Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury - PubMed (original) (raw)

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury

Katsunori Yoshida et al. Am J Pathol. 2005 Apr.

Abstract

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

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Figures

Figure 1

Antibodies selectively distinguish Smad2/3 phosphorylation at linker regions and C-terminal SSXS regions. Smad2 and Smad3 proteins as well as domain-specific Abs are shown schematically. Anti-pSmad2L (Ser 249/254) Ab and anti-pSmad3L (Ser 207/212) Ab recognize SAPK-dependent phosphorylation sites in Smad2 and Smad3, whereas anti-pSmad2C (Ser 465/467) Ab and anti-pSmad3C (Ser 423/425) Ab recognize phosphorylated C-terminal SXS sites in Smad2/3 activated by TβRI.

Figure 2

Differential localization of pSmad2L and pSmad3L in α-SMA-immunoreactive mesenchymal cells in the injured liver. Formalin-fixed, paraffin-embedded sections of normal rat liver (top) and injured liver at 36 hours after CCl4 intoxication (middle and bottom) were stained with anti-pSmad2L Ab (α pSmad2L) and anti-pSmad3L Ab (α pSmad3L). These sections paired with mirror image sections stained using anti-α-SMA Ab (α α-SMA) and anti-PCNA Ab (α PCNA). The Abs then were bounded by goat anti-mouse immunoglobulins conjugated with peroxidase-labeled polymer. Peroxidase activity was detected by 3,3′-diaminobenzidine tetrahydrochloride. Representative combinations are shown for: A, pSmad2L and α-SMA; B, pSmad2L and PCNA; C, pSmad3L and α-SMA; D, pSmad3L and PCNA. Bottom: Higher magnification of the boxed areas in middle. All sections were counterstained with hematoxylin (blue). Brown indicates specific Ab reactivity. In normal rat liver, Smad2 and Smad3 were minimally phosphorylated at linker regions (top panels in α pSmad2L and α pSmad3L). At 36 hours, pSmad2L was predominantly localized in the cytoplasm of α-SMA-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas (A and B, middle and bottom). In contrast, pSmad3L was located in nuclei of the α-SMA-immunoreactive mesenchymal cells (C and D, middle and bottom). Scale bars, 50 μm.

Figure 3

Both pSmad2C and pSmad3C are localized in nuclei of hepatocytes and mesenchymal cells in injured liver. Formalin-fixed, paraffin-embedded sections of normal rat liver and injured liver were stained with the Abs indicated, which in turn were bounded by goat anti-rabbit immunoglobulins conjugated to peroxidase-labeled polymer. Peroxidase activity then was detected by 3,3′-diaminobenzidine tetrahydrochloride. Representative examples are shown for pSmad2C (α pSmad2C) and pSmad3C (α pSmad3C) immunostaining in normal liver (top), and injured liver at 36 hours after CCl4 intoxication (middle and bottom). Bottom: Higher magnification of the boxed areas in the middle. All sections were counterstained with hematoxylin (blue). Brown indicates specific Ab reactivity. In normal rat liver, Smad2 and Smad3 were slightly phosphorylated at C-terminal regions (top). Amounts of the C-terminal phosphorylation in the nuclei of hepatocytes and mesenchymal cells surrounding centrilobular areas increased at 36 hours after CCl4 intoxication (middle and bottom). Scale bars, 50 μm.

Figure 4

Smad2 and Smad3 are phosphorylated not only at C-terminal regions but also at linker regions in the activated HSCs after CCl4 intoxication. HSCs were obtained from normal rat liver and injured livers at 36 and 72 hours after CCl4 intoxication. Cell lysates were subjected to anti-Smad2/3 immunoprecipitation (IP), and were immunoblotted with each anti-pR-Smad Ab (top). Relative amounts of endogenous Smad2/3 were determined by immunoblotting (IB) using anti-Smad2/3 Ab (bottom).

