Involvement of extracellular signal-regulated kinase (ERK) in CXCL12-induced connective tissue growth factor (CTGF) expression in WI-38 cells.

<p>A, Cells were pretreated with 10 and 30 µM PD98059 for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment. B, Cells were treated with CXCL12 (10 ng/ml) for 0∼20 min. Whole-cell lysates were prepared and immunodetected with specific antibodies for phospho-ERK Tyr 204 or ERK2. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to the control without CXCL12 stimulation.</p

Involvement of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinse (JNK) in CXCL12-induced c-Jun phosphorylation or connective tissue growth factor (CTGF) expression in WI-38 cells.

<p>A, Cells were treated with CXCL12 (10 ng/ml) for 0∼60 min. Whole-cell lysates were prepared and immunodetected with specific antibodies for phospho-c-Jun Ser 63 or c-Jun. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to the control without CXCL12 stimulation. Cells were pretreated with 10 and 30 µM PD98059 (B) or 10 and 30 µM SP600125 (C) for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 20 min. Whole-cell lysates were prepared and immunodetected with specific antibodies for phospho-c-Jun Ser 63 or c-Jun. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment. D, Cells were pretreated with SP600125 (3∼30 µM) for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment.</p

Schematic summary of the signal transduction pathway by which CXCL12 induces connective tissue growth factor (CTGF) expression in human lung fibroblasts (WI-38).

<p>CXCL12 acts on CXCR4 to activate the Rac/extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, which in turn initiates activator protein-1 (AP-1) activation, and ultimately causes CTGF expression. Moreover, CTGF mediates CXCL12-induced α-smooth muscle actin (α-SMA) expression in human lung fibroblasts.</p

Involvement of CXCR4 in CXCL12 induces connective tissue growth factor (CTGF) expression in WI-38 cells.

<p>A, Cells were pretreated with 10 µM AMD3100 for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment. B, Cells were transfected with control siRNA (Con siRNA 25 nM) or CXCR4 siRNA (25 nM) for 24 h and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 stimulation. C, Cells were transfected with control siRNA (Con siRNA 25 nM) or CXCR4 siRNA (25 nM) for 24 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for CXCR4 or α-tubulin. Traces represent results from three independent experiments.</p

Involvement of p21-activated kinase (PAK) in CXCL12-induced connective tissue growth factor (CTGF) expression in WI-38 cells.

<p>Cells were pretreated with PAK18 (10 µM) for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment.</p

Involvement of Rac1 in CXCL12-induced extracellular signal-regulated kinase (ERK) phosphorylation in WI-38 cells.

<p>Cells were transfected with pcDNA (1 µg) or RacN17 (0.5 and 1 µg) for 24 h and then stimulated with CXCL12 (10 ng/ml) for another 10 min. Whole-cell lysates were prepared and immunodetected with specific antibodies for phospho-ERK Tyr 204 or ERK2. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 stimulation.</p

CXCL12 induces activator protein-1 (AP-1) binding to the connective tissue growth factor (CTGF) promoter in WI-38 cells.

<p>Schematic diagram of AP-1-binding elements and ChIP primer locations on the CTGF promoter. ChIP primer pairs with a 128-bp PCR product was designed to amplify DNA corresponding to the putative AP-1-binding site. Cells were incubated with 10 ng/ml CXCL12 for 20 min and then cross-linked with formaldehyde at 37°C for another 10 min. Cell lysates were sonicated and prepared for the ChIP assay using antibodies specific for c-Jun and c-Fos as described in “Materials and Methods.” PCR amplification using primers designed against the AP-1-binding site was performed. Equal amounts of soluble cross-linked chromatins present in each PCR were confirmed by the product for input. A rabbit polyclonal IgG antibody was used as a negative control. Typical traces are representative of three experiments with similar results.</p

The CXCL12 induces actin stress fiber formation and Rho activity in WI-38 cells.

<p>A, Cells were pretreated with ADM3100 (10 µM) for 20 min, and then stimulated with CXCL12 (10 ng/ml) for another 24 h. In a confocal microscopic analysis, cells were stained with rhodamine-phallodin (red) and nuclei were indicated by DAPI staining (blue). Scale bar, 25 µm. Traces are representative of three experiments with similar results. B, Cells were treated with CXCL12 (10 ng/ml) for different time intervals. Whole-cell lysates were then immunoprecipitated with Rhotekin RBD-agarose. The Rho activity assay is described in “Materials and Methods”. Rho and α-tubulin protein levels were determined in total lysates by a Western blot analysis as the loading control. Immunoblots are representative of three experiments with similar results.</p

CXCL12 induces connective tissue growth factor (CTGF) expression in WI-38 cells.

<p>A, Cells were treated with various concentration of CXCL12 for 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to the control group without CXCL12 treatment. B, Cells were treated with CXCL12 (10 ng/ml) for 0∼24 h, and then immunodetected with CTGF- or α-tubulin-specific antibodies. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to the group without CXCL12 treatment. C, Cells were transfected with 0.5 µg of CTGF-Luc and 0.1 µg of pBK-CMV-Lac Z for 24 h and then stimulated with 3∼30 ng/ml of CXCL12 for another 16 h. Cells were harvested for a luciferase activity assay. Data are presented as the mean ± S.E. of four experiments. *<i>p</i><0.05, compared to the group without CXCL12 treatment. D, Cells were pretreated with 3 µM actinomycin D (Act.D) and 3 µM cycloheximide (CHX) for 30 min and then stimulated with CXCL12 (10 ng/ml) for another 2 h. Whole-cell lysates were prepared and immunodetected with specific antibodies for the CTGF or α-tubulin. Data are presented as the mean ± S.E. of three experiments. *<i>p</i><0.05, compared to CXCL12 treatment.</p