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Introduction

The annual worldwide mortality from liver cirrhosis is approximately 800,000 [1], and there is no available treatment [2]. Excessive tissue repair in chronic liver diseases induced by viral, toxic, immunologic, and metabolic disorders [3], results in the deposition of scar tissue and the development of cirrhosis [4]. Quiescent hepatic stellate cells (HSC) produce negligible amounts of extracellular matrix proteins (ECM), but after their activation, these cells develop a myofibroblastic phenotype, proliferate and become the main contributors of ECM [5] [6]. This step is required for the development of liver fibrosis and cirrhosis [7]–[9].

The mitogen-activated protein kinase (MAPK) pathway, through the extracellular signal-regulated kinase (ERK1/2), activates RSK [10]–[14], resulting in the phosphorylation of mouse C/EBPβ (NP_034013 XP_916631) on Thr217 (Thr266 in human C/EBPβ) [5] [10]. The RSK pathway may be critical for HSC activation induced by liver injury, because expression of a catalytically inactive mutant RSK [15], blocked proliferation and survival of cultured HSC upon their activation by collagen type 1 [10]. The RSK phosphoacceptor site in C/EBPβ is identical in mouse and human, it is evolutionarily conserved [5], and essential for survival of activated HSC [10].

Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis. Chronic exposure to the hepatotoxin CCl₄ can induce liver cirrhosis in humans, and it is a classical method of inducing liver injury and fibrosis in mice [10] [16]. We used this model to investigate the role of RSK and phosphorylation of C/EBPβ on Thr217 in liver fibrosis. The hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. C/EBPβ^+/+ mice with severe liver fibrosis induced by an 8-week CCl₄ treatment, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis compared to control mice treated with CCl₄. We found similar activation of RSK and phosphorylation of human C/EBPβ (NP_005185) on Thr266 (identical human phosphoacceptor) in activated HSC in the liver of patients with severe liver fibrosis. These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis, and that inhibition of the RSK pathway is a potential therapeutic strategy for the prevention and treatment of liver cirrhosis.

Results

Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are resistant to hepatotoxin-induced liver fibrosis

Given the important role of RSK in the activation of HSC [10], we hypothesized that a RSK-inhibitory transgenic protein would block phosphorylation of C/EBPβ on Thr217, induce HSC apoptosis and decrease liver fibrosis following chronic liver injury. Because the chronic exposure to the hepatotoxin CCl₄ can induce liver cirrhosis in humans, and it is a classical method of inducing liver injury and fibrosis in mice [10] [16], we analyzed whether it induces liver cirrhosis in mice expressing the dominant negative, nonphosphorylatable RSK-inhibitory C/EBPβ-Ala217 transgene. This mutation changes the phosphorylatable Thr217 to a nonphosphorylatable Ala217 within the RSK phosphoacceptor site of C/EBPβ. These animals are developmentally normal, fertile and have a normal life span [10], suggesting that the RSK-inhibitory transgene is apparently not toxic.

After the thrice-weekly intraperitoneal administration of CCl₄ for 12 weeks, we determined the degree of liver fibrosis in coded samples. This is an extensive chronic exposure for mice and comparable to established severe liver fibrosis in humans [17]. We evaluated liver fibrosis employing the following methods: i) microscopic morphology; ii) semi-quantitative METAVIR clinical grading system; iii) collagen type 1 immunofluorescence; iv) quantitative Sirius red collagen-binding assay; v) quantitative hydroxyproline collagen content; vi) RT-PCR for collagen type 1 mRNA; vii) RT-PCR for α-smooth muscle actin (α-SMA) mRNA (present in activated HSC); and viii) RT-PCR for transforming growth factor (TGF-β) mRNA (a pro-fibrotic cytokine) [4] [7] [8].

Liver samples were stained with the classical Mallory's trichrome to identify collagen in the extracellular matrix. The hepatic collagen pattern and content of C/EBPβ^+/+ (wt) mice treated with CCl₄ for 12 weeks were similar to those of patients with liver cirrhosis (Figure 1A) [4] [8]. We graded the coded liver samples with the standard 0 to 4 METAVIR clinical system [18], and found that after CCl₄ treatment, all C/EBPβ^+/+ mice had severe liver fibrosis (grade 4; n: 12), while all C/EBPβ-Ala217 mice had minimal or no liver fibrosis (grade 0; n: 6; grade 1; n: 6) (P<0.0001) (Figure 1A). In agreement with the findings with the Mallory's trichrome, the Sirius red collagen-binding stain also demonstrated decreased liver fibrosis after CCl₄ treatment in C/EBPβ-Ala217 mice (Figure 1B). Similarly, confocal scanning microscopy with specific antibodies against collagen type 1 also identify decreased liver fibrosis in C/EBPβ-Ala217 mice, compared to C/EBPβ^+/+ mice, after CCl₄ treatment (Figure S1). Although the intra- and inter-observer variability in the semi-quantitative analysis of liver fibrosis was low, we confirmed these findings using quantitative analysis of liver fibrosis.

Figure 1. Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are refractory to the induction of liver fibrosis.

