Junyu Zhang

Differential regulation of β-arrestin 1 and β-arrestin 2 gene expression in rat brain by morphine

Abstract β-Arrestins are a family of regulatory and scaffold proteins functioning in signal transduction of G protein-coupled receptors including opioid receptors. Upon agonist stimulation, β-arrestins bind to opioid receptors phosphorylated by G protein-coupled receptor kinases and promote receptor internalization and desensitization. Studies indicated that β-arrestins are required in the development of morphine tolerance in mice. In the current study, we investigated the potential regulatory effects of morphine administration on β-arrestin 1 and β-arrestin 2 mRNA levels in different brain regions in rat using in situ hybridization method. Our results showed that the acute morphine administration (10 mg/kg) resulted in approximately 30% reduction in both β-arrestin 1 and β-arrestin 2 mRNA levels in hippocampus while the chronic morphine treatment (10 mg/kg, b.i.d., for 9 days) caused no significant change in level of either β-arrestin mRNA. In locus coeruleus, both acute and chronic morphine treatments resulted in significant decreases (over 50%) in β-arrestin 1 mRNA level but failed to induce any change in the level of β-arrestin 2 gene expression. The acute morphine administration had no significant effect on β-arrestin 1 or β-arrestin 2 mRNA level in periaqueductal gray and cerebral cortex. However, after chronic morphine treatment, β-arrestin 2 mRNA level decreased by 40% in periaqueductal gray and increased by 25% in cerebral cortex, in strong contrast to the unchanged β-arrestin 1 mRNA level in these two brain regions. Furthermore, spontaneous or naloxone-precipitated withdrawal of morphine that did not affect the level of β-arrestin 1 mRNA resulted in an aberrant increase (100% over control) in β-arrestin 2 mRNA level in hippocampus. Our results thus demonstrated for the first time that opiate administration regulates level of β-arrestin mRNAs in brain and the expression of β-arrestin 1 and β-arrestin 2 subtypes is differentially regulated in locus coeruleus, periaqueductal gray, and cerebral cortex by morphine. These data suggest that β-arrestin 1 and β-arrestin 2 may play different roles in the development of opioid tolerance and dependence.

HLA-B*58:01 allele is associated with augmented risk for both mild and severe cutaneous adverse reactions induced by allopurinol in Han Chinese

AIMnAllopurinol is widely used as an effective urate-lowering drug and is one of the most frequent causes of cutaneous adverse drug reactions (cADRs). Recently, a strong association of HLA-B*58:01 with allopurinol-induced severe cADRs was identified. This study investigated the predisposition to different types of allopurinol-cADRs conferred by HLA-B*5801 in a Han population from mainland China.nnnPATIENTS & METHODSnHLA-B genotyping was performed on 38 Chinese patients with different types of allopurinol-cADRs from 2008 to 2011.nnnRESULTSnAll the allopurinol-cADR patients carried HLA-B*58:01, in contrast with only 11.11% (7/63) in the allopurinol-tolerant patients (odds ratio [OR] = 580.07; p < 0.0001) and 13.99% (80/572) in a Han Chinese population from the human MHC database (dbMHC; OR: 471.09; p < 0.0001) carried the genotype. Each type of allopurinol cADRs revealed a statistically significant association with HLA-B*58:01. In particular, the risk of allopurinol-induced maculopapular eruption was significantly higher in patients with HLA-B*58:01 (OR: 339.00; p < 0.0001).nnnCONCLUSIONnThe strong association of both the mild and severe types of allopurinol cADRs with the HLA-B*58:01 allele were observed. The results indicated that the prospective use of a genetic test of HLA-B*58:01 might reduce the prevalence of allopurinol-induced cADRs. Original submitted 7 March 2012; Revision submitted 21 May 2012.

Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain.

