Tissue Specificity Of Porcine Glucocorticoid Receptor(NR3C1)Gene Expression:Promoter Activity And Transcriptiondal Regulation Under LPS Challenge

Glucocorticoid receptor (GR), as a nuclear receptor, mediates GCs role in a number of physiological processes. GR widely existed in a wide array of cell types/tissues, however, GR expression and function are in a tissue-specific manner. GR gene is consisted of nine exons, exon 1 is untranslated exon. GR exert its biological function through GR protein, and GR protein level depends on GR mRNA level, so GR transcriptional regulation plays a important role in GR tissue-specific distribution. GR exon 1 variants exprssion is involved in GR transcriptional regulation, each of the first exon was independently controlled by its own alternative promoter. Previous research in our laboratory have identified porcine GR promoter sequence, found high homology with human and rat, and then identified seven GR exon 1 mRNA variants. But porcine GR mRNA and GR exon 1 variants tissue-specific distributions were still unclear and no report was associated with GR alternative promoter usage in GR transcriptional regulation. Studies on GR tissue-specific anti-inflammatory mechanism and GR transcriptional regulation mechanism in inflammation were limited. In this experiment, GR and its exon 1 mRNA variants tissue-specific distribution patterns were determined, GR alternative promoter activities were analyzed. Response of L1-4 and L1-10 to DEX in different cells were analyzed. Under LPS stimulation, detected GR tissue-specific expression and fuction in liver and muscle, explored the molecular mechanism of GR tissue-specific transcriptional regulation.1 Porcin GR mRNA tissue-specific distribution and promoter activity analysisIn human the distribution of exon 1 variants play an important role in GR distribution and function. Porcin GR function is in a tissue-specific manner, however, the study of GR expression distribution in different tissues is limited. We used absolute quantitative PCR to measured weaned Large White GR mRNA and its exon 1 variants expression in hippocampus, liver and muscle. First, according to GR 5’UTR sequence that was just published by our lab, the GR exonl variants to be detected were cloned using PCR, constructed the recombinant Psptl 8 plasimds, these plasmids were linearizd with restriction enzyme to make standard required for absolute quantitative PCR, copy number of GR gene and exon 1-4,1-12,1-6,1-7,1-9/10 were detected using real-time PCR. The results showed that the copy number of GR gene and exon 1 variants in the three tissues showed significant tissue-specificity:the highest expression in liver, lowest in hippocampus.Short (S1-4, S1-5,S1-6, S1-7, S1-10, S1-12) and long (L1-4, L1-5, Ll-6, L1-7, L1-10) GR promoters were cloned and connected separately with the pGL3-basic plasmid to get the promoter activity analysis plasmids. Short promoters activities were compared with their corresponding long one to analyze GR promoter activities in HepG2, SY5Y and C2C12 cells. Our results showed that porcine GR exonl variants mRNA expressions were tissue-specific and consistent with GR gene tissue specific expression, the tissue specific distribution of GR exon 1 variants were associated with long type GR alternative promoters tissue-specific usage.2 Mechanism of cell-specific GC sensitivity of porcin GR alternative promoterGR mediates GC function, while GR mRNA expression is also regulated by GC. In human and rat it has been confirmed that GR promoter activity was changed by DEX stimulation and showed cell-specificity, however, relevant studies of DEX effect on procine GR promoter is limited.Porcine GR alternative promoters activities under DEX stimulation were measured in HepG2 (liver hepatocellular cells), SY5Y (neuroblastoma cells) and C2C12 (mouse myoblast cell line) cells. Under DEX stimulation, we analyzed promoter activities of Sl-4 (containing one nGRE) and S1-6 (no nGRE) in HepG2. Result showed that only S1-4 promoter was activated under the stimulation of high or low concentration of DEX, while S1-6 showed no response to DEX. Western blot result showed that GR protein level in all three cells detected significantly reduced in DEX stimulation, meaning that endogenous GR protein levels are very low in cells within this time, so we think that at this time, the inhibition of endogenous GR on GR promoter in physiological state was significantly prevented, as a result promoter containing nGRE exhibited significantly enhanced activity. Under a series of concentrations of DEX treatment, cell viabilities that measured with CCK-8 assay kit didn’t change in HepG2, SY5Y and C2C12. Detected 1-4 and L1-10 that contain different nGRE number responsibility to a series of concentration gradient DEX in HepG2, SY5Y, C2C12, the result showed that their responsibilities to DEX were different in HepG2, indicating the number of nGRE caused distinct and cell-specific promoter sensitivity to DEX.3 Tissue-specific GR expression and function after LPS injectinLPS causes immune response and inflammation to clear pathogens that invade the body, however, excessive immune response will cause tissue damage, GR mediated GC released by adrenal within HPA axis activation, played an important role in inflammation suppression, GR anti-inflammation action showed tissue-specificity.We detected inflammatory cytokines expressions in liver and muscle by elasa kit and showed that inflammatory cytokines TNF-a and IL-6 in liver was significantly higher (P < 0.05) in the role of LPS, while invariant in muscle. To explored the mechanism of inflammation in liver and muscle, cortisol levels under LPS stimulation in those two tissue were detected, result showed those cortisol levels were significantly increased (P< 0.05) in LPS stimulation. the GR protein expression was detected by Western blot, GR mRNA expression was detected by real-time PCR, the results showed that in liver GR protein and mRNA levels were significantly reduced (P< 0.05) in LPS stimulation, but unchanged in muscle.Therefore, we used western blot to detected the critical cytokines protein expression in inflammatory signaling pathway in liver and muscle, the results showed that p-P65 NF-κB in liver was significantly activated (P< 0.01) in LPS stimulation, however, no change in muscle, p-P38 MAPK activation was significantly (P< 0.01) in the liver in LPS challenge, but invariant in muscle.The results illustrate that the systemic immune response, inflammation and injury were caused by LPS in liver, but the inflammatory role of LPS in muscle was unobvious. Cortisol level didn’t cause the tissue-specific imflammtion, GR down-regulation in liver was the cause of tissue-specific GC resistance. In liver GR protein and mRNA had positive correlation, suggested that GR transcriptional regulation played a important role in GR expression.4 Molecular mechanisms of GR tissue-specific transcriptional regulation in liver and muscle in LPS stimulationWe detected the GR exonl variants expression in LPS stimulation in liver and muscle, exon 1-4,1-5,1-6 mRNA expressions in the liver were all significantly decreased (P< 0.05), other exon 1 variants were unchanged, while the exon 1-4,1-5,1-6 mRNA expressions in muscle were significantly decreased (P< 0.05), exon 1-10 significantly increased.We analyzed the transcription factor expression and their enrichment on GR promoter. Western blot analysis showed p-CREB transcription factor protein was significantly increased (P< 0.05) in muscle, in liver unchanged, p-GR in the liver was significantly increased (P< 0.05), unchanged in muscle. ChIP result showed that p-CREB binding with S1-4 and S1-5 was significantly lower in LPS group in liver (P< 0.05), p-GR showed similar results, these results indicated that p-CREB, as P38 MAPK signaling pathway activated transcription factor, is specifically activated in the muscle and keep it binding with GR to maintain GR transcription activation.Western blot analysis showed that histone acetylation levels in LPS stimulation was significantly reduced in the liver and muscle, but ChIP showed that acetylation levels GR promoter S1-4 and S1-5 in liver were significantly decreased (P< 0.05), but only S1-5 acetylation level decreased in muscle (P< 0.05), indicated that acetylation histone H3 participated in GR tissue-specific transcriptional regulation.