14 results on '"Kotlo K"'
Search Results
2. Advances in DNA, histone, and RNA methylation mechanisms in the pathophysiology of alcohol use disorder.
- Author
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Cruise TM, Kotlo K, Malovic E, and Pandey SC
- Abstract
Alcohol use disorder (AUD) has a complex, multifactorial etiology involving dysregulation across several brain regions and peripheral organs. Acute and chronic alcohol consumption cause epigenetic modifications in these systems, which underlie changes in gene expression and subsequently, the emergence of pathophysiological phenotypes associated with AUD. One such epigenetic mechanism is methylation, which can occur on DNA, histones, and RNA. Methylation relies on one carbon metabolism to generate methyl groups, which can then be transferred to acceptor substrates. While DNA methylation of particular genes generally represses transcription, methylation of histones and RNA can have bidirectional effects on gene expression. This review summarizes one carbon metabolism and the mechanisms behind methylation of DNA, histones, and RNA. We discuss the field's findings regarding alcohol's global and gene-specific effects on methylation in the brain and liver and the resulting phenotypes characteristic of AUD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cruise, Kotlo, Malovic and Pandey.)
- Published
- 2023
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3. Increase in Chondroitin Sulfate and Decline in Arylsulfatase B May Contribute to Pathophysiology of COVID-19 Respiratory Failure.
- Author
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Tzankov A, Bhattacharyya S, Kotlo K, and Tobacman JK
- Subjects
- Chondroitin Sulfates metabolism, Glycosaminoglycans metabolism, Humans, Membrane Glycoproteins, Sulfotransferases, COVID-19, N-Acetylgalactosamine-4-Sulfatase genetics, N-Acetylgalactosamine-4-Sulfatase metabolism, Respiratory Insufficiency
- Abstract
Introduction: The potential role of accumulation of chondroitin sulfates (CSs) in the pathobiology of COVID-19 has not been examined. Accumulation may occur by increased synthesis or by decline in activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) which requires oxygen for activity., Methods: Immunostaining of lung tissue from 28 patients who died due to COVID-19 infection was performed for CS, ARSB, and carbohydrate sulfotransferase (CHST)15. Measurements of mRNA expression of CHST15 and CHST11, sulfotransferase activity, and total sulfated glycosaminoglycans (GAGs) were determined in human vascular smooth muscle cells following angiotensin (Ang) II treatment., Results: CS immunostaining showed increase in intensity and distribution, and immunostaining of ARSB was diminished in COVID-19 compared to normal lung tissue. CHST15 immunostaining was prominent in vascular smooth muscle cells associated with diffuse alveolar damage due to COVID-19 or other causes. Expression of CHST15 and CHST11 which are required for synthesis of CSE and chondroitin 4-sulfate, total sulfated GAGs, and sulfotransferase activity was significantly increased following AngII exposure in vascular smooth muscle cells. Expression of Interleukin-6 (IL-6), a mediator of cytokine storm in COVID-19, was inversely associated with ARSB expression., Discussion/conclusion: Decline in ARSB and resulting increases in CS may contribute to the pathobiology of COVID-19, as IL-6 does. Increased expression of CHSTs following activation of Ang-converting enzyme 2 may lead to buildup of CSs., (© 2021 S. Karger AG, Basel.)
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- 2022
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4. The olfactory G protein-coupled receptor (Olfr-78/OR51E2) modulates the intestinal response to colitis.
- Author
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Kotlo K, Anbazhagan AN, Priyamvada S, Jayawardena D, Kumar A, Chen Y, Xia Y, Finn PW, Perkins DL, Dudeja PK, and Layden BT
- Subjects
- Animals, Colitis genetics, Colitis pathology, Female, HT29 Cells, Humans, Intestinal Mucosa pathology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Neoplasm Proteins genetics, Receptors, Odorant genetics, Colitis metabolism, Intestinal Mucosa metabolism, Neoplasm Proteins biosynthesis, Receptors, Odorant biosynthesis
- Abstract
Olfactory receptor-78 (Olfr-78) is a recently identified G protein-coupled receptor activated by short-chain fatty acids acetate and propionate. A suggested role for this receptor exists in the prostate where it may influence chronic inflammatory response leading to intraepithelial neoplasia. Olfr-78 has also been shown to be expressed in mouse colon. Short-chain fatty acids and their receptors are well known to modulate inflammation in the gut. Considering this possibility, we first explored if colitis regulated Olfr-78 expression in the gut, where we observed a significant reduction in the expression of Olfr-78 transcript in mouse models of dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. To more directly test this, mice deficient in Olfr-78 were administered with DSS in water for 7 days and were found to have increased expression of IL-1β and inflammatory signs in colon compared with control mice. Next, we explored the expression of its human counterpart olfactory receptor family 51, subfamily E, member 2 (OR51E2) in human intestinal samples and observed that it was in fact also expressed in human colon samples. RNA sequence analysis revealed significant changes in the genes involved in infection, immunity, inflammation, and colorectal cancer between wild-type and Olfr-78 knockout mice. Collectively, our findings show that Olfr-78 is highly expressed in colon and downregulated in DSS- and TNBS-induced colitis, and DSS-treated Olfr-78 null mice had increased colonic expression of cytokine RNA levels, suggesting a potential role for this receptor in intestinal inflammation. Future investigations are needed to understand how Olfr-78/OR51E2 in both mouse and human intestine modulates gastrointestinal pathophysiology.
