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. 2016 Dec 30;291(53):27279-27288.
doi: 10.1074/jbc.M116.737353. Epub 2016 Nov 14.

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1)

Sandra J Hewett  1 , Jingxue Shi  2 , Yifan Gong  2 , Krishnan Dhandapani  3 , Carol Pilbeam  4 , James A Hewett  5
Affiliations

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1)

Sandra J Hewett et al. J Biol Chem. .

Abstract

Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.

Keywords: N-methyl-d-aspartate receptor (NMDA receptor, NMDAR); cAMP response element-binding protein (CREB); constitutive expression; cyclooxygenase (COX); neuron; specificity protein 1 (Sp1); transcription; transcriptional regulation.

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Figures

FIGURE 1.
FIGURE 1.
Comparative analysis of constitutive transgenic luciferase levels with native COX-2 mRNA levels. A, three adult male PLuc371 transgenic mice were anesthetized and transcardially perfused with ice-cold saline. Organs were rapidly dissected and frozen, and luciferase activity was measured as described under “Materials and Methods.” Data are expressed as RLU normalized to mg of protein (mean ± S.E.). B, RNA was isolated from tissue homogenates from three adult male mice, and COX-2 mRNA was quantified via quantitative PCR. COX-2 mRNA expression in kidneys was used as the comparator (set to 1).
FIGURE 2.
FIGURE 2.
Constitutive COX-2 gene transcription in cultured transgenic cells. A, basal luciferase activity was assessed in brain (cerebral cortex) homogenates from three PLuc371 transgenic mice or whole cell lysates from neurons and astrocytes cultured from PLuc371 brains. Data are expressed as RLU normalized to mg of protein (mean ± S.E.; n = 3–4 cultures per cell type). B, comparison of the temporal pattern of PLuc371-luciferase expression in purified neuronal cultures (pure neurons; black bars) or neuronal cultures plated atop non-transgenic astrocytes (mixed cultures; gray bars) at 1, 4, 7, and 10 days in vitro. Data are expressed as RLU normalized to mg of protein (mean ± S.E.). Between groups (mixed versus pure), there were no significant differences as determined by two-way ANOVA (n = 3 each).
FIGURE 3.
FIGURE 3.
Developmental profile of NMDA receptor surface expression and function. A, NMDAR surface expression. Surface proteins of mixed cortical cell cultures of PLuc371 transgenic neurons and wild-type astrocytes were labeled with biotin at 1, 4, 7, and 10 days in vitro, and the biotinylated proteins were separated from total protein. Western blotting analysis was performed using a mouse monoclonal anti-NR1 antibody. Gel films were scanned, and densitometry was performed on five independent experiments. NR1 levels are expressed as -fold increase over day 1 (set to 1) in vitro. B, NMDA receptor function. Shown is a comparison of the temporal pattern of c-Fos mRNA expression in pure wild-type neurons at 1, 3, 5, 7, and 9 days in vitro as quantified by qRT-PCR. Relative mRNA expression on DIV 1 was set to 1 and used as the comparator (n = 3).
FIGURE 4.
FIGURE 4.
COX-2 mRNA expression in pure neuronal cultures derived from NR1 wild-type and null mutant embryos. Pure cortical neurons wild-type (NR1+/+) or null (NR1−/−) were harvested 1 and 7 days after plating, and COX-2 mRNA was quantified from total RNA isolates by qRT-PCR. The graph represents the mean ± S.E.-fold change in COX-2 mRNA expression at DIV 7 relative to mRNA expression on DIV 1 (represented as a dashed line, set to 1). An asterisk (*) indicates a significant between-group difference as assessed by unpaired Student's t test of the geometric means (p = 0.0204, n = 3).
FIGURE 5.
FIGURE 5.
Effect of NMDA receptor antagonism on c-Fos and COX-2 mRNA expression. DIV 5 pure neuronal cultures were treated with 30 μm APV (+ APV) or its vehicle (−APV) and c-Fos (A) and COX-2 (B) mRNA were quantified via qRT-PCR 2 days later. Relative mRNA expression on DIV 1 (dashed lines) was set to 1 and used as comparator. An asterisk (*) indicates a significant between-group (+/−APV) difference as assessed by unpaired Student's t test of the geometric means. p < 0.0001 for both c-fos and COX-2, n = 4.
FIGURE 6.
FIGURE 6.
Lack of effect of tetanus toxin on c-Fos and COX-2 mRNA expression. Pure neurons were treated with 300 ng/ml tetanus toxin (+TeNT) or its vehicle (−TeNT) on DIV 5 and c-Fos (A) and COX-2 (B) mRNA quantified via qRT-PCR on DIV 7. Results are expressed as -fold change relative to vehicle (−TeNT, set to 1). There was no significant difference as determined via unpaired Student's t test of the geometric means (n = three from three independent experiments). Top blot: Western blot demonstrating Efficacy of TeNT-induced synaptobrevin-2 (SB2) cleavage (representative of three blots).
FIGURE 7.
FIGURE 7.
CREB is involved constitutive neuronal COX-2 transcription. Four days after plating, pure neuron cultures from non-transgenic mice were co-transfected with the PLuc371 (luciferase) reporter vector together with A-CREB (dominant negative inhibitor of CREB) and pGIPZ (GFP) expression plasmids as described under “Materials and Methods.” Comparisons were made to cells transfected in parallel with PLuc371 reporter and pGIPZ (GFP) DNA plus empty vector. Luciferase activity in cell lysates was assessed 3 days after transfection (DIV 7) and normalized to GFP intensity, which was quantified via fluorescence microscopy. Luciferase activity in cells transfected with the control plasmid was set to 1, and data are expressed as -fold change from control. The asterisks (*) indicate a value significantly different from control as assessed by Mann-Whitney U test (p = 0.0043, n = 6).
FIGURE 8.
FIGURE 8.
A, role of SP1 or CRE DNA consensus motifs in neuronal COX-2 transcription. Four days after plating, pure neuron cultures were co-transfected with pGIPZ and unmodified PLuc371 or PLuc371 having mutated SP1 or CRE elements. Three days later, GFP intensity was quantified via fluorescence microcopy, after which cells were lysed, and luciferase activity was determined. Data are expressed as RLU + S.E./GFP intensity. An asterisk (*) indicates values that are significantly different from control (−APV) (*, p < 0.02; **, p < 0.0001), whereas a pound signs (#) represents a significant SP1 mutant group difference (p < 0.0001) as determined by two-way ANOVA followed by Bonferroni's t test for multiple comparisons (n = 3). B, ChIP analysis of Sp1 and CREB binding to the native PTGS2 promoter. Top, schematic representation of CREB and Sp1-binding sites in the proximal segment of the PTGS2 promoter. Bottom, cross-linked DNA-protein complexes immunoprecipitated (IP) using antibodies to Sp1 and CREB or non-immune IgG as well as an input chromatin sample were analyzed by PCR using primers specific to the COX-2 promoter. Percent protein binding to DNA was determined by normalizing PCR products derived from non-immune and anti-SP1 or anti-CREB IgG pulldown to Input. Data are expressed as the mean ± S.E. An asterisk (*) represents a significant increase in binding over non-immune IgG as determined via two-tailed t tests (p = 0.035 and 0.0017 for SP1 and CREB, respectively, n = 4 each).
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