Biomedical subjects

Xiujun Pi

Publications and source records attributed to Xiujun Pi.

7 recordsLinked to original sources

PubMed2007

Aberrant Gi protein coupled receptor-mediated cell survival signaling in rheumatoid arthritis B cell lines.

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive lipid that transmits potent signals through a family of G protein coupled receptors with resultant anti-apoptotic and pro-angiogenic effects. We have recently reported that lymphoblastoid B cell lines (LCLs) from rheumatoid arthritis (RA) patients are resistant to Fas-mediated cell death due to over-production of S1P, secondary to over-activity of sphingosine kinase-1 (SphK1). Here we investigated the signaling events that S1P triggers in those cells. Our results show that RA-derived LCLs display increased constitutive enzymatic activity of phosphatidylinositol 3-kinase (PI3K). Incubation of LCLs with a PI3K inhibitor wortmannin reversed PI3K over-activity and the resistance to Fas-mediated cell death. Incubation of RA LCLs with nanomolar concentration of S1P triggered exaggerated activation of both SphK and PI3K in RA LCLs compared to control cells. PI3K was mapped upstream of SphK, since wortmannin could block SphK activation by S1P. S1P signaling effect could be blocked by the Gi/G0 protein inhibitor, pertussis toxin and by an inhibitor of S1P-receptor interaction, suramin. S1P receptor expression levels did not appear to be the cause of disparate S1P-triggered signaling, since LCLs from RA patients and their healthy twin controls did not show statistically significant differences in the expression levels of the five known S1P receptors, as determined by quantitative real time reverse transcription-polymerase chain reaction analyses. Thus, we conclude that Fas death signaling aberration in RA LCLs is caused by extracellular S1P, which triggers PI3K-dependent SphK over-activity through a Gi protein-coupled receptor-mediated signaling cascade.

Apoptosis↗

PubMed2006

Sphingosine kinase 1-mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells.

OBJECTIVE: It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls. METHODS: Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1-phosphate (S1P), and by investigating the ability of the SPHK inhibitor N,N-dimethylsphingosine and isozyme-specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations. RESULTS: LCLs from patients with RA displayed disease-specific Fas-mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real-time PCR analysis showed higher SPHK-1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK-1 (but not SPHK-2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fas-induced apoptosis. Additionally, resistance to Fas-mediated signaling was reversed by siRNA oligonucleotides specific for SPHK-1 but not by oligonucleotides specific for SPHK-2. CONCLUSION: These findings demonstrate disease-specific resistance to Fas-mediated death signaling in patients with RA and implicate increased SPHK-1 activity as the cause of this aberration.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

OBJECTIVE: To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. METHODS: The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. RESULTS: In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. CONCLUSION: Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2004

Decreased expression of brain cAMP response element-binding protein gene following pentylenetetrazol seizure.

The present study investigated whether the expression of the cAMP response element-binding protein (CREB) in the rat brain is altered following an acute self-limited seizure induced by pentylenetetrazol (PTZ). Male rats were injected intraperitoneally with a single convulsive dose (45 mg/kg) of PTZ, and the matched controls were given saline. For immunohistochemistry, animals were perfused with 4% parafomaldehyde at 24 h following PTZ seizures, and CREB immunoreactivity was examined in rat brain. For real-time RT-PCR, animals were sacrificed at 2 and 24 h and 1 week following PTZ seizures. Tissues from different rat brain regions were micropunched and subjected to real-time RT-PCR using Taqman probe. The CREB immunoreactive profiles were significantly decreased in CA3 and dentate gyrus of hippocampal formation, sensory cerebral cortex and thalamus at 24 h after PTZ seizures. Consistent with changes in CREB immunoreactivity, levels of CREB mRNA were significantly decreased in the hippocampus, cerebral cortex, amygdala and thalamus at 24 h after PTZ seizures. No significant change was found for CREB mRNA expression in these regions at 2 h or 1 week following PTZ seizures. These results show that a brief seizure caused a decline in CREB expression up to 24 h later.

Animals↗

PubMed2003

Promoter usage and estrogen regulation of prolactin receptor gene in the brain of the female rat.