- Published
- 2020
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5. PR65A phosphorylation regulates PP2A complex signaling.
- Author
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Kotlo K, Xing Y, Lather S, Grillon JM, Johnson K, Skidgel RA, Solaro RJ, and Danziger RS
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- Animals, Annexin A1 genetics, Annexin A1 metabolism, Chaperonin 60 genetics, Chaperonin 60 metabolism, Gene Expression Regulation, HEK293 Cells, Heart Failure genetics, Heart Failure pathology, Humans, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Myocardium pathology, NADPH Dehydrogenase genetics, NADPH Dehydrogenase metabolism, Peptide Elongation Factor 1 genetics, Peptide Elongation Factor 1 metabolism, Peptide Elongation Factor 2 genetics, Peptide Elongation Factor 2 metabolism, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphorylation, Protein Multimerization, Protein Phosphatase 2 genetics, Protein Subunits genetics, Rats, Rats, Inbred Dahl, Heart Failure metabolism, Myocardium metabolism, Protein Phosphatase 2 metabolism, Protein Subunits metabolism, Signal Transduction
- Abstract
Serine-threonine Protein phosphatase 2 A (PP2A), a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac); a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa); and one of at least 18 associated variable regulatory proteins (B subunits) classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314). Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A) and non-phosphorylated (N-PR65A) amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A), elongation factor 2, heat shock protein 60 (HSP60), NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.
- Published
- 2014
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6. Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin.
- Author
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Kotlo K, Bhattacharyya S, Yang B, Feferman L, Tejaskumar S, Linhardt R, Danziger R, and Tobacman JK
- Subjects
- Animals, Cell Line, Chlorides metabolism, Chondroitin Sulfates metabolism, Diet, Sodium-Restricted, Disaccharides metabolism, Epithelial Cells drug effects, Epithelial Cells enzymology, Epithelial Cells metabolism, Hyaluronic Acid metabolism, Kidney cytology, Kidney drug effects, Kidney metabolism, N-Acetylgalactosamine-4-Sulfatase genetics, Rats, Rats, Inbred Dahl, Sulfates urine, Bradykinin urine, Glycosaminoglycans metabolism, Kininogens metabolism, N-Acetylgalactosamine-4-Sulfatase metabolism, Sodium, Dietary pharmacology
- Abstract
N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.
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- 2013
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7. Phosphoprotein abundance changes in hypertensive cardiac remodeling.
- Author
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Kotlo K, Johnson KR, Grillon JM, Geenen DL, deTombe P, and Danziger RS
- Subjects
- Animals, Cardiomegaly metabolism, Cardiomegaly pathology, Heart Failure metabolism, Heart Failure pathology, Hypertension pathology, Hypertension physiopathology, Myocardium pathology, Myocytes, Cardiac pathology, Rats, Rats, Inbred SHR, Rats, Wistar, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Hypertension metabolism, Muscle Proteins metabolism, Myocardium metabolism, Myocytes, Cardiac metabolism, Phosphoproteins metabolism, Ventricular Remodeling
- Abstract
There is over-whelming evidence that protein phosphorylations regulate cardiac function and remodeling. A wide variety of protein kinases, e.g., phosphoinositide 3-kinase (PI3K), Akt, GSK-3, TGFβ, and PKA, MAPKs, PKC, Erks, and Jaks, as well as phosphatases, e.g., phosphatase I (PP1) and calcineurin, control cardiomyocyte growth and contractility. In the present work, we used global phosphoprotein profiling to identify phosphorylated proteins associated with pressure overload (PO) cardiac hypertrophy and heart failure. Phosphoproteins from hypertrophic and systolic failing hearts from male hypertensive Dahl salt-sensitive rats, trans-aortic banded (TAC), and spontaneously hypertensive heart failure (SHHF) rats were analyzed. Profiling was performed by 2-dimensional difference in gel electrophoresis (2D-DIGE) on phospho-enriched proteins. A total of 25 common phosphoproteins with differences in abundance in (1) the 3 hypertrophic and/or (2) the 2 systolic failure heart models were identified (CI>99%) by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Mascot analysis. Among these were (1) myofilament proteins, including alpha-tropomyosin and myosin regulatory light chain 2, cap Z interacting protein (cap ZIP), and tubulin β5; (2) mitochondrial proteins, including pyruvate dehydrogenase α, branch chain ketoacid dehydrogenase E1, and mitochondrial creatine kinase; (3) phosphatases, including protein phosphatase 2A and protein phosphatase 1 regulatory subunit; and (4) other proteins including proteosome subunits α type 3 and β type 7, and eukaryotic translation initiation factor 1A (eIF1A). The results include previously described and novel phosphoproteins in cardiac hypertrophy and systolic failure., (Copyright © 2012. Published by Elsevier B.V.)
