Seung Kyoon Woo

Cis- andtrans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

Background: Sur1-NCCa-ATP channels implicated in acute CNS injury are hypothesized to be formed by co-association of Sur1 and a nonselective cation channel. Results: Sur1 and Trpm4 form heteromers that exhibit pharmacological properties of Sur1 and biophysical properties of Trpm4. Conclusion: Sur1 and Trpm4 co-assemble to form the unique Sur1-Trpm4 channel. Significance: Identification of Sur1-Trpm4 channels has broad implications in acute CNS injuries. The sulfonylurea receptor 1 (Sur1)-NCCa-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NCCa-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and KATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.

Silencing of tonEBP/NFAT5 transcriptional activator by RNA interference

TonEBP is a transcriptional activator that is expressed throughout development in many tissues and cell types. In the kidney medulla, TonEBP appears to be an important local regulator of differentiation by virtue of stimulating several genes. To study the function of TonEBP, two small interfering RNA (siRNA) duplexes were developed that reduced TonEBP expression effectively via RNA interference. The silencing lasted only 3 d after introduction of the TonEBP-siRNAs. As expected, TonEBP-driven reporter gene expression and expression of the sodium/myo-inositol cotransproter (SMIT), aldose reductase (AR) and heat shock protein 70 (HSP70) mRNA were significantly decreased in cells where TonEBP expression was silenced. These data provide direct evidence that the SMIT, AR, and HSP70 genes are targets of TonEBP, although the potential role of other proteins, such as accessory proteins, cannot be excluded. The TonEBP-siRNA is an effective tool that should prove useful in the investigation of loss-of-function relationship in cells.

Comparison of Experimental Lung Injury from Acute Renal Failure with Injury due to Sepsis

Background: Acute renal failure (ARF) and acute respiratory distress syndrome (ARDS) coexist frequently, and the mortality rate of this combination is very high. It is well established that cytokines and chemokines play a major role in the pathogenesis of ARDS. In addition, heat shock proteins (HSPs) have been shown to be protective against ARDS. Objectives: The purpose of this study was to investigate the pathophysiology of ARDS in two different conditions, sepsis and ARF. Methods: We examined five different rat animal models including sham-operated control, sepsis and three ARF models induced by renal ischemia/reperfusion injury, bilateral nephrectomy or bilateral ligation of renal pedicles. We analyzed pulmonary histology, pulmonary vascular permeability, cellular infiltration, and expression of cytokines, chemokines and HSPs. Results: Like sepsis, the three forms of ARF led to ARDS, as manifested by increased pulmonary vascular permeability and histological changes consistent with ARDS. On the other hand, ARF and sepsis differed in that ARF was associated with markedly lower levels of pulmonary cellular infiltration. Furthermore, while pulmonary expression of tumor necrosis factor-α increased in sepsis, cytokine-induced neutrophil chemoattractant 2 increased in nephrectomized rats indicating that different inflammatory mediators were involved in the injury mechanism. Finally, pulmonary expression of multiple HSPs including HSP27-1, HSP70, HSP70-4, HSP70-8 and HSP90 was significantly different between the two conditions. Conclusions: We conclude that the pathophysiology of ARDS following ARF is distinct from that in sepsis. ARF-induced ARDS is characterized by a low level of cellular infiltration, induction of cytokine-induced neutrophil chemoattractant 2, and a discrete expression profile of HSPs.

Inhibition of the Sur1-Trpm4 Channel Reduces Neuroinflammation and Cognitive Impairment in Subarachnoid Hemorrhage

Background and Purpose— Subarachnoid hemorrhage (SAH) can leave patients with memory impairments that may not recover fully. Molecular mechanisms are poorly understood, and no treatment is available. The sulfonylurea receptor 1–transient receptor potential melastatin 4 (Sur1-Trpm4) channel plays an important role in acute central nervous system injury. We evaluated upregulation of Sur1-Trpm4 in humans with SAH and, in rat models of SAH, we examined Sur1-Trpm4 upregulation, its role in barrier dysfunction and neuroinflammation, and its consequences on spatial learning. Methods— We used Förster resonance energy transfer to detect coassociated Sur1 and Trpm4 in human autopsy brains with SAH. We studied rat models of SAH involving filament puncture of the internal carotid artery or injection of blood into the subarachnoid space of the entorhinal cortex. In rats, we used Förster resonance energy transfer and coimmunoprecipitation to detect coassociated Sur1 and Trpm4, we measured immunoglobulin G extravasation and tumor necrosis &agr; overexpression as measures of barrier dysfunction and neuroinflammation, and we assessed spatial learning and memory on days 7 to 19. Results— Sur1-Trpm4 channels were upregulated in humans and rats with SAH. In rats, inhibiting Sur1 using antisense or the selective Sur1 inhibitor glibenclamide reduced SAH-induced immunoglobulin G extravasation and tumor necrosis &agr; overexpression. In models with entorhinal SAH, rats treated with glibenclamide for 7 days after SAH exhibited better platform search strategies and better performance on incremental and rapid spatial learning than vehicle-treated controls. Conclusions— Sur1-Trpm4 channels are upregulated in humans and rats with SAH. Channel inhibition with glibenclamide may reduce neuroinflammation and the severity of cognitive deficits after SAH.

