Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells.

Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO) to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR) and hypoxia inducible factor-1α (HIF-1α). The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D) out of four potential HIF response elements of the hKOR gene (HIFA-D) synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing) produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing), suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation.

RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein-protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by (3) H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.

Effects of desferoxamine-induced hypoxia on neuronal human mu-opioid receptor gene expression.

The effect of desferoxamine (DFO)-induced hypoxia on neuronal human mu-opioid receptor (hMOR) gene expression was investigated using NMB cells. DFO decreased cell viability and increased cellular glutathione levels in a dose- and time-dependent manner. Confocal analysis using annexin-V-fluorescein and propidium iodide staining revealed that surviving/attached cells under DFO challenge were morphologically similar to control (vehicle-treated) cells. RT-PCR analysis demonstrated that the hypoxia inducible factor-1alpha (HIF-1alpha) mRNA level was augmented in these surviving neurons. DFO treatment for 8h or longer down-regulated the hMOR message, but not that of the delta-opioid receptor. Functional analysis using luciferase reporter assay showed that the hMOR 5'-regulatory region, from -357bp to translational initiation site (+1), contains the active promoter with an inhibitory region located in the -422 to -357bp region. DFO decreased hMOR promoter activity as compared to control. Mutation analysis suggested the existence of both dsDNA and ssDNA elements, located in a CT-rich region of hMOR, mediating the DFO-response. RT-PCR further revealed that DFO exhibited no effect on hMOR mRNA stability. In conclusion, DFO-induced hypoxia specifically affects neuronal hMOR gene expression at the transcriptional, not post-transcriptional, level.

Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis.

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. We show that targeting both the vascular niche and perivascular fibroblasts establishes "hospitable soil" to foster the incorporation of "seed," in this case, the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NOX4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 4] synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs (HgfiΔEC/iΔEC) aberrantly up-regulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in HgfiΔEC/iΔEC mice recapitulated the phenotype of human and mouse liver and lung fibrosis. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration.

Molecular basis underlying the poly C binding protein 1 as a regulator of the proximal promoter of mouse mu-opioid receptor gene.

Previous studies showed poly C binding protein 1 (PCBP) participating in the mu-opioid receptor (MOR) gene regulation via binding to a single-stranded (ss) DNA element. In this report, we therefore investigate the molecular basis of PCBP regulating the MOR gene expression. Various truncated PCBPs, including one domain (KH1, KH2, variable or KH3), two- (K12, K2v or Kv3) or three-sequential domains (K12v or K2v3), were constructed. The MOR ssDNA binding abilities of these truncated PCBPs were examined using electrophoretic mobility shift assay (EMSA). KH1 domain possessed a strong MOR ssDNA binding activity. Variable domain displayed no binding, and KH2 or KH3 domain possessed a weak MOR ssDNA binding activity. Binding of two-domain PCBPs indicated an additive effect of two-domain combinations. Interestingly, K2v3, a three-domain PCBP, displayed as strong ssDNA binding as that of K12v, suggesting synergism of KH2, KH3 and variable domains for the binding activity. Functional analysis demonstrated one-domain PCBPs exhibiting no transactivation on the MOR proximal promoter. Two-domain PCBPs displayed approximately 20% activity, while three-domain PCBPs displayed 70%-85% of full-length PCBP activity. Taken together, these results suggested that no single domain possessed sufficient functional activity to serve as an independent transactivation domain, and the combination of three sequential domains was necessary for its optimal activity to activate the MOR proximal promoter. In summary, our data suggested that cooperativity of three sequential domains is essential for PCBP functioning as a MOR gene regulator. Various ways in which this cooperativity could occur are discussed.

Identification of gamma-synuclein as a new PCBP1-interacting protein.

OBJECTIVES: PolyC binding protein 1 (PCBP1) is a transcriptional regulator of human mu-opioid receptor (hMOR) gene in the CNS and is also related to cancer/diseases. It possesses multi-roles that can be mediated by protein-protein interactions. To understand the mechanism controlling PCBP1 functions, PCBP1-interacting protein was investigated. METHODS: Using PCBP1 as the bait, a human brain cDNA library was screened via two-hybrid system. DNA sequence of candidate protein was confirmed using NCBI/SNP databases. Candidate protein in various cell lines was examined by RT-PCR. Glutathione-S-transferase (GST) pull-down and co-immunoprecipitation were used to validate the physical interaction. Its effects on hMOR gene regulation were examined. RESULTS: One clone was identified as gamma-synuclein110E, an SNP of gamma-synuclein110V. The interaction between PCBP1 and gamma-synuclein110E was confirmed by further validation and GST pull-down assay. Confocal analysis showed gamma-synuclein110E mainly expressing in the cytosol of human neuronal NMB cells. This interaction was confirmed by co-immunoprecipitation with NMB lysates, containing both proteins endogenously. Ectopic expression of gamma-synuclein110E or 110V did not alter hMOR mRNA level or promoter activity, suggesting no involvement of gamma-synuclein in modulating hMOR expression. Co-immunoprecipitation using gamma-synuclein110E or 110V overexpressed NMB cells with anti-PCBP1 antibody revealed a stronger intensity of co-immunoprecipitated gamma-synuclein band using gamma-synuclein110E-overexpressed cells as compared to that using gamma-synuclein110V-overexpressed cells. Synuclein110E was also identified in H292 (lung), HT29 (colon) and T47D (breast) cells, and this physical interaction was confirmed. CONCLUSION: We report a newly identified PCBP1-interacting protein, gamma-synuclein110E, and provide some insight into its complex role as well as discuss potential roles of this interaction.

Role of an AP-2-like element in transcriptional regulation of mouse mu-opioid receptor gene.

Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse mu-opioid receptor (MOR) gene. In this study, we defined another functional element located the in -450 to -400 bp (translational start site designated as +1) region of the proximal promoter, which is also essential for the full promoter activity. It is designated as the morAP-2-like element for its sequence homologous to the consensus AP-2 element. Surprisingly, electrophoretic mobility shift analysis (EMSA) revealed that Sp1 and Sp3, but not AP-2 proteins, were specifically bound to the morAP-2-like element. Mutation of the morAP-2-like element, resulting in a loss of Sp binding, led to an approximately 35% decrease in activity, further confirming the positive role of the morAP-2-like element in MOR gene expression. Dephosphorylation of Sp proteins with alkaline phosphatase also decreased Sp binding to the morAP-2-like element in EMSA, suggesting phosphorylation of Sp is essential for its binding to this element. However, direct or indirect activation of PKA, a classical G-protein coupled signaling pathway, resulted in no significant change of Sp binding to the morAP-2-like element, nor of the promoter activity the SH-SY5Y cells, MOR expressing cells, suggesting that phosphorylation of Sp does not involve PKA. These results suggest that the binding of different phosphorylated forms of Sp proteins to the morAP-2-like element may contribute to the fine tuning of MOR expression in different cells.

Differential promoter usage of mouse mu-opioid receptor gene during development.

Previously, we demonstrated that mouse mu-opioid receptor (MOR) gene expression is regulated by both distal and proximal promoters, with the latter playing a major role in controlling MOR transcription in the adult mouse brain. Here, we report studies of the relative usages of the mouse MOR dual promoters during murine development. We used the reverse transcription-polymerase chain reaction (RT-PCR) method, which gave results similar to those using binding assays or in situ hybridization. However, due to the greater sensitivity of RT-PCR method, we were able to detect the emergence of MOR as early as at embryonic day 8.5 (E8.5). We found that both proximal and distal promoters were active at E8.5. The proximal promoter initiated approximately two-thirds of total MOR transcripts at E8.5, with the distal promoter directing transcription of the remaining one-third. This is the greatest relative contribution of the distal promoter to MOR transcription we have observed during any time in development. Thereafter, the percentage of transcripts directed by the distal promoter gradually declined, and remained at a low but detectable level (approximately 5% of total MOR transcripts) throughout development and adulthood. Conversely, a progressive increase of the contribution of the proximal promoter to MOR transcription was observed during development, reaching its maximum in the adult. In summary, our results demonstrated the pivotal role of the proximal promoter in directing MOR transcription during murine development.

