Changes In mRNA Expression Profiles Research Articles

Methamphetamine-induced impairment of memory and fleeting neuroinflammation: Profiling mRNA changes in mouse hippocampus following short-term and long-term exposure

Methamphetamine (METH) has been implicated in inducing memory impairment, but the precise mechanisms underlying this effect remain unclear. Current research often limits itself to singular models or focuses on individual gene or protein functions, which hampers a comprehensive understanding of the underlying mechanisms. In this study, we established three METH mouse exposure models, extracted hippocampal nuclei, and utilized RNA sequencing to analyze changes in mRNA expression profiles. Our results indicate that METH significantly impairs the learning and memory capabilities of mice. Additionally, we observed that METH-induced inflammatory responses occur in the early phase and do not further exacerbate with repeated injections. However, RNA sequencing revealed the persistent enrichment of inflammatory pathway molecules, which correlated with worsened behaviors. This suggests that although METH-induced neuroinflammation plays a critical role in learning and memory impairment, the continued enrichment of inflammatory pathway molecules is associated with behavioral outcomes. These findings provide crucial evidence for the potential application of immune intervention in METH-related disorders.

NEK2 affects the ferroptosis sensitivity of gastric cancer cells by regulating the expression of HMOX1 through Keap1/Nrf2.

NEK2 is a serine/threonine protein kinase that is involved in regulating the progression of various tumors. Our previous studies have found that NEK2 is highly expressed in gastric cancer and suggests that patients have a worse prognosis. However, its role and mechanism in gastric cancer are only poorly studied. In this study, we established a model of ferroptosis induced by RSL3 or Erastin in AGS cells in vitro, and konckdown NEK2, HOMX1, Nrf2 by siRNA. The assay kit was used to analyzed cell viability, MDA levels, GSH and GSSG content, and FeRhoNox™-1 fluorescent probe, BODIPY™ 581/591 C11 lipid oxidation probe, CM-H2DCFDA fluorescent probe were used to detected intracellular Fe2+, lipid peroxidation, and ROS levels, respectively. Calcein-AM/PI staining was used to detect the ratio of live and dead cells, qRT-PCR and Western blot were used to identify the mRNA and protein levels of genes in cells, immunofluorescence staining was used to analyze the localization of Nrf2 in cells, RNA-seq was used to analyze changes in mRNA expression profile, and combined with the FerrDb database, ferroptosis-related molecules were screened to elucidate the impact of NEK2 on the sensitivity of gastric cancer cells to ferroptosis. We found that inhibition of NEK2 could enhance the sensitivity of gastric cancer cells to RSL3 and Erastin-induced ferroptosis, which was reflected in the combination of inhibition of NEK2 and ferroptosis induction compared with ferroptosis induction alone: cell viability and GSH level were further decreased, while the proportion of dead cells, Fe2+ level, ROS level, lipid oxidation level, MDA level, GSSG level and GSSG/GSH ratio were further increased. Mechanism studies have found that inhibiting NEK2 could promote the expression of HMOX1, a gene related to ferroptosis, and enhance the sensitivity of gastric cancer cells to ferroptosis by increasing HMOX1. Further mechanism studies have found that inhibiting NEK2 could promote the ubiquitination and proteasome degradation of Keap1, increase the level of Nrf2 in the nucleus, and thus promote the expression of HMOX1. This study confirmed that NEK2 can regulate HMOX1 expression through Keap1/Nrf2 signal, and then affect the sensitivity of gastric cancer cells to ferroptosis, enriching the role and mechanism of NEK2 in gastric cancer.

Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence.

