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Idiopathic pulmonary fibrosis (IPF) is a cryptogenic, chronic inflammatory interstitial lung disease characterized by common interstitial pneumonia[1]. Evidence exists that alveolar epithelial cells progressively reduce, with a high apoptosis index, in pulmonary fibrosis mice and IPF patients[2-5].
G1/S is a primary phase during cell cycle of most diploid cells. Cell cycle control system is composed of cyclin, cyclin dependent kinase (CDKs) and cyclin dependent kinase inhibitor (CDKI). CDK4 is the core component of cell cycle control system[6]. Cells at G0 phase enters into G1 phase under the stimulation of growth factors, and CyclinD1 binds to CDK4, resulting in cell proliferation. P16 protein, also called CDK4I, can completely bind to CDK4 with CyclinD1. Under normal circumstances, P16 protein and CyclinD1 are in the balance[7]. Under non-healthy state, P16 over-expressed would inhibit CyclinD1 binding to CDK4, finally leading to cell cycle arrest and subsequently cell number decrease. At present, correlation between P16-CyclinD1/CDK4 cell cycle regulatory pathway and progressive abnormality of alveolar epithelial cell cycle during pulmonary fibrosis remains unclear. The present study established mouse models of pulmonary fibrosis through induction of bleomycin (BLM) and dynamically observed the expression rule of P16, CyclinD1 and CDK4 to study the relationship between P16-CyclinD1/CDK4 pathway and reduction of alveolar epithelial cells during pulmonary fibrosis and further investigate the pathogenesis of IPF.

Evidence exists that during the repair process of pulmonary fibrosis, undamaged pulmonary epithelial layer can inhibit the proliferation and apposition of fibroblasts, hinder the repeat injuries and excessive apoptosis of pulmonary epithelial cells; in particular, type II epithelial cell injury can abnormally activate growth factors and result in irreversible fibrosis progression,, leading to pulmonary alveolar deteleotasis; excessively reduced type I epithelial cells would influence the function of alveolar capillary membrane, causing hyperplasia of fibrosis tissue and collagen and leading to pulmonary interstitial fibrosis[7, 11-12]. Therefore, cell cycle of epithelial cells greatly determines the occurrence of pulmonary fibrosis.
P16 has been confirmed to be one of initiating agents that cause cell aging[13-14]. Haga et al[15] reported that introduction of Bmi-1 into human mammary epithelial cells can inhibit P16 gene expression and extend the life span of human epithelial cells and that subsequent introduction of human telomerase reverse transcriptase gene into three cells, in which P16 gene is low expressed, can obviously promote cell proliferation. A study demonstrated that CyclinD1 expression in Paget's disease of the breast is obviously increased and is involved in the process of excessive hyperplasia of tumor cells[16]. Gaben et al[17] found that CDK4 promotes the proliferation of pulmonary fibroblast cultured in vitro. Gao et al[18] concluded that CDK4 mRNA expression increases from 7 days in pulmonary tissues of premature rats with hyperoxia-induced chronic lung disease.
The present study successfully established mouse models of pulmonary fibrosis induced by BLM, as evidenced by obvious alveolar inflammatory reaction at 7 days and complete formation of pulmonary fibrosis at 28 days. Results showed that with aging, pathological changes of pulmonary tissue developed from alveolitis to pulmonary fibrosis; P16, CyclinD1 and CDK4 expression was primarily located in nucleus and cytoplasm of alveolar epithelial cells; in the BLM group, P16 and CDK4 expression exhibited an increasing tendency and CyclinD1 expression showed a decreasing tendency; compared with the control group at the same time point (i.e., at 14 and 28 days), P16 and CDK4 expression was increased in alveolar epithelial cells, but CyclinD1 expression was decreased, in the BLM group, demonstrating the presence of P16 and CDK4 overexpression and absence of CyclinD1 during the progression of pulmonary fibrosis, in particular the key phase (14-28 days) for the occurrence and development of pulmonary fibrosis.
CDK4 overexpression was consistent with previous findings[19], indicating the possible organism compensative reaction of increased CDK4 expression and P16-CyclinD1/CDK4 cell cycle regulatory pathway disorder. P16 overexpression and absent CyclinD1 expression also suggest that these two produce a synergistic effect on alveolar epithelial cells P16-CyclinD1/CDK4 pathway, which leads to a fact that highly expressed CDK4 cannot sufficiently bind to CyclinD1, halting the progression of cell cycle and resulting in abnormal reduction of alveolar epithelial cells in pulmonary fibrosis, which likely contributes to the occurrence and development of pulmonary fibrosis.
In the BLM group, CyclinD1 expression decreases with time, but CyclinD1-positive cells and protein expression were greater at 3 and 7 days after modeling compared with the control group (P < 0.05). At 14 days after modeling, CyclinD1-positive cells were more in the BLM group than in the control group, which is likely to be related to abnormal activation of other signal pathways during the phase of alveolitis, such as Wnt, RhoA/Rock pathways[20-21]. Demopoulos et al[22] demonstrated the loss of heterozygosity of P16 gene in blood or sputum samples from pulmonary fibrosis patients. Correlation between P16 gene and protein expression in IPF patients needs further investigation.
Taken together, there exists abnormal expression of P16, CyclinD1 and CDK4 in the progression of pulmonary fibrosis. P16-CyclinD1/CDK4 cell cycle regulatory pathway disorder is likely an important factor of reduction of alveolar epithelial cells during pulmonary fibrosis. Determining whether alveolar epithelial cells P16-CyclinD1/CDK4 pathway participates in the reduction of alveolar epithelial cells in the progression of pulmonary fibrosis depends on in vitro intervention of alveolar epithelial cell cycle and corresponding gene level.

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