ABSTRACT
Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.
Subject(s)
Pancreatitis, Chronic , Voltage-Dependent Anion Channel 1 , Animals , Humans , Mice , Reactive Oxygen Species/metabolism , Up-Regulation , Voltage-Dependent Anion Channel 1/metabolismABSTRACT
Pancreatic ß-cell dysfunction and eventual loss are key steps in the progression of type 2 diabetes (T2D). Endoplasmic reticulum (ER) stress responses, especially those mediated by the protein kinase RNA-like ER kinase and activating transcription factor 4 (PERK-ATF4) pathway, have been implicated in promoting these ß-cell pathologies. However, the exact molecular events surrounding the role of the PERK-ATF4 pathway in ß-cell dysfunction remain unknown. Here, we report our discovery that ATF4 promotes the expression of PDE4D, which disrupts ß-cell function via a downregulation of cAMP signaling. We found that ß-cell-specific transgenic expression of ATF4 led to early ß-cell dysfunction and loss, a phenotype that resembles accelerated T2D. Expression of ATF4, rather than C/EBP homologous protein (CHOP), promoted PDE4D expression, reduced cAMP signaling, and attenuated responses to incretins and elevated glucose. Furthermore, we found that ß-cells of leptin receptor-deficient diabetic (db/db) mice had elevated nuclear localization of ATF4 and PDE4D expression, accompanied by impaired ß-cell function. Accordingly, pharmacological inhibition of the ATF4 pathway attenuated PDE4D expression in the islets and promoted incretin-simulated glucose tolerance and insulin secretion in db/db mice. Finally, we found that inhibiting PDE4 activity with selective pharmacological inhibitors improved ß-cell function in both db/db mice and ß-cell-specific ATF4 transgenic mice. In summary, our results indicate that ER stress causes ß-cell failure via ATF4-mediated PDE4D production, suggesting the ATF4-PDE4D pathway could be a therapeutic target for protecting ß-cell function during the progression of T2D.NEW & NOTEWORTHY Endoplasmic reticulum stress has been implied to cause multiple ß-cell pathologies during the progression of type 2 diabetes (T2D). However, the precise molecular events underlying this remain unknown. Here, we discovered that elevated ATF4 activity, which was seen in T2D ß cells, attenuated ß-cell proliferation and impaired insulin secretion via PDE4D-mediated downregulation of cAMP signaling. Additionally, we demonstrated that pharmacological inhibition of the ATF4 pathway or PDE4D activity alleviated ß-cell dysfunction, suggesting its therapeutic usefulness against T2D.
Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Mice , Animals , Apoptosis , Incretins/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/metabolism , Endoplasmic Reticulum Stress/genetics , Glucose/metabolism , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism , eIF-2 Kinase/metabolismABSTRACT
AIM: Doxorubicin is a highly effective anticancer agent that causes hepatotoxicity and cardiotoxicity in patients. Fibroblast growth factor 21, a well-known regulator of glucose and lipid metabolism, exerts cardioprotective effects. Gemigliptin and dipeptidyl peptidase-4 inhibitors are widely used in the treatment of patients with type 2 diabetes. The protective effects of gemigliptin on hepatotoxicity via the increase in fibroblast growth factor 21 expression has not yet been elucidated. This study was designed to investigate the protective effects of gemigliptin against doxorubicin-induced hepatotoxicity via the upregulation of fibroblast growth factor 21 expression in the cultured murine hepatocyte cell line, AML12. METHODS: Murine hepatocyte AML12 cells were treated with doxorubicin, fibroblast growth factor 21 and gemigliptin in 0.5% fetal bovine serum medium for 24 h at the indicated doses. Cells were transfected with the fibroblast growth factor 21 small interfering RNA for 24 h, followed by protein isolation. RESULTS: Fibroblast growth factor 21 expression levels were increased during doxorubicin-induced hepatotoxicity in the murine hepatocyte AML12 cells. Fibroblast growth factor 21 treatment prevented doxorubicin-induced hepatotoxicity by attenuating apoptosis. Gemigliptin prevented doxorubicin-induced hepatotoxicity by upregulating fibroblast growth factor 21 expression. However, the protective effects of gemigliptin were blocked by fibroblast growth factor 21 inhibition in doxorubicin-treated AML12 cells. CONCLUSION: These results indicate that gemigliptin exhibits protective effects against doxorubicin-induced hepatotoxicity by upregulating the fibroblast growth factor 21 expression levels in the cultured murine hepatocyte AML12 cells.