Mechanisms underlying hormonal regulation of prolactin receptor (PRL-R) gene in the brain are unknown. The 5'-untranslated region of PRL-R mRNA in peripheral tissues contains at least three alternative first exons (1A, B, C) that are expressed as tissue-specific, suggesting the differential usage of PRL-R gene promoters. The present study aimed to investigate: (1). the possible regulation of PRL-R mRNA levels by estrogen in in vitro and in vivo tissues; (2). which exon (1A, or 1B, or 1C)-containing PRL-R mRNA transcript is expressed in the brain, and (3). how the specific exon 1-containing mRNA is affected by estrogen by using RT-PCR, Southern blot and 5'Race PCR techniques. The RT-PCR results showed that PRL-R mRNA was detected in the cerebral cortex and pons medulla in addition to the choroid plexus and hypothalamus in the female rat. The expression of PRL-R mRNA was up-regulated by estrogen treatment in the rat brain tissue and in the GT1-7 cell culture. Both exon 1A- and 1C-containing transcripts were expressed in all four regions, suggesting that promoters 1A and 1C for the PRL-R gene are utilized in the rat brain. Exon 1A-containing transcript was up-regulated by estrogen treatment in all four brain regions, whereas Exon 1C-containing transcript was up-regulated by estrogen treatment in 3 of the 4 brain regions, cerebral cortex being the exception. Exon 1B-containing transcript was neither detectable nor induced by estrogen treatment in any of the brain regions examined. The RT-PCR results were confirmed by partial isolation of 5'-untranslated regions of exon 1A- and 1C-containing PRL-R mRNA transcripts from brain tissue by using 5'Race PCR. The present result confirms the expression of PRL-R mRNA in the cerebral cortex and pons medulla in the female rat. The levels of PRL-R mRNA were up-regulated by estrogen in rat brain tissue and GT1-7 cell cultures. Detection of exon 1A- and 1C-containing transcripts implies that the promoter 1A and 1C are active in the female rat brain. Estrogen differentially regulates expression of the PRL-R mRNA in the different brain regions by increasing the utilization of PRL-R gene promoters 1A and 1C in the female rat.

Alternative Splicing↗

PubMed2002

Sex difference and estrous cycle: expression of prolactin receptor mRNA in rat brain.

The short and long forms of prolactin receptor (PRL-R) mRNA have been detected in the female rat brain. The present study aimed to investigate: (1) if the PRL-R mRNA is expressed in the male rat brain; (2) if expression levels in the female brain vary during the estrous cycle. All animals were sacrificed between 12:00 and 14:00 h. Radioactive RNase protection assay was used to measure mRNA levels. The results showed that both forms of PRL-R mRNA were expressed to varying degrees in the choroid plexus (ChP), preoptic area (POA), mediobasal hypothalamus (MBH), cerebral cortex (CTX) and pons-medulla PON) in both male and female rats. The average amount of both forms of PRL-R mRNA in the ChP, POA, MBH of cycling females was significantly higher than in the male rat. Among cycling female rats, the expression levels of both forms of PRL-R mRNA in the ChP, POA and MBH during proestrous were significantly greater than during diestrous or estrous. In proestrous females, the ChP expressed the highest levels of mRNA whereas the CTX contained the lowest. The ratios of short:long form mRNA were not significantly changed according to sex, estrous stage or brain regions although a slightly higher amount of the short form was observed. The detection of PRL-R mRNA in the male rat implicates that PRL may be involved in regulation of brain function in the male subject. The higher levels of PRL-R mRNA in female rats on proestrous suggest that PRL-R may be regulated by PRL or steroid hormones that show a surge on this day.

Animals↗

PubMed2002

Detection of prolactin receptor mRNA in the corpus striatum and substantia nigra of the rat.

The observation of prolactin modulation of the nigrostriatal dopaminergic system suggests the expression of prolactin receptor in the corpus striatum or substantia nigra. The present study investigated expression of prolactin receptor mRNA in tissues microdissected from the corpus striatum and substantia nigra of the rat. By using reverse transcription PCR combined with Southern hybridization, the long form of prolactin receptor mRNA was detected in the substantia nigra, caudate putamen, globus pallidus, and ventral pallidum in ovariectomized rats, whereas the short form was not detectable in any of these areas. Estrogen had no effect on expression of the long-form mRNA in the substantia nigra and corpus striatum. By using the RNase protection assay, the expression of both short and long forms of prolactin receptor mRNA was observed in the corpus striatum in ovariectomized rats. Again, levels of expression were not significantly altered by estrogen treatment. Both forms of prolactin receptor mRNA were clearly expressed in the choroid plexus and were up-regulated by estrogen treatment. The expression of both forms of prolactin receptor mRNA in nigrostriatal areas may help to support the hypothesis that prolactin has direct actions on these brain regions.

Animals↗