cyclin-d1 and Asthma

Abnormal airway smooth muscle cell (ASMC) proliferation and migration contribute significantly to increased ASM mass associated with asthma. MicroRNA (miR)-638 is a primate-specific miRNA that plays important roles in development, DNA damage repair, hematopoiesis, and tumorigenesis. Although it is highly expressed in ASMCs, its function in ASM remodeling remains unknown. In the current study, we found that in response to various mitogenic stimuli, including platelet-derived growth factor-two B chains (PDGF-BB), transforming growth factor β1, and fetal bovine serum, the expression of miR-638, as determined by quantitative real-time polymerase chain reaction (qRT-PCR), was significantly downregulated in the proliferative human ASMCs. Both gain- and loss-of-function studies were performed to study the role of miR-638 in ASMC proliferation and migration. We found that adenovirus-mediated miR-638 overexpression markedly inhibits ASMC proliferation and migration, while ablation of miR-638 by anti-miR-638 markedly increases cell proliferation and migration, as determined by WST-8 proliferation and scratch wound assays. Dual-luciferase reporter assay, qRT-PCR, and immunoblot analysis were used to investigate the effects of miR-638 on the expression of the downstream target genes in ASMCs. Our results demonstrated that miR-638 overexpression significantly reduced the expression of downstream target cyclin D1 and NOR1, both of which have been shown to be essential for cell proliferation and migration. Together, our study provides the first in vitro evidence highlighting the antiproliferative and antimigratory roles of miR-638 in human ASMC remodeling and suggests that targeted overexpression of miR-638 in ASMCs may provide a novel therapeutic strategy for preventing ASM hyperplasia associated with asthma.

Topics: Airway Remodeling; Asthma; Becaplermin; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation; Humans; Membrane Transport Proteins; MicroRNAs; Myocytes, Smooth Muscle

Topics: Asthma; Cell Proliferation; Cyclin D1; Humans; Myocytes, Smooth Muscle; RNA-Binding Proteins

Excessive proliferation and migration of airway smooth muscle cell (ASMC) contribute to asthma pathogenesis. Long noncoding RNAs (lncRNAs) are reported to take part in asthma pathogenesis. This study is targeted at deciphering the role of the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) in ASMC proliferation, migration and extracellular matrix (ECM) deposition. qRT-PCR was performed to determine ANRIL, miR-98-5p, and cyclin D1 (CCND1) mRNA expression levels in transforming growth factor-β1 (TGF-β1)-treated ASMCs. CCK-8 and Transwell assays were employed to examine ASMC proliferation and migration, respectively. Dual-luciferase reporter gene assay and RNA immunoprecipitation assay were carried out for analyzing the targeted relationship of miR-98-5p with ANRIL or CCND1 mRNA 3'-UTR. The levels of CCND1 and ECM proteins (such as fibronectin, COL3A1, and COL1A2) in ASMCs were detected through Western blot. In this work, we found that ANRIL and CCND1 were up-regulated in TGF-β1-treated ASMCs, whereas miR-98-5p was down-regulated. ANRIL overexpression facilitated the proliferation, ECM deposition and migration of TGF-β1-induced ASMCs, while knocking down ANRIL had the opposite effect. Furthermore, ANRIL targeted miR-98-5p directly, and CCND1 was miR-98-5p's downstream target. ANRIL indirectly increased CCND1 expression in ASMCs via competitively binding to miR-98-5p. MiR-98-5p inhibition or CCND1 overexpression counteracted the inhibiting effect that ANRIL knockdown had on TGF-β1-stimulated ASMC proliferation, migration and ECM deposition. In conclusion, ANRIL indirectly up-regulates CCND1 expression by targeting miR-98-5p to promote ASMC proliferation, migration and ECM deposition, thus facilitating the pathogenesis of asthma.

Topics: Asthma; Cell Movement; Cell Proliferation; Cyclin D1; Extracellular Matrix; Humans; MicroRNAs; Muscle, Smooth; Myocytes, Smooth Muscle; RNA, Long Noncoding; RNA, Messenger; Transforming Growth Factor beta1

This study aims to investigate mechanism of "Ephedrae Herba-Descurainiae Semen Lepidii Semen" combination(MT) in the treatment of bronchial asthma based on network pharmacology and in vivo experiment, which is expected to lay a theoretical basis for clinical application of the combination. First, the potential targets of MT in the treatment of bronchial asthma were predicted based on network pharmacology, and the "Chinese medicine-active component-target-pathway-disease" network was constructed, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the potential targets. Molecular docking was used to determine the binding activity of key candidate active components to hub genes. Ovalbumin(OVA, intraperitoneal injection for sensitization and nebulization for excitation) was used to induce bronchial asthma in rats. Rats were classified into control group(CON), model group(M), dexamethasone group(DEX, 0.075 mg·kg~(-1)), and MT(1∶1.5) group. Hematoxylin and eosin(HE), Masson, and periodic acid-Schiff(PAS) staining were performed to observe the effect of MT on pathological changes of lungs and trachea and goblet cell proliferation in asthma rats. The levels of transforming growth factor(TGF)-β1, interleukin(IL)6, and IL10 in rat serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein levels of mitogen-activated protein kinase 8(MAPK8), cyclin D1(CCND1), IL6, epidermal growth factor receptor(EGFR), phosphatidylinositol 3-kinase(PI3 K), and protein kinase B(Akt) by qRT-PCR and Western blot. Network pharmacology predicted that MAPK8, CCND1, IL6, and EGFR were the potential targets of MT in the treatment of asthma, which may be related to PI3 K/Akt signaling pathway. Quercetin and β-sitosterol in MT acted on a lot of targets related to asthma, and molecular docking results showed that quercetin and β-sitosterol had strong binding activity to MAPK, PI3 K, and Akt. In vivo experiment showed that MT could effectively alleviate the symptoms of OVA-induced asthma rats, improve the pathological changes of lung tissue, reduce the production of goblet cells, inhibit the inflammatory response of asthma rats, suppress the expression of MAPK8, CCND1, IL6, and EGFR, and regulate the PI3 K/Akt signaling pathway. Therefore, MT may relieve the symptoms and inhibit inflammation of asthma rats by regulating the PI3 K/Akt signaling pathway, and quercetin and β-sitos

Topics: Animals; Asthma; Cyclin D1; Dexamethasone; Drug Combinations; Drugs, Chinese Herbal; Eosine Yellowish-(YS); Ephedra; ErbB Receptors; Hematoxylin; Interleukin-10; Interleukin-6; Mitogen-Activated Protein Kinase 8; Molecular Docking Simulation; Network Pharmacology; Ovalbumin; Periodic Acid; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quercetin; Rats; RNA, Messenger

