Genetic insights into biological mechanisms governing human ovarian ageing.

Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

Tumor miRNA expression profile is related to vestibular schwannoma growth rate.

OBJECTIVE: Our objective was to investigate if the tumor microRNA (miRNA) expression profile was related to tumor growth rate. Growth-related miRNAs might be potential targets for future therapeutic intervention. MATERIAL AND METHODS: Tumor tissue was sampled during surgery of patients with a sporadic vestibular schwannoma. Tumor growth rate was determined by tumor measurement on the two latest pre-operative MRI scans. Tumor miRNA expression was analyzed using the Affymetrix Gene Chip® protocol, and CEL files were generated using GeneChip® Command Console® Software and normalized using Partek Genomics Suite 6.5. The CEL files were analyzed using the statistical software program R. Principal component analysis, affected gene ontology analysis, and analysis of miRNA expression fold changes were used for analysis of potential relations between miRNA expression profile and tumor growth rate. RESULTS AND CONCLUSION: Tumor miRNA expression is related to the growth rate of sporadic vestibular schwannomas. Rapid tumor growth is associated with deregulation of several miRNAs, including upregulation of miR-29abc, miR-19, miR-340-5p, miR-21, and miR-221 and downregulation of miR-744 and let-7b. Gene ontologies affected by the deregulated miRNAs included neuron development and differentiation, gene silencing, and negative regulation of various biological processes, including cellular and intracellular signaling and metabolism.

Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans.

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

BACKGROUND: Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. METHODS: PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. CONCLUSION: PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use.

Gene expression, signal transduction pathways and functional networks associated with growth of sporadic vestibular schwannomas.

The objective of this study was to determine global gene expression in relation to Vestibular schwannomas (VS) growth rate and to identify signal transduction pathways and functional molecular networks associated with growth. Repeated magnetic resonance imaging (MRI) prior to surgery determined tumor growth rate. Following tissue sampling during surgery, mRNA was extracted from 16 sporadic VS. Double stranded cDNA was synthesized from the mRNA and used as template for in vitro transcription reaction to synthesize biotin-labeled antisense cRNA, which was hybridized to Affymetrix HG-U133A arrays and analyzed by dChip software. Differential gene expression was defined as a 1.5-fold difference between fast and slow growing tumors (><0.5 ccm/year), employing a p-value <0.01. Deregulated transcripts were matched against established gene ontology. Ingenuity Pathway Analysis was used for identification of signal transduction pathways and functional molecular networks associated with tumor growth. In total 109 genes were deregulated in relation to tumor growth rate. Genes associated with apoptosis, growth and cell proliferation were deregulated. Gene ontology included regulation of the cell cycle, cell differentiation and proliferation, among other functions. Fourteen pathways were associated with tumor growth. Five functional molecular networks were generated. This first study on global gene expression in relation to vestibular schwannoma growth rate identified several genes, signal transduction pathways and functional networks associated with tumor progression. Specific genes involved in apoptosis, cell growth and proliferation were deregulated in fast growing tumors. Fourteen pathways were associated with tumor growth. Generated functional networks underlined the importance of the PI3K family, among others.

Gene expression profiling in patients with polymyalgia rheumatica before and after symptom-abolishing glucocorticoid treatment.