Sequential activation of hypoxia-inducible factor 1 and specificity protein 1 is required for hypoxia-induced transcriptional stimulation of Abcc8

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

Methemoglobin Is an Endogenous Toll-Like Receptor 4 Ligand—Relevance to Subarachnoid Hemorrhage

Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NFκB) in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb), heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS) contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα), from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.

The Sur1-Trpm4 channel regulates NOS2 transcription in TLR4-activated microglia

BackgroundHarmful effects of activated microglia are due, in part, to the formation of peroxynitrite radicals, which is attributable to the upregulation of inducible nitric oxide (NO) synthase (NOS2). Because NOS2 expression is determined by Ca2+-sensitive calcineurin (CN) dephosphorylating nuclear factor of activated T cells (NFAT), and because Sur1-Trpm4 channels are crucial for regulating Ca2+ influx, we hypothesized that, in activated microglia, Sur1-Trpm4 channels play a central role in regulating CN/NFAT and downstream target genes such as Nos2.MethodsWe studied microglia in vivo and in primary culture from adult rats, and from wild type, Abcc8−/− and Trpm4−/− mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation.ResultsIn microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca2+]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca2+]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca2+/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation.ConclusionsSur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca2+-sensitive gene expression, including Nos2.

Complex N-Glycosylation Stabilizes Surface Expression of Transient Receptor Potential Melastatin 4b Protein

Background: N-Glycosylation is important for the function and regulation of ion channels but has not been examined for Trpm4. Results: N-Glycosylation is not required for surface expression, but complex N-glycosylation stabilizes Trpm4b surface expression. Conclusion: Complex N-glycosylation of Trpm4b has important functional implications. Significance: By promoting surface expression, N-glycosylation contributes importantly to calcium regulation byTrpm4b. N-Glycosylation is important for the function and regulation of ion channels. We examined the role of N-glycosylation of transient receptor potential melastatin (Trpm) 4b, a membrane glycoprotein that regulates calcium influx. Trpm4b was expressed in vivo in all rat tissues examined. In each tissue, Trpm4b had a different molecular mass, between ∼129 and ∼141 kDa, but all reverted to ∼120 kDa following treatment with peptide:N-glycosidase F, consistent with N-glycosylation being the principal form of post-translational modification of Trpm4b in vivo. In six stable isogenic cell lines that express different levels of Trpm4b, two forms were found, high mannose, core-glycosylated and complex, highly glycosylated (HG), with HG-Trpm4b comprising 85% of the total Trpm4b expressed. For both forms, surface expression was directly proportional to the total Trpm4b expressed. Complex N-glycosylation doubled the percentage of Trpm4b at the surface, compared with high mannose N-glycosylation. Mutation of the single N-glycosylation consensus sequence at Asn-988 (Trpm4b-N988Q), located near the pore-forming loop between transmembrane helices 5 and 6, prevented glycosylation, but did not prevent surface expression, impair formation of functional membrane channels, or alter channel conductance. In transfection experiments, the time courses for appearance of HG-Trpm4b and Trpm4b-N988Q on the surface were similar. In experiments with cycloheximide inhibition of protein synthesis, the time course for disappearance of HG-Trpm4b from the surface was much slower than that for Trpm4b-N988Q. We conclude that N-glycosylation is not required for surface expression or channel function, but that complex N-glycosylation plays a crucial role in stabilizing surface expression of Trpm4b.

SUR1-TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling

Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin‐4 (AQP4), an astrocyte water channel, or of Na+‐permeable channels, which mediate cellular osmolyte influx. However, the spatio‐temporal functional interactions between AQP4 and Na+‐permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na+‐permeable channels that are newly upregulated. Here, using co‐immunoprecipitation and Förster resonance energy transfer, we report that AQP4 physically co‐assembles with the sulfonylurea receptor 1—transient receptor potential melastatin 4 (SUR1‐TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell‐swelling studies using calcein fluorescence imaging of COS‐7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1‐TRPM4‐AQP4, was required for fast, high‐capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold‐injury to the cerebellum, we found that astrocytes newly upregulate SUR1‐TRPM4, that AQP4 co‐associates with SUR1‐TRPM4, and that genetic inactivation of the solute pore of the SUR1‐TRPM4‐AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1‐TRPM4‐AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4‐mediated pathological conditions.