The Tissue Specific Expression Of CRF And IGF In IGFBP2 Knock-out Mouse

Background and ObjectiveCRF (Corticotrophin releasing factor) is a kind of neuropeptides related stress, synthesized and secreted from the hypothalamus neurons of PVN area. CRF has two receptors, CRFR1 and CRFR2, play important role in the process of adjusting the HPA axis.Insulin-like growth factor (IGF) family including two kinds of ligands (IGF-1 and IGF-2) and the corresponding receptor (IGF-1R and IGF-2R) with six kinds of binding protein (IGFBP1～6). IGF family has important biological function in regulating cell proliferation, survival, differentiation and migration.Our lab works showed, CRF and its receptor play the main role in hypoxia neuroendocrine network adjustment mechanism, where hypoxia affect liver and brain tissue expression of IGF family. Experiment through the whole animal and cytological experiments showed that, CRF and IGFBP family possibly regulate the underlying mechanism of hypoxia and the effect of hypoxia on related cytokines. Using IGFBP2 knockout mice we compared tissue specific expression of CRF and IGF family. Transcription factors AP-1 and NF-kappaB are studied on AtT-20 cell line for CRFR1 and IGF-1R transcriptional regulation.Overall expression of CRF, CRFR1 and IGF family gene in the cortex and pituitary were altered during exposure to low levels of oxygen, such as 7 km hypoxia environment and acute hypoxia (1,4,8 and 24 h) and treatment with antagonist (PDTC, pyrrolidine dithiocarbamate and CP154,526, CRFR1 antagonists). IGFBP2 knockout mice are used to test the gene expression of CRFs- and IGFs family in cortex and hippocampus.At cellular level, AtT-20 cell lines with CRF incubation revealed that reverse transcription factors NF-κB and AP-1 regulate the transcription of CRFR1 and IGF-1R.Research Design and MethodsAnimal experiment:1. Rat experiment:rats exposed to low oxygen tank simulated hypoxia (7 km,1,4,8 and 24 h), animal groups:control group, different time of hypoxic exposure group, antagonist group, after hypoxia, collection of pituitary, cortex and blood leukocytes.2. Mice experiment:different development age of C57 and IGFBP2 knock-out C57 mice, born in 15 d,45 d collection of pituitary and cortex, respectively.Cell culture:Using mice pituitary tumor cell lines (AtT-20), grouped into control group, CRF treatment group (10 nM), antagonist group (AP-1 and NF-kappa B inhibitor) and CRF (10 nM)+antagonist (AP-1 and NF-kappa B inhibitor) group. Research the CRF through the transcription factors AP-1 and NF-κB to regulate CRFR1 and IGF-1R transcription.Real time Q-PCR and Western-blot is the test method.Results1. Acute hypoxia (7 km,1,4 and 8 h) increase pituitary mRNA expression of the CRF in rats, but acute hypoxia 7 km 24 h showed a down trend. Moreover, acute hypoxia 7 km 1 h,4 h,8 h significantly reduced CRFR1 mRNA expression in the rat pituitary, but acute hypoxia 7 km 24 h returned to normal levels.2. Acute hypoxia 7 km 8 h significantly reduced IGF-1 mRNA expression in the rat pituitary and cortex; but IGF-1 R mRNA expression in 4 h,8 h after hypoxia increased significantly.3. NF-κB- and CRFR1-antagonists partially blocking the CRF mRNA expression which was increased in pituitary induced by acute hypoxia; blocking the CRFR1 mRNA expression decreased and IGF-1R mRNA expression increased in pituitary induced by acute hypoxia. But the NF-κB antagonists can’t block the IGF-1 mRNA expression drop in pituitary induced by acute hypoxia; CRFR1 antagonists can block the IGF-1 mRNA expression drop in pituitary induced by acute hypoxia.4. Acute hypoxia in related to time (7 km,1,4 and 8 h) increase TNF-alpha, IL-1beta and IL-6 mRNA expression in rat cortex and blood leukocyte.5. NF-κB antagonists blocked TNF-alpha, IL-1beta and IL-6 mRNA expression increases caused by acute hypoxia in rats blood leukocyte; CRFR1 antagonists blocked IL-1beta and IL-6 mRNA expression increase caused by acute hypoxia in rats blood leukocyte, but can’t block TNF-alpha mRNA expression increase caused by acute hypoxia.6. In hippocampus of C57 mice, IGFBP2, CRFR1 and IGF-1R mRNA expression was decreased with ageing, but CRF, SPAR and NR-2B mRNA expression showed no obvious change. IGFBP2, CRF, CRFR1, IGF-1R, SPAR and NR-2B mRNA expression were significantly decreased in IGFBP2 KO mice compared with control C57. In cortex of C57 mice IGFBP2, CRF and NR-2B mRNA expression decreased along with ageing, but SPAR and IGF-1R mRNA expression increased and CRFR1 mRNA expression has no obvious change. In addition, IGFBP2, IGF-1R, SPAR and NR-2B mRNA expression were significantly decreased in IGFBP2 KO compared with control C57; the mRNA expression of CRF decline with ageing and CRFR1 mRNA expression showed no obvious change.7. In control, CRF significantly reduced AtT-20 cells CRFR1 mRNA expression, increase the mRNA expression of IGF-1R, AP-1 antagonists could block the CRFR1 gene expression decrease caused by CRF, could not block the IGF-1R gene expression increase caused by CRF. NF-κB antagonists cannot block the CRFR1 gene expression decrease and IGF-1R gene expression increase caused by CRF. During hypoxia, CRF significantly increase AtT-20 cells CRFR1 and IGF-1R mRNA expression, AP-1 antagonists could not block the CRFR1 and IGF-1R gene expression increase caused by CRF. NF-κB antagonists could block the CRFR1 and IGF-1R gene expression increase caused by CRF.SummaryAfter acute hypoxia, CRFR1 and IGF-1 mRNA expression decreased significantly in rat pituitary and CRF and IGF-1R mRNA considerably increase.In rat cortex and blood leukocyte, TNF-alpha, IL-lbeta and IL-6 mRNA expression increases significantly under acute hypoxia, regulated by CRFR1 and NF-κB.At different developmental age in mice, the expression of CRF and IGF family has different patterns. IGFBP2 may play an important role in the development of neurons in hippocampus.In the normoxic condition, AP-1 negatively regulated CRFR1 transcription as transcript factor. In the hypoxic condition, NF-κB positively regulated CRFR1 transcription.