cyclin-d1 and Idiopathic-Pulmonary-Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible and the most common fatal interstitial lung disease, which is characterized by damaged alveolar structure, the massive proliferation of fibroblasts and deposition of extracellular matrix (ECM). While the pathogenesis of IPF remains unclear, it has been clearly established that the excessive proliferation of lung fibroblasts is the most direct cause of fibrogenesis. Numerous proliferating fibroblasts form fibrous foci and secrete a large amount of ECM to aggravate the process of pulmonary fibrosis. Tissue plasminogen activator (tPA) is a kind of serine protease, its main function is to activate zymogens into active enzymes involved in fibrinolysis. Our study found tPA functioned as a cytokine to promote the proliferation of lung fibroblasts through intracellular signaling events involving Erk1/2, p90RSK, GSK-3β phosphorylation, and cyclinD1 induction. We also uncovered that tPA indirectly activated the Wnt/β-catenin signaling pathway by regulating the GSK-3β phosphorylation level. It's well-known that Wnt/β-catenin signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis, in which the accumulation of β-catenin in the cytoplasm is an important signal of the activation of Wnt/β-catenin signaling pathway. Our study unveiled that tPA can serve as a cytokine involved in Wnt/β-catenin signaling pathway and be implicated in pulmonary fibrosis.

Topics: Animals; beta Catenin; Cell Proliferation; Cells, Cultured; Cyclin D1; Extracellular Matrix; Fibroblasts; Glycogen Synthase Kinase 3 beta; Idiopathic Pulmonary Fibrosis; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ribosomal Protein S6 Kinases, 90-kDa; Tissue Plasminogen Activator; Wnt Signaling Pathway

Distinction of idiopathic pulmonary fibrosis (IPF) from other chronic fibrosing interstitial pneumonitides, such as hypersensitivity pneumonitis (HP) and connective tissue diseases, is critical due to varied biological and clinical outcomes. However, their histologic overlaps often pose diagnostic challenges. A recent study suggested an association of herpesvirus saimiri infection with IPF. Productive viral infection is associated with coexpression of pirated mammalian protein cyclin D1, shown to be overexpressed by immunohistochemistry (IHC) in the regenerating alveolar epithelium in IPF but not in normal lungs. We evaluated the diagnostic utility of cyclin D1 to discriminate between IPF and other fibrosing interstitial lung diseases.. A retrospective study of cyclin D1 IHC expression in 27 consecutive cases of chronic fibrosing interstitial lung diseases from 2011 to 2017: 12 usual interstitial pneumonia (UIP) pattern; 5 nonspecific interstitial pneumonia pattern; 3 HP pattern; 7 unclassifiable was performed. Five cases of normal lung obtained from lobectomy specimen for malignancy are included as control. Immunoreactivity was graded semiquantitatively on a scale of 0 to 3.. Cyclin D1 staining was uniformly strongly positive in all cases evaluated in the study, particularly in proliferating type II pneumocytes in the region of fibrosing areas. There was no statistical difference in the extent of cyclin D1 expression between UIP and non-UIP groups (2.7 vs. 2.5) and IPF versus non-IPF groups (2.7 vs. 2.4). Cyclin D1 expression is lower in control group compared with UIP groups (1.2 vs. 2.7).. Cyclin D1 is not a specific marker of UIP pattern/IPF. The high expression of cyclin D1 in lung tissue of fibrosing interstitial pneumonitides regardless of etiology most likely correlates with proliferation in type II pneumocytes.

Topics: Adult; Aged; Biomarkers; Cyclin D1; Diagnosis, Differential; Female; Herpesviridae Infections; Herpesvirus 2, Saimiriine; Humans; Idiopathic Pulmonary Fibrosis; Immunohistochemistry; Lung; Lung Diseases, Interstitial; Male; Middle Aged; Retrospective Studies; Tumor Virus Infections; Up-Regulation

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Topics: Adult; Aged; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin A1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Fibroblasts; Humans; Hypoxia; Idiopathic Pulmonary Fibrosis; Lung; Male; Middle Aged; NFATC Transcription Factors; Oncogene Proteins

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease of unknown aetiology with no current cure. The pathogenesis of IPF remains unclear but repeated alveolar epithelial cell (AEC) injuries and subsequent apoptosis are believed to be among the initiating/ongoing triggers. However, the precise mechanism of apoptotic induction is hitherto elusive. In this study, we investigated expression of a panel of pro-apoptotic and cell cycle regulatory proteins in 21 IPF and 19 control lung tissue samples. We reveal significant upregulation of the apoptosis-inducing ligand TRAIL and its cognate receptors DR4 and DR5 in AEC within active lesions of IPF lungs. This upregulation was accompanied by pro-apoptotic protein p53 overexpression. In contrast, myofibroblasts within the fibroblastic foci of IPF lungs exhibited high TRAIL, DR4 and DR5 expression but negligible p53 expression. Similarly, p53 expression was absent or negligible in IPF and control alveolar macrophages and lymphocytes. No significant differences in TRAIL expression were noted in these cell types between IPF and control lungs. However, DR4 and DR5 upregulation was detected in IPF alveolar macrophages and lymphocytes. The marker of cellular senescence p21(WAF1) was upregulated within affected AEC in IPF lungs. Cell cycle regulatory proteins Cyclin D1 and SOCS3 were significantly enhanced in AEC within the remodelled fibrotic areas of IPF lungs but expression was negligible in myofibroblasts. Taken together these findings suggest that, within the remodelled fibrotic areas of IPF, AEC can display markers associated with proliferation, senescence, and apoptotosis, where TRAIL could drive the apoptotic response. Clear understanding of disease processes and identification of therapeutic targets will direct us to develop effective therapies for IPF.

Topics: Apoptosis; Biomarkers; Case-Control Studies; Cell Proliferation; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; Humans; Idiopathic Pulmonary Fibrosis; Lymphocytes; Macrophages, Alveolar; Myofibroblasts; Pulmonary Alveoli; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53; Up-Regulation

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-β. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, β and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Topics: Adult; Cell Growth Processes; Cells, Cultured; Class I Phosphatidylinositol 3-Kinases; Collagen; Cyclin D1; Female; Fibroblasts; Gene Silencing; Humans; Idiopathic Pulmonary Fibrosis; Immunohistochemistry; Lung; Male; Middle Aged; Myofibroblasts; Proliferating Cell Nuclear Antigen; Protein Isoforms; Protein Kinase Inhibitors; RNA, Small Interfering; Up-Regulation