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1.
FEBS Open Bio ; 14(6): 906-921, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38604990

ABSTRACT

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.


Subject(s)
Endothelial Cells , Mice, Knockout , rho-Associated Kinases , Animals , rho-Associated Kinases/metabolism , rho-Associated Kinases/genetics , Mice , Endothelial Cells/metabolism , Intercellular Junctions/metabolism , Lung/metabolism , Lung/pathology , Cadherins/metabolism , Cadherins/genetics , beta Catenin/metabolism , beta Catenin/genetics , Male , Antigens, CD
2.
Chronobiol Int ; 39(8): 1132-1143, 2022 08.
Article in English | MEDLINE | ID: mdl-35603436

ABSTRACT

Eating during a rest phase disrupts the biological clock system and leads to obesity and metabolic diseases. Although a rest phase restricted feeding (RF) is reported to enhance hepatic lipid accumulation, the mechanism(s) of the phenomenon is still unknown. This study evaluated the potential involvement of the CD36-related transport of lipids into the liver in mice with the RF procedure. This study showed that hepatic lipid accumulation was more significant in the RF group compared with mice under an active phase restricted feeding (AF). The RF procedure also elevated the expression of CD36 mRNA and its protein on the cellular membrane throughout the day. The transcription factor profiling array revealed that the RF activated the proliferator-activated receptor-γ (PPARγ), one of the CD36 transcript enhancers. In the liver of RF mice, the expression of miR-27b-3p, which is known to interfere with PPARγ gene expression, significantly decreased. These results suggest that the RF procedure inhibits the expression of miR-27b-3p in the liver and subsequently elevates PPARγ activity. Activated PPARγ might lead to CD36 upregulation, which, in turn, stimulates the transport of lipids into the liver.


Subject(s)
MicroRNAs , PPAR gamma , Animals , Circadian Rhythm , Lipids , Liver/metabolism , Mice , MicroRNAs/genetics , PPAR gamma/metabolism
3.
Genes Cells ; 25(4): 270-278, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32050049

ABSTRACT

The expression levels of many cell-surface proteins vary with the time of day. Glycoprotein 2 (Gp2), specifically expressed on the apical surface of M cells in Peyer's patches, functions as a transcytotic receptor for mucosal antigens. We report that cAMP response element-binding protein (CREB) regulates the transcription of the Gp2 gene, thereby generating the circadian change in its expression in mouse Peyer's patches. The transcytotic receptor activity of Gp2 was increased during the dark phase when the Gp2 protein abundance increased. Rhythmic expression of clock gene mRNA was observed in mouse Peyer's patches, and expression levels of Gp2 mRNA also exhibited circadian oscillation, with peak levels during the early dark phase. The promoter region of the mouse Gp2 gene contains several cAMP response elements (CREs). Chromatin immunoprecipitation assays revealed that CREB bound to the CREs in the Gp2 gene in Peyer's patches. Forskolin, which promotes CREB phosphorylation, increased the transcription of the Gp2 gene in Peyer's patches. As phosphorylation of CREB protein was increased when Gp2 gene transcription was activated, CREB may regulate the rhythmic expression of Gp2 mRNA in Peyer's patches. These findings suggest that intestinal immunity is controlled by the circadian clock system.


Subject(s)
Biological Clocks , Circadian Rhythm , GPI-Linked Proteins/metabolism , Peyer's Patches/metabolism , Animals , GPI-Linked Proteins/genetics , Male , Mice , Mice, Inbred ICR , Mice, Mutant Strains
4.
Biochem Biophys Res Commun ; 519(3): 613-619, 2019 11 12.
Article in English | MEDLINE | ID: mdl-31540689

ABSTRACT

P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17ß-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17ß-estradiol, but not testosterone. Similar 17ß-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Digoxin/pharmacokinetics , Epithelial Cells/drug effects , Estradiol/pharmacology , Estrogens/pharmacology , Kidney Tubules/drug effects , Kidney/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Cells, Cultured , Digoxin/administration & dosage , Digoxin/analysis , Epithelial Cells/metabolism , Female , Humans , Injections, Intravenous , Kidney/metabolism , Kidney Tubules/metabolism , Male , Mice , Mice, Inbred ICR , Tissue Distribution
5.
J Biol Chem ; 294(2): 547-558, 2019 01 11.
Article in English | MEDLINE | ID: mdl-30429219