Rabies is a lethal encephalitis caused by the rabies virus (RABV) with a fatality rate near 100% after the onset of clinical symptoms in humans and animals. Microglia are resident immune cells in the central nervous system. Few studies have been conducted on the functional role of microglia in RABV infection. Here, we performed a transcriptomic analysis of mRNA expression profiles in the microglia of mouse brains intracerebrally infected with RABV. We successfully isolated single microglial cells from the mouse brains. The survival rate of dissociated microglial cells was 81.91%-96.7%, and the purity was 88.3%. Transcriptomic analysis revealed 22,079 differentially expressed mRNAs identified in the microglia of mouse brains infected with RABV strains (rRC-HL, GX074, and CVS-24) of varying virulence at 4 and 7 days post-infection (dpi) compared to the control group. The numbers of DEGs versus the control at 4 and 7 dpi in mice infected with rRC-HL, GX074, and CVS-24 were 3622 and 4590, 265 and 4901, and 4079 and 6337. The GO enrichment analysis showed that response to stress, response to external stimulus, regulation of response to stimulus, and immune system process were abundant during RABV infection. The KEGG analysis indicated that the Tlr, Tnf, RIG-I, NOD, NF-κB, MAPK, and Jak-STAT signaling pathways were involved in RABV infection at both 4 and 7 dpi. However, some phagocytosis and cell signal transduction processes, such as endocytosis, p53, phospholipase D, and oxidative phosphorylation signaling pathways, were only expressed at 7 dpi. The involvement of the Tnf and Tlr signaling pathways prompted us to construct a protein-protein interaction (PPI) network of these pathways. The PPI revealed 8 DEGs, including Mmp9, Jun, Pik3r1, and Mapk12. Notably, Il-1b interacted with Tnf and Il-6 with combined scores of 0.973 and 0.981, respectively. RABV causes significant changes in mRNA expression profiles in the microglia in mice. 22,079 differentially expressed mRNAs were identified in the microglia of mice infected with RABV strains of varying virulence at 4 and 7 dpi. The DEGs were evaluated using GO, KEGG, and PPI network analysis. Many immune pathways were up-regulated in RABV-infected groups. The findings will help elucidate the microglial molecular mechanisms of cellular metabolism dysregulated by RABV and may provide important information for investigating RABV pathogenesis and therapeutic methods.

Bmp5 Mutation Alters mRNA Expression During External Ear Development.

To understand changes in gene regulation and mRNA expression in external ear development, we used a bone morphogenetic protein 5 (BMP5) short-ear mouse model. External ear tissues at E15.5 and E17.5 were collected, and mRNA expression profiles were analyzed. Upregulated and downregulated mRNA expression was identified using find_circ and CIRI2 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the differentially expressed mRNAs. Alterations in related signal pathways were identified from the upregulated and downregulated mRNA transcripts. The results showed a correlation between the mRNA expression during external ear development in BMP5 short-ear mice, including key regulatory mRNA changes after point mutations of the Bmp5 gene. This study provides evidence for the mechanism underlying mRNA regulation during external ear development. Changes in mRNA expression profiles also provide clues for future studies regarding the regulatory mechanisms underlying external ear development.

Involvement of melatonin in transducing moon-related signals into the reproductive network of the female honeycomb grouper Epinephelus merra

Most groupers (genus Epinephelus) inhabiting tropical and subtropical waters exhibit lunar-related reproductive cycles. Their gametes develop synchronously toward and are released around the species-selected moon phase. Periodical changes in cues from the moon are likely used as zeitgeber, and the hypothalamic-pituitary-gonadal (HPG) axis may be activated after cues are perceived by the sensory organ and transduced as internal signals. The objective of this study was to examine weekly changes in mRNA expression profiles of gonadotropin-releasing hormones (gnrh1 and gnrh2) and the β-subunit of gonadotropins (fshβ and lhβ) during the spawning season (May to June) of the female honeycomb grouper Epinephelus merra, which spawns around the full moon period. When mature females were collected based on the lunar cycle, the gonadosomatic index peaked around the full moon. Ovarian histology revealed that oocytes laden with yolk developed toward the full moon and, subsequently, ovulatory follicles appeared around the last quarter moon, confirming lunar-related spawning with a full moon preference. Real-time quantitative polymerase chain reaction analyses revealed high abundances of fshβ and lhβ toward the first quarter moon, whereas concentrations of gnrh1 and gnrh2 increased around the last quarter moon and the first quarter moon, respectively, suggesting that transcription levels of these hormones fluctuate with the lunar cycle. The measurement of melatonin in the eye around the new moon and the full moon revealed that the ocular melatonin content was higher around the new moon than around the full moon, suggesting that the honeycomb grouper can perceive changes in moonlight. In addition, implantation of an osmotic pump containing melatonin into the body cavity of E. merra reduced the transcription levels of gonadotropins, suggesting that melatonin negatively affects hormonal synthesis at the HPG axis. We concluded that melatonin plays an essential role in transducing periodical changes in moonlight and that decreases in melatonin levels from the new moon to the full moon activate the HPG axis for entrainment of gonadal development and spawning.

Evaluation of Sensitivity of Two Triple Negative Breast Cancer Cell Lines to 2, 3‐ dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone and 4 Hydroxy‐Tamoxifen

Breast cancer (BC) is a heterogeneous disease and it is one of the leading causes of mortality worldwide in women each year. However, in the United States, there is a disparity in survival between African American (AA) and Caucasian women with AA women having a higher rate of mortality. BC is typically classified based on the expression of the following receptors: Estrogen receptor α (ERα), Progesterone receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2). Conventional treatment for BC relies on the expression of ERα for anti‐estrogenic therapy and HER2 for HER2 inhibition. Triple negative breast cancer (TNBC) is a form of breast cancer which lacks expression of ERα, PR and HER2 and affects minority population disproportionately. Potential mechanisms for the loss of ERα expression include hyperactivation of MAPK pathways due to EGFR or HER2 overexpression, estrogen withdrawal, hypoxia and methylation of the CpG island, cytosine‐guanine abundant area, on the ER gene promoter region. Due to lack of targetted treatment for TNBC, alternative treatment options are essential to increase patient survivability. 2, 3‐ dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone (Z285), a 1,4 napthoquinone analog, has demonstrated cytotoxic effects in estrogen positive and estrogen negative cell lines. Previous in silico data showed that this compound may function as an MAPK allosteric inhibitor. MCF7 cells were used as an ERα positive control and two TNBC cell lines, HCC1806, from a AA woman, and MDA MB 231, from a Caucasian woman, were used. Cell viability studies were carried out on cells treated with Z285 alone (0.5–10μM), 4 Hydroxy‐Tamoxifen (4OH‐Tam) alone (3–30μM) for 24,72 and 120hrs. In combination studies, the cells were pretreated with Z285 (2 or 5μM) for 6 hrs followed by 4OH‐Tam (1–15μM) for 24, 72 and 120hrs. These results showed a dose and time‐dependent response with individual treatment. Combinatory studies showed significant decreases in cell viability when compared to individual studies. Cells were treated with 5 μM Z285 and mRNA was collected for RNA microarray analysis to determine changes in mRNA expression profile. These results showed modulation of several genes involved in the MAPK pathway and suggest a mechanism of increased sensitivity of the TNBC cells to 4OH‐Tamoxifen. Therefore, this novel compound which targets the MAPK pathway may offer a model for treating TNBC breast cancers by reactivating ERα expression in these cancers and thus rendering them susceptible to anti‐estrogen therapy.Support or Funding InformationCharles and Mary Latham FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic deletion of soluble 5′-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5′-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2−/−) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2−/− mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2−/− mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2−/− versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2−/− versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.

Altered function and maturation of primary cortical neurons from a 22q11.2 deletion mouse model of schizophrenia

Given its high penetrance, clearly delineated and evolutionary conserved genomic structure, mouse models of the 22q11.2 deletion provide an ideal organism-based and cell-based model of this well-established disease mutation for schizophrenia. In this study we examined the development of changes in intrinsic properties, action potential firing and synaptic transmission using whole-cell patch-clamp recordings of cultured embryonic cortical neurons from Df(16)A+/− and WT mice at DIV7 and DIV14, respectively. Compared to neurons from the WT littermates, significantly increased input resistance and decreased rising rate of action potential was observed in Df(16)A+/− mice at DIV7 but not at DIV14 indicative of delayed neuronal maturation. Neurons from Df(16)A+/− mice also showed significantly higher cellular excitability at both DIV7 and DIV14. Evaluation of Ca2+ homeostasis perturbation caused by 22q11.2 deletion using calcium imaging revealed a significantly lower amplitude of calcium elevation and a smaller area under the curve after depolarization in neurons from Df(16)A+/− mice at both DIV7 and DIV14. Furthermore, the properties of inhibitory synaptic events were significantly altered in Df(16)A+/− mice. We identified changes in mRNA expression profiles, especially in ion channels, receptors, and transporters that may underlie the neurophysiological effects of this mutation. Overall, we show a number of alterations in electrophysiological and calcium homeostatic properties of embryonic cortical neurons from a 22q11.2 deletion mouse model at different culture times and provide valuable insights towards revealing disease mechanisms and discovery of new therapeutic compounds.

Alveolar macrophages from tuberculosis patients display an altered inflammatory gene expression profile

Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.

Acute toxicity of CCl4 but not of paracetamol induces a transcriptomic signature of fibrosis in precision-cut liver slices

In rat in vivo, both paracetamol (APAP) and carbon tetrachloride (CCl4) induce liver necrosis, but long-term treatment with CCl4, in contrast to paracetamol, causes liver fibrosis. The aim of this study was to perform transcriptomic analysis to compare the early changes in mRNA expression profiles induced by APAP and CCl4 in the rat precision-cut liver slice model (PCLS) and to identify early markers that could predict fibrosis-inducing potential.Microarray data of rat PCLS exposed to APAP andCCl4was generated using a toxic dose based on decrease in ATP levels. Toxicity pathway analysis using a custom made fibrosis-related gene list showed fibrosis as one of the predominant toxic endpoints in CCl4-treated, but not in APAP-treated PCLS. Moreover, genes which have a role in fibrosis such as alpha-B crystallin, jun proto-oncogene, mitogen-activated protein kinase 6, serpin peptidase inhibitor and also the transcription factor Kruppel-like-factor-6 were up-regulated by CCl4, but not by APAP. Predicted activation or inhibition of several upstream regulators due to CCl4 is in accordance with their role in fibrosis.In conclusion, transcriptomic analysis of PCLS successfully identified the fibrotic potential of CCl4 as opposed to APAP. The application of PCLS as an ex vivo model to identify early biomarkers to predict the fibrogenic potential of toxic compounds should be further explored.

