EZH2 promotes angiogenesis in peritoneal dialysis by epigenetically activating SP4 expression in the IL-6/sIL-6R signalling pathway.

Background: Interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) promotes peritoneal angiogenesis by stimulating SP4-mediated vascular endothelial growth factor (VEGF) production in peritoneal dialysis (PD). Moreover, histone methyltransferase enhancer of zeste homologue 2 (EZH2) is involved in IL-6/sIL-6R signalling via the acceleration of vascular endothelial growth factor (VEGF)-induced angiogenesis. However, the molecular mechanism underlying how EZH2 epigenetically activates VFGF expression in IL-6/sIL-6R signalling during PD is still unclear. Methods and Results: In this study, we measured the expression of EZH2, DNMT3B and SP4 in human peritoneal mesothelial cells (HPMCs) treated with IL-6/sIL-6R stimulation and/or EZH2 overexpression, silencing or inhibition. Methylation of the CpG site in the SP4 promoter region and VEGF production were measured under these treatments in HPMCs. Moreover, tube formation in human umbilical vein endothelial cells (HUVECs) was detected following treatment with conditioned media from these stimulated HPMCs. The 5/6 nephrectomy (5/6Nx) rat model was established, and the rats were injected with peritoneal dialysate. EZH2, DNMT3B and SP4 expression and microvessels were analysed in 5/6Nx + PD rats treated with IL-6/sIL-6R and EZH2 overexpression. The results showed that IL-6/sIL-6R and EZH2 overexpression enhanced the expression of EZH2, DNMT3B and SP4, but EZH2 silencing/inhibition reduced these expression levels. The results for VEGF production and tube formation in vitro followed the same trend. IL-6/sIL-6R and EZH2 overexpression increased the methylation percentage of the -170 bp CpG site in the SP4 promoter region in HPMCs. Moreover, IL-6/sIL-6R and EZH2 overexpression stimulated EZH2, DNMT3B and SP4 expression and promoted angiogenesis in 5/6Nx + PD rats. Conclusions: Thus, this study indicated that EZH2 is involved in IL-6/sIL-6R signalling and epigenetically regulates SP4 expression, thereby stimulating VEGF production and angiogenesis in PD. Targeting EZH2 is expected to be a novel therapeutic approach for end-stage renal disease (ESRD) patients with PD treatment.

Over-representation of potential SP4 target genes within schizophrenia-risk genes.

Reduction of Sp4 expression causes age-dependent hippocampal vacuolization and many other intermediate phenotypes of schizophrenia in Sp4 hypomorphic mice. Recent human genetic studies from both the Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) and the Genome-Wide Association Study (GWAS) validated SP4 as a schizophrenia-risk gene over the exome-wide or the genome-wide significance. Truncation of the human SP4 gene has an odds ratio of 9.37 (3.38-29.7) for schizophrenia. Despite successful identification of many schizophrenia-risk genes, it is unknown whether and how these risk genes may interact with each other in the development of schizophrenia. By taking advantage of the specific localization of the GC-boxes bound by SP4 transcription factors, I analyzed the relative abundance of these GC-boxes in the proximal promoter regions of schizophrenia-risk genes. I found that the GC-box containing genes are significantly over-represented within schizophrenia-risk genes, suggesting that SP4 is not only a high-risk gene for schizophrenia, but may also act as a hub of network in the regulation of many other schizophrenia-risk genes via these GC-boxes in the pathogenesis of schizophrenia.

Sp4 controls constitutive expression of neuronal serine racemase and NF-E2-related factor-2 mediates its induction by valproic acid.

