Jian Ying Chuang

Dynamic Interaction between Sigma-1 Receptor and Kv1.2 Shapes Neuronal and Behavioral Responses to Cocaine

The sigma-1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone protein, is an interorganelle signaling modulator that potentially plays a role in drug-seeking behaviors. However, the brain site of action and underlying cellular mechanisms remain unidentified. We found that cocaine exposure triggers a Sig-1R-dependent upregulation of D-type K(+) current in the nucleus accumbens (NAc) that results in neuronal hypoactivity and thereby enhances behavioral cocaine response. Combining ex vivo and in vitro studies, we demonstrated that this neuroadaptation is caused by a persistent protein-protein association between Sig-1Rs and Kv1.2 channels, a phenomenon that is associated to a redistribution of both proteins from intracellular compartments to the plasma membrane. In conclusion, the dynamic Sig-1R-Kv1.2 complex represents a mechanism that shapes neuronal and behavioral response to cocaine. Functional consequences of Sig-1R binding to K(+) channels may have implications for other chronic diseases where maladaptive intrinsic plasticity and Sig-1Rs are engaged.

Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Sumoylation of Specificity Protein 1 Augments Its Degradation by Changing the Localization and Increasing the Specificity Protein 1 Proteolytic Process

Although specificity protein 1 (Sp1) accumulation has been found in various tumor strains, its mechanism is still not very clear. Herein, we found that modification of Sp1 by SUMO-1 facilitates Sp1 degradation. Our findings revealed that, although the amounts of Sp1 and Sp1 mutant (K16R) [Sp1(K16R)] mRNA in cells were equal, the protein level of Sp1(K16R) was higher than that of wild-type Sp1. We also proved that this sumoylation site was not the residue at which ubiquitination occurred. Invitro and in vivo pull-down assays revealed that more sumoylated Sp1 was localized in the cytoplasm, and the interaction between SUMO-1-Sp1 and the proteasome subunit rpt6 in HeLa cells was enhanced. In addition, although Sp1 accumulated in the tumorous cervical tissue, it was not prone to sumoylation. Finally, by overexpression of HA (hemagglutinin)-SUMO-1-Sp1-myc, HA-Sp1-myc, and HA-Sp1(K16R), we found that modification of Sp1 by SUMO-1 was important for Sp1 proteolysis. In conclusion, modification of Sp1 by SUMO-1 altered its localization and then increased its interaction with rpt6. This interaction increased the efficiency of Sp1 proteolytic processing and ubiquitination and then resulted in Sp1 degradation. Therefore, sumoylation of Sp1 is attenuated during tumorigenesis in order to increase Sp1 stability.

Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells.

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro‐apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP‐Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub‐G1 fraction, caspase‐3 cleavage, and annexin‐V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53‐dependent apoptotic pathways in the wild‐type p53 cells but not in the p53‐mutated or p53‐deleted cell lines when these cells were infected with adeno‐GFP‐Sp1 virus. In addition, A549 (p53+/+) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53−/−) cell viability was significantly inhibited by adeno‐GFP‐Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression‐induced apoptotic cell death in transforming cells.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

Significance The endoplasmic reticulum (ER), although functioning as protein synthesis machinery in the cell, plays other important roles that have yet to be fully unveiled. We found here that the ER can directly send an envoy protein, the sigma-1 receptor (Sig-1R), to the nuclear envelope (NE), where the Sig-1R begins to recruit chromatin-remodeling molecules through the NE integral protein emerin to control gene transcription. Thus, the Sig-1R represents a molecule that shapes the functional connection between the NE and the DNA. We also demonstrate in this study that cocaine, a Sig-1R agonist, down-regulates the critical enzyme monoamine oxidase B that influences the cocaine-induced dopamine level in a dopamine transporter-independent manner via this never-before-described, to our knowledge, Sig-1R-linked genomic action of cocaine. The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