Airway remodeling in asthma involves the hyperproliferation of airway smooth muscle (ASM) cells. However, the molecular signals that regulate ASM growth are not completely understood. Gq-coupled G protein-coupled receptor and receptor tyrosine kinase signaling regulate ASM cell proliferation via activation of phospholipase C, generation of inositol triphosphate (IP

Topics: Airway Remodeling; Animals; Asthma; Cell Proliferation; Cyclin D1; Diacylglycerol Kinase; Diglycerides; Humans; Inositol; Mice; Mitogens; Myocytes, Smooth Muscle; Phosphatidic Acids; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Receptors, G-Protein-Coupled; TOR Serine-Threonine Kinases; Type C Phospholipases

The purpose of this study was to establish a rat asthma model and extract MUC5AC to explore the mechanism of mucin 5AC (MUC5AC) signaling pathway regulating the function of asthmatic airway smooth muscle cells (ASMC) and participating in asthmatic airway remodeling. Western blot was used to detect β-catenin (β-catenin), glycogen synthase kinase-3β (GSK-3β), proto-oncogene MUC5AC and cyclin D1 (cyclin D1) in MUC5AC of asthmatic and normal groups. After inhibiting the interaction between β-catenin and transcription cofactor p300 / CBP in ASMC of the asthma group and control group, the cell viability and cycle changes of ASMC were detected by the CCK-8 method and flow cytometry. After inhibiting the activity of P38 mitogen-activated protein kinase (MAPK), the protein expression changes of c-Myc and cyclin D1 were detected by Western blot. Results showed that comprehensive HE staining results of lung tissue sections indicate that the experimental rat model of asthma airway remodeling was successfully established. Compared with the control group, 100 fxmol and L1 Efaroxan promoted insulin secretion (P <0.01), and administration of the MUC5AC antagonist KU14R significantly inhibited the effect of MUC5AC.Western blot showed that the protein expression levels of β-catenin, c-Myc and cyclin D1 in ASMC of the obese asthma group were significantly higher than those of the control group (P <0.05), while the protein expression level of GSK-3β was lower than Control group (P <0.05). After inhibiting the interaction between β-catenin and p300 / CBP, the decrease in cell viability and the degree of cell cycle change of ASMC in the asthma group were more obvious than those in the control group (P <0.05). After inhibiting the activity of P38 MAPK, the expressions of the target proteins c-Myc and cyclin D1 in the MUC5AC signaling pathway in ASMC model rats and control rats were down-regulated, and the difference was statistically significant (P <0.05). The conclusion was that the Wnt/β-catenin signaling pathway can regulate the proliferation and differentiation of ASMC by up-regulating the expression level of cMyc. Cyclin D1 interacts with the MAPK signaling pathway, thereby affecting the function of ASMC and participating in asthma airway remodeling.

Topics: Airway Remodeling; Animals; Asthma; beta Catenin; Cell Proliferation; Cyclin D1; Glycogen Synthase Kinase 3 beta; Mucin 5AC; Obesity; p38 Mitogen-Activated Protein Kinases; Rats; Wnt Signaling Pathway

Asthma is a prevalent chronic inflammatory airway disease that is characterised by airway remodelling and airway hyperresponsiveness. Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) contribute to airway remodelling in asthma. However, the molecular mechanism underlying an increased ASMC mass in asthma remains elusive. Herein, we aimed at investigating the regulation of lncRNA PVT1 on ASMCs and focussing on the mechanism in the proliferation and migration.. Expression levels of lncRNA PVT1 and miR-590-5p in the serum collected from 24 children with asthma and 10 control children were determined by qRT-PCR. ASMCs proliferation and migration prior to and post platelet-derived growth factor subunit B (PDGF-BB) stimulation were examined by CCK-8 test and transwell assay. Dual-luciferase reporter assay was performed to determine miR-590-5p interaction with lncRNA PVT1 and follistatin-like 1 (FSTL1). Expression of lncRNA PVT1, miR-590-5p, FSTL1, C-Myc, cyclin D1, and cyclin-dependent kinase 1 (CDK1) was tested by quantitative real-time PCR (qRT-PCR) and immunoblotting analysis.. The expression level of lncRNA PVT1 was higher but the expression level of miR-590-5p was lower in the serum of children with asthma than in control children. The expression level of lncRNA PVT1 was negatively correlated with the expression level of miR-590-5p in asthma. LncRNA PVT1 was upregulated upon PDGF-BB stimulation. LncRNA PVT1 knockdown by its specific shRNA repressed PDGF-BB-induced promotion of proliferation and migration in ASMCs and triggered an elevated miR-590-5p along with declined C-Myc, cyclin D1, and CDK1. The effects of lncRNA PVT1 knockdown on PDGF-BB-induced ASMCs were lost upon miR-590-5p inhibition. MiR-590-5p targeted FSTL1 gene and declined its expression, thus suppressing ASMC proliferation and migration following PDGF-BB stimulation and downregulating C-Myc, cyclin D1, and CDK1 expressions. The effects of miR-590-5p on PDGF-BB-induced ASMCs were lost upon FSTL1 overexpression.. These results support the notion that the lncRNA PVT1/miR-590-5p/FSTL1 axis modulates ASMCs proliferation and migration following PDGF-BB stimulation, providing a potential therapeutic target to attenuate airway remodelling in asthma.

Topics: Apoptosis; Asthma; Cell Line; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Follistatin-Related Proteins; HEK293 Cells; Humans; Lung; MicroRNAs; Myocytes, Smooth Muscle; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction

Abnormal proliferation of airway smooth muscle cells (ASMCs) is a hallmark of airway remodeling. Platelet-derived growth factor (PDGF) is known to be a major stimulus inducing the proliferation of ASMCs. It has been reported that triptolide demonstrates protective effects against airway remodeling. In this study, we investigated the antiproliferative effects of triptolide on PDGF-induced ASMCs and its underlying mechanisms. Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Quantitative real-time PCR and Western blot analysis were employed to detect the expression of proliferating cell nuclear antigen (PCNA), cyclinD1 and cyclin dependent kinase 4 (CDK4). Proteins involved in the protein kinase B (AKT) and nuclear factor kappa B (NF-κB) signaling pathways were evaluated using Western blot analysis. Triptolide could significantly inhibit cell proliferation, induce cell cycle arrest in the G0/G1 phase, and reduce the expression of PCNA, cyclinD1, and CDK4 in PDGF-treated ASMCs. Levels of phosphorylated AKT, p65 and NF-κB inhibitor α (IκBα) stimulated by the presence of PDGF were markedly suppressed after triptolide treatment. Moreover, triptolide cotreatment with the phosphatidylinositol 3 kinase (PI3k) inhibitor, 2-(4-morpholinyl)-8-phenylchromone (LY294002), could further suppress the proliferation, NF-κB activation and cyclinD1 expression. Similar results were observed after triptolide cotreatment with the NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC). Our results suggest that triptolide could inhibit the PDGF-induced proliferation of ASMCs through G0/G1 cell cycle arrest and suppression of the AKT/NF-κB/cyclinD1 signaling pathway.