C/EBPβ^+/+ [wt], C/EBPβ-Ala217 and C/EBPβ^−/− [ko] mice received weekly IP injections of CCl₄ or mineral oil (control) for 12 weeks as described in Methods. A. Representative Mallory's trichrome stain for liver fibrosis (in blue; arrowheads). All C/EBPβ^+/+ (wt) mice (n: 12) developed severe liver fibrosis. The C/EBPβ-Ala217 (n: 12; P<0.0001) and C/EBPβ^−/− [ko] (n: 6; P<0.01) mice had either no fibrosis or only minimal liver fibrosis. B. Representative Sirius red immunohistochemistry for collagen (in red; arrowhead). Marked increase in liver collagen in a cirrhotic pattern was observed in C/EBPβ^+/+, but not in C/EBPβ-Ala 217 or C/EBPβ^−/− [ko], mice. C. Analysis of hepatic collagen content by the Sirius red collagen–binding assay, showed a ~2.5-fold increase in C/EBPβ^+/+ mice treated with CCl₄ (n: 12), compared to C/EBPβ-Ala217 mice treated with CCl₄ (n: 12; P<0.001). C/EBPβ^−/− mice were also refractory to the induction of liver fibrosis by CCl₄ (n: 6; P<0.01). D. Analysis of hepatic collagen content by the hydroxyproline assay, showed a ~2-fold increase in C/EBPβ^+/+ mice treated with CCl₄ (n: 7), compared to C/EBPβ-Ala217 mice treated with CCl₄ (n: 6; P<0.01). C/EBPβ^−/− mice were also refractory to the induction of liver fibrosis by CCl₄ (n: 6; P<0.01).

doi:10.1371/journal.pone.0001372.g001

The quantitative analysis of liver collagen, the major extracellular matrix protein in liver fibrosis [4], with the Sirius red collagen-binding assay [19] (Figure 1C), demonstrated that C/EBPβ-Ala217 mice were refractory to the development of liver fibrosis after chronic exposure to the hepatotoxin. The liver collagen content increased approximately 2.5-fold from baseline in C/EBPβ^+/+ (wt) mice (P<0.001) while remaining unchanged in C/EBPβ-Ala217 mice (NS) (Figure 1C). Similarly, chronic administration of CCl₄ increased liver hydroxyproline collagen content in C/EBPβ^+/+ (wt) mice (P<0.01) while remaining unchanged in C/EBPβ-Ala217 mice (NS) (Figure 1D).

We have reported that by lacking the essential C/EBPβ-PhosphoThr217, C/EBPβ−/− HSC are also unable to survive the activation signals [10]. Thus, we postulated that C/EBPβ−/− mice would be refractory to the induction of liver fibrosis. Indeed, C/EBPβ^−/− (ko) mice had a lower fibrotic response than C/EBPβ^+/+ mice to the chronic treatment with CCl₄ (P<0.01) (Figure 1 A, B, C and D).

Further, the expression of the liver fibrogenic indicators such as collagen α 1 type 1 mRNA (newly synthesized collagen) (NM_007742 )(P<0.01), α-SMA mRNA (activated HSC) (NM_007392.2) (P<0.05), and TGF-β mRNA (fibrogenic cytokine) (NM_009370.2) (P<0.01), were induced by CCl₄ treatment significantly more in C/EBPβ^+/+ mice than in C/EBPβ-Ala217 mice as measured by RT-PCR (Figure 2).

Figure 2. Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are resistant to hepatotoxin-induced liver fibrogenesis.

Mice were treated with CCl₄ or control mineral oil for 12 or 16 weeks and RT-PCR was performed as described in Materials and Methods. A. RT-PCR for collagen α1 type 1 was induced in C/EBPβ ^+/+ [wt] mice treated with CCl₄ for 12 or 16 weeks (P<0.01). Treatment of these animals after week 8 with the RSK-inhibitory peptide while continuing the exposure to the hepatotoxin, as described in Materials and Methods blocked this increase at week 16 (P<0.05). Collagen α1 type 1 was not increased in livers of C/EBPβ-Ala217 mice at 12 or 16 weeks (P<0.01). B. RT-PCR for α-SMA was induced in C/EBPβ ^+/+ [wt] mice treated with CCl₄ for 12 or 16 weeks (P<0.05). Treatment of these animals after week 8 with the RSK-inhibitory peptide while continuing the exposure to the hepatotoxin, as described in Materials and Methods blocked this increase at week 16 (P<0.05). α-SMA was not increased in livers of C/EBPβ-Ala217 mice at 12 or 16 weeks (P<0.05). C. RT-PCR for TGF-β was induced in C/EBPβ ^+/+ [wt] mice treated with CCl₄ for 12 or 16 weeks (P<0.01). Treatment of these animals after week 8 with the RSK-inhibitory peptide while continuing the exposure to the hepatotoxin, as described in Materials and Methods, blocked this increase at week 16 (P<0.01). TGF-β was not increased in livers of C/EBPβ-Ala217 mice at 12 or 16 weeks (P<0.01).

doi:10.1371/journal.pone.0001372.g002

In summary, blocking phosphorylation of C/EBPβ-Thr217 through the inhibition of RSK activity with the C/EBPβ-Ala217 transgene or by C/EBPβ gene knock-out decreases the fibrotic response of the liver to chronic injury.

Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are resistant to hepatotoxin-induced liver inflammation

Because liver inflammation, at least in part through the activation of macrophages [20]–[22] and loss of hepato-trophic factors from HSC and liver endothelial cells [23] [24] is a major contributor to liver injury and liver fibrosis, we assessed the degree of liver injury and inflammation in response to the hepatotoxin. Liver injury was determined by measuring serum alanine aminotransferase (ALT) levels in mice after exposure to the hepatotoxin. Serum ALT is a sensitive and specific indicator of hepatocellular injury in humans and animals, and it is the standard clinical test used by the U.S. Food and Drug Administration to ascertain hepatotoxicity of herbal products and drugs [25] [26]. We found that C/EBPβ-Ala217 mice had less liver injury than C/EBPβ^+/+ mice after CCl₄ treatment, judging by the ALT serum levels (Table S1).