Serine racemase (SR) synthesizes l-type serine to its enantimor, d-serine which participates in physiological processes and in pathological conditions. In the central nervous system, SR is highly expressed in neurons and astrocytes but expressed at relatively lower amount in microglia. However, the mechanism by which SR is highly expessed in neurons is hitherto unknown. We report that the SR mRNA and protein levels in Neuro-2a were increased by valproic acid (VPA), a neuron differentiation stimulator as well as a histone deacetylase inhibitor. SR proximal promoter contained nine putative Sp-binding elements and in the exon 1, three putative anti-oxidant elements (AREs) were conservative among human, rat, and mouse genome. The promoter constructs including 5'-, 3'-fragment, and full length fragment from mouse were individually cloned into a luciferase reporter. Using dual-luciferase assay, the promoter harboring 3'-fragment contained much lower activity than the construct containing 5'-fragment which was though resistant to VPA induction, relative to 3'-fragment. Overexpression of Sp4 or Nrf2 increased whereas knockdown of either decreased Srr mRNA and SR protein. Using site-directed mutagenesis, mutation of Sp-binding elements or AREs in the constructs significantly decreased luciferase activity of the corresponding promoter construct. With chromatin immunoprecipitation, Sp4 was demonstrated to interact directly with the Sp-binding elements whereas Nrf2 bound AREs in Srr mRNA promoter. Altogether, our study highlights that Sp4 controls constitutive expression of SR in neuron and VPA mediates SR expression in N2A cells which is associated with its effect on neuron differentiation, that is, the effect is mediated via Nrf2.

Whole Genome Expression Analyses of miRNAs and mRNAs Suggest the Involvement of miR-320a and miR-155-3p and their Targeted Genes in Lithium Response in Bipolar Disorder.

Lithium is the mainstay in the maintenance of bipolar disorder (BD) and the most efficacious pharmacological treatment in suicide prevention. Nevertheless, its use is hampered by a high interindividual variability and important side effects. Genetic and epigenetic factors have been suggested to modulate lithium response, but findings so far have not allowed identifying molecular targets with predictive value. In this study we used next generation sequencing to measure genome-wide miRNA expression in lymphoblastoid cell lines from BD patients excellent responders (ER, n = 12) and non-responders (NR, n = 12) to lithium. These data were integrated with microarray genome-wide expression data to identify pairs of miRNA/mRNA inversely and significantly correlated. Significant pairs were prioritized based on strength of association and in-silico miRNA target prediction analyses to select candidates for validation with qRT-PCR. Thirty-one miRNAs were differentially expressed in ER vs. NR and inversely correlated with 418 genes differentially expressed between the two groups. A total of 331 of these correlations were also predicted by in-silico algorithms. miR-320a and miR-155-3p, as well as three of their targeted genes (CAPNS1 (Calpain Small Subunit 1) and RGS16 (Regulator of G Protein Signaling 16) for miR-320, SP4 (Sp4 Transcription Factor) for miR-155-3p) were validated. These miRNAs and mRNAs were previously implicated in psychiatric disorders (miR-320a and SP4), key processes of the central nervous system (CAPNS1, RGS16, SP4) or pathways involved in mental illnesses (miR-155-3p). Using an integrated approach, we identified miRNAs and their targeted genes potentially involved in lithium response in BD.

Krüppel-like factor 5 (Klf5) regulates expression of mouse T1R1 amino acid receptor gene (Tas1r1) in C2C12 myoblast cells.

T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.

Transcription factor Sp4 is required for hyperalgesic state persistence.

Understanding how painful hypersensitive states develop and persist beyond the initial hours to days is critically important in the effort to devise strategies to prevent and/or reverse chronic painful states. Changes in nociceptor transcription can alter the abundance of nociceptive signaling elements, resulting in longer-term change in nociceptor phenotype. As a result, sensitized nociceptive signaling can be further amplified and nocifensive behaviors sustained for weeks to months. Building on our previous finding that transcription factor Sp4 positively regulates the expression of the pain transducing channel TRPV1 in Dorsal Root Ganglion (DRG) neurons, we sought to determine if Sp4 serves a broader role in the development and persistence of hypersensitive states in mice. We observed that more than 90% of Sp4 staining DRG neurons were small to medium sized, primarily unmyelinated (NF200 neg) and the majority co-expressed nociceptor markers TRPV1 and/or isolectin B4 (IB4). Genetically modified mice (Sp4+/-) with a 50% reduction of Sp4 showed a reduction in DRG TRPV1 mRNA and neuronal responses to the TRPV1 agonist-capsaicin. Importantly, Sp4+/- mice failed to develop persistent inflammatory thermal hyperalgesia, showing a reversal to control values after 6 hours. Despite a reversal of inflammatory thermal hyperalgesia, there was no difference in CFA-induced hindpaw swelling between CFA Sp4+/- and CFA wild type mice. Similarly, Sp4+/- mice failed to develop persistent mechanical hypersensitivity to hind-paw injection of NGF. Although Sp4+/- mice developed hypersensitivity to traumatic nerve injury, Sp4+/- mice failed to develop persistent cold or mechanical hypersensitivity to the platinum-based chemotherapeutic agent oxaliplatin, a non-traumatic model of neuropathic pain. Overall, Sp4+/- mice displayed a remarkable ability to reverse the development of multiple models of persistent inflammatory and neuropathic hypersensitivity. This suggests that Sp4 functions as a critical control point for a network of genes that conspire in the persistence of painful hypersensitive states.

Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons.

Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na(+)/K(+)-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons.

O-GlcNAc modification of Sp3 and Sp4 transcription factors negatively regulates their transcriptional activities.

The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome.

Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein.

The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1-4 bound to IE62 and amino acids 238-258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner.

Histone Deacetylase Inhibitors Inhibit Rhabdomyosarcoma by Reactive Oxygen Species-Dependent Targeting of Specificity Protein Transcription Factors.

The two major types of rhabdomyosarcoma (RMS) are predominantly diagnosed in children, namely embryonal (ERMS) and alveolar (ARMS) RMS, and patients are treated with cytotoxic drugs, which results in multiple toxic side effects later in life. Therefore, development of innovative chemotherapeutic strategies is imperative, and a recent genomic analysis suggested the potential efficacy of reactive oxygen species (ROS)-inducing agents. Here, we demonstrate the efficacy of the potent histone deacetylase (HDAC) inhibitors, panobinostat and vorinostat, as agents that inhibit RMS tumor growth in vivo, induce apoptosis, and inhibit invasion of RD and Rh30 RMS cell lines. These effects are due to epigenetic repression of cMyc, which leads to decreased expression of cMyc-regulated miRs-17, -20a, and -27a; upregulation of ZBTB4, ZBTB10, and ZBTB34; and subsequent downregulation of Sp transcription factors. We also show that inhibition of RMS cell growth, survival and invasion, and repression of Sp transcription factors by the HDAC inhibitors are independent of histone acetylation but reversible after cotreatment with the antioxidant glutathione. These results show a novel ROS-dependent mechanism of antineoplastic activity for panobinostat and vorinostat that lies outside of their canonical HDAC-inhibitory activity and demonstrates the potential clinical utility for treating RMS patients with ROS-inducing agents.

Transcription factor SP4 phosphorylation is altered in the postmortem cerebellum of bipolar disorder and schizophrenia subjects.

Transcription factors play important roles in the control of neuronal function in physiological and pathological conditions. We previously reported reduced levels of transcription factor SP4 protein, but not transcript, in the cerebellum in bipolar disorder and associated with more severe negative symptoms in schizophrenia. We have recently reported phosphorylation of Sp4 at S770, which is regulated by membrane depolarization and NMDA receptor activity. The aim of this study was to investigate SP4 S770 phosphorylation in bipolar disorder and its association with negative symptoms in schizophrenia, and to explore the potential relationship between phosphorylation and protein abundance. Here we report a significant increase in SP4 phosphorylation in the cerebellum, but not the prefrontal cortex, of bipolar disorder subjects (n=10) (80% suicide) compared to matched controls (n=10). We found that SP4 phosphorylation inversely correlated with SP4 levels independently of disease status in both areas of the human brain. Moreover, SP4 phosphorylation in the cerebellum positively correlated with negative symptoms in schizophrenia subjects (n=15). Further, we observed that a phospho-mimetic mutation in truncated Sp4 was sufficient to significantly decrease Sp4 steady-state levels, while a non-phosphorylatable mutant showed increased stability in cultured rat cerebellar granule neurons. Our results indicate that SP4 S770 phosphorylation is increased in the cerebellum in bipolar disorder subjects that committed suicide and in severe schizophrenia subjects, and may be part of a degradation signal that controls Sp4 abundance in cerebellar granule neurons. This opens the possibility that modulation of SP4 phosphorylation may contribute to the molecular pathophysiology of psychotic disorders.

Restoration of Sp4 in Forebrain GABAergic Neurons Rescues Hypersensitivity to Ketamine in Sp4 Hypomorphic Mice.