Topics: Airway Remodeling; Animals; Asthma; Bronchi; Cell Proliferation; Cells, Cultured; Chromones; Cyclin D1; Diterpenes; Epoxy Compounds; G1 Phase Cell Cycle Checkpoints; Humans; Morpholines; Myocytes, Smooth Muscle; NF-kappa B; Phenanthrenes; Phosphatidylinositol 3-Kinases; Phosphorylation; Platelet-Derived Growth Factor; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Pyrrolidines; Rats; Signal Transduction; Thiocarbamates

YKL-40 is a chitinase-like protein which was significantly elevated in asthma patients and related closely to asthma severity and airway remodeling. Airway remodeling in asthma involves complicated physical and pathological processes, including increased airway smooth muscle mass due to proliferation, migration of airway smooth muscle cells, epithelial-mesenchymal transition (EMT) and sub-epithelial fibrosis. However, the precise effect and underlying mechanism of YKL-40 in this pathological alteration remained unelucidated. In this study, we demonstrated that YKL-40 could promote asthma airway remodeling by increasing airway smooth muscle mass, inducing EMT and sub-epithelial fibrosis. Furthermore, we identified that FAK and MAPK signaling pathways are activated in the process. Inhibiting FAK or MAPK pathway could significantly ameliorate airway remodeling induced by excessive secretion of YKL-40

Topics: Airway Remodeling; Animals; Asthma; Cell Movement; Cell Proliferation; Chitinase-3-Like Protein 1; Cyclin D1; Cyclin-Dependent Kinases; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Focal Adhesion Protein-Tyrosine Kinases; Humans; MAP Kinase Signaling System; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; src-Family Kinases

BACKGROUND Asthma is a chronic disease with high morbidity rates. Brain-derived neurotrophic factor (BDNF) has been proven to induce airway hyper-responsiveness, but the function of BDNF in the wound-healing process of asthmatic human airway epithelial cells (HAECs) remains unclear. This study investigated the effects of BDNF in asthmatic children with injured HAECs. MATERIAL AND METHODS HAECs were obtained from healthy children and asthmatic children through bronchoscopy, and then cultured in air-liquid (ALI) interface with or without BDNF. A mechanical injury model was established for the wound-healing assay. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to measure BDNF mRNA expressions, while western blot assay was used for the measurement of BDNF and CCND1 protein expressions. Cell proliferation of impaired HAECs was assayed in a ³H-thymidine incorporation experiment. RESULTS The mRNA and protein levels of BDNF were overexpressed, and the wound-healing ability of HAECs decreased in asthma samples. Also, the cell proliferation of HAECs was suppressed in the asthmatic injury model and the injury-induced increase of CCND1 protein expressions was inhibited in asthma. Although mRNA and protein expressions of BDNF remained unchanging in healthy HAECs, there was an increase in impaired asthmatic HAECs. Upregulating BDNF led to a decrease in wound-healing ability of HAECs in both healthy children and children with asthma. Simultaneously, overexpressed BDNF reduced the CCND1 protein expressions in healthy HAECs, but had little impact on asthmatic HAECs. CONCLUSIONS Brain-derived neurotrophic factor (BDNF) inhibited wound-healing and cell proliferative ability of human airway epithelial cells (HAECs) in asthmatic children.

Topics: Adolescent; Asthma; Brain-Derived Neurotrophic Factor; Cell Proliferation; Child; Cyclin D1; Epithelial Cells; Female; Gene Expression Regulation; Humans; Male; Respiratory Mucosa; Wound Healing

Airway smooth muscle (ASM) cell hyperplasia contributes to airway wall remodeling (AWR) in asthma. Glucocorticoids, which are used as first-line therapy for the treatment of inflammation in asthma, have limited impact on AWR, and protracted usage of high doses of glucocorticoids is associated with an increased risk of side effects. Moreover, patients with severe asthma often show reduced sensitivity to glucocorticoids. Artesunate, a semisynthetic artemisinin derivative used to treat malaria with minimal toxicity, attenuates allergic airway inflammation in mice, but its impact on AWR is not known. We examined the effects of artesunate on ASM proliferation in vitro and in vivo. Primary human ASM cells derived from nonasthmatic donors were treated with artesunate before mitogen stimulation. Artesunate reduced mitogen-stimulated increases in cell number and cyclin D1 protein abundance but had no significant effect on ERK1/2 phosphorylation. Artesunate, but not dexamethasone, inhibited phospho-Akt and phospho-p70(S6K) protein abundance. Artesunate, but not dexamethasone, inhibited mitogen-stimulated increases in cell number, cyclin D1, and phospho-Akt protein abundance on ASM cells derived from asthmatic donors. In a murine model of allergic asthma, artesunate reduced the area of α-smooth muscle actin-positive cells and decreased cyclin D1 protein abundance. Our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the PI3K/Akt/p70(S6K) pathway and suggests that artesunate may be used as combination therapy with glucocorticoids.

Topics: Airway Remodeling; Animals; Antimalarials; Artemisinins; Artesunate; Asthma; Cell Proliferation; Cells, Cultured; Cyclin D1; Dexamethasone; Female; Glucocorticoids; Humans; Mice; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Respiratory System

In the present study, we determined whether Phosphoinositide 3-kinase (PI3K) and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 which were key molecules in controlling cell cycling from CD4(+) T lymphocyte in animal model of asthma.. Ovalbumin (OVA) sensitized murine model of asthma was used to investigate the expression of cyclin D1, cyclin A, and p27kip1 by splenic CD4(+) T lymphocytes. We further observed the effect of specific inhibitor of PI3K(LY294002) and specific inhibitor of Notch(DAPT) on the proliferation of such CD4(+) T lymphocytes.. We found that the expression of cyclinD1 and cyclinA was upregulated at both protein and mRNA levels in asthma group while p27kip1 was down-regulated. Both LY294002 and DAPT inhibit the proliferation of CD4(+) T lymphocytes in a time- and dose-dependent manner. Furthermore, LY294002 and DAPT have additive effect in down-regulation of cyclinD1 and upregulation of p27kip1. An upregulation of cyclinA, although not statistically significant, was also observed.. These data suggested that PI3K signal pathway and Notch signal pathway may coordinately regulate the cell proliferation and differentiation processes through up-regulating cyclinD1 and down-regulating p27kip1 of CD4(+) T lymphocytes.

