Id1 is a critical mediator in TGF-β–induced transdifferentiation of rat hepatic stellate cells

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Transforming growth factor (TGF)-β is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-β antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and α-smooth muscle actin (α-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-β pathways that control the transdifferentiation process. In an attempt to elucidate TGF-β target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with α-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-β1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-β/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. (HEPATOLOGY 2006;43:1032–1041.)

Liver injury and subsequent hepatic fibrogenesis may be provoked by various causes, including infection with hepatitis viruses, aflatoxin, or chronic alcohol abuse. An early event in the development of hepatic fibrosis is the activation of the hepatic stellate cells (HSCs), a liver-specific type of pericyte residing in the subendothelial space of Disse. On activation, HSCs change their phenotype into extracellular matrix (ECM) producing myofibroblasts (MFBs).1, 2 A cohort of cytokines is involved in the regulation of profibrogenic processes in the liver, amongst which transforming growth factor beta (TGF-β) plays a prominent role.3 Quiescent HSCs display TGF-β receptors I, II, and III4 on their surface and are therefore responsive to TGF-β. On ligand binding, signaling cascades are activated, namely, activin-like kinase (ALK)-5 and subsequent phosphorylation of Smad2 and 3, which form complexes with Smad4 and translocate to the nucleus. TGF-β–mediated signal transduction via ALK-1 with subsequent activation of Smads1, -5, and -8 has not been described in HSCs. In the nucleus, activated Smad-complexes inhibit DNA synthesis and stimulate Smad7 and collagen (Col1) expression.5, 6 In the process of transdifferentiation to MFBs, the TGF-β responsiveness is modulated. As a consequence, Smad7 induction as well as DNA synthesis control is lost, and features of excessive profibrogenic TGF-β effects predominate. With perpetuated activation of HSCs, fibrous tissue substitutes for normal liver architecture, and chronic liver disease develops. The aim of the current study was to understand the molecular events underlying TGF-β signaling in HSCs and MFBs. Recently, we were able to demonstrate that ectopic overexpression of the TGF-β antagonist Smad7 blocks HSC transdifferentiation and inhibits experimentally induced liver fibrosis in rats.7 Because Smad7 has a relatively broad spectrum of activities and may inhibit all type I receptors of the TGF-β family, our interest was to determine an expression profile of HSCs after overexpression of Smad7 and compare this with control cells to identify TGF-β/Smad7 target genes. In similar approaches, ECM components including collagen type I8, 9 and plasminogen activator inhibitor10 have been identified. Our study indicates that the helix-loop-helix (HLH) protein inhibitor of differentiation 1 (Id1) is a novel TGF-β target gene in HSCs. In contrast to the basic (b) HLH proteins that bind either as homo- or heterodimers to E-box elements (CAnnTG motifs) and regulate cell-specific gene transcription, Id proteins 1 to 4 lack the basic DNA binding domain and function as naturally occurring dominant negative inhibitors of gene transcription by forming nonfunctional heterodimers with bHLH proteins.11, 12 In the current study, expression and function of ALK1 in HSCs was identified, and TGF-β signaling via ALK1/Smad1 induced expression of Id1. Ectopic Id1 overexpression was sufficient to overcome the inhibitory effects of Smad7 on HSC activation and alpha smooth muscle actin (α-SMA) fiber formation in vitro. Our data indicate that Id1 might represent a novel objective to more specifically block profibrogenic TGF-β effects.

Abbreviations

HSCs, hepatic stellate cells; ECM, extracellular matrix; TGF-β, transforming growth factor beta; ALK, activin like kinase; bHLH, basic helix-loop-helix; Id1, inhibitor of differentiation 1; MOI, multiplicity of infection; BMP, bone morphogenetic proteins; SMA, smooth muscle actin; PCR, polymerase chain reaction; MFB, myofibroblasts; siRNA, short interfering RNA; CFSC, cirrhotic fat storing cells; BDL, bile duct ligation; EC, endothelial cells.

Materials and Methods

Cell Culture and Adenovirus Infection.

HSCs were prepared from male Sprague-Dawley rats by the pronase/collagenase method followed by single-step density gradient centrifugation with Nycodenz (Nyegaard Co. AS, Oslo, Norway) as previously described.5 The mean purity was higher than 95%, and the yield ranged from 30 to 50 × 106 HSCs/liver. Cells were cultured as previously described.7 Primary HSCs were infected with recombinant adenoviruses at day 2 after plating at a dose of 50 to 250 multiplicities of infection (MOI). For cytokine stimulation, 16 hours' serum starvation was performed, followed by stimulation with 5 ng/mL TGF-β1 or bone morphogenetic (BMP)-2. To inhibit ALK5, serum-starved HSCs (day 2) were pre-treated with 5 μmol/L SB431542 (Sigma, St. Louis, MO) before stimulation with TGF-β1.

Virology.