Effects of trichostatin A on neuronal mu-opioid receptor gene expression.

In this study, we determined the effects of a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on neuronal mu-opioid receptor (MOR) gene expression using human neuronal NMB cells, endogenously expressing MOR. Recruitment of two classes of HDAC, HDAC1 and HDAC2, to MOR promoter region in situ was detected via chromatin immunoprecipitation (ChIP) analysis with NMB cells. Functional analysis using the luciferase reporter gene system showed that TSA induced an approximately 3-fold increase of the promoter activity as compared to the vehicle treated group. Mutation analysis demonstrated that TSA response was mediated by both dsDNA (Sp1/Sp3 binding site) and ssDNA (PolyC binding protein1, PCBP, binding site) elements located in mouse MOR proximal core promoter region, further suggesting the functional importance of this cis-element, which shows high sequence homology between human and mouse MOR genes. ChIP analysis further suggested that TSA enhanced the recruitment of Sp1/Sp3 and PCBP to the promoter region, whereas no significant changes of total proteins were observed in response to TSA using Western blot analysis. Moreover, confocal images showed TSA-induced nuclear hot spots of endogenous PCBP in neuronal cells, whereas no obvious nuclear PCBP hotspot was observed in vehicle treated cells. Taken together, these results suggested that TSA enhanced neuronal MOR gene expression at the transcriptional level. RT-PCR analysis further revealed that TSA also decreased the steady-state level of MOR mRNA in a time-dependent manner by enhancing its instability. Thus, data suggest that TSA, an epigenetic regulator, affects neuronal MOR gene expression at both transcriptional and post-transcriptional levels.

Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells.

Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

Interplay of Sps and poly(C) binding protein 1 on the mu-opioid receptor gene expression.

The proximal promoter of mouse mu-opioid receptor (MOR) gene is the dominant promoter for directing MOR-1 gene expression in brain. Sp1/Sp3 (Sps) and poly(C) binding protein 1 (PCBP) bind to a cis-element of MOR proximal promoter. Functional interaction between Sps and PCBP and their individual roles on MOR proximal core promoter were investigated using SL2 cells, devoid of Sps and PCBP. Each factor contributed differentially to the promoter, with a rank order of activity Sp1>Sp3>PCBP. Functional analysis suggested the interplay of Sps and PCBP in an additive manner. The in vivo binding of individual Sps or PCBP to MOR proximal promoter was demonstrated using chromatin immunoprecipitation (ChIP). Re-ChIP assays further suggested simultaneous bindings of Sps and PCBP to the proximal promoter, indicating physiologically relevant communication between Sps and PCBP. Collectively, results documented that a functional coordination between Sps and PCBP contributed to cell-specific MOR gene expression.

Poly C binding protein, a single-stranded DNA binding protein, regulates mouse mu-opioid receptor gene expression.

Previously, a single-stranded (ss) DNA element, polypyrimidine (PPy) element, was found to be important for the proximal promoter activity of mouse micro-opioid receptor (MOR) gene in a neuronal cell model. In this study, we identified the presence of unknown ssDNA binding proteins specifically bound to MOR ssPPy element in the mouse brain, implicating the physiological significance of these proteins. To identify the ssDNA binding proteins, yeast one-hybrid system with PPy element as the bait was used to screen a mouse brain cDNA library. The clone encoding poly C binding protein (PCBP) was obtained. Its full-length cDNA sequence and protein with molecular weight approximately 38 kDa were confirmed. Electrophoretic mobility shift analysis (EMSA) revealed that PCBP bound to ssPPy element, but not doubled-stranded, in a sequence-specific manner. EMSA with anti-PCBP antibody demonstrated the involvement of PCBP in MOR ssPPy/proteins complexes of mouse brain and MOR expressing neuroblastoma NMB cells. Functional analysis showed that PCBP trans-activated MOR promoter as well as a heterologous promoter containing MOR PPy element. Importantly, ectopic expression of PCBP in NMB cells up-regulated the expression level of endogenous MOR gene in vivo in a dose-dependent manner. Collectively, above results suggest that PCBP participates in neuronal MOR gene expression.