Topics: Animals; Asthma; CD4-Positive T-Lymphocytes; Cell Proliferation; Chromones; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Male; Mice; Mice, Inbred BALB C; Morpholines; Ovalbumin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Notch; RNA, Messenger; Signal Transduction; Spleen; Time Factors; Up-Regulation

To investigate the effects of peroxisome proliferator-activated receptor-gamma (PPARγ) agonist rosiglitazone on the expression of cyclin D1 in lung tissue, and the proliferation of airway smooth muscle cells (ASMCs) in mice with bronchial asthma.. Thirty clean BALB/c mice were randomly divided into control group (n = 10), asthma group (n = 10), and rosiglitazone treatment group (n = 10). A mouse model of asthma was established by ovalbumin (OVA) sensitization and challenge. The treatment group received rosiglitazone (5 mg/kg) by gavage 1 hour before each challenge and the control group received saline instead of OVA sensitization and challenge. Leukocytes and eosinophils in bronchoalveolar lavage fluid (BALF) were counted under a microscope. Airway structural changes were observed by hematoxylin-eosin staining. Protein and mRNA expression levels of cyclin D1 were measured by immunohistochemical staining and RT-PCR. Perimeter of the basement membrane (Pbm), total bronchial wall area (WAt), airway smooth muscle area (WAm), and number of nuclei in ASMCs (N) were determined using image analysis software, and WAt/Pbm, WAm/Pbm, and N/Pbm were calculated.. Compared with the control group, the asthma group showed significant increases in the total number of leukocytes and percentage of eosinophils in BALF, as well as in the mRNA and protein expression of cyclin D1, but changes in these indices were significantly reduced in the rosiglitazone treatment group (P < 0.05). In addition, compared with the control group, the asthma group had significantly increased WAt/Pbm, WAm/Pbm, and N/Pbm, but rosiglitazone significantly decreased these ratios (P < 0.05).. Rosiglitazone may delay the process of airway remodeling by inhibiting the proliferation of ASMCs, so it can be used for preventing and treating chronic asthma.

Topics: Airway Remodeling; Animals; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cyclin D1; Female; Lung; Mice; Mice, Inbred BALB C; Myocytes, Smooth Muscle; PPAR gamma; RNA, Messenger; Rosiglitazone; Thiazolidinediones

To explore whether the signal pathways of phosphoinositide 3-kinase (PI3K) and Notch can realize coordinated regulation on the activation and proliferation of CD4(+)T lymphocytes.. Male BALB/c mice were randomly divided into control and asthma groups. Then the murine model of asthma was established by the method of ovalbumin (OVA) challenge. The CD4(+)T lymphocytes were isolated by magnetic activated cell sorter (MACS) and then activated with phytohaemagglutinin (PHA) (10 µg/ml) and IL-2 (1000 U/ml) for 6 h. Those cells were then divided into Group A: without any treatment; Group B: treatment with PI3K inhibitor (LY294002); Group C: treatment with Notch inhibitor (gamma-secretase inhibitor, DAPT); Group D: treatment with PI3K inhibitor and Notch inhibitor. The protein and transcription levels of Cyclin A, Cyclin D1 and P27(kip1) of CD4(+)T lymphocytes were assessed by flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR).. The results of flow cytometry showed that the purity of MACS-isolated CD4(+)T lymphocytes was 90.0% ± 5.2% and the survival rate 94.8% ± 3.2%. The protein (28.0% ± 3.5%, 14.9% ± 3.4%) and mRNA levels (0.55 ± 0.16, 1.38 ± 0.42) of Cyclin A and Cyclin D1 in CD4(+)T lymphocytes of asthma group were significantly higher than those of the control group (13.4% ± 3.5%, 7.7% ± 1.8% and 0.32 ± 0.10, 0.92 ± 0.37) (P = 0.002, 0.036 and P = 0.007, 0.042). The protein and mRNA levels (23.3% ± 3.9% and 0.16 ± 0.03) of P27(kip1) of asthma group were significantly lower than those of control group (37.5% ± 5.8% and 0.32 ± 0.03, P = 0.006 and P = 0.000). The protein and mRNA levels of Cyclin D1 in groups A, B, C and D-treated CD4(+)T lymphocytes were 12.2% ± 3.7%, 7.3% ± 3.0%, 8.1% ± 2.3%, 4.2% ± 1.7% and 1.71 ± 0.44, 1.07 ± 0.31, 1.21 ± 0.32 and 0.62 ± 0.20 respectively; groups B, C and D decreased markedly compared with group A (all P < 0.01) while group D decreased significantly compared with groups B and C (all P < 0.05). The protein levels of P27(kip1) in groups A, B, C and D were 22.9% ± 3.0%, 31.6% ± 5.3%, 28.4% ± 5.6% and 44.6% ± 2.8% respectively; group B was significantly higher than that of group A (P = 0.016) while group D was significantly higher than those of groups A, B and C (P = 0.003, 0.004, 0.000). Meanwhile P27(kip1) mRNA levels in each group were 0.16 ± 0.07, 0.36 ± 0.09, 0.63 ± 0.08 and 0.99 ± 0.21 respectively; groups B, C and D were much higher than that of group A (P = 0.016, 0.000, 0.000) while group D was significantly higher than those of groups B and C (P = 0.000, 0.023). The protein and mRNA levels of CylinA showed no statistical significance among different experimental groups (all P > 0.05).. The signal pathways of PI3K and Notch may coordinately up-regulate the expression of positive regulatory factor cylinD1 and down-regulation the expression of negative regulatory factor P27(kip1) of CD4(+)T lymphocytes.

Topics: Animals; Asthma; CD4-Positive T-Lymphocytes; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Notch; Signal Transduction

It is well established that hyperplasia and decreased apoptosis of airway smooth muscle cells (ASMCs) play an important role in the asthmatic airway remodeling. Tumor suppressor PTEN gene with phosphatase activity plays an important regulatory role in embryonic development, cell proliferation, and apoptosis, cell cycle regulation, migration (invasion) of the cytoskeleton. We hypotheses that PTEN gene could affect the growth and viability of ASMCs through the regulation of PI3K/Akt, MAPK, and cell cycle-related gene expression. We constructed a recombinant adenovirus to transfect ASMCs. Cells were divided into the overexpression of PTEN gene group (Ad-PTEN-GFP), negative control group (Ad-GFP), and blank control group (DMEM). The cell apoptosis of ASMCs were evaluated by Hoechst-33342 staining and PE-7AAD double-labeled flow cytometry. The cell cycle distribution was observed by flow cytometry with PI staining. The expression of PTEN, p-Akt, total-Akt, p-ERK1/2, total-ERK1/2, cleaved-Caspases-3, Caspases-9, p21, and Cyclin D1 were tested by the Western blotting. Our study revealed that overexpression of PTEN gene did not induce apoptosis of human ASMCs cultured in vitro. However, overexpression of PTEN inhibited proliferation of human ASMCs cultured in vitro and was associated with downregulation of Akt phosphorylation levels, while did not affect ERK1/2 phosphorylation levels. Moreover, overexpression of PTEN could induce ASMCs arrested in the G0/G1 phase through the downregulation of Cyclin D1 and upregulation of p21 expressions.