Aging-induced changes in sex-steroidogenic enzymes and sex-steroid receptors in the cortex, hypothalamus and cerebellum.

We investigated age-induced changes in mRNA expression profiles of sex-steroidogenic enzymes and sex-steroid receptors in 3-, 12-, and 24-month-old male rat brain subregions [cerebral cortex (CC), hypothalamus (Hy) and cerebellum (CL)]. In many cases, the expression levels of mRNA decreased with age for androgen synthesis enzyme systems, including Cyp17a1, Hsd17b and Srd5a in the CC and CL, but not in the Hy. Estradiol synthase Cyp19a1 did not show age-induced decline in the Hy, and nearly no expression of Cyp19a1 was observed in the CC and CL over 3-24 m. Androgen receptor Ar increased in the Hy but decreased in the CC with age. Estrogen receptor Esr1 increased in the CC and Hy, and did not change in the CL with age. Esr2 did not change in the CC and Hy, but decreased in the CL with age. As a comparison, age-induced changes of brain-derived neurotrophic factor mRNA were also investigated.

Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients

BackgroundThe risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population. Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs. The aim of this genome-wide study was to identify pathways and genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs).MethodsTo perform the analysis in an isogenic background, RNA and DNA were isolated from SCC, AK and normal (unexposed) epidermis (NS) from each of 13 OTRs. Samples were subjected to genome-wide expression analysis and genome SNP analysis using Illumina’s HumanWG-6 BeadChips and Infinium II HumanHap550 Genotyping BeadChips, respectively. mRNA expression results were verified by quantitative PCR.ResultsHierarchical cluster analysis of mRNA expression profiles showed SCC, AK and NS samples to separate into three distinct groups. Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were hyperproliferation related genes and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17. Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFκB and TNF already in AKs. In contrast to what has been reported previously, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles.ConclusionVast differences in gene expression profiles exist between SCC, AK and NS from immunosuppressed OTRs. Moreover, several pathways activated in SCCs were already activated in AKs, confirming the assumption that AKs are the precursor lesions of SCCs. Since the drastic changes in gene expression appeared unlinked to specific genomic gains or losses, the causal events driving SCC development require further investigation. Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs. Further study is required to identify the mechanisms of early activation of NFκB and TNF, and to establish whether these pathways offer a feasible target for preventive intervention among OTRs.

Dynamics of the transcriptome in the primate ovulatory follicle

Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24 and 36 h) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n= 4-8/timepoint), with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1 and HSD11B2), steroidogenic acute regulatory protein (StAR) and gonadotrophin receptors [LH/choriogonadotrophin receptor (LHCGR), FSH receptor (FSHR)] as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for epidermal-growth-factor-like factors (amphiregulin, epiregulin) and processes [hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6)] implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g. CYP17A), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell and surface epithelium (HSD11B)-related genes in the rodent/primate pre-ovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response and angiogenesis), and for identifying novel gene products controlling mammalian fertility.

Three-dimensional culture alters primary cardiac cell phenotype.

The directed formation of complex three-dimensional (3D) tissue architecture is a fundamental goal in tissue engineering and regenerative medicine. The growth of cells in 3D structures is expected to influence cellular phenotype and function, especially relative cell distribution, expression profiles, and responsiveness to exogenous signals; however, relatively few studies have been carried out to examine the effects of 3D reaggregation on cells from critical target organs, like the heart. Accordingly, we cultured primary cardiac ventricular cells in a 3D model system using a serum-free medium to test the hypothesis that expression profiles, multicellular organizational pathways, tissue maturation markers, and responsiveness to hormone stimulation were significantly altered in stable cell populations grown in 3D versus 2D culture. We found that distinct multi-cellular structures formed in 3D in conjunction with changes in mRNA expression profile, up-regulation of endothelial cell migratory pathways, decreases in the expression of fetal genes (Nppa and Ankrd1), and increased sensitivity to tri-iodothyronine stimulation when compared to parallel 2D cultures comprising the same cell populations. These results indicate that the culture of primary cardiac cells in 3D aggregates leads to physiologically relevant alterations in component cell phenotype consistent with cardiac ventricular tissue formation and maturation.

