Silicon dioxide-upregulated plasminogen activators inhibitor-1 protein expression is dependent on activation of extracellular signal-regulated kinase/activator protein-1 signaling pathway / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) signaling pathway in SiO(2)-induced plasminogen activators inhibitor-1 (PAI-1) protein expression in human lung epithelial cells A549.</p><p><b>METHODS</b>A549 cells were cultured and then stimulated with 200 microg/ml SiO(2) for 0 approximately 24 h. To prevent AP-1 activity, Curcumin was added into culture medium before incubating with SiO(2) and transient TAM-67 transfection was performed. In addition, PD98059 was pretreated with cells to prevent ERK activity. The PAI-1 protein expression and ERK activity were evaluated by Western blot. The AP-1 DNA binding activity was tested by EMSA.</p><p><b>RESULTS</b>(1) At 4, 8 and 16 h after exposure to SiO(2), the fold change of AP-1 DNA binding activity (relative to the control group) were 1.3, 1.3, and 2.1, respectively (P < 0.05). 10, 25, 50 micromol/L Curcumin inhibited SiO(2)-induced PAI-1 protein expression (inhibition ratio: 20%, 63%, 65%; P < 0.05). TAM-67 downregulated SiO(2)-induced PAI-1 protein expression (inhibition ratio: 59%, P < 0.05). (2) SiO(2) activated ERK and PD98059 downregulated SiO(2)-induced PAI-1 protein expression (inhibition ratio: 51%, P < 0.05). (3) PD98059 downregulated SiO(2)-induced AP-1 DNA binding activity (inhibition ratio: 73%, P < 0.05).</p><p><b>CONCLUSION</b>ERK/AP-1 signaling pathway is responsible for SiO(2)-induced PAI-1 protein expression.</p>

AP-1 regulates TGF-beta1-induced secretion of Type I collagen in human lung fibroblasts / 中南大学学报(医学版)

OBJECTIVE@#To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts.@*METHODS@#Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR.@*RESULTS@#TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05).@*CONCLUSION@#AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.

Effects of silicon dioxide on expression of alpha-smooth muscle actin in human lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of SiO(2) on the expression of alpha-smooth muscle actin (alpha-SMA) in human lung fibroblasts in vitro and vivo.</p><p><b>METHODS</b>The experimental group comprised 32 rats while 32 rats were included in the control. In vivo, the expression of alpha-SMA in lung tissues of rats exposed to SiO(2), the supernate of RAW264.7 cells, SiO(2) and the growth factor beta(1) (TGF-beta(1)) were investigated, respectively.</p><p><b>RESULTS</b>(1) alpha-SMA positive myofibroblasts appeared in the lung tissues of the 28th day groups exposed to SiO(2). (2) The expression of alpha-SMA in HLF-02 cells was unregulated by TGF-beta(1) and supernate of RAW264.7 cells exposed to SiO(2). (3) The expression of alpha-SMA in HLF-02 cells was not induced by SiO(2).</p><p><b>CONCLUSION</b>Myofibroblasts related to silicosis, and the appearance of myofibroblasts (in vitro) are independent on direct stimulation by SiO(2), but related to the mediator (TGF-beta(1)) secreted by SiO(2) stimulated macrophages.</p>

p38/ERK signal pathways regulating the expression of type I collagen and activity of MMP-2 in TGF-beta1-stimulated HLF-02 cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of TGF-beta(1)/MAPK signaling pathways in the expression of type I collagen and activity of MMP-2, 9 in human lung fibroblasts.</p><p><b>METHODS</b>Human lung fibroblasts cell line (HLF-02) was cultured and and then stimulated with 10 ng/ml TGF-beta(1) for different time; SB203580 or PD98059 was added into culture medium to block p38 or ERK kinase pathway before incubated with TGF-beta(1); the expression of type I collagen was detected by Western blotting and RT-PCR; zymogram analysis was used to analyze the activity of MMP-2 and MMP-9.</p><p><b>RESULTS</b>(1) In the process of stimulation by TGF-beta(1), the type I collagen mRNA level of 24 h, 48 h and 72 h group was: 1.33 +/- 0.07, 2.46 +/- 0.09 and 2.39 +/- 0.08 respectively; and the type I collagen protein level of 24 h, 48 h and 72 h group was: 114.89 +/- 8.95, 208.16 +/- 6.75 and 211.46 +/- 8.05 respectively; and the activity of MMP-2 of 24 h, 48 h and 72 h group was: 190.33 +/- 5.86, 214.33 +/- 8.39 and 212.67 +/- 11.59 respectively. (2) SB203580 significantly inhibited the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 51%, 24% and 20%); (3) PD98059 also significantly attenuated the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 42%, 13% and 16%).</p><p><b>CONCLUSION</b>TGF-beta(1) is capable of inducing the expression of type I collagen mRNA and protein and up-regulating MMP-2 activity in HLF-02 cells. p38 and ERK kinase signaling pathways play important role in regulation and control for this process.</p>

Transforming growth factor-beta1 induced phenotypic differentiation of human lung fibroblasts through mitogen activated protein kinase-dependent pathway / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of MAPK signal transduction in TGF-beta1 induced phenotypic differentiation of human lung fibroblasts.</p><p><b>METHOD</b>Human lung fibroblasts cell line (HLF-02) were cultured and then stimulated with 10 ng/ml TGF-beta1 for different time; SB203580 or PD98059 was added into culture medium to prevent p38 or Erk kinase pathway before incubating with TGF-beta1; the expression of alpha-smooth muscle actin (alpha-SMA) was detected by Western blotting and RT-PCR; Western blotting was used to assay phosphorylation of p38, Erk, and JNK kinase.</p><p><b>RESULTS</b>(1) In the process of stimulation by TGF-beta1, the alpha-SMA mRNA expression levels of 24, 48 and 72 h groups were 1.87 +/- 0.11, 2.49 +/- 0.10, 3.02 +/- 0.15 respectively; and the alpha-SMA protein expression levels of 24, 48 and 72 h groups were 3.20 +/- 0.14, 3.96 +/- 0.21, 4.57 +/- 0.13 respectively. (2) TGF-beta1 induced p38, Erk kinase phosphorylation but not JNK kinase. (3) The inhibitors SB203580 and PD98059 suppressed TGF-beta1-induced p38 kinase and Erk phosphorylation respectively. (4) SB203580 significantly attenuated TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 30% and 40%); PD98059 also significantly inhibited TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 10% and 20%).</p><p><b>CONCLUSION</b>TGF-beta1 is capable of inducing the phenotypic differentiation of HLF-02, which is regulated by p38 and Erk kinase signal pathway.</p>

Expression of early growth response gene-1 in macrophages stimulated by silicon dioxide / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression and localization of early growth response gene-1 (Egr-1) in macrophages after stimulation by silicon dioxide in vivo and in vitro and to discuss the role of Egr-1 in the development of silicosis.</p><p><b>METHODS</b>The expression of Egr-1 in animal model of silicosis was analyzed by using immunohistochemistry. Western-blot, immunofluorescence and RT-PCR analysis were used to detect the expression and localization of Egr-1 protein and the dynamic changes of Egr-1 mRNA in cultured macrophages RAW264.7, after stimulation by silicon dioxide.</p><p><b>RESULTS</b>In animal model with induced silicosis, there was an increased expression of Egr-1 in pulmonary macrophages. The expression levels peaked at the 14th day. In vitro, the transcription of Egr-1 increased in RAW264.7 macrophages during 15 to 240 minutes after the administration of silicon dioxide. The response peaked at 15 minutes and diminished to a minimal level at 480 minutes. Nuclear translocation was most apparent at 60 minutes, lasted till 120 minutes and diminished gradually. During the period from 60 to 120 minutes, the expression of Egr-1 protein also reached a peak.</p><p><b>CONCLUSIONS</b>Silicon dioxide can activate the nuclear transcription factor Egr-1 in vivo and in vitro in macrophages. Egr-1 may thus play an important pathogenetic role in the development of silicosis.</p>