Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (− 83 to − 67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.     

Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival

Recently, mutations in the progranulin (PGRN) gene were found to cause familial and apparently sporadic frontotemporal lobe dementia (FTLD). Moreover, missense changes in PGRN were identified in patients with motor neuron degeneration, a condition that is related to FTLD. Most mutations identified in patients with FTLD until now have been null mutations. However, it remains unknown whether PGRN protein levels are reduced in the central nervous system from such patients. The effects of PGRN on neurons also remain to be established. We report that PGRN levels are reduced in the cerebrospinal fluid from FTLD patients carrying a PGRN mutation. We observe that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons. These results demonstrate that PGRN/GRN is a neurotrophic factor with activities that may be involved in the development of the nervous system and in neurodegeneration.     

Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice

Background  

The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model.     

Novel trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines as mu opioid receptor antagonists with improved opioid receptor selectivity profiles

A series of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines, mu opioid receptor antagonists, analogs of alvimopan, were prepared using solid phase methodology. This study led to the identification of a highly selective mu opioid receptor antagonist, which interacts selectively with mu peripheral receptors.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

ARA67/PAT1 functions as a repressor to suppress androgen receptor transactivation

The androgen receptor (AR) may recruit multiple coregulators for proper or optimal transactivation. Here we report the identification and characterization of ARA67/PAT1 as an AR coregulator from a prostate cDNA library. ARA67/PAT1 was screened out as an AR N terminus interacting protein. Interaction mapping shows that the cooperation of multiple domains within ARA67/PAT1 may be required for the maximal interaction with AR. ARA67/PAT1 functions as a repressor with better suppressive effects on AR compared to glucocorticoid receptor and estrogen receptor. Further mechanism dissection reveals that the interrupted AR cytoplasmic-nuclear shuttling may play a major role in ARA67/PAT1 mediated suppression on AR. Together, these results suggest that ARA67/PAT1 may function as a novel repressor that can modulate AR function in prostate cancer.