FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3–induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB–FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness

Fn14, a Downstream Target of the TGF-β Signaling Pathway, Regulates Fibroblast Activation.

Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-β, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-β signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-β signaling was activated by TGF-β1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-β signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-β signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis

Fn14 expression in human tissue and cultured cells in response to TGF-β signaling.

<p>(A) Fn14 expression in leukocytes from healthy donors and burn patients was detected by RT–qPCR. n = 3–5. (B) Fn14 expression in skin tissue from healthy donors (normal) and burn patients (scar) was detected by RT–qPCR. n = 3–5. (C) Fn14 mRNA expression was detected by RT–qPCR in human dermal fibroblasts (HDFs) in response to TGF-β signaling. (D) Fn14 protein expression in human dermal fibroblasts (HDFs) in response to TGF-β signaling was detected by western blotting. Human dermal fibroblasts (HDFs) were treated with TGF-β1, SB431542 or TGF-β1 plus SB431542. The protein expression data in the histogram was calculated using gray scale western blots of HDFs cultured in six-well plates. Total RNA was isolated from human tissues and cultured cells using TRIzol Reagent, and cDNA was synthesized for RT–qPCR. Data from at least three independent experiments are shown. Data are presented as the mean ± SD (standard deviation).</p

Detection of SMAD4 binding to the human Fn14 promoter in HDFs.

<p>(A) The human Fn14 gene promoter was analyzed, and primers for chromatin immunoprecipitation (ChIP) were designed according to the sequences of potential binding sites. (B)-(I) ChIP-qPCR was performed with primer sets P-1 (B), P-2 (C), P-3 (D), P-4 (E), P-5 (F), P-6 (G), P-7 (H) and P-8 (I). Data from at least three independent experiments are shown. Data are presented as the mean ± SD.</p

Fn14 overexpression in HDFs promotes fibroblast activation.

<p>(A) Cultured HDFs were transfected with pcDNA3.1 (control) or pcDNA3.1-hFn14 (p-Fn14). (B) Fn14 mRNA expression in HDFs was detected by RT–qPCR after Fn14 overexpression. (C) Fn14 protein expression in HDFs was detected by western blotting after Fn14 overexpression. (D) COL1A1 mRNA expression was detected by RT–qPCR. (E) COL3A1 mRNA expression was detected by RT–qPCR. (F) COL1 protein expression was detected by western blotting. (G) αSMA protein expression was detected by western blotting. The protein expression data in the histogram were calculated using gray scale western blots. Data from at least three independent experiments are shown. Data are presented as the mean ± SD.</p

Fn14, a downstream target of the TGF-β signaling pathway, regulates dermal fibroblast activation.

<p>The TGF-β signaling pathway increases Fn14 expression through SMAD4 binding to the Fn14 promoter in human dermal fibroblasts. Then, upregulated Fn14 expression promotes ECM synthesis and fibroblast activation.</p