BACKGROUND: Ketamine produces schizophrenia-like behavioral phenotypes in healthy people. Prolonged ketamine effects and exacerbation of symptoms after the administration of ketamine have been observed in patients with schizophrenia. More recently, ketamine has been used as a potent antidepressant to treat patients with major depression. The genes and neurons that regulate behavioral responses to ketamine, however, remain poorly understood. Sp4 is a transcription factor for which gene expression is restricted to neuronal cells in the brain. Our previous studies demonstrated that Sp4 hypomorphic mice display several behavioral phenotypes relevant to psychiatric disorders, consistent with human SP4 gene associations with schizophrenia, bipolar disorder, and major depression. Among those behavioral phenotypes, hypersensitivity to ketamine-induced hyperlocomotion has been observed in Sp4 hypomorphic mice. METHODS: In the present study, we used the Cre-LoxP system to restore Sp4 gene expression, specifically in either forebrain excitatory or GABAergic inhibitory neurons in Sp4 hypomorphic mice. Mouse behavioral phenotypes related to psychiatric disorders were examined in these distinct rescue mice. RESULTS: Restoration of Sp4 in forebrain excitatory neurons did not rescue deficient sensorimotor gating nor ketamine-induced hyperlocomotion. Restoration of Sp4 in forebrain GABAergic neurons, however, rescued ketamine-induced hyperlocomotion, but did not rescue deficient sensorimotor gating. CONCLUSIONS: Our studies suggest that the Sp4 gene in forebrain GABAergic neurons regulates ketamine-induced hyperlocomotion.

GlyT-1 Inhibition Attenuates Attentional But Not Learning or Motivational Deficits of the Sp4 Hypomorphic Mouse Model Relevant to Psychiatric Disorders.

Serious mental illness occurs in 25% of the general population, with many disorders being neurodevelopmental, lifelong, and debilitating. The wide variation and overlap in symptoms across disorders increases the difficulty of research and treatment development. The NIMH Research Domain of Criteria initiative aims to improve our understanding of the molecular and behavioral consequences of specific neurodevelopmental mechanisms across disorders, enabling targeted treatment development. The transcription factor Specificity Protein 4 (SP4) is important for neurodevelopment and is genetically associated with both schizophrenia and bipolar disorder. Reduced Sp4 expression in mice (hypomorphic) reproduces several characteristics of psychiatric disorders. We further tested the utility of Sp4 hypomorphic mice as a model organism relevant to psychiatric disorders by assessing cognitive control plus effort and decision-making aspects of approach motivation using cross-species-relevant tests. Sp4 hypomorphic mice exhibited impaired attention as measured by the 5-Choice Continuous Performance Test, an effect that was attenuated by glycine type-1 transporter (GlyT-1) inhibition. Hypomorphic mice also exhibited reduced motivation to work for a reward and impaired probabilistic learning. These deficits may stem from affected anticipatory reward, analogous to anhedonia in patients with schizophrenia and other psychiatric disorders. Neither positive valence deficit was attenuated by GlyT-1 treatment, suggesting that these and the attentional deficits stem from different underlying mechanisms. Given the association of SP4 gene with schizophrenia and bipolar disorder, the present studies provide support that personalized GlyT-1 inhibition may treat attentional deficits in neuropsychiatric patients with low SP4 levels.

Class I histone deacetylase-mediated repression of the proximal promoter of the activity-regulated cytoskeleton-associated protein gene regulates its response to brain-derived neurotrophic factor.

We examined the transcriptional regulation of the activity-regulated cytoskeleton-associated protein gene (Arc), focusing on BDNF-induced Arc expression in cultured rat cortical cells. Although the synaptic activity-responsive element (SARE), located -7 kbp upstream of the Arc transcription start site, responded to NMDA, BDNF, or FGF2, the proximal region of the promoter (Arc/-1679) was activated by BDNF or FGF2, but not by NMDA, suggesting the presence of at least two distinct Arc promoter regions, distal and proximal, that respond to extracellular stimuli. Specificity protein 4 (SP4) and early growth response 1 (EGR1) controlled Arc/-1679 transcriptional activity via the region encompassing -169 to -37 of the Arc promoter. We found that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, significantly enhanced the inductive effects of BDNF or FGF2, but not those of NMDA on Arc expression. Inhibitors of class I/IIb HDACs, SAHA, and class I HDACs, MS-275, but not of class II HDACs, MC1568, enhanced BDNF-induced Arc expression. The enhancing effect of TSA was mediated by the region from -1027 to -1000 bp, to which serum response factor (SRF) and HDAC1 bound. The binding of HDAC1 to this region was reduced by TSA. Thus, Arc expression was suppressed by class I HDAC-mediated mechanisms via chromatin modification of the proximal promoter whereas the inhibition of HDAC allowed Arc expression to be markedly enhanced in response to BDNF or FGF2. These results contribute to our understanding of the physiological role of Arc expression in neuronal functions such as memory consolidation.

