Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells.

BACKGROUND: The major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation. RESULTS: The designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins. CONCLUSION: The current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.

Peripheral lipopolysaccharide administration transiently affects expression of brain-derived neurotrophic factor, corticotropin and proopiomelanocortin in mouse brain.

Peripheral inflammation induced by intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS) is known to cause functional impairments in the brain affecting memory and learning. One of mechanisms may be the interference with neurotrophin (NT) expression and function. In the current study we administered a single, high dose of LPS (3mg/kg, i.p.) into mice and investigated changes in brain-derived neurotrophic factor (BDNF) gene expression within 1-6 days after LPS injection. Crude synaptosomes were isolated from brain tissue and subjected to Western-blot analyses. We found transient reductions in synaptosomal proBDNF- and BDNF protein expression, with a maximal decrease at day 3 as compared to saline injected controls. The time course of reduction of BDNF mRNA in whole brain extracts parallels the decrease in protein levels in synaptosomes. LPS effects in the central nervous system (CNS) are known to crucially involve the activation of the hypothalamic-pituitary-adrenal (HPA) axis. We analysed the time course of corticotropin releasing hormone (CRH)- and proopiomelanocortin (POMC) mRNA expression. As observed for BDNF-, CRH- and POMC mRNA levels are also significantly reduced on day 3 indicating a comparable time course. These results suggest that peripheral inflammation causes a reduction of trophic supply in the brain, including BDNF at synaptic sites. The mechanisms involved could be a negative feedback of the activated HPA axis.

Genetic variants of the DDR1 gene are associated with vitiligo in two independent Brazilian population samples.

Vitiligo is a chronic disease characterized by macules devoid of melanin and identifiable melanocytes. Adhesion of melanocytes to the basement membrane by integrin CCN3 is mediated through collagen IV receptor DDR1. We hypothesize that genetic variants of the DDR1 gene are associated with the occurrence of vitiligo. To test this hypothesis, we genotyped 10 DDR1 tag single-nucleotide polymorphisms (SNPs) in 212 trios composed of an affected child and both parents. Associated markers were then genotyped in 134 independent, unrelated individuals with vitiligo and 134 unrelated controls. Allele T of tag SNP rs4618569 was associated with an increased risk for vitiligo in the family trios (P=0.002, odds ratio (OR)=5.27; 95% confidence interval (CI)=1.59-17.40), whereas allele C of tag SNP rs2267641 was associated with an increased risk for vitiligo in both family-based and case-control populations (P=0.01, OR=3.47; 95% CI=1.22-9.17; P=0.04, OR=6.00; 95% CI=1.73-52.33, respectively). The best evidence for association in the trios was obtained for a haplotype composed of risk alleles of markers rs4618569 and rs2267641 (P=0.0006). There was an age-dependent enrichment of rs4618569 T allele and rs2267641 C allele in early-onset affected individuals. In conclusion, we propose DDR1 as a susceptibility gene for vitiligo, possibly implicating a defective cell adhesion in vitiligo pathogenesis.

Characterization of behavioral response to amphetamine, tyrosine hydroxylase levels, and dopamine receptor levels in neurokinin 3 receptor knockout mice.

The neurokinin 3 (NK3) receptor is a novel target under investigation for improvement of symptoms of schizophrenia, because of its ability to modulate dopaminergic signaling. To further understanding of the function of this receptor, sensitivity to dopaminergic stimuli and levels of dopaminergic receptors and tyrosine hydroxylase in NK3 receptor knockout mice were studied. Knockout of the receptor was confirmed by lack of NK3 protein and lack of electrophysiological responsivity of presumed dopaminergic neurons to senktide. NK3 receptor knockout mice showed mild hyperlocomotion and deficits on the rotarod. NK3 receptor knockout mice did not show significant differences in sensitivity to locomotor effects of acute amphetamine (0.3, 1, and 3 mg/kg subcutaneously) or significant alterations in sensitization to locomotor effects of amphetamine, but did show nonsignificant hyperreactivity to 1 mg/kg amphetamine and a nonsignificantly increased propensity to develop sensitization. A small decrease in D1 receptor binding was seen in the dorsal striatum and olfactory tubercle, and a small decrease of in tyrosine hydroxylase in the olfactory tubercle, but no change was seen in D2 receptor binding. Together, these results support a role for the NK3 receptor in reactivity to dopaminergic stimuli, but the lack of robust changes indicates that the sensitivity to dopamine may be activity-dependent or benign in nature.