MEKK1, JNK, and SMAD3 mediate CXCL12-stimulated connective tissue growth factor expression in human lung fibroblasts

Abstract

BackgroundIn idiopathic pulmonary fibrosis, the interaction of CXCL12 and CXC receptor 4 (CXCR4) plays a critical role in lung fibrosis. Connective tissue growth factor (CTGF) overexpression underlies the development of pulmonary fibrosis. Our previous report showed that the Rac1-dependent extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein (AP)-1 pathways are involved in CXCL12-generated CTGF expression in human lung fibroblasts (WI-38). In present study, we additionally inspected the involvement of mitogen-activated protein kinase kinase kinase 1 (MEKK1)/JNK-dependent SMAD3 in CXCL12-triggered CTGF expression in WI-38 cells.MethodsWI-38 cells were stimulated with CXCL12 in the absence or presence of specific inhibitors or small interfering RNAs (siRNAs). CTGF expression and signaling transduction molecules were assessed by Western blot, luciferase activity assay, or ChIP assay.ResultsCXCL-12-induced CTGF expression was attenuated by SIS3 (a SMAD3 inhibitor) and SMAD3 siRNA, but not by SB431542 (an activin receptor-like kinase 5, ALK5, inhibitor). CXCL12-stimulated CTGF expression was also attenuated by MEKK1 siRNA. Treatment of cells with CXCL12 caused an increase in SMAD3 phosphorylation at Ser208, translocation to nuclei, SMAD3-luciferase activity, and recruitment of SMAD3 to the CTGF promoter. Stimulation of cells with CXCL12 resulted in increase in JNK phosphorylation at Thr183/Tyr185 and MEKK1 phosphorylation at Thr261. Moreover, CXCL12-mediated SMAD3 phosphorylation or SMAD3-luciferase activity was inhibited by MEKK1 siRNA or SP600125. Finally, CXCL12-mediated JNK phosphorylation was attenuated by MEKK1 siRNA.ConclusionIn conclusion, results of this study suggest that CXCL12 activates the MEKK1/JNK signaling pathway, which in turn initiates SMAD3 phosphorylation, its translocation to nuclei, and recruitment of SMAD3 to the CTGF promoter, which ultimately induces CTGF expression in human lung fibroblasts.

Abundance of Non-Polarized Lung Macrophages with Poor Phagocytic Function in Chronic Obstructive Pulmonary Disease (COPD)

“…However, with a chronic persistent inflammatory stimulus such as cigarette smoking, it could promote proliferation of structural cells such as fibroblasts and their production of pro-inflammatory cytokines such as IL-8, vascular endothelial growth factor (VEGF), and transforming growth factor alpha (TGF-α) with augmented expression in chronic inflammatory conditions such as rheumatoid arthritis [50]. Mitogen-activated protein kinase 5 (MAP3K5) is a member of MAP kinase family that activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx [51][52][53]. It has been implicated in the pathogenesis of chronic inflammatory conditions such as rheumatoid arthritis, cardiopulmonary diseases and diabetes [54,55].…”

CXCR4 knockdown prevents inflammatory cytokine expression in macrophages by suppressing activation of MAPK and NF-κB signaling pathways

“…Furthermore, CXCL12/CXCR4 activation through multiple downstream pathways leads to increased cell proliferation and migration, such as NF-κB [31] and PI3K/Akt [32] pathway activation. CXCL12/CXCR4 can act on MAPK pathway through G protein and then act on its downstream ERK1/2 and FAK to cause cell chemotaxis and inflammatory cytokine accumulation [33–36].…”

<p>Role of the CXCR4/ALK5/Smad3 Signaling Pathway in Cancer-Induced Bone Pain</p>

“… 21 – 24 Signaling via the CXCL12/CXCR4 axis also resulted in the activation of Smad3 via MAPK. 42 The results presented here revealed that ALK5 may be among the downstream targets of CXCR4 and may be capable of CXCR4 transactivating ALK5. However, whether the activation of Smad3 in the non-canonical pathway depends on MAPK also remains unclear; further experiments will be needed in order to clarify this point.…”