Figure 5

JNK is phosphorylated in the activated HSCs after CCl4 intoxication. HSCs were obtained from normal rat liver and injured livers at 36 and 72 hours after CCl4 intoxication. Phosphorylation of endogenous JNK1/2 was analyzed by immunoblotting with anti-pJNK1/2 Ab (α pJNK1/2; top). Expression of endogenous JNK1/2 was monitored by immunoblotting using anti-JNK1/2 Ab (α JNK1/2; bottom).

Figure 6

JNK in the activated HSCs after CCl4 intoxication directly phosphorylates Smad2/3 at linker regions. HSCs were extracted from normal rat liver and injured livers at 36 and 72 hours after CCl4 intoxication. Immunoprecipitate of cell extracts with Ab against phosphorylated JNK were mixed with bacterially expressed GST-Smad2 (A) and GST-Smad3 (B). Phosphorylation of Smad2 and Smad3 was analyzed by immunoblotting (IB) using each anti-pSmad2/3 Ab (top). Total Smad2 and Smad3 were monitored by immunoblotting (IB) using anti-Smad2/3 Ab (bottom).

Figure 7

Both TGF-β and PDGF signals phosphorylate linker regions of Smad2/3 in HSCs. HSCs were incubated with or without TGF-β1 and PDGF for 30 minutes as indicated. Followed by immunoprecipitation (IP) of cell lysates with anti-Smad2/3 Ab, phosphorylation of Smad2 and Smad3 was analyzed by immunoblotting (IB) using each anti-pSmad2/3 Ab. Expression of endogenous Smad2 and Smad3 was monitored by immunoblotting using anti-Smad2/3 Ab (bottom).

Figure 8

Both TGF-β and PDGF signals stimulate JNK phosphorylation in HSCs. HSCs were incubated with TGF-β1 and/or PDGF for 15 minutes. Phosphorylation of endogenous JNK1/2 was analyzed by immunoblotting using anti-pJNK1/2 Ab (α pJNK1/2; top). Expression of endogenous JNK1/2 was monitored by immunoblot using anti-JNK1/2 Ab (α JNK1/2; bottom).

Figure 9

JNK activated by TGF-β and PDGF signals in HSCs directly phosphorylates Smad2/3 at linker regions. HSCs were extracted at 15 minutes after TGF-β1 and/or PDGF treatment, and cell extracts were immunoprecipitated with anti-pJNK1/2 Ab. In vitro kinase assays were performed using GST-Smad2 (A) and GST-Smad3 (B) as substrates. Phosphorylation states of Smad2/3 were analyzed by immunoblotting (IB) using each anti-pSmad2/3 Ab (top). Total Smad2 and Smad3 were monitored by immunoblot (IB) using anti-Smad2/3 Ab (bottom).

Figure 10

A JNK inhibitor SP600125 treatment causes the reduction of migratory response of HSCs to TGF-β and PDGF stimulation. HSCs were cultured on Matrigel for 12 hours with TGF-β1 and/or PDGF in the absence or presence of SP600125. After fixation with 100% methanol, the cells were stained by hematoxylin. The number of infiltrating cells was counted in five regions selected at random, and the extent of invading cells was determined by the mean count. Duplicate filters were used, and the experiments were repeated three times.

Figure 11

Both TGF-β and PDGF signals stimulate PAI-1 transcriptional activity through pSmad3L in HSCs. HSCs were transiently co-transfected of PF1-Luc with Smad3WT, Smad3EPSM, Smad3(3S-A), or empty control vector (pcDNA3). After changing the medium, cells were incubated for another 30 hours. Under serum-free conditions, the cells were incubated for 12 hours with TGF-β1, PDGF, or a combination of both. Luciferase activity was determined and normalized to transfection efficiency. Values of samples from cells transfected with PF1-Luc alone and those left untreated were set arbitrarily at 1. Results shown are the mean ± SD (n = 4) from a representative experiment. *, P < 0.05; **, P < 0.01, by one-way analysis of variance.

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Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury - PubMed (original) (raw)