Subject(s)
Chemical and Drug Induced Liver Injury , Diabetes Mellitus, Type 2 , Dipeptidyl-Peptidase IV Inhibitors , Piperidones , Animals , Apoptosis/drug effects , Chemical and Drug Induced Liver Injury/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Dipeptidyl-Peptidase IV Inhibitors/therapeutic use , Doxorubicin/adverse effects , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Humans , Mice , Piperidones/pharmacology , Piperidones/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic useABSTRACT
A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34 % similarity). Optimal growth was observed at 30-40 °C and pH 7. Growth occurred in the presence of 0-6 % (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6 %. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).
Subject(s)
Actinomycetales/classification , Fingers/microbiology , Phylogeny , Suppuration/microbiology , Actinomycetales/genetics , Actinomycetales/isolation & purification , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Fingers/pathology , Humans , Necrosis , Nucleic Acid Hybridization , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Republic of Korea , Sequence Analysis, DNA , Vitamin K 2/chemistryABSTRACT
We analyzed national data on blood lead levels (BLL) and blood cadmium levels (BCL) in residents living near 38 abandoned metal mining areas (n = 5,682, 18-96 years old) in Korea that were collected by the first Health Effect Surveillance for Residents in Abandoned Metal mines (HESRAM) from 2008 to 2011. The geometric mean BCL and BLL were 1.60 µg/L (95 % CI = 1.57-1.62 µg/L) and 2.87 µg/dL (95 % CI = 2.84-2.90 µg/dL), respectively, notably higher than levels in the general population in Korea and other countries. We found significantly higher BLL and BCL levels in people living within 2 km of an abandoned metal mine (n = 3,165, BCL = 1.87 µg/L, BLL = 2.91 µg/dL) compared to people living more than 2 km away (n = 2,517, BCL = 1.31 µg/L, BLL = 2.82 µg/dL; P < 0.0001) and to the general population values reported in the literature.
Subject(s)
Cadmium/blood , Environmental Exposure/statistics & numerical data , Environmental Pollutants/blood , Lead/blood , Mining , Adolescent , Adult , Aged , Aged, 80 and over , Environmental Monitoring , Female , Humans , Male , Metals/blood , Middle Aged , Republic of Korea , Young AdultABSTRACT
BACKGRUOUND: Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. METHODS: To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-ß-treated renal cells in vitro. RESULTS: Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-ßstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-ß-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-ß secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. CONCLUSION: Alantolactone improves renal fibrosis by inhibiting the TGF-ß/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.
Subject(s)
Kidney Diseases , Lactones , Sesquiterpenes, Eudesmane , Ureteral Obstruction , Mice , Animals , Fibronectins/pharmacology , Fibronectins/therapeutic use , Plasminogen Activator Inhibitor 1/pharmacology , Plasminogen Activator Inhibitor 1/therapeutic use , Collagen Type I/pharmacology , Collagen Type I/therapeutic use , Kidney Diseases/drug therapy , Kidney Diseases/etiology , Ureteral Obstruction/complications , Ureteral Obstruction/drug therapy , Ureteral Obstruction/metabolism , Transforming Growth Factor beta/metabolism , Signal Transduction , FibrosisABSTRACT
Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Glucose/toxicity , Insulin-Secreting Cells/drug effects , Palmitates/toxicity , Animals , Basic-Leucine Zipper Transcription Factors/agonists , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Rats , Sterol Regulatory Element Binding Protein 1/agonists , Sterol Regulatory Element Binding Protein 1/metabolism , Transcription Factors/metabolismABSTRACT