Increased SP4 and SP1 transcription factor expression in the postmortem hippocampus of chronic schizophrenia.

Altered levels of transcription factor specificity protein 4 (SP4) and 1 (SP1) in the cerebellum, prefrontal cortex and/or lymphocytes have been reported in severe psychiatric disorders, including early psychosis, bipolar disorder, and chronic schizophrenia subjects who have undergone long-term antipsychotic treatments. SP4 transgenic mice show altered hippocampal-dependent psychotic-like behaviours and altered development of hippocampal dentate gyrus. Moreover, NMDAR activity regulates SP4 function. The aim of this study was to investigate SP4 and SP1 expression levels in the hippocampus in schizophrenia, and the possible effect of antipsychotics and NMDAR blockade on SP protein levels in rodent hippocampus. We analysed SP4 and SP1 expression levels in the postmortem hippocampus of chronic schizophrenia (n = 14) and control (n = 11) subjects by immunoblot and quantitative RT-PCR. We tested the effect of NMDAR blockade on SP factors in the hippocampus of mouse treated with an acute dose of MK801. We also investigated the effect of subacute treatments with haloperidol and clozapine on SP protein levels in the rat hippocampus. We report that SP4 protein and both SP4 and SP1 mRNA expression levels are significantly increased in the hippocampus in chronic schizophrenia. Likewise, acute treatment with MK801 increased both SP4 and SP1 protein levels in mouse hippocampus. In contrast, subacute treatment with haloperidol and clozapine did not significantly alter SP protein levels in rat hippocampus. These results suggest that SP4 and SP1 upregulation may be part of the mechanisms deregulated downstream of glutamate signalling pathways in schizophrenia and might be contributing to the hippocampal-dependent cognitive deficits of the disorder.

Store-operated calcium entry promotes the degradation of the transcription factor Sp4 in resting neurons.

Calcium (Ca(2+)) signaling activated in response to membrane depolarization regulates neuronal maturation, connectivity, and plasticity. Store-operated Ca(2+) entry (SOCE) occurs in response to depletion of Ca(2+) from endoplasmic reticulum (ER), mediates refilling of this Ca(2+) store, and supports Ca(2+) signaling in nonexcitable cells. We report that maximal activation of SOCE occurred in cerebellar granule neurons cultured under resting conditions and that this Ca(2+) influx promoted the degradation of transcription factor Sp4, a regulator of neuronal morphogenesis and function. Lowering the concentration of extracellular potassium, a condition that reduces neuronal excitability, stimulated depletion of intracellular Ca(2+) stores, resulted in the relocalization of the ER Ca(2+) sensor STIM1 into punctate clusters consistent with multimerization and accumulation at junctions between the ER and plasma membrane, and induced a Ca(2+) influx with characteristics of SOCE. Compounds that block SOCE prevented the ubiquitylation and degradation of Sp4 in neurons exposed to a low concentration of extracellular potassium. Knockdown of STIM1 blocked degradation of Sp4, whereas expression of constitutively active STIM1 decreased Sp4 abundance under depolarizing conditions. Our findings indicated that, in neurons, SOCE is induced by hyperpolarization, and suggested that this Ca(2+) influx pathway is a distinct mechanism for regulating neuronal gene expression.