Development of the osteoblastic phenotype in human alveolar bone‐derived cells grown on a collagen type I‐coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappaB ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

MICROARRAY ANALYSIS OF GENE EXPRESSION IN THE PRIMATE OVULATORY FOLLICLE: VALIDATION OF SELECTED GENE TRANSCRIPTS BY REAL-TIME PCR

The recent development of genomic approaches permits, for the first time, a complete evaluation of LH-regulated genes in the ovulatory, luteinizing follicle. Therefore, experiments were designed to evaluate gene expression, using the Affymetrix rhesus macaque cDNA microarray, in a controlled ovulation (COv) model which permits evaluation of the naturally selected, dominant follicle at specific timepoints after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle in rhesus monkeys. The preovulatory follicle (n = 4–8/timepoint) was collected before (0 hr) and 12, 24, and 36 hr post-hCG injection. Since ovulated follicles were observed at 36 hr, samples at this timepoint were divided (n = 4/group) into unruptured and ruptured follicles. Total RNA was prepared from individual follicles, with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder stored for real-time PCR assays. Initial results indicate that: (a) RNA preparation, labeling and hybridization met high quality standards, (b) microarray data from individual samples distinctly clustered according to timepoint, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 hr, and (c) between timepoint comparisons revealed profound changes in mRNA expression profiles. In every instance, the dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B, HSD11B1, HSD11B2), StAR, and receptors for gonadotropin receptors (LHCGR, FSHR) determined by microarray analysis correlated precisely with those from blinded real-time PCR assays. Notably, mRNA levels for gonadotropin receptors declined (p<0.05) markedly to low levels within 12 hr post-hCG injection; those for CYP17A1 and CYP19A also declined (p<0.05) but did not reach low levels until 24 hr post-hCG. However, levels for LHCGR and CYP19A later increased (p<0.05) in ruptured, compared to unruptured, follicles at 36 hr post-hCG. Levels of HSD11B1 mRNA displayed a different pattern, increasing (p<0.05) initially between 0 and 12 hrs post-hCG and again in the ruptured follicle at 36 hr post-hCG. In contrast, mRNA levels for HSD11B2 declined (p<0.05) after hCG injection and remained low after follicle rupture. Thus, several mRNAs displayed the expected expression pattern for purported theca-cell specific (e.g., CYP17A), granulosa-cell specific (CYP19A, FSHR), and surface epithelium-related (HSD11B) genes in the rodent/primate ovulatory follicle. This genomic database is of great value in analyzing molecular and cellular pathways associated with follicle rupture and other periovulatory events in the primate follicle (e.g. luteinization, neuronal differentiation, inflammatory response, hypoxia, and angiogenesis), and for identifying novel gene products controlling mammalian fertility. Supported by Schering Female AMPPA, U54 SCCPIR HD18185, RR00163 (platform)

Seasonal changes in peptide, receptor and ion channel mRNA expression in the caudal neurosecretory system of the European flounder ( Platichthys flesus)

The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August–October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, κ-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca 2+ and Ca-activated K + channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish migrate between sea and estuaries in winter and spring.

MRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. We further show that the N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. Our findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets.

Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes.

Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

Changes in mRNA expression profile underlie phenotypic adaptations in creatine kinase-deficient muscles

We have studied the mechanisms that regulate the remodeling of the glycolytic, mitochondrial and structural network of muscles of creatine kinase M (M-CK)/sarcomeric mitochondrial creatine kinase (ScCKmit) knockout mice by comparison of wild-type and mutant mRNA profiles on cDNA arrays. The magnitudes of changes in mRNA levels were most prominent in M-CK/ScCKmit (CK −/−) double mutants but did never exceed those of previously observed changes in protein level for any protein examined. In gastrocnemius of CK −/− mice we measured a 2.5-fold increase in mRNA level for mitochondrial encoded cytochrome c oxidase (COX)-III which corresponds to the increase in protein content. The level of the nuclear encoded mRNAs for COX-IV, H +-ATP synthase-C, adenine nucleotide translocator-1 and insulin-regulatable glucose transporter-4 showed a 1.5-fold increase, also in agreement with protein data. In contrast, no concomitant up-regulation in mRNA and protein content was detected for the mitochondrial inorganic phosphate-carrier, voltage-dependent anion channel and certain glycolytic enzymes. Our results reveal that regulation of transcript level plays an important role, but it is not the only principle involved in the remodeling of mitochondrial and cytosolic design of CK −/− muscles.