BACKGROUND: Integrin-mediated interaction of neuronal cells with extracellular matrix (ECM) is important for the control of cell adhesion, morphology, motility, and differentiation in both in vitro and in vivo systems. Arg-Gly-Asp (RGD) sequence is one of the most potent integrin-binding ligand found in many native ECM proteins. An elastin-mimetic recombinant protein, TGPG[VGRGD(VGVPG)6]20WPC, referred to as [RGD-V6]20, contains multiple RGD motifs to bind cell-surface integrins. This study aimed to investigate how surface-adsorbed recombinant protein can be used to modulate the behaviors and differentiation of neuronal cells in vitro. For this purpose, biomimetic ECM surfaces were prepared by isothermal adsorption of [RGD-V6]20 onto the tissue culture polystyrene (TCPS), and the effects of protein-coated surfaces on neuronal cell adhesion, spreading, migration, and differentiation were quantitatively measured using N2a neuroblastoma cells. RESULTS: The [RGD-V6]20 was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V6]20 or fibronectin followed hyperbolic binding kinetics saturating around 2 µM protein concentration. The apparent maximum cell binding to [RGD-V6]20 was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V6]20 and fibronectin occurring at a coating concentration of 2.4 × 10-7 and 1.4 × 10-7 M, respectively. The percentage of spreading cells was in the following order of proteins: fibronectin (84.3% ± 6.9%) > [RGD-V6]20 (42.9% ± 6.5%) > [V7]20 (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V6]20 was similar to that of cells on fibronectin. The expression of neuronal marker proteins Tuj1, MAP2, and GFAP was approximately 1.5-fold up-regulated by [RGD-V6]20 relative to TCPS. Moreover, by the presence of both [RGD-V6]20 and RA, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated. CONCLUSION: We have shown that an elastin-mimetic protein consisting of alternating tropoelastin structural domains and cell-binding RGD motifs is able to stimulate neuronal cell behaviors and differentiation. In particular, adhesion-induced neural differentiation is highly desirable for neural development and nerve repair. In this context, our data emphasize that the combination of biomimetically engineered recombinant protein and isothermal adsorption approach allows for the facile preparation of bioactive matrix or coating for neural tissue regeneration.
Subject(s)
Biomimetic Materials/metabolism , Oligopeptides/biosynthesis , Adsorption , Animals , Biomimetic Materials/pharmacology , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Elastin/genetics , Elastin/metabolism , Escherichia coli/metabolism , Fibronectins/chemistry , Fibronectins/metabolism , Mice , Neurons/cytology , Oligopeptides/genetics , Oligopeptides/pharmacology , Polystyrenes/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
To investigate the association of pathogenic Escherichia coli fimbrial adhesins with the development of diarrhoea in piglets of different age groups and to test their relative competitiveness, piglets were orally inoculated with a mixture of E. coli strains harbouring F4, F5, F6, F18 and F41 fimbrial genes. A total of 537 E. coli strains with haemolytic activity were isolated from 36 diarrhoeic piglets. The F4 fimbrial gene was observed in 98.5%, 97.6% and 80.6% strains carrying fimbrial genes isolated from diarrhoeic piglets that were infected at 1, 3 and 5 weeks of age, respectively. These data demonstrate that F4 fimbriae are highly associated with diarrhoea in piglets of all age groups. Interestingly, the F18 fimbrial gene was observed in 2.4% and 25.4% strains carrying fimbrial genes isolated from the 3- and 5-week-old groups, respectively, which confirms that F18 fimbriae are associated with diarrhoea in piglets from late stages of suckling to post-weaning, and are more related to diarrhoea in weaned than in unweaned piglets.
Subject(s)
Aging , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/veterinary , Fimbriae, Bacterial/metabolism , Swine Diseases/microbiology , Animals , Animals, Suckling , Diarrhea/microbiology , Diarrhea/veterinary , Escherichia coli Infections/microbiology , Fimbriae, Bacterial/genetics , Gene Expression Regulation, Bacterial , Swine , Swine Diseases/pathologyABSTRACT
An excessive and prolonged increase in glucose levels causes ß-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicityinduced ß-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced ß-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in ß-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirusmediated Lin28a overexpression in ß-cells reversed the glucotoxicity- induced reduction of insulin secretion and insulin mRNA expression via regulation of ß-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic ß-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway. [BMB Reports 2021; 54(4): 215-220].
Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/metabolism , RNA-Binding Proteins/metabolism , Animals , Apoptosis/drug effects , Cells, Cultured , Glucose/toxicity , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/pathology , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Paeonia japonica, distributed throughout Asia, is a traditional medicinal herb in Korea, with many potential beneficial effects including pain-relieving, anti-inflammatory, and anti-cancer activities. Despite its high pharmacological value, the genetic information on Paeonia japonica remains limited. In this study, the chloroplast genome of P. japonica was sequenced using next-generation sequencing (NGS) technology and genome and phylogeny were analyzed using multiple tools. The chloroplast genome of P. japonica was 152,731 bp in length with an inverted repeat region of 26,656 bp, including a large single-copy region of 84,389 bp and a small single copy region of 17,030 bp. The P. japonica chloroplast genome included 113 genes comprising 80 protein-coding genes, 27 tRNA, and 5 rRNA genes. Phylogenetic analysis indicated that P. japonica and P. obovata share a close evolutionary relationship.
ABSTRACT
We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel ß-helix domain structure; this new gene was designated vatH. [corrected] The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatH, [corrected] was detected together with vatH [corrected] in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatH-containing [corrected] E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatH-containing [corrected] E. faecium isolates differed for isolates from humans and nonhumans.
Subject(s)
Enterococcus faecium/drug effects , Streptogramin A/pharmacology , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Blotting, Southern , DNA Transposable Elements/genetics , Drug Resistance, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecium/genetics , Enterococcus faecium/metabolism , Genotype , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
The accumulation of extracellular matrix proteins is a common feature of fibrotic kidney diseases. Accumulating evidence suggests that TGF-beta and plasminogen activator inhibitor type 1 (PAI-1) promote the development of renal fibrosis by stimulating the generation and inhibiting the removal of matrix proteins. The small heterodimer partner (SHP) represses PAI-1 expression in the liver by inhibiting TGF-beta signaling, but whether SHP inhibits renal fibrosis is unknown. Here, unilateral ureteral obstruction (UUO) markedly increased the expression of PAI-1, type I collagen, and fibronectin but decreased SHP gene expression. Moreover, in kidneys of SHP-/- mice, the expression of PAI-1, type I collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA) were higher compared with those in kidneys of wild-type mice. In addition, loss of SHP accelerated renal fibrosis after UUO. Adenovirus-mediated overexpression of SHP in cultured rat mesangial cells and renal tubular epithelial cells inhibited TGF-beta-stimulated expression of PAI-1, type I collagen, and fibronectin. SHP inhibited TGF-beta- and Smad3-stimulated PAI-1 promoter activities as well as TGF-beta-stimulated binding of Smad3 to its consensus response element on the PAI-1 promoter. Similarly, in vivo, adenovirus-mediated overexpression of SHP in the kidney inhibited the expression of UUO-induced PAI-1, type I collagen, fibronectin, and alpha-SMA. In summary, SHP attenuates renal fibrosis in obstructive nephropathy, making its pathway a possible therapeutic target for chronic kidney disease.
Subject(s)
Kidney/pathology , Receptors, Cytoplasmic and Nuclear/physiology , Actins/genetics , Animals , Collagen Type I/genetics , Fibronectins/genetics , Fibrosis , Male , Plasminogen Activator Inhibitor 1/genetics , Promoter Regions, Genetic , Rats , Rats, Sprague-Dawley , Smad3 Protein/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/physiology , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathologyABSTRACT
OBJECTIVE: Enhancement of ß-cell proliferation plays an important role in maintaining ß-cell mass and function, and in improving pancreatic ß-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of ß-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance ß-cell adhesion and proliferation and elucidated the signaling pathways involved. METHODS: We investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. RESULTS: REP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of ß-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When ß-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced ß-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. CONCLUSIONS: Our results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic ß-cell adhesion and proliferation.
ABSTRACT
Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-ß-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-ß-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-ß-stimulated SMAD3 activity, via inhibition of SMAD3 phosphorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease. [BMB Reports 2020; 53(11): 594-599].
