cyclin-d1 and Diabetes-Mellitus--Type-1

The purpose of this study was to explore the role of microRNA-451a (miR-451a) in diabetic retinopathy through activating transcription factor 2 (ATF2). The epiretinal membrane samples from patients with proliferative diabetic retinopathy (PDR) were immunolabeled with an antibody for Ki-67 to identify the proliferative cells. The expression of miR-451a was measured by qRT-PCR in the retina of Akita mice and in RPE cells under diabetic conditions. The potential downstream targets of miR-451a were predicted by bioinformatics and confirmed by dual luciferase assay, qRT-PCR, and Western blotting. Mitochondrial function, cell proliferation, and migration assays were used to detect the functional change after transfection of miR-451a mimic and inhibitor. Proliferative RPE cells were identified in the epiretinal membrane from PDR patients. The expression of miR-451a was downregulated both in the retina of Akita mice and 4-hydroxynonenal (4-HNE)-treated RPE cells. Bioinformatic analysis and luciferase assay identified ATF2 as a potential target of miR-451a. miR-451a inhibited proliferation and migration of RPE cells. The mitochondrial function was enhanced by miR-451a mimic, but suppressed by miR-451a inhibitor. In diabetic conditions, miR-451a showed a protective effect on mitochondrial function. The results of qRT-PCR and Western blotting revealed that overexpression of miR-451a downregulated the expression of ATF2 and its downstream target genes CyclinA1, CyclinD1, and MMP2. In conclusion, miR-451a/ATF2 plays a vital role in the regulation of proliferation and migration in RPE cells through regulation of mitochondrial function, which may provide new perspectives for developing effective therapies for PDR.

Topics: Activating Transcription Factor 2; Adult; Aged; Animals; Cell Movement; Cell Proliferation; Cyclin A1; Cyclin D1; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Disease Models, Animal; Female; Humans; Male; Matrix Metalloproteinase 2; Mice; MicroRNAs; Middle Aged; Mitochondria; Retinal Pigment Epithelium

It is well known that diabetes imparts high sensitivity to numerous hepatotoxicants. Previously, we have shown that a normally non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats due to inhibited tissue repair allowing progression of liver injury. On the other hand, DB rats exposed to 30 mg TA/kg exhibit delayed tissue repair and delayed recovery from injury. The objective of this study was to investigate the mechanism of impaired tissue repair and progression of liver injury in TA-treated DB rats by using cDNA microarray. Gene expression pattern was examined at 0, 6, and 12 h after TA challenge, and selected mechanistic leads from microarray experiments were confirmed by real-time RT-PCR and further investigated at protein level over the time course of 0 to 36 h after TA treatment. Diabetic condition itself increased gene expression of proteases and decreased gene expression of protease inhibitors. Administration of 300 mg TA/kg to DB rats further elevated gene expression of proteases and suppressed gene expression of protease inhibitors, explaining progression of liver injury in DB rats after TA treatment. Inhibited expression of genes involved in cell division cycle (cyclin D1, IGFBP-1, ras, E2F) was observed after exposure of DB rats to 300 mg TA/kg, explaining inhibited tissue repair in these rats. On the other hand, DB rats receiving 30 mg TA/kg exhibit delayed expression of genes involved in cell division cycle, explaining delayed tissue repair in these rats. In conclusion, impaired cyclin D1 signaling along with increased proteases and decreased protease inhibitors may explain impaired tissue repair that leads to progression of liver injury initiated by TA in DB rats.

Topics: Animals; Blotting, Western; Cyclin D1; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; DNA, Complementary; Expressed Sequence Tags; Gene Library; Image Processing, Computer-Assisted; Male; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA; Thioacetamide

Previously we reported that a nonlethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats because of irreversible acute liver injury owing to inhibited hepatic tissue repair, primarily due to blockage of G(0) to S phase progression of cell division cycle. On the other hand, DB rats receiving 30 mg TA/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic (NDB) rats receiving 300 mg TA/kg, resulting in a delay in recovery from liver injury and survival. The objective of the present study was to test the hypothesis that impaired cyclin-regulated progression of G(1) to S phase of the cell cycle may explain inhibited liver tissue repair, hepatic failure, and death, contrasted with delayed liver tissue repair but survival observed in the DB rats receiving 300 in contrast to 30 mg TA/kg. In the TA-treated NDB rats sustained MAPKs and cyclin expression resulted in higher phosphorylation of retinoblastoma (pRb), explaining prompt tissue repair and survival. In contrast, DB rats receiving the same dose of TA (300 mg/kg) exhibited suppressed MAPKs and cyclin expression that led to inhibition of pRb, inhibited tissue repair, and death. On the other hand, DB rats receiving 30 mg TA/kg exhibited delayed up regulation of MAPK signaling that delayed the expression of CD1 and pRb, explaining delayed stimulation of tissue repair observed in this group. In conclusion, the hepatotoxicant TA has a dose-dependent adverse effect on cyclin-regulated pRb signaling: the lower dose causes a recoverable delay, whereas the higher dose inhibits it with corresponding effect on the ultimate outcomes on hepatic tissue repair; this dose-dependent adverse effect is substantially shifted to the left of the dose response curve in diabetes.