Topics: Adenoviridae; Airway Remodeling; Apoptosis; Asthma; Caspase 3; Caspase 9; Cell Proliferation; Cell Shape; Cell Survival; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Lung; Myocytes, Smooth Muscle; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transfection

The molecular mechanisms underlying the association between obesity (BMI ≥ 30 kg/m(2)) and asthma are poorly understood. Since shifts in the fate of bronchial cells due to low-grade systemic inflammation may provide a possible explanation, we investigated whether two of the best documented functional variants in cell cycle control genes modify the obesity-asthma association.. We genotyped 5930 SAPALDIA cohort participants for the single-nucleotide polymorphisms (SNPs) rs9344 in the cyclin D1 gene (CCND1) and rs1042522 in the gene encoding tumor protein 53 (TP53). We assessed the independent association of these SNPs and obesity with asthma prevalence and incidence.. The CCND1 SNP modified the association between obesity and asthma prevalence (p(interaction )= 0.03). The odds ratios (ORs) and 95% confidence intervals (CIs) for reporting a physician diagnosis of asthma at baseline, comparing obese with non-obese participants, were 1.09 (0.51-2.33), 1.64 (0.94-2.88), and 3.51 (1.63-7.53) for GG, GA, and AA genotypes, respectively. We found comparable genotype differences for incident asthma within the 11 years of follow-up. As for the TP53 SNP, the interactions with obesity status with respect to asthma were not statistically significant.. Our results suggest that obesity may contribute to asthma and associated tissue remodeling by modifying the processes related to the CCND1 gene activity.

Topics: Adolescent; Adult; Asthma; Cohort Studies; Cyclin D1; DNA; DNA-Binding Proteins; Female; Genotype; Humans; Incidence; Male; Middle Aged; Obesity; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prevalence; Regression Analysis; Switzerland; Young Adult

To investigate the effects of H(2) Relaxin (Relaxin) on airway remodeling and the expression of cyclin D(1) in a murine model of chronic asthma.. Forty BALB/c mice were randomly divided into 4 groups:a normal control group, an asthma group, a vehicle control group and a relaxin treatment group, with 10 mice in each. The mice were sensitized and challenged with ovalbumin (OVA) to establish the chronic asthmatic model. The vehicle control group and the relaxin treatment group were subcutaneously injected with saline and relaxin (0.25 mg × kg(-1)× d(-1))respectively. Alteration of the airway inflammation and collagen deposition were observed by haematoxylin-eosin (HE) and Masson staining. Hydroxyproline in the lung was measured by enzyme linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) in lungs was evaluated by immunohistochemistry. The protein expression and the mRNA of cyclin D(1) were detected by Western blot and RT-PCR respectively.. There were inflammatory cell infiltration, airway stenosis, bronchial smooth muscle hypertrophy and increased collagen deposition in the asthmatic group and the vehicle control group; but these changes were significantly ameliorated in the relaxin treatment group. The area of the α-SMA-stained smooth muscle layer in the asthmatic group and the vehicle control group was significantly greater than that in the control group (all P < 0.05), while administration of relaxin decreased the α-SMA immunostained area (all P < 0.05). The lung hydroxyproline content in the asthmatic and the vehicle groups [(0.68 ± 0.10) mg/g lung tissue, (0.67 ± 0.10) mg/g lung tissue] was significantly greater than that in the control group [(0.26 ± 0.05) mg/g lung tissue] (q = 16.61, 16.01 respectively, all P < 0.01). In contrast, treatment with relaxin significantly reduced the lung hydroxyproline content [(0.40 ± 0.06) mg/g lung tissue] compared with aforementioned 2 groups (q = 10.88, 10.26 respectively, all P < 0.05). The results of the Western blot analysis showed that the expression level of cyclin D(1) in the asthmatic and the vehicle groups [(1.38 ± 0.18), (1.50 ± 0.10)] was higher than that in the control group (0.38 ± 0.10) (q = 13.00, 14.65 respectively, all P < 0.05), while it was significantly decreased in the relaxin group (0.72 ± 0.13) (q = 8.51, 10.16 respectively, all P < 0.05). There were no differences in all of the parameters between the asthmatic group and the vehicle group (P > 0.05).. Relaxin alleviated airway inflammation, airway smooth muscle thickening and airway remodelling in a murine model of chronic asthma, partially by down-regulating the expression level of cyclin D(1).

Topics: Actins; Airway Remodeling; Animals; Asthma; Cyclin D1; Female; Lung; Mice; Mice, Inbred BALB C; Relaxin

To study the effect of cigarette smoke extract (CSE) on the proliferation of human airway smooth muscle cells (HASMCs) sensitized by serum from asthmatic patients and the underlying mechanisms.. HASMCs were cultured from primary generation. Cells between passage 4 and 8 were used in the study. HASMCs were sensitized by 10% serum from asthmatic patients and were divided into an asthmatic serum group, an asthmatic serum + CSE group, an asthmatic serum + GW8510 (inhibitor of cyclin-dependent kinase-4) group and an asthmatic serum + CSE + GW8510 group. Non-asthmatic human serum treated HASMCs served as the control. The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and [(3)H] thymidine incorporation. The expression of cyclinD(1) was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.. The percentage of S + G(2)/M phase, the absorbance (A) value and the DNA synthesis value in asthmatic serum group were significantly increased compared with those of the control group (q = 6.25, 5.61, 6.82, respectively, all P < 0.01). The percentage of S + G(2)/M phase, the absorbance (A) value and DNA synthesis value in the asthmatic serum group were (21.4 ± 1.1)%, 0.392 ± 0.124 and 2669 ± 138, respectively. Their value in the asthmatic serum + CSE group were (33.3 ± 1.3)%, 0.612 ± 0.201 and 3552 ± 303, respectively, which were significantly increased compared with those of the asthmatic serum group (q = 5.67, 6.32, 5.56, respectively, all P < 0.01). Their value in the asthmatic serum + GW8510 group were (14.7 ± 1.4)%, 0.301 ± 0.097 and 1812 ± 109, respectively, which were significantly decreased compared with those of the asthmatic serum group (q = 6.02, 5.53, 5.79, respectively, all P < 0.01). The ratios of A value of cyclinD(1) mRNA and the expression of cyclinD(1) protein in the asthmatic serum group were 0.291 ± 0.112 and 0.186 ± 0.002, respectively. The ratios of A value in the asthmatic serum + CSE group were 0.521 ± 0.102 and 0.312 ± 0.002, respectively, which were significantly increased compared with those of the asthmatic serum group (q = 12.09, 9.26, respectively, all P < 0.01). The ratios of A value in the asthmatic serum + GW8510 group were 0.223 ± 0.038 and 0.150 ± 0.002, respectively, which were significantly decreased compared with those of the asthmatic serum group (q = 6.86, 5.60, respectively, all P < 0.01).. HASMCs sensitized by serum from asthmatic patients showed accelerated proliferation after intervention by CSE, with increased expression of cyclinD(1). CSE may increase the proliferation of HASMCs sensitized by serum from asthmatic patients via regulating cyclinD expression.