- Published
- 2012
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8. Non-histone lysine acetylated proteins in heart failure.
- Author
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Grillon JM, Johnson KR, Kotlo K, and Danziger RS
- Subjects
- Acetylation, Animals, Muscle Proteins chemistry, Rats, Rats, Inbred SHR, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Heart Failure metabolism, Histones metabolism, Lysine metabolism, Muscle Proteins metabolism
- Abstract
Both histone-acetylations and histone deacetylases have been shown to play a key role in cardiac remodeling. Recently, it has become abundantly clear that many non-histone proteins are modified by post-translational lysine acetylations and that these acetylations regulate protein activity, conformation, and binding. In the present study, non-histone acetylated proteins associated with heart failure were identified. Global screening for lysine acetylated proteins was performed using 2-dimensional gel electrophoresis coupled with immunoblotting with a primary monoclonal anti-acetyl-lysine antibody. Lysine acetylated proteins were compared in two rodent models of hypertensive heart failure, the Dahl salt-sensitive (SS) and spontaneously hypertensive heart failure prone (SHHF) rats with those in corresponding controls, i.e., the Dahl salt-resistant (SR) and W (W) rat strains, respectively. Forty-one and 66 acetylated proteins were detected in SS and SHHF failing hearts, respectively, but either not detected or detected with less abundance in corresponding control hearts. Twelve of these acetylated proteins were common to both models of heart failure. These were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry followed by Mascot Analysis and included mitochondrial enzymes: ATP synthase, long-chain acyl-CoA dehydrogenase, creatine kinase, malate dehydrogenase, and pyruvate dehydrogenase. The abundance of NAD-dependent deacetylase sirtuin-3 (Sirt3), a mitochondrial deacetylase was reduced in SS and SHHF failing hearts. This is the first description of non-histone protein acetylations associated with heart failure and raises the prospect that acetylations of mitochondrial proteins linked to reduced Sirt3 mediate, in part, metabolic changes in heart failure., (Copyright © 2011. Published by Elsevier B.V.)
- Published
- 2012
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9. Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells.
- Author
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Bhattacharyya S, Kotlo K, Danziger R, and Tobacman JK
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- Animals, Bradykinin pharmacology, Cells, Cultured, Epithelial Cells drug effects, Glycosaminoglycans metabolism, Immunoprecipitation, Kidney cytology, Kidney drug effects, RNA, Small Interfering pharmacology, Rats, Vasodilator Agents pharmacology, Chondroitin Sulfates metabolism, Epithelial Cells metabolism, Kidney metabolism, Kininogens metabolism, N-Acetylgalactosamine-4-Sulfatase physiology
- Abstract
The enzyme arylsulfatase B (N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen-bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen-C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen-bradykinin axis and, thereby, may influence blood pressure. Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation., (Published by Elsevier B.V.)
- Published
- 2010
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10. Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates.
- Author
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Bhattacharyya S, Kotlo K, Shukla S, Danziger RS, and Tobacman JK
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- Antibodies chemistry, Cell Line, Tumor, Chondroitin ABC Lyase chemistry, Chondroitin Sulfates chemistry, Chondroitin Sulfates genetics, Chondroitinsulfatases genetics, Decorin, Dermatan Sulfate biosynthesis, Dermatan Sulfate chemistry, Dermatan Sulfate genetics, Extracellular Matrix Proteins biosynthesis, Extracellular Matrix Proteins genetics, Female, Gene Silencing, Heparin biosynthesis, Heparin chemistry, Heparin genetics, Heparitin Sulfate biosynthesis, Heparitin Sulfate chemistry, Heparitin Sulfate genetics, Humans, Mucopolysaccharidosis VI enzymology, Mucopolysaccharidosis VI genetics, N-Acetylgalactosamine-4-Sulfatase genetics, Proteoglycans biosynthesis, Proteoglycans genetics, RNA, Small Interfering, Syndecan-1 biosynthesis, Syndecan-1 genetics, Chondroitin Sulfates biosynthesis, Chondroitinsulfatases metabolism, Gene Expression Regulation, Enzymologic genetics, N-Acetylgalactosamine-4-Sulfatase metabolism
- Abstract
The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.
- Published
- 2008
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11. Renal phosphodiesterase 4B is activated in the Dahl salt-sensitive rat.
