TGF-β and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions

Abstract

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-b (TGF-b) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-b treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-b or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-b signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-b allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-bmediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-b signals. Moreover, a combined treatment with HGF and TGF-b led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-bdependent activation of p15 INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-b transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor β signaling, promoting cirrhosis and hepatocellular carcinoma

“…Phosphorylation of Smad3 was analyzed using rabbit polyclonal anti-pSmad3L Ab and antipSmad3C Ab as described previously. 15 Reverse-Transcription PCR. Isolation of RNA, reverse transcription, and PCR for T␤RII, Smad2, Smad4, PAI-1, p21 WAF1 , and GAPDH was performed as described previously.…”

“…Infiltrating cells were counted in 5 regions selected at random as described previously. 15 [ 3 H] Thymidine Incorporation. DNA synthesis was measured by incorporation of 1 Ci/ml [ 3 H] thymidine (Amersham Pharmacia Biotech) into 5% trichloroacetic acid-precipitable material after a 4-hour pulse as described previously.…”

“…10,11 Although several studies of EMT have suggested that the process involves Smad-independent pathways, 12 recent studies using Smad3 knockout mice have indicated that signaling through the Smad3-dependent pathway is required for injury-dependent multistage transition of an epithelial cell to a mesenchymal phenotype. 13 We therefore have focused on Smad3 signaling, 14,15 and have recently reported different roles of Smad3 phosphoisoform-mediated signaling in epithelial cells and mesenchymal cells. 16,17 Thus, TGF-␤ activates not only TGF-␤ type I receptor (T␤RI) but also c-Jun N-terminal kinase (JNK), converting Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L).…”

To (TGF)β or not to (TGF)β: Fine-tuning of Smad signaling via post-translational modifications

“…However, in contrast, ERK-dependent phosphorylation of Smad2 at the N-terminal Thr8 enhances its transcriptional activity [29]. Phosphorylation of Smad3 by p38 MAPK and ROCK (Ser204, Ser208, and Ser213) and c-Jun N-terminal kinase (JNK) (Ser208 and Ser213; analogous Ser250 and Ser255 in Smad2) may also enhance Smad2/3 transcriptional activity, suggesting that Smads and the p38/ROCK/JNK signaling pathways might cooperate in generating a more robust TGF-β response [30][31][32]. In accordance, a significant increase in Ser208/Ser213 phosphorylation of Smad3 is associated with late stage colorectal tumors, suggesting that the linkerphosphorylated Smad3 may mediate the tumor-promoting role of TGF-β in late tumorigenesis [33].…”

“…Additional kinases, e.g. MEKK-1, CaMKII, protein kinase C, and CKε, target R-Smads and regulate Smad-dependent transcriptional responses [36][37][38][39], and TGF-β may also induce Smad phosphorylation in the linker region as well as at the SXS motif [31,32]. Thus, the Smad linker region is emerging as an important and critical regulatory platform in the fine-tuning of TGF-β signaling.…”