Biomedical subjects

Seung Kyoon Woo

Publications and source records attributed to Seung Kyoon Woo.

12 recordsLinked to original sources

PubMed2006

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-alpha 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.

Acute Kidney Injury↗

PubMed2006

Mre11-Rad50-Nbs1 complex is activated by hypertonicity.

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including gammaH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and gammaH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.

ATP-Binding Cassette Transporters↗

PubMed2004

Maturation of TonEBP expression in developing rat kidney.

Tonicity-responsive enhancer binding protein (TonEBP) is a transcriptional activator of the Rel family. In the renal medulla, TonEBP stimulates genes encoding proteins involved in cellular accumulation of organic osmolytes, the vasopressin-regulated urea transporters (UT-A), and heat shock protein 70. To understand the role of TonEBP in the development of urinary concentrating ability, TonEBP expression during rat kidney development was investigated. In embryonic kidneys, TonEBP immunoreactivity was detected 16 days postcoitus in the cytoplasm of the endothelial cells surrounding the medullary collecting ducts (MCD). By 20 days, TonEBP was detected in most tubular profiles in the medulla, including the loop of Henle and MCD, and interstitial cells. The intensity of TonEBP immunoreactivity was much higher in the vasa recta than the tubules. In addition, immunoreactivity was localized predominantly to the cytoplasm. On postnatal day 1, two major changes were observed. TonEBP immunoreactivity shifted to the nucleus, and the intensity of TonEBP immunoreactivity of the tubules increased dramatically. These changes were associated with an increase in TonEBP and sodium-myo-inositol cotransporter mRNA abundance. Thereafter, TonEBP expression in tubular profiles increased moderately. The adult pattern of TonEBP expression was established at postnatal day 21 coincident with full maturation of the renal medulla. Thus expression of TonEBP in developing kidneys occurred predominantly in the medulla and preceded expression of its target genes, including UT-A. These data suggest that TonEBP contributes to the development of urine-concentrating ability.

Animals↗

PubMed2003

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-siRNA's. 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.

Aldehyde Reductase↗

PubMed2002

Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity.

In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.

Animals↗

PubMed2002

Dimerization is required for phosphorylation and DNA binding of TonEBP/NFAT5.

TonEBP (NFAT5) is a newly identified member of the Rel family of transcriptional activators that include NF-kappaB and NFAT1 to NFAT4. Activated in response to hypertonicity, TonEBP stimulates transcription of transporters of organic osmolytes, certain cytokines, and a molecular chaperone. We provide biochemical data demonstrating that full-length TonEBP dimerizes via the C-terminus of the Rel-homology domain (CRHD). The two polyglutamine motifs were not involved. The dimerization was not affected by nucleocytoplasmic shifts in TonEBP in response to changes in ambient tonicity. Preventing the dimer formation by deleting the CRHD did not affect the nucleocytoplasmic shifts. On the other hand, deletion of the CRHD prevented DNA binding and eliminated the dominant negative activity of a C-terminal truncated TonEBP. Furthermore, phosphorylation was dramatically reduced especially in hypertonic conditions by deletion of the CRHD. We conclude that dimerization is required for proper phosphorylation of TonEBP as well as DNA binding.

Amino Acid Motifs↗

PubMed2002

TonEBP transcriptional activator in the cellular response to increased osmolality.

TonEBP is a member of the Rel family of transcriptional activators, distinct from other members NFkappaB and NFAT. In the medulla of mammalian kidney, TonEBP is a local differentiation factor that stimulates the transcription of several genes. The activity of TonEBP is regulated by changes in diuretic status mainly at the level of nucleocytoplasmic distribution. Some of the genes stimulated by TonEBP encode transporters and a synthetic enzyme for cellular accumulation of compatible osmolytes, which protect cells from the deleterious effects of hypertonicity by lowering cellular ionic strength. Other genes include the vasopressin-regulated urea transporters that play a key role in building up high urea concentrations in the renal medulla during antidiuresis and a heat shock protein 70 that protects cells from the deadly effects of urea. TonEBP is also abundantly expressed in other tissues such as brain, heart, liver, and activated T-cells. TonEBP plays a significant role in immunity by stimulating several cytokine genes in the T-cells. A lot more needs to be learned about the genes regulated by TonEBP and signaling pathways from hypertonicity to the activation of TonEBP.

Animals↗

PubMed2002

Adaptation of kidney medulla to hypertonicity: role of the transcription factor TonEBP.

The osmolality of the mammalian kidney medulla is very high. The high osmolality provides the driving force for water reabsorption and urinary concentration, key functions of the kidney for maintaining proper body fluid volume and blood pressure. Salt and urea are the major solutes in the renal medullary interstitium. Unfortunately, high salt (hypertonicity) causes DNA damage and cell death. In response, the renal medullary cells adapt to the hypertonicity by accumulating compatible osmolytes. A regulatory protein, tonicity-responsive enhancer binding protein (TonEBP), plays a central role in the accumulation of these compatible osmolytes by stimulating genes whose products either actively transport or synthesize the appropriate osmolytes. TonEBP is active under isotonic conditions. It responds to both an increase and a decrease in ambient tonicity, in opposite directions, which involves changes in its abundance and nucleocytoplasmic distribution. In the kidney medulla, however, nucleocytoplasmic distribution is the major site of control, under normal conditions of diuresis and antidiuresis.

Animals↗

PubMed2002

TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity.

While hyperosmolality of the kidney medulla is essential for urinary concentration, it imposes a great deal of stress. Cells in the renal medulla adapt to the stress of hypertonicity (hyperosmotic salt) by accumulating organic osmolytes. Tonicity-responsive enhancer (TonE) binding protein (TonEBP) (or NFAT5) stimulates transcription of transporters and a synthetic enzyme for the cellular accumulation of organic osmolytes. We found that dominant-negative TonEBP reduced expression of HSP70 as well as the transporters and enzyme. Near the major histocompatibility complex class III locus, there are three HSP70 genes named HSP70-1, HSP70-2, and HSC70t. While HSP70-1 and HSP70-2 were heat inducible, only HSP70-2 was induced by hypertonicity. In the 5' flanking region of the HSP70-2 gene, there are three sites for TonEBP binding. In cells transfected with a reporter plasmid containing this region, expression of luciferase was markedly stimulated in response to hypertonicity. Coexpression of the dominant-negative TonEBP reduced the luciferase expression. Mutating all three sites in the reporter plasmid led to a complete loss of induction by hypertonicity. Thus, TonEBP rather than heat shock factor stimulates transcription of the HSP70-2 gene in response to hypertonicity. We conclude that TonEBP is a master regulator of the renal medulla for cellular protection against high osmolality via organic osmolytes and molecular chaperones.

5' Flanking Region↗

PubMed2002

Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity.

Osmolality of the mammalian renal medulla is high because of the operation of the urinary concentrating mechanism. To understand molecular events during the early phase of cellular adaptation to hypertonicity, we performed comprehensive searches for genes induced in response to hypertonicity using a cell line (mIMCD3) derived from the inner medullary collecting duct of mouse kidney. PCR-based subtractive hybridization of cDNA pools and cDNA microarray analysis were used. We report 12 genes whose mRNA expression is significantly increased within 4 h after exposure to hypertonicity. The increase in mRNA expression was the result of increased transcription. Many are either stress response genes or growth regulatory genes, supporting the notion that hypertonicity evokes the stress response and growth regulation in cells. Experiments using inhibitors revealed that mitogen-activated protein kinases were commonly involved in signaling for the induction of genes by hypertonicity. Tyrosine kinases and phosphatidylinositol 3-kinase also play a significant role. Signaling pathways for stimulation of transcription appeared quite diverse in that each gene was sensitive to different combinations of inhibitors.

Animals↗

PubMed2001

Hydration status affects nuclear distribution of transcription factor tonicity responsive enhancer binding protein in rat kidney.

Tonicity responsive enhancer binding protein (TonEBP) is the transcription factor that regulates tonicity responsive expression of proteins that catalyze cellular accumulation of compatible osmolytes. In cultured MDCK cells, hypertonicity stimulates the activity of TonEBP via a combination of increased protein abundance and increased nuclear localization. For investigating regulation of TonEBP in the kidney, rats were subjected to water loading or dehydration. Water loading lowered urine osmolality and mRNA expression of sodium/myo-inositol cotransporter (SMIT), a target gene of TonEBP, in the renal medulla; dehydration doubled the urine osmolality and increased SMIT mRNA expression. In contrast, overall abundance of TonEBP and its mRNA measured by immunoblot and ribonuclease protection assay, respectively, was not affected. Immunohistochemical analysis, however, revealed that nuclear distribution of TonEBP is generally increased throughout the medulla in dehydrated animals compared with water loaded animals. Increased nuclear localization was particularly dramatic in thin limbs. Notable exceptions were the middle to terminal portions of the inner medullary collecting ducts and blood vessels, where a change in TonEBP distribution was not evident. Immunohistochemical detection of SMIT mRNA revealed that the changes in nuclear distribution of TonEBP correlate with expression of SMIT. It is concluded that under physiologic conditions, nucleocytoplasmic distribution is the dominant mode of regulation of TonEBP in the renal medulla.

Animals↗

PubMed1998

A Method for Assaying Deubiquitinating Enzymes.

A general method for the assay of deubiquitinating enzymes was described in detail using (125)I-labeled ubiquitin-fused alphaNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.

Journal Article↗