CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.

Ribosome distribution in HeLa cells during the cell cycle.

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope.