compared to fibroblasts and, pre-treatment of tachyzoites with heparin decreased the number of infected cells and parasite burden particularly for E8 retinal cells.

The Histone Deacetylase Inhibitors MS-275 and SAHA Suppress the p38 Mitogen-Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells

MS-275 (entinostat) and SAHA (vorinostat), two histone deacetylase (HDAC) inhibitors currently in oncological trials, have displayed potent anti-rheumatic activities in rodent models of rheumatoid arthritis (RA). To further elucidate their anti-inflammatory mechanisms, the impact of MS-275 and SAHA on the p38 mitogen-activated protein kinase (MAPK) signaling pathway and chemotaxis was assessed in human rheumatoid arthritic synovial fibroblastic E11 cells. MS-275 and SAHA significantly suppressed the expression of p38α MAPK, but induced the expression of MAPK phosphatase-1 (MKP-1), an endogenous suppressor of p38α in E11 cells. At the same time, the association between p38α and MKP-1 was up-regulated and consequently, the activation (phosphorylation) of p38α was inhibited. Moreover, MS-275 and SAHA suppressed granulocyte chemotactic protein-2 (GCP-2), monocyte chemotactic protein-2 (MCP-2) and macrophage migration inhibitory factor (MIF) in E11 cells in a concentration-dependent manner. Subsequently, E11-driven migration of THP-1 and U937 monocytes was inhibited. In summary, suppression of the p38 MAPK signaling pathway and chemotaxis appear to be important anti-rheumatic mechanisms of action of these HDAC inhibitors.

Histone deacetylase inhibitors MS-275 and SAHA induced growth arrest and suppressed lipopolysaccharide-stimulated NF- B p65 nuclear accumulation in human rheumatoid arthritis synovial fibroblastic E11 cells

MS-275 and suberoylanilide hydroxamic acid (SAHA) are histone deacetylase (HDAC) inhibitors currently tested in oncology trials. They have also been found to display potent anti-rheumatic activities in rodent models for RA. However, the anti-rheumatic mechanisms of action remain unknown. The study was carried out with the intent of determining the anti-inflammatory and anti-rheumatic mechanisms of the HDAC inhibitors. In this study, the anti-rheumatic mechanisms of MS-275 and SAHA were investigated in several cell culture models. MS-275 and SAHA inhibited human RA synovial fibroblastic E11 cell proliferation in a non-cytotoxic manner. The anti-proliferative activities were associated with G(0)/G(1) phase arrest and induction of cyclin-dependent kinase inhibitor p21. In addition, MS-275 and SAHA suppressed lipopolysaccharide (LPS)-induced NF-kappaB p65 nuclear accumulation, IL-6, IL-18 and nitric oxide (NO) secretion as well as down-regulated pro-angiogenic VEGF and MMP-2 and MMP-9 production in E11 cells at sub-micromolar levels. At similar concentrations, MS-275 and SAHA suppressed LPS-induced NF-kappaB p65 nuclear accumulation and IL-1beta, IL-6, IL-18 and TNF-alpha secretion in THP-1 monocytic cells. Moreover, NO secretion in RAW264.7 macrophage cells was also inhibited. In summary, MS-275 and SAHA exhibited their anti-rheumatic activities by growth arrest in RA synovial fibroblasts, inhibition of pro-inflammatory cytokines and NO, as well as down-regulation in angiogenesis and MMPs. Their anti-rheumatic activities may be mediated through induction of p21 and suppression of NF-kappaB nuclear accumulation.

Involvement of CREM in CYP1A1 induction through ligand-independent activation pathway of aryl hydrocarbon receptor in HepG2 cells

Using a radiation-hybrid cell, E11, produced by the fusion of Hepa-1c1c7 and X-ray irradiated HepG2 cells, we located the omeprazole (OP) responsive region on chromosome 10p. The cDNA of 12 genes expressed in E11 cells were cloned from HepG2 mRNA by RT-PCR. The cDNA was transfected into Hepa-1c1c7 cells to check the increased expression of Cyp1a1 by reporter gene (luciferase) assay. Finally, one gene (τ CREM: cAMP responsive element modulator, τ-isoform) was identified as a candidate gene for the gene responsive to OP. The regulatory sequence of Cyp1a1 in response to τCREM transfection was identified in one region (−60 to −52 bp relative to the transcription start site), which was the basic transcription element (BTE). Electromobility shift assay with the BTE sequence showed an increase in the band intensity when the cells were treated with OP. Decreased level of endogenous CREM by siRNA transfection inhibited the induction of CYP1A1-mRNA in HepG2 cells by OP.

Glucocorticoid induction of lactotrophs and prolactin gene expression in chicken embryonic pituitary cells: a delayed response relative to stimulated growth hormone production.

We reported that corticosterone (CORT) induces GH cell differentiation in chicken embryonic pituitary cells in culture and in living embryos. The present study tested whether CORT could also induce prolactin (PRL) cell differentiation in vitro. CORT increased the percentage of GH cells in cultures of embryonic d (e) 13 pituitary cells after 1 d of treatment and PRL cells after 3 d of incubation. Dual immunofluorescence showed that the PRL cells induced by CORT did not contain GH, indicating they were separate cell populations. Similar PRL cell responses were also observed in cultures of e11, e15, and e17 pituitary cells. ZK98299, a glucocorticoid receptor (GR) antagonist, suppressed CORT effects, verifying involvement of GR. Northern blot analysis indicated that CORT increased GH mRNA levels after 1 d of treatment. In contrast, increases in PRL mRNA levels were delayed and observed after 3 d of treatment. Induction of a luciferase reporter driven by the PRL promoter was also delayed until 3 d of CORT treatment. Dual-labeling immunofluorescence indicated that the majority of PRL cells induced by CORT were not labeled with bromodeoxyuridine, suggesting that lactotrophs induced by CORT do not result from cell proliferation. Proportions of pituitary-specific transcription factor (Pit-1)-containing cells and the total amount of Pit-1 protein spontaneously increased with increasing culture time. However, no effect of CORT on Pit-1 levels or the number of Pit-1-containing cells were observed. We conclude that CORT can induce lactotroph differentiation in culture and that longer CORT exposure is needed for lactotroph induction compared with somatotroph induction. The effects of CORT on PRL cell induction are GR dependent and involve an increase in PRL gene expression. PRL cell induction by CORT is not associated with an increase in Pit-1-containing cells.

Adenosine downregulates cytokine‐induced expression of intercellular adhesion molecule‐1 on rheumatoid synovial fibroblasts independently of adenosine receptor signaling

AbstractAdhesion of fibroblast‐like synoviocytes (FLSs) to T cells through the interaction of lymphocyte function‐associated antigen‐1 (LFA‐1) and intercellular adhesion molecule‐1 (ICAM‐1) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). We therefore used flow cytometry and quantitative polymerase chain reaction (PCR) to examine the effect of adenosine and its derivatives on expression of ICAM‐1 induced by tumor necrosis factor‐alpha and interferon‐gamma in primary rheumatoid FLSs (RA‐FLSs) and E11 cells, an RA‐FLS line. Exposing cells to adenosine (5–500 µM) for 24 h in the presence of coformycin, an adenosine deaminase inhibitor, concentration‐dependently inhibited cytokine‐induced transcription of ICAM‐1 mRNA, as well as subsequent surface expression of the protein. Although transcription of all four adenosine receptor isoforms has been detected in FLSs, neither the A1 receptor agonist R‐PIA, the A2A receptor agonist CGS21680 nor the A3 agonist Cl‐IB‐MECA had any effect on cytokine‐induced ICAM‐1 expression. Conversely, A1/A2 receptor antagonist xanthine amine congener and A2A antagonist ZM240385 both failed to suppress the effect of adenosine. Adenosine appears to inhibit cytokine‐induced ICAM‐1 expression in FLSs independently of adenosine receptor‐mediated signaling. By contrast, the effect of adenosine was neutralized by nitrobenzylmercaptopurin, a nucleoside transporter inhibitor, or by ABT702, an adenosine kinase inhibitor. This suggests that adenosine taken up via the nucleoside transporter is phosphorylated by adenosine kinase, and the resultant phospho‐adenosine interferes with the ICAM‐1 transcription and cell surface expression. Downregulation of T cell–FLS interaction by adenosine may thus represent a novel approach to the treatment of RA. Drug Dev. Res. 58:368–376, 2003. © 2003 Wiley‐Liss, Inc.