Topics: Alanine Transaminase; Animals; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; ErbB Receptors; G1 Phase; Genes, p16; Liver; Male; MAP Kinase Signaling System; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; S Phase; Signal Transduction; Streptozocin; Thioacetamide

Previously we reported that an ordinarily nonlethal dose of thioacetamide (300 mg/kg) causes liver failure and 90% mortality in type 1 diabetic rats, primarily because of inhibited tissue repair. On the other hand, the diabetic rats receiving 30 mg thioacetamide/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic rats receiving 300 mg thioacetamide/kg, resulting in a delay in recovery from that liver injury and survival. These data indicate that impaired tissue repair in diabetes is a dose-dependent function of diabetes. The objective of the present study was to test the hypothesis that disrupted nuclear factor-kappaB (NF-kappaB)-regulated cyclin D1 signaling may explain dose-dependent impaired tissue repair in the thioacetamide-treated diabetic rats. Administration of 300 mg thioacetamide/kg to nondiabetic rats led to sustained NF-kappaB-regulated cyclin D1 signaling, explaining prompt compensatory tissue repair and survival. For the first time, we report that NF-kappaB-DNA binding is dependent on the dose of thioacetamide in the liver tissue of the diabetic rats. Administration of 300 mg thioacetamide/kg to diabetic rats inhibited NF-kappaB-regulated cyclin D1 signaling, explaining inhibited tissue repair, liver failure and death, whereas remarkably higher NF-kappaB-DNA binding but transient down regulation of cyclin D1 expression explains delayed tissue repair in the diabetic rats receiving 30 mg thioacetamide/kg. These data suggest that dose-dependent NF-kappaB-regulated cyclin D1 signaling explains inhibited versus delayed tissue repair observed in the diabetic rats receiving 300 and 30 mg thioacetamide/kg, respectively.

Topics: Alanine Transaminase; Analysis of Variance; Animals; Cyclin D1; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; Dose-Response Relationship, Drug; I-kappa B Kinase; I-kappa B Proteins; Liver; Liver Regeneration; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Streptozocin; Thioacetamide; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha

To determine the role of cell-cycle proteins in regulating pathological renal hypertrophy, diabetes was induced in mice expressing a human retinoblastoma (RB) transgene and in wild-type littermates. Whole-kidney and glomerular hypertrophy caused by hyperglycemia was associated with specific G1 phase cell-cycle events: early and sustained increase in expression of cyclin D1 and activation of cyclin D1-cdk4 complexes, but no change in expression of cyclin E or cdk2 activity. Overexpression of RB alone likewise caused hypertrophy and increased only cyclin D1-cdk4 activity; these effects were not further augmented by high glucose. Identical observations were made when isolated mesangial cells conditionally overexpressing RB from a tetracycline-repressible system hypertrophied in response to high glucose. A mitogenic signal in the same cell-culture system, in contrast, transiently and sequentially activated both cyclin D1-cdk4 and cyclin E-cdk2. In vivo and in cultured mesangial cells, high glucose resulted in persistent partial phosphorylation of RB, an event catalyzed specifically by cyclin D1-cdk4. These data indicate that mesangial hypertrophy caused by hyperglycemia in diabetes results in sustained cyclin D1-cdk4-dependent phosphorylation of RB and maintenance of mesangial cells in the early-to-middle G1 phase of the cell cycle.

Topics: Animals; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Glomerular Mesangium; Humans; Hypertrophy; Mice; Mice, Transgenic; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein

Cultured skin fibroblasts from type 1 diabetic patients with nephropathy have a hyperplastic growth phenotype in comparison with diabetics without nephropathy and controls. We studied the G1 phase cyclins in skin fibroblasts from control subjects to define the temporal profile of serum-induced pRB phosphorylation, cyclin D1 protein expression, and cyclin D1/CDK4(6) kinase activity as well as S-phase entry by FACS analysis. Our preliminary studies indicate that cultured skin fibroblasts from type 1 diabetic patients with nephropathy have an enhanced pRB phosphorylation, cyclin D1 protein expression, and cyclin D1/CDK4(6) kinase activity. This finding may become useful to identify patients at risk for the development of nephropathy.

Topics: Blood Proteins; Cells, Cultured; Cyclin D1; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibroblasts; G1 Phase; Humans; Phosphorylation; Retinoblastoma Protein; S Phase; Skin; Thymidine; Time Factors; Tritium