ABSTRACT

Disruption of circadian rhythms has been implicated in an increased risk for cancer development. The Period2 (Per2) gene encodes one of the major components of the mammalian circadian clock, which plays a key role in controlling the circadian rhythms in physiology and behavior. PER2 has also been reported to suppress the malignant transformation of cells, but its role in the regulation of cancer susceptibility to chemotherapeutic drugs remains unclear. In this study, we found that oncogene-transformed embryonic fibroblasts prepared from Per2-mutant (Per2m/m ) mice, which are susceptible to both spontaneous and radiation-induced tumorigenesis, were resistant against common chemotherapeutic drugs and that this resistance is associated with up-regulation of the aldehyde dehydrogenase 3a1 (Aldh3a1) gene. Co-expression of the oncogenes H-rasV12 and SV40 large T-antigen induced malignant transformation of both WT and Per2m/m cells, but the cytotoxic effects of the chemotherapeutic agents methotrexate, gemcitabine, etoposide, vincristine, and oxaliplatin were significantly alleviated in the oncogene-transformed Per2m/m cells. Although introduction of the two oncogenes increased the expression of Aldh3a1 in both WT and Per2m/m cells, the ALDH3A1 protein levels in the Per2m/m cells were ∼7-fold higher than in WT cells. The elevated ALDH3A1 levels in the oncogene-transformed Per2m/m cells were sufficient to prevent chemotherapeutic drug-induced accumulation of reactive oxygen species. Consequently, shRNA-mediated suppression of Aldh3a1 expression relieved the chemoresistance of the Per2m/m cells. These results suggest a role for mutated PER2 in the development of multiple drug resistance and may inform therapeutic strategies for cancer management.


Subject(s)
Aldehyde Dehydrogenase/genetics , Carcinogenesis/genetics , Drug Resistance, Neoplasm , Period Circadian Proteins/genetics , Up-Regulation , Animals , Carcinogenesis/drug effects , Cells, Cultured , Circadian Clocks , Mice, Inbred ICR , Mutation , Neoplasms/drug therapy , Neoplasms/etiology , Neoplasms/genetics
6.
Jpn J Ophthalmol ; 62(6): 677-685, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30250985

ABSTRACT

PURPOSE: Clock genes are components of the molecular clock. Their malfunction is thought to increase the risk of numerous diseases, including cancer. Vascular endothelial growth factor (VEGF) has a pivotal role in angiogenesis, and its expression levels are controlled by clock genes in tumor cells. Ophthalmic diseases such as age-related macular degeneration, proliferative diabetic retinopathy, and neovascular glaucoma are also associated with abnormal angiogenesis followed by upregulation of VEGF in the eye. In the present study, we aimed to uncover the relationship between clock genes and VEGF in the eye. STUDY DESIGN: Laboratory investigation METHODS: Oxygen-induced retinopathy (OIR) mice were prepared to mimic hypoxic conditions in the eye. Deferoxamine (DFO) was used to mimic hypoxic conditions in human Müller cell line MIO-M1 cells. Expression levels of mRNA and protein were quantified by quantitative reverse transcription polymerase chain reaction and Western blot analysis, respectively. RESULTS: In the retinas of OIR mice, the expression levels of Vegf and the clock gene Dec2 increased transiently, and their temporal profiles were correlated. Knockdown of DEC2 resulted in a significant (26.7%) reduction of VEGF expression in MIO-M1 cells under hypoxia-mimicking conditions induced by DFO (P < .05). Levels of HIF1α protein were also reduced significantly, by 60.2%, in MIO-M1 cells treated with siRNA against the DEC2 gene (P < .05). Moreover, HIF1α levels showed a significant (2.5-fold) increase in MIO-M1 cells overexpressing DEC2 (P < .05). CONCLUSION: DEC2 could upregulate retinal VEGF gene expression through modulation of HIF1α levels under hypoxic conditions.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Ependymoglial Cells/metabolism , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , RNA, Messenger/genetics , Retinal Diseases/genetics , Vascular Endothelial Growth Factor A/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Cell Line , Cells, Cultured , Ependymoglial Cells/pathology , Humans , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Mice , Retinal Diseases/metabolism , Retinal Diseases/pathology , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation , Vascular Endothelial Growth Factor A/biosynthesis
7.
Biochem Biophys Res Commun ; 503(1): 181-187, 2018 09 03.
Article in English | MEDLINE | ID: mdl-29864425