Using a microarray assay to assess expression of 66 inflammation genes in the liver of control and transgenic mice, we found that the expression of 21 inflammation genes was decreased, while the expression of other 45 inflammation genes was unchanged in C/EBPβ-Ala217 mice after CCl₄–induced liver injury, when compared to C/EBPβ^+/+ animals treated with CCl₄ (Table 1).

Table 1. Decreased Inflammation in the Livers of C/EBPβ- Ala217 mice after CCl₄ treatment.

doi:10.1371/journal.pone.0001372.t001

These data suggest that partial resistance to liver injury and inflammation may contribute to the prevention of liver fibrosis in C/EBPβ-Ala217 mice. A decreased inflammatory response, mediated at least in part by monocytes/macrophages in the livers of C/EBPβ^+/+ mice (Figure 3A), may be responsible for the decreased liver injury in C/EBPβ-Ala217 mice, since RSK inhibition also affected the recruitment of CD-68+ inflammatory cells to the liver (Figure 3B).

Figure 3. Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are resistant to hepatotoxin-induced liver inflammation.

Mice were treated with CCl₄ or control mineral oil for 16 weeks as described in Materials and Methods. A. Activated monocytes/macrophages, identified by confocal microscopy for CD-68 (green), were increased in livers of C/EBPβ ^+/+ [wt] mice treated with CCl₄ for 16 weeks (middle panels). Treatment of these animals after week 8 with the RSK-inhibitory peptide while continuing the exposure to the hepatotoxin, as described in Material and methods, blocked the monocytes/macrophage inflammatory reaction at week 16 (lower panels). Nuclei are identified with TO-PRO-3 (blue). Only background staining was observed when omitting the first antibody. Microscopy shown is representative of six animals in each group. B. Activated monocytes/macrophages, were not increased in livers of C/EBPβ-Ala217 mice as much as in the livers of C/EBPβ ^+/+ [wt] mice after treatment with CCl₄ for 16 weeks (lower panel). Microscopy shown is representative of six animals in each group.

doi:10.1371/journal.pone.0001372.g003

Given that CCl₄ is metabolized by the cythocrome-P-450 2E-1(Cyp-2E-1) (ABH07947) to form a free radical hepatotoxic metabolite [27], we evaluated the possibility that C/EBPβ-Ala217 affects Cyp-2E1 expression and/or activity. The Cyp-2E1 mRNA expression was similarly inhibited by CCl₄ in the livers of C/EBPβ^+/+ and C/EBPβ-Ala217 mice (Figure S2A). In addition, the Cyp-2E-1 protein expression was also similar in the livers of C/EBPβ^+/+ and C/EBPβ-Ala217 mice after the hepatotoxin treatment (Figure S2B). Moreover, the Cyp-2E1 activity as measured in a cell-free system was unaltered by a RSK-inhibitory C/EBPβ-Ala217 peptide, even at µM concentrations (Figure S2C).

These results indicate that the protective effects of the RSK-inhibitory transgene in CCl₄ -induced liver injury and fibrosis are not due to the spurious blockade of the production of a toxic CCl₄ metabolite.

Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are resistant to hepatotoxin-induced HSC activation and proliferation

Quiescent HSC produce negligible amounts of ECM, but after their activation, these cells develop a myofibroblastic phenotype, proliferate, and become the main contributors of ECM [5] [6]. Because this step is required for the development of liver fibrosis and cirrhosis [7]–[9], we analyzed the activation and proliferation of HSC in the livers of mice chronically exposed to the hepatotoxin.

As expected, chronic CCl₄ administration to C/EBPβ^+/+ (wt) mice, induced marked activation of HSC, as indicated by the positive immunofluorescence for α-SMA within the scar tissue [5] [6] (Figure 4A), and proliferation of HSC, as indicated by the presence of proliferating cell nuclear antigen (PCNA; DNA polymerase δ auxiliary protein), an S-phase marker [28] (Figure 4B). By contrast, C/EBPβ-Ala217 mice were refractory to the induction of HSC activation and proliferation by CCl₄ treatment (Figure 4A and B). Moreover, chronic CCl₄ treatment induced the apoptotic cascade in HSC in the livers of C/EBPβ-Ala217 mice, but not C/EBPβ^+/+ mice, as determined by the presence of active caspase 3 immunofluorescence (Figure 4B). After chronic CCl₄ administration, C/EBPβ was phosphorylated on Thr217 in HSC of C/EBPβ^+/+ mice, but not in C/EBPβ-Ala217 mice, as determined by confocal microscopy (Figure 4A and B), using specific antibodies against this phosphorylated epitope [10].

Figure 4. Mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene are refractory to hepatic stellate cell activation and proliferation.

Mice received CCl₄ or mineral oil injections for 12 weeks as described in Materials and methods. A. Activated stellate cells, identified by confocal microscopy for α-smooth muscle actin (α-SMA; red), displayed C/EBPβ-PhosphoThr217 (green) in livers of C/EBPβ ^+/+ [wt], but not C/EBPβ-Ala217, mice treated with CCl₄. Colocalization of α-SMA and C/EBPβ-PhosphoThr217 is shown in yellow (merge). Nuclei are identified with TO-PRO-3 (blue). Only background staining was observed when omitting the first antibody. B. Proliferating cell nuclear antigen (PCNA; red) was present in activated stellate cells only in livers of C/EBPβ^+/+ [wt] mice treated with CCl₄, while active caspase 3 (green) was found in HSC only in livers of C/EBPβ-Ala217 mice treated with CCl₄. Nuclei are identified with TO-PRO-3 (blue).

doi:10.1371/journal.pone.0001372.g004

Because the mixed cell population of the liver limits the evaluation of signaling cascades in a specific cell type, we studied the fibrogenic pathway in purified HSC. After chronic CCl₄ or control mineral oil administration, we analyzed proteins associated with C/EBPβ, immunopurified from HSC, freshly isolated from C/EBPβ^+/+ and C/EBPβ-Ala217 mice. C/EBPβ-Ala217 binding to, and blocking, RSK phosphorylation (Figure 5A) results in decreased phosphorylation of C/EBPβ on Thr217, and presumably, other target survival proteins by activated RSK [5],[10],[11]. The Ac-KAla217VD-CHO or C/EBPβ216-253-Ala217 (0.25 nM) peptides inhibited RSK activity in a cell-free system, suggesting a direct effect (Figure 5B). The Ac-KThr217VD-CHO wt peptide also inhibited RSK, probably, because it binds to the kinase but cannot be phosphorylated by RSK given its small size (Figure 5B).