Topics: Adult; Asthma; Cell Proliferation; Cells, Cultured; Cyclin D1; Female; Humans; Male; Middle Aged; Myocytes, Smooth Muscle; Nicotiana; Respiratory System; Serum; Signal Transduction; Smoke; Young Adult

PPARgamma levels in asthma- and non-asthma-derived airway smooth muscle cells and PPARgamma activation-induced cell proliferation were investigated. In the presence of FBS, PPARgamma levels were higher in subconfluent asthma-derived cells but lower in confluent cells compared with non-asthma-derived. However, PPARgamma activation did not alter cell proliferation.. Airway remodelling involves thickening of the airway smooth muscle (ASM) bulk. Proliferation of asthma-derived ASM cells is increased in vitro, but underlying mechanisms remain unknown. Peroxisome proliferators activated receptor-gamma (PPARgamma) regulates the cell cycle. It is suggested that PPARgamma agonists have anti-inflammatory effects, which may be valuable in the treatment of asthma, but information regarding their antiproliferative properties in ASM is lacking. Although corticosteroids reduce airway inflammation, in vitro they inhibit proliferation in only non-asthma ASM cells by reducing cyclin D1. We therefore investigated the effects of mitogenic stimulation (foetal bovine serum (FBS)), and a PPARgamma ligand (ciglitazone), on PPARgamma and cyclin D1 expression and proliferation of ASM cells. In addition, we examined the effects of ciglitazone on ASM cell proliferation.. We assessed PPARgamma and cyclin D1 mRNA and protein levels using quantitative PCR and immunoblotting. Cell proliferation was assessed using bromodeoxyuridine uptake.. In the presence of 5% FBS, PPARgamma and cyclin D1 expression decreased over time in non-asthmatic cells but increased in asthmatic cells (compared with sub-confluent cells). FBS-induced proliferation of asthmatic cells increased at all time points, but occurred only at day 7 with non-asthmatic cells (compared with unstimulated time-matched control). Ciglitazone increased PPARgamma expression in both groups, but did not alter cell proliferation, while fluticasone increased PPARgamma protein only in asthmatic cells.. Although in the presence of a mitogenic stimulus, PPARgamma was differentially expressed in asthma- and non-asthma-derived ASM; its expression was not related to the increased proliferation observed in asthmatic ASM.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Androstadienes; Asthma; Bronchi; Bronchodilator Agents; Cell Proliferation; Cells, Cultured; Cyclin D1; Female; Fluticasone; Humans; Male; Middle Aged; Mitogens; Myocytes, Smooth Muscle; PPAR gamma; RNA, Messenger; Thiazolidinediones; Young Adult

Abnormal hypertrophy and hyperplasia of airway smooth muscle cells play an important role in airway remodeling in chronic asthma. The authors' previous studies have indicated that protein kinase C alpha (PKC alpha) is involved in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). However, the underlying mechanisms remain unknown. Here, the authors examined the possible role of the alpha isoform of PKC in the control of cyclin D1 expression, using HASMCs passively sensitized on human atopic asthmatic serum as a model system. Cultured HASMCs were passively sensitized with serum from atopic asthmatic patients. Cell proliferation was measured by [(3)H]thymidine incorporation and an MTT assay. Cell cycle status was analyzed by flow cytometry. The mRNA and protein expression profiles of cyclin D1 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, the authors assessed the role of cyclin D1 in PKC alpha-induced HASMC proliferation by transfection with a recombinant cyclin D1 antisense construct. The activation of PKC alpha with phorbol myristate acetate (PMA), a PKC activator, up-regulated cyclin D1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly decreased by specific inhibition of PKC alpha with Go6976. In addition, the authors showed that transfection with antisense cyclin D1 abolished PMA-induced G1/S progression and HASMC proliferation. These results demonstrate that PKC alpha promotes the proliferation of HASMCs sensitized with atopic asthmatic serum via up-regulation of cyclin D1 expression.

Topics: Adult; Asthma; Bronchi; Carbazoles; Cell Proliferation; Cells, Cultured; Cyclin D1; Female; Humans; Isoenzymes; Male; Middle Aged; Myocytes, Smooth Muscle; Protein Kinase C-alpha; Serum; Tetradecanoylphorbol Acetate; Up-Regulation; Young Adult

Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle.. ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE + pcDNA3.1 group and CSE + pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting.. (1) The percentage of S + G2M phase, absorbance value at 490 nm wavelength (A(490)) and the expression rate of PCNA protein in CSE group were (31.22 +/- 1.17)%, 0.782 +/- 0.221, (90.2 +/- 7.0)% respectively, which were significantly increased compared with those of control group ((18.36 +/- 1.02)%, 0.521 +/- 0.109, and (54.1 +/- 3.5)%, respectively) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S + G2M phase, A(490) and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P < 0.01). (2) The ratios of A(490) of cyclin D1 mRNA in CSE group was 0.288 +/- 0.034, which was significantly increased compared with that of control group (0.158 +/- 0.006) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P < 0.01). (3) The ratios of A(490) of cyclin D1 protein expression in CSE group was 0.375 +/- 0.008, which was significantly increased compared with that of control group (0.268 +/- 0.004) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P < 0.01).. CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

Topics: Animals; Asthma; Blotting, Western; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; Female; Flow Cytometry; Immunohistochemistry; Microscopy, Phase-Contrast; Myocytes, Smooth Muscle; Nicotiana; Plant Extracts; Rats; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Smoking

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

Topics: Animals; Asthma; Cell Cycle; Cell Proliferation; Codon; Cyclin D1; Disease Models, Animal; DNA, Antisense; Gene Expression; Genetic Vectors; Male; Myocytes, Smooth Muscle; Rats; Rats, Sprague-Dawley; Recombination, Genetic; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Transduction, Genetic; Transfection