ABSTRACT

The intensity of pain in diabetic neuropathy varies in a circadian time-dependent manner. It is well known that diabetes has two distinct types, which are differentiated based on the cause of the disease. Previous studies have yet to compare the circadian properties of the pain intensity of diabetic neuropathy between type I and type II diabetes. In this study, we demonstrated that the pain intensity of diabetic peripheral neuropathy in a db/db mouse model of type II diabetes showed a significant diurnal oscillation, but such time-dependent oscillation was not detected in a streptozotocin (STZ)-induced type I diabetic mouse model. The polyol pathway-induced accumulation of sorbitol in peripheral nerve cells suppresses Na+/K+-ATPase activity, which is associated with the intensity of pain in diabetic neuropathy. In db/db mice, this accumulation of sorbitol in peripheral nerve cells showed significant diurnal oscillation. In addition, pain intensity and Na+/K+-ATPase activity were decreased at the peak time of sorbitol accumulation in these mice. Although STZ-induced diabetic mice also showed sorbitol accumulation and Na+/K+-ATPase dysfunction, these measures did not oscillate in a time-dependent manner. These findings reveal differences in the circadian properties of pain hypersensitivity in mouse models of type I and type II diabetes, and also provide ideas for developing novel approaches to the management of diabetic neuropathy.


Subject(s)
Diabetic Neuropathies/physiopathology , Neuralgia/physiopathology , Sciatic Nerve/metabolism , Sorbitol/metabolism , Animals , Circadian Rhythm/physiology , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Diabetic Neuropathies/drug therapy , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Inbred ICR , Mice, Mutant Strains , Neuralgia/drug therapy , Rhodanine/analogs & derivatives , Rhodanine/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Thiazolidines/pharmacology
8.
Nat Commun ; 7: 13102, 2016 10 14.
Article in English | MEDLINE | ID: mdl-27739425

ABSTRACT

Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain.


Subject(s)
Adenosine Triphosphate/metabolism , Astrocytes/drug effects , Glucocorticoids/pharmacology , Spinal Cord/drug effects , Adrenalectomy , Animals , Animals, Newborn , Astrocytes/cytology , Astrocytes/metabolism , Cells, Cultured , Circadian Rhythm , Corticosterone/blood , Corticosterone/pharmacology , Gene Expression Profiling , Glucocorticoids/blood , Hyperalgesia/physiopathology , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Ligation , Male , Mice, Inbred ICR , Neuralgia/physiopathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Sciatic Nerve/surgery , Spinal Cord/cytology , Spinal Cord/metabolism
9.
Biochem Biophys Res Commun ; 467(1): 157-63, 2015 Nov 06.
Article in English | MEDLINE | ID: mdl-26403971

ABSTRACT

Clock gene regulates the circadian rhythm of various physiological functions. The expression of clock gene has been shown to be attenuated by certain drugs, resulting in a rhythm disorder. Mitomycin C (MMC) is often used in combination with ophthalmic surgery, especially in trabeculectomy, a glaucoma surgical procedure. The purpose of this study was to investigate the influence of MMC on clock gene expression in fibroblasts, the target cells of MMC. Following MMC treatment, Bmal1 mRNA levels was significantly decreased, whereas Dbp, Per1, and Rev-erbα mRNA levels were significantly increased in the mouse fibroblast cell line NIH3T3 cells. Microarray analysis was performed to explore of the gene(s) responsible for MMC-induced alteration of clock gene expression, and identified Nr3c1 gene encoding glucocorticoid receptor (GR) as a candidate. MMC suppressed the induction of Per1 mRNA by dexamethasone (DEX), ligand of GR, in NIH3T3 cells. MMC also modulated the DEX-driven circadian oscillations of Per2::Luciferase bioluminescence in mouse-derived ocular fibroblasts. Our results demonstrate a previously unknown effect of MMC in GR signaling and the circadian clock system. The present findings suggest that MMC combined with trabeculectomy could increase the risk for a local circadian rhythm-disorder at the ocular surface.


Subject(s)
CLOCK Proteins/genetics , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Mitomycin/pharmacology , ARNTL Transcription Factors/genetics , Animals , Chronobiology Disorders/etiology , Chronobiology Disorders/genetics , Chronobiology Disorders/metabolism , Circadian Clocks/drug effects , Circadian Clocks/genetics , Circadian Clocks/physiology , DNA-Binding Proteins/genetics , Dexamethasone/pharmacology , Eye/metabolism , Gene Expression/drug effects , Mice , Mice, Transgenic , Mitomycin/adverse effects , NIH 3T3 Cells , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Period Circadian Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Glucocorticoid/genetics , Signal Transduction/drug effects , Trabeculectomy/adverse effects , Transcription Factors/genetics
10.
J Pharmacol Exp Ther ; 354(1): 65-72, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25962390