Figure 5. C/EBPβ-Ala217 inhibits RSK activation in hepatic stellate cells.

A. A phospho-RSK immunoblot was performed on RSK immunoprecipitates from protein lysates of purified HSC in an experiment conducted as described in (Fig. 1). Phosphorylated RSK (RSKp380) was decreased in HSC isolated from C/EBPβ-Ala217 mice. C/EBPβ and RSK were similar in the different experimental groups. β-Actin was used as an internal control for the immunoprecipitations. Results from triplicate samples of three independent experiments are shown. B. RSK activity was determined in a cell-free system as described in Methods. Recombinant RSK was activated with ATP (125 µM) in the presence or absence of Ac-KThrVD-CHO (200 µM), Ac-KAlaVD-CHO (200 µM), or C/EBPβ216-253-Ala217 (0.25 nM) peptides. Staurosporine was used as a control inhibitor (0.01 nM). All C/EBPβ peptides inhibited RSK activity to a similar extent as staurosporine (P<0.01). Results from triplicate samples of two independent experiments are shown. C. Primary human C/EBPβ^+/+ HSC were transfected with vectors (1 µg each) expressing green fluorescent protein with control wt RSK (GFP), a dominant negative RSK mutant, or C/EBPβ-Ala217. Transfected HSC were selected by sorting for GFP, and cell lysates were immunoprecipitated with C/EBPβ specific antibodies. C/EBPβ-PhosphoThr217 (C/EBPβp217), and caspase 8 immunoblots were performed in C/EBPβ immunoprecipitates. Dominant negative RSK or C/EPBβ-Ala217 prevented C/EPBβ phosphorylation and stimulated the association of unphosphorylated C/EBPβ with active caspase 8. β-Actin was used as an internal control for the immunoprecipitations. Results from triplicate samples of three independent experiments are shown.

doi:10.1371/journal.pone.0001372.g005

C/EBPβ-Ala217 transgene and unphosphorylated C/EBPβ-Thr217 are associated with active caspase 8 and death receptor complex II proteins in HSC

Unexpectedly, we found that C/EBPβ-Ala217 was present with active caspase 8 in HSC from C/EBPβ-Ala217 mice after chronic CCl₄ administration and, to a lesser extent, after mineral oil administration (Figure 6A). In contrast, the association between inactive procaspase 8 and C/EBPβ-PhosphoThr217 (Figure 6A), as well as that between C/EBPβ and activated phospho-RSK (Figure 5A), increased in HSC of C/EBPβ^+/+ mice after chronic CCl₄ administration. Reciprocal immunoprecipitation with caspase 8 antibodies confirmed the presence of C/EBPβ- Ala217 with active caspase 8 (Figure 6A). RSK phosphorylation was inhibited in HSC from C/EBPβ-Ala217 mice, indicating that C/EBPβ-Ala217 not only associates with RSK but that it also decreases its phosphorylation and activation (Figure 5A). The hypothesis that inhibition of RSK by nonphosphorylatable C/EBPβ-Ala217 is critical for caspase 8 activation is supported by the increased caspase 8 activation in HSC from C/EBPβ-Ala217 mice after chronic CCl₄ administration (Figure 6A).

Figure 6. C/EBPβ-Ala217 associates with active caspase 8 in hepatic stellate cells.

A. A caspase 8 immunoblot was performed on C/EBPβ immunoprecipitates from protein lysates from samples described in (Fig 5 A). The association between C/EBPβ-Ala217 with active caspase 8 was increased in HSC isolated from C/EBPβ-Ala217 mice. Phosphorylated C/EBPβ-Thr217 was decreased in HSC from C/EBPβ-Ala217 mice treated with CCl₄. C/EBPβ and RSK were similar in the different experimental groups. β-Actin was used as an internal control for the immunoprecipitations. B. Reciprocal caspase 8 immunoprecipitation of experiment described in (A) confirmed the association of C/EBPβ-Ala217 with active caspase 8. β-Actin was used as an internal control for the immunoprecipitations. C. Reciprocal caspase 8 immunoprecipitation of experiment described in (Figure 5C), confirmed the association of C/EBPβ-Ala217 with active caspase 8. β-Actin was used as an internal control for the immunoprecipitations.

doi:10.1371/journal.pone.0001372.g006

Next, we studied whether blocking RSK activity results in activation of caspase 8. Activated primary human C/EBPβ^+/+ HSC were transfected with vectors expressing green fluorescent protein with a control wt RSK (GFP), to facilitate cell sorting, with a dominant negative RSK mutant, or with the RSK-inhibitory dominant negative C/EBPβ-Ala217. The HSC were activated on a collagen type 1 matrix, a condition that recapitulates the activation of HSC in vivo [9], and identified by their expression of GFAP [10].