Airway smooth muscle (ASM) is suspected to be a determining factor in the structural change of asthma. However, the role of protein kinase C alpha (PKCalpha) and cyclin D1 involved in the dysfunction of ASM leading to asthmatic symptoms is not clear. In this study, the central role of PKCalpha and cyclin D1 in ASM proliferation in asthmatic rats was explored.. Thirty-six pathogen-free male Brown Norway (BN) rats were randomly divided into 2 groups: control groups (group N1, N2 and N3) and asthmatic groups (group A1, A2, and A3). Groups A1, A2 and A3 were challenged with ovalbumin (OA) for 2 weeks, 4 weeks and 8 weeks respectively. Control animals were exposed to an aerosolized sterile phosphate buffered saline (PBS). The ASM mass and nucleus numbers were studied to estimate the degree of airway remodeling by the hematoxylin-eosin staining method. PKCalpha and cyclin D1 expression in the ASM cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relation between PKCalpha and cyclin D1 was assessed by linear regression analysis. PKC agonist phorbol 12-myristate 13-acetate (PMA), PKC inhibitor Ro31-8220 and an antisense oligonucleotide against cyclin D1 (ASOND) were used to treat ASM cells (ASMCs) obtained from the 2 weeks asthmatic rats. The cyclin D1 protein expression level was detected by Western blotting.. Compared with the control group, the PKCalpha and cyclin D1 mRNA levels were increased in the asthmatic group. Similar to RT-PCR results, immunohistochemistry analysis for PKCalpha and cyclin D1 expression revealed an increased production in ASMCs after allergen treatment for 2, 4 and 8 weeks compared with the respective control groups. No difference in expression of PKCalpha and cyclin D1 in ASM were found in the 2, 4 or 8 weeks asthmatic rats. There were significant positive correlations between PKCalpha and cyclin D1 expression, both transcriptionally (r = 0.944, P < 0.01) and translationally (r = 0.826, P < 0.01), in ASM. The content of cyclin D1 in asthmatic ASMCs increased after being stimulated by PMA, and decreased when induced by Ro31-8220. ASOND targeting for cyclin D1 lowered the expression of cyclin D1 induced by PMA.. Increased expression of PKCalpha and cyclin D1 in ASM along with smooth muscle structure changes might implicate PKCalpha and cyclin D1 participation in the proliferation of ASM and contribute to the pathogenesis of asthma after repeated allergen exposure in rats. The results suggested that cyclin D1 might be downstream of PKC signal transduction pathway.

Topics: Animals; Asthma; Cell Proliferation; Cyclin D1; Lung; Male; Myocytes, Smooth Muscle; Protein Kinase C-alpha; Rats; Rats, Inbred BN; RNA, Messenger

To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics.. HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups: the control group, PMA treated group, PMA and PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay.. Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) protein increased correspondingly (the A value % control was 2.10 +/- 0.29, 1.67 +/- 0.19, 2.20 +/- 0.27, 1.99 +/- 0.22 and 3.11 +/- 0.29 respectively; q value was 9.87, 7.06, 10.57, 11.10 and 20.33 respectively; all P < 0.05) in PMA treated group, and cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) decreased correspondingly (the A value % control was 1.23 +/- 0.19, 1.34 +/- 0.18, 1.52 +/- 0.20, 1.45 +/- 0.18 and 1.49 +/- 0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (21.2 +/- 2.8)%, A(490) value was 0.51 +/- 0.04; q = 6.07, 12.63; all P < 0.05], as compared with those of the PMA treated group. In PMA and U0126 treated group, the level of p-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) reduced (the A value % control was 0.95 +/- 0.21, 1.15 +/- 0.19, 1.37 +/- 0.15 and 1.96 +/- 0.21 respectively; q value was 7.79, 9.16, 6.92 and 11.16 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (22.0 +/- 3.2)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group.. ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.

Topics: Adult; Asthma; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Protein Kinase C-alpha; Signal Transduction; Up-Regulation; Young Adult

Airway mesenchymal cells, such as myofibroblasts and airway smooth muscle cells, contribute to inflammation, airway remodelling and hyperresponsiveness in asthma by excessive proliferation and inflammatory mediator production. Using endobronchial biopsies obtained from both nonasthmatic and asthmatic subjects, in situ proliferation was assessed by immunostaining for cyclin D1. The number of immunoreactive cells increased with asthma severity and was restricted to the epithelium and subepithelial connective tissue. Despite increases in smooth muscle area, cyclin D1 was not detected in cells in intact muscle bundles. Biopsy-derived cell cultures were characterised as predominantly myofibroblasts, and were assessed to determine whether proliferation and cytokine production varied with asthma status. Cell enumeration showed that basal proliferation was similar in cells from nonasthmatics and asthmatics, and mitogenic responses to fibroblast growth factor-2, thrombin or serum were either reduced or unchanged in cells from asthmatics. Interleukin (IL)-1-dependent granulocyte-macrophage colony-stimulating factor and IL-8 release was increased in cell supernatants from asthmatics. Thus, increased rates of cellular proliferation identified in situ in the asthmatic airway occurred outside the expanded smooth muscle compartment. Although reduced proliferative responses were observed in cultured myofibroblasts from asthmatics, the increased cytokine production by these cells suggests that this contributes to and may perpetuate ongoing inflammation in asthma.

Topics: Adult; Albuterol; Androstadienes; Asthma; Bronchodilator Agents; Cell Proliferation; Cyclin D1; Female; Fibroblasts; Fluticasone; Humans; Male; Middle Aged; Muscle, Smooth; Muscles; Salmeterol Xinafoate; Trachea

To determine whether protein kinase C (PKC) has any effect on the expression of cyclinD1, a key regulator of growth control and G1/S transition, and to investigate the underlying molecular mechanisms of PKC involving the remodeling of the asthmatic airway smooth muscle (ASM).. The treatment of synchronized ASM cells from asthmatic rats with PKC-specific agonist phorbol 12-myristate 13-acetate (PMA) and antagonist 2-{1-[3-(amidinothio) propyl]-1Hindol-3-yl}-3-(1-methylindol-3-yl) maleimide methanesulfonate salt (Ro31-8220) was followed by the proliferation assay. PKCalpha and cyclinD1 expressions in ASM cells (ASMC) were detected by RT-PCR and Western blotting. The relation between PKCalpha and cyclinD1 was assessed by linear regression analysis. The effect of the construct recombinant plasmid pcDNA3.1-antisense cyclinD1 (pcDNA3.1-ascyclinD1) on the proliferation of ASMC was found to be induced by PMA.. The data showed phorbol ester-dependent PKCalpha promoted the proliferation of ASMC. The closely-positive correlation existed between the expression of PKCalpha and cyclinD1 at the transcriptional (r=0.821, P<0.01) and translational (r=0.940, P<0.01) levels. pcDNA3.1-ascyclinD1 could inhibit the proliferation of ASMC. pcDNA3.1-ascyclinD1 almost completely attenuated the PMA-induced proliferation effect as Ro31-8220+pcDNA3.1.. The proliferation of ASMC by PKC might by regulated by the cyclinD1 expression in asthmatic rats.