Bile acids stimulate invasion and haptotaxis in human colorectal cancer cells through activation of multiple oncogenic signaling pathways.

Bile acids are implicated in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. We examined whether bile acids stimulate cellular invasion of human colorectal and dog kidney epithelial cells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidney MDCKT23 cells were seeded on top of collagen type I gels and invasive cells were counted after 24 h incubation. Activation of the Rac1 and RhoA small GTPases was investigated by pull-down assays. Haptotaxis was analysed with modified Boyden chambers. Lithocholic acid, chenodeoxycholic acid, cholic acid and deoxycholic acid stimulated cellular invasion of SRC- and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originating from a sporadic tumor, but were ineffective in premalignant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation of haptotaxis and was dependent on the RhoA/Rho-kinase pathway and signaling cascades using protein kinase C, mitogen-activated protein kinase, and cyclooxygenase-2. Accordingly, BA-induced invasion was associated with activation of the Rac1 and RhoA GTPases and expression of the farnesoid X receptor. We conclude that bile acids stimulate invasion and haptotaxis in colorectal cancer cells via several cancer invasion signaling pathways.

Functional Nicotinic Acetylcholine Receptor Expression in Stem and Progenitor Cells of the Early Embryonic Mouse Cerebral Cortex

The adult cerebral cortex contains nicotinic acetylcholine (ACh) receptors vital to cortical function. However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role in cortical development. We now report evidence of functional nicotinic acetylcholine receptor channels in fetal mouse cerebral cortex as early as embryonic day 10 (E10), when the cortex consists of dividing stem and progenitor cells. Patch–clamp electrophysiological measurements indicate that nicotine and ACh evoke sizable inward currents characteristic of nicotinic receptors, that are strongly rectifying with a reversal potential near 0 mV. Three different nicotinic agonists, ACh, nicotine, and dimethylphenylpiperazinium, evoked cytosolic Ca2+ signals. Agonist-evoked Ca2+ signals and electrophysiological responses were found in greater than 70% of all E10–E11 cells tested and were blocked by nicotinic receptor antagonists. The Ca2+ response to nicotinic agonists was markedly prolonged in cells from early embryonic stages relative to later stages of development. α3, α4, and α7 receptor subunit proteins were detected immunocytochemically in cortical cells from E10 to birth. The incidence of each subunit declined with embryonic age, suggesting a role in early development. We discuss the possible function of nicotinic receptors in early cortical development and their role as a target for nicotine in the developmental pathologies associated with the fetal tobacco syndrome.

Development of the Avian Iris and Ciliary Body: Mechanisms of Cellular Differentiation during the Smooth-to-Striated Muscle Transition

The avian iris and ciliary body undergoes a transition from smooth-to-striated muscle during embryonic development. Using antibodies specific for smooth muscle-specific α-actin and myosin heavy chain, we confirm that a smooth-to-striated muscle transition occurs between E8 and E17 in both iris and ciliary body of the chick. To study the mechanisms regulating the transition in muscle type, we analyzed the fate of quail clones derived from E7 iris cells. When cells were cloned alone, 45/71 colonies differentiated into smooth muscle and 10/71 became striated muscle. None of the colonies were mixed with respect to muscle phenotype, indicating a lack of pluripotent stem cells. Furthermore, clones giving rise to nonstriated muscle could not be forced to incorporate into myotubes when cocultured with chick myocytes. Clones grown in coculture with chick embryo fibroblasts or E11 iris cells had very high cloning efficiencies (>98%). Significantly more clones differentiated into striated muscle when cocultured with E11 cells (60/156) than when cocultured with fibroblasts (29/108). This was due to an increased recruitment of undifferentiated cells into striated muscle, rather than a change in the percentage of cells differentiating into smooth muscle.In vivoandin vitro,various smooth and striated muscle-specific markers including contractile proteins, acetylcholine receptor subtypes, and transcription factors were colocalized in cells. Although our data argue against a multipotent stem cell for smooth and striated muscle cells, they cannot exclude a role for transdifferentiation. Cumulatively these results suggest that both smooth muscle and migratory myoblasts contribute to the development of myotubes in the avian iris and that this process is regulated in a non-cell-autonomous fashion by locally generated signals.