As expected, C/EBPβ-PhosphoThr266 (identical to mouse phosphoacceptor Thr217) was markedly decreased in activated human HSC expressing either the RSK mutant or C/EBPβ-Ala217. Moreover, we found that unphosphorylated C/EBPβ was associated active caspase 8 (Figure 5C). The RSK phosphoacceptor site in C/EBPβ is identical in mouse and human, it is evolutionarily conserved [5], and essential for HSC survival upon their activation [10].

C/EBPβ-Ala266 associated with active caspase 8, in cells expressing the dominant negative RSK (Figure 5C), which prevents C/EBPβ phosphorylation on Thr266 by RSK. In contrast, in the control cells expressing GFP and RSK wt, C/EBPβ was phosphorylated and associated with procaspase 8 (Figure 5C), as we reported previously [10]. Reciprocal immunoprecipitation with caspase 8 specific antibodies confirmed the presence of unphosphorylated C/EBPβ with active caspase 8 (Figure 6 B and C). After blocking the RSK-C/EBPβ phosphorylation cascade in activated HSC, with either an ERK1/2 inhibitor or a cell permeant Ac-KAla217VD-CHO peptide, which contains the mutated C/EBPβ-Ala217 phosphoacceptor, unphosphorylated C/EBPβ became associated with other members of the death receptor complex II, such as TNFR1, TRAF2, TRADD and RIP [29] (Figure 7A, B, and C). These associations were identified in C/EBPβ, TNFR1, TRAF2, TRADD and RIP immunoprecipitations (Figure 7 and Figure S3). The Ac-KAla217VD-CHO peptide is cell permeant due to its N-terminus acetyl group, as it has been documented for other peptides used as substrate or inhibitors of caspases [30] [31].

Figure 7. RSK inhibition induces the association of C/EBPβ with active caspase 8, TNFR1, TRAF2, TRADD and RIP.

A. TRADD, C/EBPβ, RIP and caspase 8 immunoblots were performed on C/EBPβ immunoprecipitates from primary human HSC treated with an ERK1/2 inhibitor (10 µM) or the cell permeant Ac-KA217VD-CHO peptide (200 µM). Blocking the phosphorylation of C/EBPβ by RSK with the ERK1/2 inhibitor or the cell permeant Ac-KA217VD-CHO (KAVD) peptide, increased the association between C/EBPβ, active caspase 8, TRADD and RIP. β-Actin was used as an internal control for the immunoprecipitations. B. TNFR1 and C/EBPβ immunoblots were performed on C/EBPβ immunoprecipitates from HSC isolated from mice treated with CCl₄ for 12 or 16 weeks as described in Materials and Methods. Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between C/EBPβ and TNFR1. β-Actin was used as an internal control for the immunoprecipitations. C. TRAF2 and C/EBPβ immunoblots were performed on C/EBPβ immunoprecipitates from HSC isolated from mice treated with CCl₄ for 12 or 16 weeks as described in Materials and Methods. Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between C/EBPβ and TRAF2. β-Actin was used as an internal control for the immunoprecipitations. D. Cytochrome C and Apaf1 immunoblots were performed on cytochrome C immunoprecipitates in livers from mice treated with CCl₄ for 12 or 16 weeks as described in Materials and Methods. Blocking the phosphorylation of C/EBPβ by RSK with C/EBPβ-Ala217 transgene or the cell permeant Ac-KA217VD-CHO peptide increased the association between cytochrome C and Apaf1. β-Actin was used as an internal control for the immunoprecipitations.

doi:10.1371/journal.pone.0001372.g007

A cell-permeant C/EBPβ-Ala217 peptide stimulates active caspase 8 and cell death in collagen type 1-induced activation of cultured HSC

We corroborated the activation of caspase 8 in HSC, under conditions that prevented phosphorylation of C/EBPβ, as described above, by demonstrating the association between cytochrome C with Apaf-1 (Figure 7D), which reflects a downstream amplification effect of active caspase 8 on mitochondrial apoptotic pathways [32]. The reciprocal analysis could not be performed since Apaf-1 antibodies are not suitable for immunoprecipitation. Moreover, inhibition of the RSK-C/EBPβ phosphorylation cascade with the cell permeant C/EBPβ-Ala217 peptide, Ac-KAla217VD-CHO induced expression of the apoptosis effector caspase 3 in cultured, activated mouse HSC, but not in primary mouse hepatocytes (Figure S4). These findings suggest a selective induction of apoptotic pathways by C/EBPβ-Ala217 peptides in cultured activated HSC but not in cultured primary hepatocytes. This conclusion is supported by the apoptotic changes in activated HSC but not hepatocytes of C/EBPβ-Ala217 transgenic mice treated with CCl₄, and by the lack of apoptosis of normal quiescent HSC and hepatocytes of C/EBPβ-Ala217 transgenic mice not treated with CCl₄. In addition, after CCl₄ treatment, hepatocyte injury and hepatic inflammation are decreased in C/EBPβ-Ala217 transgenic mice compared to control C/EBPβ^+/+ mice, arguing for a protective rather than an apoptotic effect on hepatocytes.

Our findings in cell-free, cellular and animal models strongly support the main hypothesis, and suggest a novel, physiological role for unphosphorylated C/EBPβ on caspase 8 activation. Indeed, we found that caspase 8 activation is stimulated by C/EBPβ-Ala217 in HSC from C/EBPβ-Ala217 mice treated with CCl₄ (Figure 8A). In addition, we determined that the peptide enhanced the activity of recombinant caspase 8 in a cell-free system at picomolar concentrations (Figure 8B). To our knowledge, the C/EBPβ-Ala217 peptides are the first reported compounds to directly enhance caspase 8 activation (G Salvesen, personal communication).