ABSTRACT

Patients with diabetes often develop peripheral nerve complications, including numbness and pain in the extremities. Diabetes-induced peripheral neuropathic pain is characterized by hypersensitivity to innocuous stimuli, known as tactile allodynia. Pregabalin (PGN) is currently used to treat diabetes-induced peripheral neuropathy and alleviates allodynia. In the present study, we demonstrated that the antiallodynic effect of PGN on diabetic mice was modulated by circadian changes in its intestinal absorption. A single intraperitoneal administration of 200 mg/kg streptozotocin (STZ) to mice induced type I diabetic pathologic changes that were accompanied by tactile allodynia. The intensity of tactile allodynia in STZ-induced diabetic mice was alleviated by the oral administration of PGN; however, the antiallodynic effect varied according to its dosing time. The analgesic effect of PGN was enhanced by its administration at the times of day when its intestinal absorption was accelerated. Organic cation transporter novel type 1 (Octn1) mediated the uptake of PGN into intestinal epithelial cells. The expression of Octn1 in the small intestine of STZ-induced diabetic mice oscillated in a circadian time-dependent manner. This oscillation in Octn1 appeared to cause the time of day-dependent changes in the intestinal absorption of PGN. Similar dosing time dependencies of the antiallodynic effect of PGN and oscillation in Octn1 expression were also detected in type II diabetic db/db mice. These results suggested that the dosing time-dependent differences in the analgesic effect of PGN were attributable to circadian oscillations in the intestinal expression of Octn1 and also that optimizing its dosing schedule may assist in achieving rational pharmacotherapy for diabetes-induced peripheral neuropathic pain.


Subject(s)
Analgesics/administration & dosage , Diabetic Neuropathies/drug therapy , gamma-Aminobutyric Acid/analogs & derivatives , Analgesics/pharmacokinetics , Animals , Calcium Channels/metabolism , Carrier Proteins/metabolism , Circadian Rhythm , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/physiopathology , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/physiopathology , Diabetic Neuropathies/etiology , Diabetic Neuropathies/physiopathology , Epithelial Cells/metabolism , Humans , Hyperalgesia/drug therapy , Hyperalgesia/etiology , Hyperalgesia/physiopathology , Intestinal Absorption , Jejunum/metabolism , Membrane Proteins/metabolism , Mice, Inbred ICR , Organic Cation Transport Proteins , Pregabalin , Spinal Cord/metabolism , Symporters , Time Factors , Touch , gamma-Aminobutyric Acid/administration & dosage , gamma-Aminobutyric Acid/pharmacokinetics
11.
Mol Pharmacol ; 87(2): 314-22, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25422143

ABSTRACT

In addition to their digestive actions, bile acids modulate gene expression by altering the activity of peroxisome proliferator-activated receptor-α (PPARα). The modulatory effects of bile acids have been shown to affect the expression of genes responsible for lipid metabolism as well as membrane transporters. Bile acids are secreted in response to food intake and accumulate in intestinal epithelial cells. In the present study, we identified soluble carrier protein family 22 member 4 (Slc22a4), encoding organic cation transporter novel type-1 (Octn1), as a PPARα-regulated gene and its intestinal expression exhibited circadian oscillations in a bile acid-dependent manner. Nocturnally active mice mainly consumed their food around the early dark phase, during which bile acids accumulated in intestinal epithelial cells. PPARα activated the intestinal expression of Slc22a4 mRNA during the light period, and protein levels of Octn1 peaked before the start of the dark phase. The bile acids that accumulated in intestinal epithelial cells suppressed the PPARα-mediated transactivation of Slc22a4 in the dark phase. The time-dependent suppression of PPARα-mediated transactivation by bile acids regulated oscillations in the intestinal expression of Octn1/Slc22a4 during the daily feeding cycle. The results of a pharmacokinetic analysis also revealed that oscillations in the expression of Octn1 caused dosing time-dependent differences in the intestinal absorption of gabapentin (2-[1-(aminomethyl)cyclohexyl]acetic acid). These results suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal organic cation transporters. This mechanism could also account for interindividual variations in the pharmacokinetics of drugs that are substrates of Octn1.


Subject(s)
Bile Acids and Salts/pharmacology , Carrier Proteins/biosynthesis , Circadian Rhythm/physiology , Gene Expression Regulation , Intestinal Mucosa/metabolism , Membrane Proteins/biosynthesis , PPAR alpha/metabolism , Animals , Caco-2 Cells , Circadian Rhythm/drug effects , Humans , Intestinal Mucosa/drug effects , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Organic Cation Transport Proteins , Symporters
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