BACKGROUND: The pathophysiology, including the impact of gene expression, of polymyalgia rheumatica (PMR) remains elusive. We profiled the gene expression in muscle tissue in PMR patients before and after glucocorticoid treatment. METHODS: Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 arrays in muscle biopsies from 8 glucocorticoid-naive patients with PMR and 10 controls before and after prednisolone-treatment for 14 days. For 14 genes, quantitative real-time PCR (qRT-PCR, n = 9 in both groups) was used to validate the microarray findings and to further investigate the expression of genes of particular interest. RESULTS: Prednisolone normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in PMR patients. A total of 165 putatively clinically relevant, differentially expressed genes were identified (cut-off: fold difference > ±1.2, difference of mean > 30, and p < 0.05); of these, 78 genes differed between patients and controls before treatment, 131 genes responded to treatment in a given direction only in patients, and 44 fulfilled both these criteria. In 43 of the 44 genes, treatment counteracted the initial difference. Functional clustering identified themes of biological function, including regulation of protein biosynthesis, and regulation of transcription and of extracellular matrix processes. Overall, qRT-PCR confirmed the microarray findings: Microarray-detected group differences were confirmed for 9 genes in 17 of 18 comparisons (same magnitude and direction of change); lack of group differences in microarray testing was confirmed for 5 genes in 8 of 10 comparisons. Before treatment, using qRT-PCR, expression of interleukin 6 (IL-6) was found to be 4-fold higher in patients (p < 0.05). CONCLUSIONS: This study identifies genes in muscle, the expression of which may impact the pathophysiology of PMR. Moreover, the study adds further evidence of the importance of IL-6 in the disease. Follow-up studies are needed to establish the exact pathophysiological relevance of the identified genes. The study was retrospectively listed on the ISRCTN registry with study ID ISRCTN69503018 and date of registration the 26th of July 2017.

p38 MAPK activation upregulates proinflammatory pathways in skeletal muscle cells from insulin-resistant type 2 diabetic patients.

Skeletal muscle is the key site of peripheral insulin resistance in type 2 diabetes. Insulin-stimulated glucose uptake is decreased in differentiated diabetic cultured myotubes, which is in keeping with a retained genetic/epigenetic defect of insulin action. We investigated differences in gene expression during differentiation between diabetic and control muscle cell cultures. Microarray analysis was performed using skeletal muscle cell cultures established from type 2 diabetic patients with a family history of type 2 diabetes and clinical evidence of marked insulin resistance and nondiabetic control subjects with no family history of diabetes. Genes and pathways upregulated with differentiation in the diabetic cultures, compared with controls, were identified using Gene Spring and Gene Set Enrichment Analysis. Gene sets upregulated in diabetic myotubes were associated predominantly with inflammation. p38 MAPK was identified as a key regulator of the expression of these proinflammatory gene sets, and p38 MAPK activation was found to be increased in the diabetic vs. control myotubes. Although inhibition of p38 MAPK activity decreased cytokine gene expression from the cultured diabetic myotubes significantly, it did not improve insulin-stimulated glucose uptake. Increased cytokine expression driven by increased p38 MAPK activation is a key feature of cultured myotubes derived from insulin-resistant type 2 diabetic patients. p38 MAPK inhibition decreased cytokine expression but did not affect the retained defect of impaired insulin action in the diabetic muscle cells.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function.

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.

BACKGROUND: Cancers of unknown primary (CUPs) constitute ~5% of all cancers. The tumors have an aggressive biological and clinical behavior. The aim of the present study has been to uncover whether CUPs exhibit distinct molecular features compared to metastases of known origin. METHODS: Employing genome wide transcriptome analysis, Linear Discriminant Analysis (LDA) and Quadratic Discriminant Analysis (QDA), we defined the putative origins of a large series of CUP and how closely related a particular CUP was to corresponding metastases of known origin. LDA predictions were subsequently used to define a universal CUP core set of differentially expressed genes, that by means of gene set enrichment analysis was exploited to depict molecular pathways characterizing CUP. RESULTS: The analyses show that CUPs are distinct from metastases of known origin. CUPs exhibit inconsistent expression of conventional cancer biomarkers and QDA derived outlier scores show that CUPs are more distantly related to their primary tumor class than corresponding metastases of known origin. Gene set enrichment analysis showed that CUPs display increased expression of genes involved in DNA damage repair and mRNA signatures of chromosome instability (CIN), indicating that CUPs are chromosome unstable compared to metastases of known origin. CONCLUSIONS: CIN may account for the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP and warrant selective diagnostic strategies and treatment.

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II.

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.

Genome-wide analysis of cytogenetic aberrations in ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia.

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1-positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0-14). Concordance with available G-band karyotyping and comparative genomic hybridization was 93%. Based on three major protein-protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour-suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1-positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.

Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells.

In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration.

Gene expression profiling of placentas affected by pre-eclampsia.

Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events effectuated throughout gestation. They were associated with transcriptional regulation and vasoregulative pathways, along with a number of hypothetical proteins and gene sequences with unknown functions.