Opioid agonist stimulates activation of G protein-coupled receptor kinase (GRK) and causes desensitization of opioid signaling, which plays an important role in opioid tolerance. The current study investigated the potential regulatory effects of acute and chronic morphine administration and withdrawal on GRK2 and GRK5 gene expression in rat brain. Our results showed that the initial morphine treatment (10 mg/kg) significantly increased GRK mRNA levels in cerebral cortex, hippocampus, and lateral thalamic nuclei. A significant decrease in GRK5 mRNA levels was observed in periaqueductal gray. In strong contrast, repeated administration of morphine for 9 days failed to cause any significant increase in GRK5 mRNA in any of these brain regions. Chronic morphine treatment resulted in 30-70% down-regulation of GRK2 expression in cerebral cortex, hippocampus, thalamus, and locus coeruleus, opposite to what observed with the single morphine administration. Moreover, spontaneous and naloxone-precipitated morphine withdrawal resulted in aberrant increases in GRK2 and GRK5 mRNA levels in these brain regions. Taken together, our study suggests that opioid not only induces rapid negative feedback regulation on opioid signals through activation of GRK but also exerts its impact, via controlling levels of GRK gene expression, on the regulatory machinery itself over a longer period of time in brain.

Chronic morphine treatment and withdrawal induce up-regulation of c-Jun N-terminal kinase 3 gene expression in rat brain.

Chronic opiate applications produce long-term impacts on many functions of the brain and induce tolerance, dependence, and addiction. It has been demonstrated that opioid drugs are capable to induce apoptosis of neuronal cells, but the mechanism is not clear. c-Jun N-terminal kinase 3 (JNK3), specifically expressed in brain, has been proved to mediate neuronal apoptosis and is involved in opiate-induced cell apoptosis in vitro. The present study investigated the effect of opioid administration on expression of JNK3, an important mediator involved in apoptosis of neurons, in rat brain. Our results showed that single or chronic injection of morphine resulted in a 45-50% increase in the level of JNK3 mRNA in frontal cortex, while no significant change was detected in other brain regions such as thalamus, hippocampus and locus coeruleus. Similar to what was observed after the acute or chronic morphine administration, no significant change in JNK3 expression was detected in locus coeruleus following cessation of the chronic morphine administration. However, interestingly, sustained elevation of JNK3 expression peaked on day 14 after cessation of morphine treatment was observed in the brain regions such as hippocampus and thalamus, where acute or chronic morphine treatment did not cause any significant change in JNK3 gene expression. The increased JNK3 mRNA in these brain areas returned to the control levels in 28 days following cessation of chronic morphine treatment. Taken together, these results demonstrated for the first time that the expression of JNK3 gene is regulated by opioids and that chronic opioid administration and withdrawal could induce sustained elevation of JNK3 mRNA in many important brain areas. The changes in JNK3 gene expression in brain induced by chronic opioid treatment may play a role in opioid-induced apoptosis and neurotoxicity.

Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro.

The SAM and SH3 domain containingxa01 (SASH1) gene was originally identified as a potential tumor suppressor gene in breast cancer, mapped on chromosome 6q24.3. The expression of SASH1 plays a prognostic role in human colon cancer. Its expression is frequently downregulated in several human malignancies. However, the biological function of SASH1 in melanoma cells is yet to be determined. In this study, in order to investigate the tumor suppressive effects of the SASH1 gene, an A-375 stable melanoma cell line was established, overexpressing the SASH1 gene. The stable cell line was examined using proliferation assay, apoptosis assay, cell cycle analysis and real-time PCR. The results indicated that the tumor suppressive activity of SASH1 derived from G2/M arrest in A-375 cells, and that the phosphorylation of Cdc2 or the disruption of cyclinxa0B-Cdc2 binding may be responsible for the G2/M arrest.

SASH1 regulates melanocyte transepithelial migration through a novel Gαs–SASH1–IQGAP1–E-Cadherin dependent pathway

One important function of melanocytes (MCs) is to produce and transfer melanin to neighbouring keratinocytes (KCs) to protect epithelial cells from UV radiation. The mechanisms regulating the specific migration and localisation of the MC lineage remain unknown. We have found three heterozygous mutations that cause amino acid substitutions in the SASH1 gene in individuals with a kind of dyschromatosis. In epidermal tissues from an affected individual, we observed the increased transepithelial migration of melanocytes. Functional analyses indicate that these SASH1 mutations not only cause the increased migration of A375 cells and but also induce intensive bindings with two novel cell adhesion partners IQGAP1 and Gαs. Further, SASH1 mutations induce uniform loss of E-Cadherin in human A375 cells. Our findings suggest a new scaffold protein SASH1 to regulate IQGAP1-E-Cadherin signalling and demonstrate a novel crosstalking between GPCR signalling, calmodulin signalling for the modulation of MCs invasion.