Figure 8. The RSK-inhibitory peptide blocks hepatic stellate cell activation and liver fibrosis induced by CCl₄.

A Caspase 8 activity was measured in lysates from HSC isolated from C/EBPβ^+/+ [wt] and C/EBPβ-Ala217 mice untreated or treated with CCl₄ for 24 hr. Caspase activity was increased in C/EBPβ-Ala217 mice treated with CCl₄. Results from triplicate samples of two independent experiments are shown. B. Caspase activation in a cell-free system was determined as described in Methods. The Ac-KAla217VD-CHO peptide enhanced the activation of caspase 8 at picomolar concentrations. Baseline caspase 8 activity was 3.8 U (100%). Results from triplicate samples of three independent experiments are shown (P<0.01 for the Ac-KAla217VD-CHO peptide). C. Animals received a single injection of CCl₄ or mineral oil as described in Methods. α-SMA (red) and C/EBPβ-PhosphoThr217 (green) were identified as described in Materials and methods. Treatment with the cell permeant Ac-KAla217VD-CHO peptide blocked the expression of α-SMA and C/EBPβ-PhosphoThr217. D. PCNA (red) and active caspase 3 (green) were identified as described in Materials and methods. Treatment with the Ac-KAla217VD–CHO peptide blocked the expression of PCNA and induced active caspase 3. E. C/EBPβ^+/+ (wt) mice with severe liver fibrosis after treatment with CCl₄ for 8 weeks, while continuing on CCl₄, received the RSK inhibitory peptide (5 µg IP, three times/week, for week 9 followed by 1 µg IP, three times/week for weeks 10–12 or 10–16). These are representative Mallory's trichrome stain for liver fibrosis (in blue). All control mice (n: 8 at 8-weeks; n: 8 at 12-weeks; and n: 8 at 16-weeks) developed severe liver fibrosis, while mice receiving the RSK-inbitory peptide (n: 8 at 12-weeks; and n: 8 at 16-weeks) had no fibrosis or only minimal liver fibrosis (P<0.01). F. Analysis of hepatic collagen content by the Sirius red collagen–binding assay showed a ~2.5 to 3-fold increase in C/EBPβ^+/+ mice treated with CCl₄ (n: 8 for 8 weeks; n: 8 for 12 weeks and n: 8 for the 16 weeks), compared to animals also receiving the RSK-inbitory peptide (n: 6 for 12weeks; n: 8 for 16 weeks; P<0.01). G. Analysis of hepatic collagen content by the hydroxyproline assay, showed a 2.2-, 3.5- and 5.3-fold increase in C/EBPβ^+/+ mice treated with CCl₄, respectively (n: 8 for 8 weeks; n: 7 for 12 weeks and n: 7 for the 16 weeks), compared to animals also receiving the RSK-inbitory peptide (n: 6 for 12weeks; n: 8 for 16 weeks; P<0.01).

doi:10.1371/journal.pone.0001372.g008

These experiments provided a strong proof-of-principle that RSK activation mediates the signaling required for liver inflammation and liver fibrosis.

A cell-permeant C/EBPβ-Ala217 peptide stimulates cell death in hepatotoxin-induced activation of HSC

Because in preliminary studies the cell permeant tetrapeptide Ac-KAla217VD-CHO was effective in inducing apoptosis of cultured activated HSC [10], we studied whether this peptide would also be effective in inducing apoptosis of activated HSC in a physiologically relevant animal model of liver injury [10] [16]. To activate HSC, we administered a single dose of CCl₄ to C/EBPβ^+/+ (wt) mice, while control mice received the mineral oil vehicle [16]. Six hours later, animals received an intraperitoneal injection of the cell permeant Ac-KAla217VD-CHO peptide [10] (100 µg) or saline vehicle (100 µl). In preliminary studies, we found that 1–100 µg of peptide dose provided adequate bioavailability (M B, unpublished observations). Animals were sacrificed at 24 h. Acute CCl₄ administration induced both activation and proliferation of HSC (among other hepatic cells), judging by the expression of αSMA and PCNA, as determined by confocal microscopy (Figure 8C and D).

HSC activation and proliferation were blocked by treatment with the Ac-KAla217VD-CHO peptide, but not by treatment with saline (Figure 8C and D). Similarly to our findings of RSK inhibition in cell-free, cultured primary stellate cells and in C/EBPβ-Ala217 transgenic mice, the Ac-KAla217VD-CHO peptide prevented the phosphorylation of C/EBPβ on Thr217 in HSC activated by the liver injury induced by CCl₄ (Figure 8C). Further, and in agreement to our findings of the increased caspase 8 activation in cell-free, cultured primary stellate cells and in C/EBPβ-A217 transgenic mice, the Ac-KAla217VD-CHO peptide stimulated the apoptotic pathway of C/EBPβ wt HSC as indicated by the presence of active caspase 3 (Figure 8 D).

A cell-permeant C/EBPβ-Ala217 peptide inhibits progression and stimulates regression of hepatotoxin-induced liver fibrosis

Activation of stellate cells is responsible for the development of liver fibrosis in chronic liver diseases of all causes [3],[4] [8], and remarkably, HSC clearance by apoptosis may allow recovery from liver injury and reversal of liver fibrosis [33] [34].

Given the effective blocking by the RSK-inhibitory peptide of molecular pathways leading to liver fibrosis in an acute CCl₄ model of liver injury and fibrogenesis, we asked whether these effects would occur in a model of established liver fibrosis due to chronic liver injury, reproducing the disease state of patients with severe liver injury and fibrosis.