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.

Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development.

Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.

Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts.

Single-chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 (insulin-like growth factor 1) receptors. We have shown previously that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signalling cascades despite similar binding affinity and in vivo hypoglycaemic potency. It is still unclear how two ligands can initiate different signalling responses through the IR (insulin receptor). To investigate further how the activation of the IR by insulin and S597 differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK (mitogen-activated protein kinase) pathway. These findings support our signalling results obtained previously and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion, we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.

Human granulosa cells function as innate immune cells executing an inflammatory reaction during ovulation: a microarray analysis.

Ovulation has been compared to a local inflammatory reaction. We performed an in silico study on a unique, PCR validated, transcriptome microarray study to evaluate if known inflammatory mechanisms operate during ovulation. The granulosa cells were obtained in paired samples at two different time points during ovulation (just before and 36 hours after ovulation induction) from nine women receiving fertility treatment. A total of 259 genes related to inflammation became significantly upregulated during ovulation (2-80 fold, p<0.05), while specific leukocyte markers were absent. The genes and pathway analysis indicated NF-KB-, MAPK- and JAK/STAT signalling (p<1.0E-10) as the major pathways involved in danger recognition and cytokine signalling to initiate inflammation. Upregulated genes further encoded enzymes in eicosanoid production, chemo-attractants, coagulation factors, cell proliferation factors involved in tissue repair, and anti-inflammatory factors to resolve the inflammation again. We conclude that granulosa cells, without involvement from the innate immune system, can orchestrate ovulation as a complete sterile inflammatory reaction.

Calprotectin is released from human skeletal muscle tissue during exercise.

Skeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle. IL-6 was infused for 3 h into healthy young males (n = 7) and muscle biopsies obtained at time points 0, 3 and 6 h in these individuals and in resting controls. Affymetrix microarray analysis of gene expression changes in skeletal muscle biopsies identified a small set of genes changed by IL-6 infusion. RT-PCR validation confirmed that S100A8 and S100A9 mRNA were up-regulated 3-fold in skeletal muscle following IL-6 infusion compared to controls. Furthermore, S100A8 and S100A9 mRNA levels were up-regulated 5-fold in human skeletal muscle following cycle ergometer exercise for 3 h at approximately 60% of in young healthy males (n = 8). S100A8 and S100A9 form calprotectin, which is known as an acute phase reactant. Plasma calprotectin increased 5-fold following acute cycle ergometer exercise in humans, but not following IL-6 infusion. To identify the source of calprotectin, healthy males (n = 7) performed two-legged dynamic knee extensor exercise for 3 h with a work load of approximately 50% of peak power output and arterial-femoral venous differences were obtained. Arterial plasma concentrations for calprotectin increased 2-fold compared to rest and there was a net release of calprotectin from the working muscle. In conclusion, IL-6 infusion and muscle contractions induce expression of S100A8 and S100A9 in skeletal muscle. However, IL-6 alone is not a sufficient stimulus to facilitate release of calprotectin from skeletal muscle.

Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells.

Cholecystokinin (CCK) is abundantly expressed in the CNS, in which it regulates feeding behavior and long-term memory. Moreover, CCK has been implicated in mental disorders, such as anxiety and schizophrenia. Despite its manifest physiological and pathophysiological role, the molecular targets of neuronal CCK are incompletely understood. To identify genes regulated by neuronal CCK, we generated neuronal PC12 cells stably expressing the CCK-2 receptor (CCK-2R) and treated the cells with sulphated CCK-8 for 2-16 h, before the global expression profile was examined. The changes in gene expression peaked after 2 h, with 67 differentially expressed transcripts identified. A pathway analysis indicated that CCK was implicated in the regulation of the circadian clock system, the plasminogen system and cholesterol metabolism. But transcripts encoding proteins involved in dopamine signaling, ornithine decarboxylase (ODC) regulation, memory and epidermal growth factor receptor (EGFR) signaling were also found. Several target genes contained cAMP response elements (CREs), serum response elements (SREs), activator protein 1 (AP1) elements and GC-rich regions, but otherwise no common regulatory promoter element could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol and in the regulation of the plasminogen system.