Topics: Animals; Asthma; Cell Proliferation; Cells, Cultured; Cyclin D1; DNA Fingerprinting; Gene Expression Regulation; Male; Myocytes, Smooth Muscle; Plasmids; Proliferating Cell Nuclear Antigen; Protein Kinase C; Rats; Rats, Inbred BN

Glucocorticoids seem to mediate the effect of stimulant drugs such as nicotine. Several studies have pointed to an association between the BclI polymorphism in the glucocorticoid receptor gene and increased glucocorticoid effects. We analysed the association of smoking behaviour and the BclI polymorphism using a case-control design within the framework of a larger pharmacogenetic study. A total of 327 Caucasian patients with asthma or chronic obstructive pulmonary disease from 39 German general practices gave informed consent to take part in the study. They filled in questionnaires concerning their smoking behaviour and were genotyped for the BclI polymorphism. The genotype frequencies for non-smokers (n = 251; CC, 0.42; CG, 0.46; GG, 0.12) as well as for smokers (n = 76; CC, 0.29; CG, 0.55; GG, 0.16) were consistent with the Hardy-Weinberg equilibrium. The proportion of smokers was significantly lower among carriers of the CC-genotype (22/127 = 17%) compared with carriers of the G-allele (54/200 = 27%; chi2 = 4.08; P = 0.04). Within the group of smokers, the proportion of heavy smokers (> 19 cigarettes/day; median) was reduced in C-homozygous patients when compared with carriers of the G-allele (7/22 = 32% versus 31/54 = 57%; chi2 = 4.09; P = 0.04). Stepwise logistic regression analysis also pointed to an association between the CC-genotype and a reduced probability of being a smoker (odds ratio = 0.55; 95% confidence interval = 0.30-1.00; P = 0.05) controlling for other predictors. In summary, this study provides evidence that the BclI polymorphism might play a role in the maintenance and severity of nicotine dependence.

Topics: Aged; Alleles; Asthma; Cyclin D1; Female; Gene Frequency; Genetic Carrier Screening; Genotype; Germany; Homozygote; Humans; Male; Middle Aged; Polymorphism, Genetic; Pulmonary Disease, Chronic Obstructive; Receptors, Glucocorticoid; Smoking; Tobacco Use Disorder

To evaluate the expression of cyclin D(1) in asthmatic mouse lungs, and to explore the role of cyclin D(1) in bronchial asthma and airway remodeling.. Forty BALB/c mice were randomized to group A (normal), group B (sensitized for 2 weeks), group C (sensitized for 4 weeks) and group D (sensitized for 8 weeks), 10 mice each group. The mice were sensitized with 10% ovalbumin and challenged with 1% ovalbumin to establish the asthmatic model. The number of eosinophils and the cell percentages in bronchoalveolar lavage fluid (BALF) were counted by cytology method. Pulmonary functions were measured to evaluate the resistance of expiration. Airway inflammation and eosinophil infiltration were evaluated by HE staining, and the airway wall thickness (WAt/Pi), smooth muscle thickness (WAm/Pi) and smooth nucleus counts (N/Pi) were quantified by computer-assisted image analysis system. The mRNA expression of cyclin D(1) was measured by RT-PCR and Real-time PCR. The protein expression of cyclin D(1) was assayed by Western blot. The correlation between airway resistance of expiration and the expression of cyclin D(1) was studied.. The eosinophil count and differential in BALF of group B, C, and D [(42.6 +/- 0.9) x 10(4)/L, (54.7 +/- 1.4) x 10(4)/L, (44.8 +/- 2.4) x 10(4)/L] were higher than those of group A (3.4 +/- 0.5) x 10(4)/L (q = 79.75, 91.42, 84.82, all P < 0.01). The airway resistance of expiration after challenge with 45 microg/kg acetylcholine in group B, C, and D [(5.27 +/- 0.16) cmxL(-1)xmin(-1), (6.68 +/- 0.20) cmxL(-1)xmin(-1), (7.14 +/- 0.41) cmxL(-1)xmin(-1)] was higher than that in group A [(4.11 +/- 0.15) cmxL(-1)xmin(-1), q = 5.58, 6.39, 7.11, all P < 0.05]. Eosinophil infiltration, cilium loss, formation of mucus plug and smooth muscle cell layer thickening were observed in group B, C, and D. The morphological changes of the airways became evident following airway remodeling. WAm/Pi in group B, C, and D (2.8 +/- 0.6, 4.8 +/- 0.6, 6.4 +/- 0.7) were higher than in group A (2.4 +/- 0.4, q = 6.40, 8.28, 9.27, all P < 0.05), and WAt/Pi in group B, C, and D (6.4 +/- 0.8, 8.3 +/- 1.2, 9.3 +/- 1.0) were higher than in group A (5.6 +/- 1.0, q = 2.80, 4.83, 6.37, all P < 0.05). The protein expression of Cyclin D(1) in group B, C, and D (0.587 +/- 0.015, 0.808 +/- 0.029, 0.826 +/- 0.022) were higher than in group A (0.404 +/- 0.016, q = 5.87, 8.08, 8.26, all P < 0.01). There was a positive correlation between the expression of cyclin D(1) and airway resistance (r = 0.83, P < 0.05).. The expression of cyclin D(1) in the asthmatic mouse lung was increased, and positively correlated to airway reactivity. Cyclin D(1) might be essential in the airway remodeling of asthma through ERK signaling pathway.

Topics: Airway Remodeling; Animals; Asthma; Cyclin D1; Eosinophils; Female; Lung; Mice; Mice, Inbred BALB C

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 microg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 microM), p38 kinase inhibitor (SB203580, 10 microM) or elastase inhibitor II (100 microg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.

Topics: Asthma; Cell Division; Cell Size; Cells, Cultured; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Humans; Leukocyte Elastase; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Muscle, Smooth; Time Factors; Trachea

Airway smooth muscle proliferation may contribute to the airway wall remodeling seen in asthma. In this study we tested for the presence of airway smooth muscle mitogenic activity in bronchoalveolar lavage (BAL) fluid obtained from 12 atopic asthmatics before and serially after segmental allergen challenge, and from four normal subjects who did not undergo allergen challenge. Mitogenic effect was assessed by coincubating BAL fluid with human airway smooth muscle cells, and measuring its effect on (3)[H]thymidine incorporation and cell number. Induction of ERK phosphorylation and cyclin D(1) protein abundance were also assessed. Compared with serum-free medium alone, BAL fluid obtained from normal subjects increased thymidine incorporation, cell number, ERK phosphorylation, and cyclin D(1) abundance. BAL fluid from asthmatic subjects prior to allergen challenge induced even greater increases in all measures, except for cell number, which was similar to that observed with normal subjects' BAL fluid. Incubation with lavage fluid obtained 48 h after segmental allergen challenge in atopic asthmatics caused yet further increases in thymidine incorporation, cell number, and cyclin D(1) protein abundance. Molecular sieving of prechallenge BAL fluid from three asthmatic subjects demonstrated that mitogenic activity was present exclusively in the > 10 kD fraction. These results provide the first direct demonstration that fluid lining the airways of asthmatics contains excess mitogenic activity for human airway smooth muscle, and that this activity increases further after allergen challenge.

Topics: Adolescent; Adult; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Cell Count; Cell Division; Cells, Cultured; Cyclin D1; Female; Humans; Hypersensitivity, Immediate; Male; Mitogen-Activated Protein Kinases; Mitogens; Muscle, Smooth; Phosphorylation