Association study of miR‑149 rs2292832 and miR‑608 rs4919510 and the risk of hepatocellular carcinoma in a large‑scale population.

Polymorphisms in pre‑microRNAs (miRNAs) or mature miRNAs may influence miRNA processing or target binding, thus contributing to tumorigenesis and cancer development. The present study aimed to evaluate whether miR‑149 rs2292832 (C>T) and miR‑608 rs4919510 (G>C) are associated with the risk and clinical characteristics of hepatocellular carcinoma (HCC) in a large‑scale population. miR‑149 rs2292832 and miR‑608 rs4919510 were genotyped in a total of 993 patients with HCC and 992 unrelated healthy subjects by Sequenom MassARRAY. The results showed that, compared with the reference CC genotype, the TC+TT genotype of miR‑149 was more highly associated with HCC [CC vs. TC+TT: Odds ratio (OR)=1.384, 95% confidence interval (CI)=1.013‑1.892, P=0.041], and was also associated with an increased risk of hepatitisxa0B virus (HBV)‑associated HCC (CC vs. TC+TT: OR=1.453, 95% CI=1.034‑2.042, P=0.031). However, no significant association between miRNA‑608 rs4919510 and the risk of HCC/HBV‑associated HCC was found. In addition, these two SNPs were shown not to be correlated with a range of clinical characteristics. The present study may provide an indicator for identification of the high risk of HCC in patients.

Up-Regulated Expression and Aberrant DNA Methylation of LEP and SH3PXD2A in Pre-Eclampsia

The primary mechanism underlying pre-eclampsia (PE) remains one of the most burning problems in the obstetrics and gynecology. In this study, we performed an expression profiling screen and detected 1312 genes that were differentially expressed (p<0.05 and fold change >1.5) in PE placentas, including LEP and SH3PXD2A. After validating the microarray results, we conducted the quantitative methylation analysis of LEP and SH3PXD2A in preeclamptic (nu200a=u200a16) versus normal placentas (nu200a=u200a16). Our results showed that many CpG sites close to the transcriptional start site (TSS) of LEP gene were hypomethylated in placentas from pregnancies with PE compared with those of in controls, including the TSS position (pu200a=u200a0.001), the binding sites of Sp1 (pu200a=u200a1.57×10−4), LP1 (pu200a=u200a0.023) and CEBPα (pu200a=u200a0.031). Luciferase reporter analysis confirmed the aberrant methylation of LEP promoter and CEBPα co-transfection had a role in the regulation of gene expression. Our results indicated the aberrant LEP promoter methylation was involved in the development of PE. We did not find a significant methylation differences between groups in the promoter region of SH3PXD2A, however, a CGI region in the gene body (CGI34) presented a higher methylation in preeclamptic placentas (pu200a=u200a1.57×10−4), which might promote the efficiency of gene transcription. We speculated that SH3PXD2A may take part in the pathogenesis of PE through its role in the regulation of trophoblast cell invasion in the period of placenta formation.

A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1.

Methylenetetrahydrofolate reductase gene polymorphisms and cerebral palsy in Chinese infants

Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) have been suggested as being associated with cerebral palsy (CP) but the evidence is uncertain. The purpose of this study was to investigate whether MTHFR gene polymorphisms contribute to the development of CP in Chinese infants. For this study, 169 health controls and 159 infants with CP including 43 cases also suffering from mental retardation (MR) were recruited. Genomic DNA was prepared from venous blood and all five single nucleotide polymorphisms in MTHFR (rs4846049, rs1476413, rs1801131, rs1801133 and rs9651118) were genotyped using TaqMan technology. There were no significant differences in allele or genotype frequencies between the CP patients and controls at any of the five genetic polymorphisms. Subgroup analysis found statistically significant difference in allele and genotype frequencies between cases with both CP and MR (CP + MR) compared with both CP-only cases and controls at rs4846049, rs1476413 and rs1801131. The frequencies of the T alleles of rs4846049, rs1476413 and the G allele of rs1801131 were greater in the CP + MR patients than in the CP-only patients and controls. This study provides the first evidence pointing to a MTHFR gene polymorphism as a potential risk factor for CP combined with MR.