Specificity protein 4 (Sp4) regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are important glutamatergic receptors mediating fast excitatory synaptic transmission in the brain. The regulation of the four subunits of AMPA receptors, GluA1-4, is poorly understood. Excitatory synaptic transmission is highly energy-demanding, and this energy is derived mainly from the oxidative pathway. Recently, we found that specificity factor regulates all subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme. COX is also regulated by nuclear respiratory factor 1 (NRF-1), which transcriptionally controls the Gria2 (GluA2) gene of AMPA receptors. The goal of the present study was to test our hypothesis that Sp-factors (Sp1, Sp3, and/or Sp4) also regulate AMPA subunit genes. If so, we wish to determine if Sp-factors and NRF-1 function via a complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel mechanism. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, we found that Sp4, but not Sp1 or Sp3, regulates the Gria2, but not Gria1, 3, or 4, subunit gene of the AMPA receptor in a concurrent and parallel manner with NRF-1. Thus, Sp4 and NRF-1 both mediate the tight coupling between neuronal activity and energy metabolism at the transcriptional level.

Phosphorylation of the transcription factor Sp4 is reduced by NMDA receptor signaling.

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites.

The anticancer antibiotic mithramycin-A inhibits TRPV1 expression in dorsal root ganglion neurons.

Activation of peripheral nociceptors by products of inflammation has been shown to be dependent on specific sensory transducing elements such as the capsaicin receptor, TRPV1. The development of high-affinity antagonists to TRPV1 as well as to other receptors capable of detecting noxious stimuli has now become a major focus in analgesic development. Another critical feature of nociception is the relative abundance of a particular pain transducing receptor under normal or pathophysiologic conditions. Increases in expression and/or changes in distribution of nociceptive receptors such as TRPV1 have been correlated with progression of tissue injury and persistence of pain behaviors. Although some details are emerging as to what regulates nociceptor-specific gene expression, compounds that could potentially be used to block or reverse over-expression of nociceptive gene expression are essentially absent. In our efforts to better understand the transcriptional regulation of TRPV1 in sensory neurons, we identified an anticancer agent, mithramycin-A, that decreased TRPV1 expression in primary rat dorsal root ganglion (DRG) neurons. Mithramycin-A dose-dependently (10-50 nM) decreased endogenous TRPV1 mRNA content and appeared to decrease TRPV1-like protein expression in DRG neurons. We also observed that mithramycin-A directed a decrease in the number of capsaicin-responsive DRG neurons without a significant change in the capsaicin-response magnitudes. Interestingly, mithramycin-A also reduced the mRNA encoding Sp1 and Sp4 in DRG neurons, transcription factors previously found to positively regulate TRPV1 expression in sensory neurons. Taken together, we propose that mithramycin-A directs an inhibitory effect on a subpopulation of capsaicin-responsive DRG neurons that utilize Sp1-like factors for TRPV1 expression. Given the therapeutic correlate of mithramycin-A effectiveness in the treatment of certain cancers, small molecule transcriptional inhibitors such as mithramycin-A may serve as useful tools of discovery in pain transduction and possibly future analgesic development.

Regulation of Na(+)/K(+)-ATPase by neuron-specific transcription factor Sp4: implication in the tight coupling of energy production, neuronal activity and energy consumption in neurons.

A major source of energy demand in neurons is the Na(+)/K(+)-ATPase pump that restores the ionic gradient across the plasma membrane subsequent to depolarizing neuronal activity. The energy comes primarily from mitochondrial oxidative metabolism, of which cytochrome c oxidase (COX) is a key enzyme. Recently, we found that all 13 subunits of COX are regulated by specificity (Sp) factors, and that the neuron-specific Sp4, but not Sp1 or Sp3, regulates the expression of key glutamatergic receptor subunits as well. The present study sought to test our hypothesis that Sp4 also regulates Na(+)/K(+)-ATPase subunit genes in neurons. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutational analysis, over-expression, and RNA interference studies, we found that Sp4, with minor contributions from Sp1 and Sp3, functionally regulate the Atp1a1, Atp1a3, and Atp1b1 subunit genes of Na(+)/K(+)-ATPase in neurons. Transcripts of all three genes were up-regulated by depolarizing KCl stimulation and down-regulated by the impulse blocker tetrodotoxin (TTX), indicating that their expression was activity-dependent. Silencing of Sp4 blocked the up-regulation of these genes induced by KCl, whereas over-expression of Sp4 rescued them from TTX-induced suppression. The effect of silencing or over-expressing Sp4 on primary neurons was much greater than those of Sp1 or Sp3. The binding sites of Sp factors on these genes are conserved among mice, rats and humans. Thus, Sp4 plays an important role in the transcriptional coupling of energy generation and energy consumption in neurons.