Subject(s)
Fibrosis/physiopathology , Kidney Tubules/metabolism , Animals , Cell Line , Collagen Type I/metabolism , Fibronectins/metabolism , Fibrosis/metabolism , Humans , Kidney/pathology , Kidney Tubules/physiology , Male , Mice , Mice, Inbred C57BL , Phosphorylation , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiology , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Ureteral Obstruction/drug therapy , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathologyABSTRACT
Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-beta-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-beta/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-beta-induced PAI-1 expression.
Subject(s)
Forkhead Transcription Factors/metabolism , Hyperinsulinism/enzymology , Insulin/metabolism , Plasminogen Activator Inhibitor 1/biosynthesis , Transforming Growth Factor beta/metabolism , Cell Line, Tumor , Forkhead Box Protein O1 , Gene Expression , Humans , Insulin/pharmacology , Plasminogen Activator Inhibitor 1/genetics , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/biosynthesis , Transforming Growth Factor beta/pharmacologyABSTRACT
We investigated the species distribution and antifungal susceptibility of Candida isolates from tertiary and non-tertiary hospitals in South Korea from 2002-2004. Of the 612 Candida isolates that were collected, Candida albicans, C. parapsilosis, C. tropicalis, and C. glabrata occurred most frequently, accounting for 97.3% and 96.8% of the isolates in tertiary and non-tertiary hospitals, respectively. C. albicans was the most common isolate, but the incidence of non-C. albicansCandida species was higher than that of C. albicans in tertiary hospitals. The Candida species had much lower MIC(90) to voriconazole (tertiary hospitals: 0.5 microg/ml, non-tertiary hospitals: 0.25 microg/ml) than to fluconazole (tertiary hospitals: 8 microg/ml, non-tertiary hospitals: 4 microg/ml). The MIC(90) of Candida isolates to 5-flucytosine in non-tertiary hospitals was two times higher than that observed in tertiary facilities. The C. glabrata isolates showed a tendency toward strong resistance to fluconazole, but C. parapsilosis isolates were susceptible to all of the evaluated antifungal agents. Voriconazole showed strong in vitro activity against Candida species, especially C. krusei, which is resistant to fluconazole and 5-flucytosine. To our knowledge, this is the first report of Candida antifungal susceptibility that includes non-tertiary hospitals in South Korea.
Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Candidiasis/microbiology , Candida/isolation & purification , Candidiasis/epidemiology , Drug Resistance, Fungal , Fluconazole/pharmacology , Flucytosine/pharmacology , Hospitals , Humans , Microbial Sensitivity Tests , Population Surveillance/methods , Pyrimidines/pharmacology , Republic of Korea/epidemiology , Triazoles/pharmacology , VoriconazoleABSTRACT
SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDA-MB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ER-mediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogen-induced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-type-specific manner and act as a novel transcriptional co-regulator in ER signalling.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Protein Isoforms/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Estrogen/metabolism , Transcriptional Activation , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Cell Line , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Humans , Leucine Zippers , Mice , Protein Isoforms/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Estrogen/genetics , Tissue Distribution , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
Of 143 clinical isolates of Klebsiella pneumoniae collected from Korean non-tertiary hospitals, 24 (16.8%) showed an extended-spectrum beta-lactamase-positive phenotype. PCR and sequence analysis revealed the presence of TEM-116 (n=13), CTX-M-3 (n=5), CTX-M-14 (n=2), CTX-M-15 (n=3), and SHV-12 (n=16). Each of the 24 isolates encoded more than one beta-lactamase, and seven isolates (29%) harbored two different SHV-type beta-lactamase genes (blaSHV-11 and blaSHV-12) bounded by insertion sequence IS26 in a single transferable plasmid.
Subject(s)
Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , beta-Lactamases/genetics , Blotting, Southern/methods , DNA Primers , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Hospitals/standards , Humans , Klebsiella Infections/drug therapy , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Korea , Microbial Sensitivity Tests , Phenotype , Plasmids/genetics , Sputum/microbiology , beta-Lactamases/metabolismABSTRACT
Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced ß-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. STATEMENT OF SIGNIFICANCE: 1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices. 2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways. 3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity. 4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.