Therefore, C/EBPβ^+/+ mice with severe liver fibrosis, after receiving CCl₄ for 8 weeks, were treated with the RSK inhibitory peptide for an additional 4 or 8 weeks (5 µg IP, three times/week, for week 9, followed by 1 µg IP, three times/week for weeks 10–12 or 10–16), while continuing to induce liver injury and fibrosis with CCl₄. Treatment of animals with liver fibrosis with the peptide, while continuing to receive CCl₄, prevented the progression and induced regression of liver fibrosis compared to control mice treated with CCl₄. At week-12 or week-16, there was a marked regression of liver fibrosis judging by the trichrome stain (Figure 8E) and Sirius red staining (Figure S5). All control mice (n: 22) had severe liver fibrosis, while all mice that received the RSK-inhibitory peptide had minimal or no liver fibrosis. We confirmed these findings by quantitative analysis of liver collagen with the Sirius red binding assay (P<0.01) [19] (Figure 8F). Analysis of hepatic collagen content by the hydroxyproline assay (Figure 8G), showed a 2.2-, 3.5-, and 5.3-fold increase in C/EBPβ^+/+ mice treated with CCl₄ for 8, 12 and 16 weeks, respectively (n: 8 for 8 weeks; n: 7 for 12 weeks and n: 7 for the 16 weeks), compared to animals treated with the RSK-inhibitory peptide (n: 6 for 12 weeks; n: 8 for 16 weeks; P<0.01).

In agreement with the results observed after CCl₄ treatment in mice expressing the C/EBPβ-Ala217 transgene, treatment with the RSK-inhibitory peptide of C/EBPβ^+/+ mice with severe liver fibrosis induced by CCl₄, while continuing to receive CCl_4, reduced the following: i) expression of liver fibrogenic indicators such as collagen α 1 type 1 mRNA (P<0.05), α-SMA mRNA (activated HSC) (P<0.05), and TGF-β mRNA (fibrogenic cytokine) (P<0.01) (Figure 2A, B, and C); and ii) recruitment of CD-68+ inflammatory cells to the liver (Figure 3A).

Increased expression of active RSK and C/EBPβ-PhosphoThr266 in activated HSC of human liver fibrosis

The RSK phosphoacceptor site in C/EBPβ is identical in mouse and human, it is evolutionarily conserved [5], and necessary for HSC survival upon their activation [10]. The RSK pathway may be critical for HSC activation induced by liver injury, because expression of a catalytically inactive mutant RSK [15], blocked proliferation and survival of cultured HSC upon their activation by collagen type 1 [10].

To assess the relevance to human liver fibrosis of the cellular and animal models of liver fibrosis, we analyzed, in preliminary studies, the role of activated RSK and phosphorylated C/EBPβ on Thr266 (identical to mouse Thr217 phosphoacceptor) as possible mechanisms leading to increased liver fibrosis in four patients with chronic hepatitis C viral infection that resulted in severe liver fibrosis (53±17 years) (see Materials and methods). Liver biopsies from hepatitis C patients afflicted with severe liver fibrosis (METAVIR scores of 3 or 4) displayed a high level expression of both active, phosphorylated RSK and phosphorylated human C/EBPβ on Thr266 in activated HSC within the fibrous tissue, compared with samples from three control patients (60±13 years) as identified by confocal scanning microscopy with specific antibodies against RSK-PhosphoSer380, C/EBPβ-PhosphoThr266, and glial fibrillary protein for HSC [10] (Figure 9 A and B).

Figure 9. Increased expression of active RSK and C/EBPβ-PhosphoThr217 in activated hepatic stellate cells of human liver fibrosis.

Representative confocal microscopy of 4 patients with severe liver fibrosis and 3 control subjects. A. Activated HSC, identified by confocal microscopy for their morphology and fluorescence for glial fibrillary acidic protein (GFAP; red), displayed activated RSK-PhosphoSer380 in livers of patients with severe liver fibrosis (lower panel) but not in the livers of control subjects (upper panel). Colocalization of RSK-PhosphoSer380 and GFAP is shown in yellow (merge). Nuclei are identified with TO-PRO-3 (blue). Only background staining was observed when omitting the first antibody. B. C/EBPβ-PhosphoThr217 (green) was present in activated HSC only in livers of patients with liver fibrosis (lower panel). Colocalization of C/EBPβ-PhosphoThr217 and GFAP (red) is shown in yellow (merge). Nuclei are identified with TO-PRO-3 (blue).

doi:10.1371/journal.pone.0001372.g009

Thus, our findings in liver biopsies from patients with liver fibrosis are congruent with the hypothesis we developed in cell-free, cellular and animal models that RSK activation and its phosphorylation of C/EBPβ in activated HSC may be important in the development of human liver fibrosis.

Discussion

Activation of HSC is responsible for the development of liver fibrosis in chronic liver diseases of all causes [3] [4] [8], and remarkably, HSC clearance by apoptosis may allow recovery from liver injury and reversal of liver fibrosis [33] [34].

In this study we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis. We used the classical CCl₄-induced liver injury and fibrosis model in mice [10] [16], primary mouse and human HSC [10], and cell-free systems to investigate the role of RSK and phosphorylation of C/EBPβ on Thr217 in the activation of HSC and liver fibrosis. It would be important to determine whether RSK and phosphorylation of C/EBPβ are also critical in other animal models that reflect other causes of human liver fibrosis, such as biliary cirrhosis, alcoholic liver disease, immune liver injury and genetic iron overload [3]. Any one of these studies will require as extensive an analysis as that performed with the CCl₄ model of liver fibrosis.