Heat Shock Protein 90 Is Important for Sp1 Stability during Mitosis

Our previous study has revealed that heat shock protein (Hsp) 90 can interact with Sp1 to regulate the transcriptional activity of 12(S)-lipoxygenase. Herein, we further found that the interaction between Hsp90 and Sp1 occurred during mitosis. By geldanamycin (GA) treatment and knockdown of Hsp90, we found that this interaction during mitosis was involved in the maintenance of Sp1 stability, and that the phospho-c-Jun N-terminal kinase (JNK)-1 level also decreased. As the JNK-1 was knocked down by the shRNA of JNK-1, Sp1 was degraded through a ubiquitin-dependent proteasome pathway. In addition, for mutation of the JNK-1 phosphorylated residues of Sp1, namely, Sp1(T278/739A) and Sp1(T278/739D), the effect of GA on Sp1 stability was reversed. Finally, based on the involvement of Hsp90 in Sp1 stability, the transcriptional activities of p21(WAF1/CIP1) and 12(S)-lipoxygenase under GA treatment were observed to have decreased. Taken together, Hsp90 is important for maintaining Sp1 stability during mitosis by the JNK-1-mediated phosphorylation of Sp1 to enable division into daughter cells and to regulate the expression of related genes in the interphase.

Hydrogen peroxide induces Sp1 methylation and thereby suppresses cyclin B1 via recruitment of Suv39H1 and HDAC1 in cancer cells

Sp1 is an important transcription factor for a number of genes that regulate cell growth and survival. Sp1 is an anchor protein that recruits other factors to regulate its target genes positively or negatively, but the mechanism of its functional switch by which positive or negative coregulators are recruited is not clear. In this study, we found that Sp1 could be methylated and that methylation was maintained by treatment with pargyline, a lysine-specific demethylase 1 (LSD1) inhibitor or knock LSD1 down directly. Hydrogen peroxide treatment increased the methylation of Sp1 and repressed Sp1 transcriptional activity. Investigation of the mechanism by which methylation decreased Sp1 activity found that methylation of Sp1 increased the recruitment of Su(var) 3-9 homologue 1(Suv39H1) and histone deacetylase 1 (HDAC1) to the cyclin B1 promoter, resulting in deacetylation and methylation of histone H3 and subsequent downregulation of cyclin B1. Finally, downregulation of cyclin B1 led to cell cycle arrest at the G2 phase. These results show that methylation of Sp1 causes it to act as a negative regulator by recruiting Suv39H1 and HDAC1 to induce chromatin remodeling. This finding that methylation acts as a functional switch provides new insight into the modulation of Sp1 transcriptional activity.

Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

Phosphorylation of p300 increases its protein degradation to enhance the lung cancer progression

p300 is a transcription cofactor for a number of nuclear proteins. Most studies of p300 have focused on the regulation of its function, which primarily includes its role as a transcription co-factor for a number of nuclear proteins. In this study, we found that p300 was highly phosphorylated and its level was decreased during mitosis and tumorigenesis. In vitro and in vivo experiments aimed showed that cyclin-dependent kinase 1 (CDK1) and ERK1/2 phosphorylated p300 on Ser1038 and Ser2039. Mutations of Ser1038 and Ser2039 increased p300 protein stability and levels. Inhibition of p300 degradation by blocking its phosphorylation decreased the proliferation and metastasis activity of lung cancer cells, indicating that p300 acts as a tumor suppressor in lung cancer tumorigenesis. Investigation of the molecular mechanism showed that blocking p300 phosphorylation disrupted chromatin condensation and the increased the acetylation of histone H3. Analysis of cell cycle progression in HA-p300-S2A-expressing cells by flow cytometry showed that the p300 mutants arrested the cells at S-phase or delayed the mitotic entry and exit. The expression of several important oncogenes, MMP-9, vimentin, β-catenin, N-cadherin and c-myc, was negatively regulated by p300. In conclusion, during lung tumorigenesis, a phosphorylation-mediated decrease in p300 level enhanced oncogene expression during interphase and decreased histone H3 acetylation during mitosis, which promoted lung cancer progression.