- Author
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Tawar U, Kotlo K, Jain S, Shukla S, Setty S, and Danziger RS
- Subjects
- Animals, Cyclic AMP metabolism, Disease Models, Animal, Endothelium, Vascular enzymology, Endothelium, Vascular pathology, Enzyme Activation, Hypertension, Renal pathology, Isoenzymes metabolism, Kidney blood supply, Kidney Tubules enzymology, Kidney Tubules pathology, Phosphorylation, Rats, Rats, Inbred SHR, Receptors, Adrenergic, beta metabolism, Signal Transduction physiology, Cyclic Nucleotide Phosphodiesterases, Type 4 metabolism, Hypertension, Renal enzymology, Kidney enzymology, Rats, Inbred Dahl metabolism
- Abstract
Reduced beta-adrenoreceptor signaling is associated with increased sympathoadrenal activity in hypertensive patients and animal models of hypertension. However, the mechanism that accounts for this characteristic decline in beta-adrenergic signaling is unclear. In the present study, we investigated renal phosphodiesterase 4B, which metabolizes cAMP. Immunoblot analysis detected only the phosphodiesterase 4B4 isoform present in kidney tissue from spontaneously hypertensive rats, hypertensive Dahl salt-sensitive (SS) rats, and Dahl salt-resistant rats. The phosphorylated (activated) form of the protein was present at 2-fold greater levels in Dahl SS rats than in spontaneously hypertensive rats and Dahl salt-resistant rats, whereas the unphosphorylated form of the protein was reduced by approximately one half in SS animals. In accord with immunoblot data, rolipram-inhibitable cAMP hydrolyzing activity, a measure of PDE4 activity, was approximately 3-fold greater in kidney cytosolic extracts from SS rats than in extracts from spontaneously hypertensive rats and salt-resistant rats. Phosphodiesterase 4B expression was detected by immunohistochemistry in the renal vasculature, proximal tubules, and distal tubules. These results raise the possibility that increased PDE4 activity, specifically phosphodiesterase 4B4 activity, reduces beta-adrenergic signaling in the kidney and contributes to salt-sensitive hypertension in the Dahl SS rat.
- Published
- 2008
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12. Aminopeptidase N reduces basolateral Na+ -K+ -ATPase in proximal tubule cells.
- Author
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Kotlo K, Shukla S, Tawar U, Skidgel RA, and Danziger RS
- Subjects
- Animals, CD13 Antigens genetics, Cells, Cultured, Gene Expression Regulation, Enzymologic drug effects, Kidney Tubules, Proximal cytology, Kidney Tubules, Proximal drug effects, RNA, Small Interfering pharmacology, Receptors, Angiotensin metabolism, Signal Transduction physiology, Swine, CD13 Antigens metabolism, Kidney Tubules, Proximal enzymology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na(+)-K(+)-ATPase, a preeminent basolateral Na(+) transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na(+)-K(+)-ATPase activity and basolateral Na(+)-K(+)-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na(+)-K(+)-ATPase activity and basolateral abundance by approximately 50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na(+)-K(+)-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na(+)-K(+)-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.
- Published
- 2007
- Full Text
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13. Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat.
- Author
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Kotlo K, Hughes DE, Herrera VL, Ruiz-Opazo N, Costa RH, Robey RB, and Danziger RS
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-alpha genetics, Disease Models, Animal, Male, Polymorphism, Genetic, Polymorphism, Single Nucleotide, Promoter Regions, Genetic genetics, Rats, Rats, Inbred Dahl, Rats, Inbred Lew, CD13 Antigens genetics, Hypertension genetics
- Abstract
We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximately 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.
- Published
- 2007
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14. Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis.
- Author
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Scheuner D, Patel R, Wang F, Lee K, Kumar K, Wu J, Nilsson A, Karin M, and Kaufman RJ
- Subjects
- Animals, Caspase 3, Caspases metabolism, Cell Line, DNA chemistry, HeLa Cells, Homozygote, Humans, Mice, Phosphorylation, RNA, Double-Stranded chemistry, Transfection, Apoptosis, Eukaryotic Initiation Factor-2 physiology, eIF-2 Kinase chemistry
- Abstract
As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2alpha)atSer(51) is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2alpha stress kinases. We used gene targeting to replace the wild-type Ser(51) allele with an Ala in the eIF2alpha gene to test the hypothesis that translational control through eIF2alpha phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2alpha mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-alpha, and serum deprivation. TNFalpha treatment induced eIF2alpha phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2alpha kinase PKR. In addition, expression of a phospho-mimetic Ser(51) to Asp mutant eIF2alpha-activated caspase 3, indicating that eIF2alpha phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2alpha phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2alpha kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2alpha. We propose that eIF2alpha phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.
- Published
- 2006
- Full Text
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