We found that mice expressing the RSK-inhibitory C/EBPβ-Ala217 transgene were refractory to the induction of HSC activation and proliferation by CCl₄ treatment. After chronic CCl₄ administration, C/EBPβ was phosphorylated on Thr217 in HSC of C/EBPβ^+/+ mice, but not of C/EBPβ-Ala217 mice. C/EBPβ-Ala217 binding to, and blocking, RSK phosphorylation results in decreased phosphorylation of C/EBPβ on Thr217, and presumably, other target survival proteins by activated RSK. Moreover, chronic CCl₄ treatment induced the apoptotic cascade in HSC in the livers of C/EBPβ-Ala217 mice, but not C/EBPβ^+/+ mice, as determined by the presence of active caspase 8 and 3.

C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and was linked to apoptosis of activated HSC, freshly isolated from transgenic mice. After blocking the RSK-C/EBPβ phosphorylation cascade in activated human HSC in culture, with either an ERK1/2 inhibitor or a cell permeant RSK-inhibitory peptide, Ac-KAla217VD-CHO, unphosphorylated C/EBPβ became associated with other members of the death receptor complex II, such as TNFR1, TRAF2, TRADD and RIP [29]. The combined results suggest a functional link between inactive RSK and the active caspase 8 complex II. Further, inactive RSK and active caspase 8 are co-immunoprecipitated with C/EBPβ-Ala217 or unphosphorylated C/EBPβ, suggesting also a physical link. However, identification of a putative RSK/caspase 8 complex would require crystallographic analysis.

In support of our proposed role of unphosphorylated and phosphorylated C/EBPβ-Thr217 on the modulation of HSC survival following their activation by liver injury, expression of the dominant positive phosphorylation mimic C/EBPβ-Glu217 [5] enhances survival of cultured progenitor neuronal cells [35] while C/EBPβ^−/− macrophages display defective bacterial killing and tumor cytotoxicity [36]. A corollary of our study is that mice expressing the C/EBPβ-Glu217 transgene would be more susceptible to HSC activation and liver fibrosis induced by liver injury and inflammation. Indeed, this seems to be the case in preliminary studies with these novel transgenic mice (M.B, unpublished observations).

Although the association between C/EBPβ-PhosphoThr217 and inactive procaspase 8 is linked to the inhibition of active caspase 8 [10], the precise molecular mechanisms by which phosphorylated C/EBPβ prevents liver injury-induced HSC apoptosis have not been characterized yet. Phosphorylated C/EBPβ could induce the inhibition of pro-apoptotic proteins, such as p53 [37] or the activation of survival proteins, such as MnSOD [38], or FLIP [29]. Alternatively, granzyme B rather than caspase 8 could be the major target of phosphorylated C/EBPβ-Thr217, as we suggested previously for HSC apoptosis/survival [10].

The hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene as detected by morphological, semi quantitative and quantitative assays. Blocking phosphorylation of C/EBPβ-Thr217 through the inhibition of RSK activity with the C/EBPβ-Ala217 transgene or by C/EBPβ gene knock-out decreases the fibrotic response of the liver to chronic injury. These findings indicate that the RSK phosphoacceptor site in C/EBPβ, which is identical in mouse and human [5], is essential for HSC survival upon their activation in chronic liver injury in mice. Further, the decreased fibrotic response of the liver to the hepatotoxin in C/EBPβ ^−/− mice suggests that the critical target of RSK in activated HSC is C/EBPβ-Thr217 rather than other RSK phosphoacceptors in c-Fos, CREB, CBP or other proteins [10]–[14].

Because liver inflammation induces liver injury and liver fibrosis [34] we assessed the degree of liver injury and the inflammatory response to the hepatotoxin. We found decreased liver injury and inflammation in response to CCl₄ treatment in mice expressing the C/EBPβ-Ala217 transgene. Our data suggest that in addition to increased HSC apoptosis, partial resistance to liver injury and inflammation may contribute to the prevention of liver fibrosis in C/EBPβ-Ala217 mice. A decreased inflammatory response, mediated at least in part by monocytes/macrophages in the livers of C/EBPβ^+/+ mice, may be responsible for the decreased liver injury in C/EBPβ-Ala217 mice, since RSK inhibition also affected the recruitment of CD-68+ inflammatory cells to the liver. Our results are congruent with the proposed role of macrophages as a major contributor to liver injury and liver fibrosis [20] [22].

Similarly to our findings of RSK inhibition in cell-free, cultured primary stellate cells and in C/EBPβ-Ala217 transgenic mice, the cell permeant Ac-KAla217VD-CHO peptide prevented the phosphorylation of C/EBPβ on Thr217 in HSC activated by the liver injury induced by CCl₄. Further, and in agreement to our findings of the increased caspase 8 activation in cell-free, cultured primary stellate cells and in C/EBPβ-A217 transgenic mice, the Ac-KAla217VD-CHO peptide stimulated the apoptotic pathway of C/EBPβ^+/+ HSC as indicated by the presence of active caspase 8 and 3.

C/EBPβ^+/+ mice with severe liver fibrosis induced by an 8-week CCl₄ treatment, while continuing on CCl₄, were treated with the cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulated apoptosis of HSC and blocked active fibrogenesis, preventing progression and inducing regression of liver fibrosis compared to control mice treated with CCl₄. Because the resulting activation of HSC and liver fibrosis is similar in any type of chronic liver injury, these results suggest that the inhibition of both RSK and its phosphorylation of C/EBPβ may be effective in preventing/regressing liver fibrosis in animal models that reflect other causes of human liver fibrosis, such as biliary cirrhosis, alcoholic liver disease, immune liver injury, and genetic iron overload [3].

To assess the relevance of the cellular and animal models of liver fibrosis to human liver fibrosis, we analyzed, in preliminary studies, the role of activated RSK and phosphorylated C/EBPβ on Thr266 (ident