Recombinant E1-deleted adenoviral vectors carrying cDNAs encoding mouse Id1 (Ad-Id1), constitutively active mutants for the TGF-β type I receptor family (Ad-caALK3, Ad-caALK1, Ad-caALK5) or Smad7 (Ad-Smad7) under the control of a cytomegalovirus promoter or a cassette of BMP response elements from the mouse Id1 promoter driving a luciferase reporter gene (AdBRE-Luc) were prepared and used as previously described.13 Virus amplification, purification, and infections were performed as described.14, 15

Animal Treatment.

All animal protocols were in full compliance with the guidelines for animal care and were approved by the Animal Care Committee. Sprague-Dawley rats were bile duct ligated as described.7 Control animals were sham operated. Rats were killed 1, 2, 3, 4, and 5 weeks after surgery. Liver samples were fixed in 4% buffered paraformaldehyde for immunostaining.

Preparation of Cell Lysates and Immunoblotting.

Lysates from cell cultures were prepared as described.16 Protein concentrations were determined using DC Protein Assay (Biorad, München, Germany) and 20- to 40-μg protein lysates were separated by 4% to 12% SDS-PAGE and transferred to nitrocellulose membrane (PIERCE, Bonn, Germany). Western blot analysis was performed as described.16 Antibodies used in Western blots were as follows: Id1, C-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-Smad1/5/8, phospho-Smad2, Smad1 (Cell Signaling Technology, Beverly, MA), Smad2 (Zymed, South San Francisco, CA). Antiserum against endoglin was provided by A. Lux,17 and the HA-antibody was from Sigma. All expression data were confirmed in at least two independent experiments.

Immunofluorescence Microscopy.

Rat HSCs were cultured on glass coverslips, 2 × 104 cells in 35-mm dishes. HSCs were subjected to adenoviral infection at day 2 after plating. On day 7, cells were stained as previously described.7 α-SMA was detected using mouse antibody (Sigma, 1:100), followed by incubation with Cy3-conjugated goat anti-mouse immunoglobulin G (Zymed, 1:400). Nuclei were stained with 4,6-diamidino-2-phenylindole. Staining was documented using an LSM 510 laser scanning microscope (Zeiss).

Immunohistochemical Staining.

Four-micron paraffin sections were used for immunohistochemical staining as previously described.7 Antibodies against Id1 and phospho-Smad1/5/8 were as mentioned for Western blot. As a secondary antibody, peroxidase-conjugated swine-anti-rabbit immunoglobulin (DAKO, Carpinteria, CA) was used. Sections were counterstained with Mayer's hemalaun solution (DAKO). Control experiments included incubation of sections with normal rabbit immunoglobulin G or primary antibodies neutralized with an excess of blocking peptide, respectively. Collagen staining was performed with 0.1% Sirius red F3Ba (Waldeck GmbH & Co.KG, Muenster, Germany) in picric acid as previously described.7

RNA Isolation.

Total RNA was purified from HSC with an RNA purification kit (Qiagen, Hilden, Germany) according to the manufacturers' instructions, and the concentration was measured spectrophotometrically.

Real-Time Polymerase Chain Reaction.

A cDNA fragment was amplified and column-purified with a QIAquick polymerase chain reaction (PCR) purification kit (Qiagen). Primer sequences for ALK5: (GI: 416397) were as follows: forward 5′-CGTCTGCATTGCACTTATGC-3′, reverse 5′-AGCAGTGGTAAACCTGATCC-3′. Gene-specific standards were produced as previously reported18 and sequenced on a semiautomated sequencer (310A, Applied Biosystems, Foster City, CA). Serial 10 times logarithmic dilutions of the cRNA and 1 μg total RNA from cell culture samples were reverse transcribed. Real-time PCR was performed using LightCycler FastStart DNA Master SYBR Green I (Roche) according to manufacturers' protocol, and the following primers were used: rat Id1 (GI: 516116): Forward 5′-TGGACGAACAGCAGGTGAAC-3′, reverse 5′-TCTCCACCTTGCTCACTTTGC-3′; endoglin (GI: 45550266): Forward 5′-CTGGGCATCACCTTTGGTGC-3′, reverse 5′-GCCATGCTGCTGGTGGAG-3′; ALK3 (GI: 13540660): Forward 5′-AGATTACTGGGAGCCTGTCT- 3′, reverse 5′-ACGTCACTCCAT TTTCCGGC-3′; ALK1 (GI: 11967972): Forward 5′-GTCAAGAAGCCTCCAGCAAC-3′, reverse 5′-CAT CAACTCAGGCTTCGGG- 3′; Betaglycan (GI: 8394445): Forward 5′- AGGTCCATGTCCTAAACCTC-3′, reverse 5′-GAGTTGAGCAGGAACACGAT-3′. The light cycler conditions were: Initial denaturation at 95°C for 600 s, followed by 40 cycles with denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and elongation at 72°C for 15 seconds. By comparing with the ALK5 standard curve, relative quantities of target mRNA in the unknown sample were determined or the crossing points were plotted.

Id1-Promoter Reporter Gene Analysis.

A mouse Id1-promoter-luciferase construct containing fragment −1231/+88 from the mouse genomic region, which has been subcloned in KpnI and XmaI sites upstream of a minimal adenoviral major late promoter reporter construct, was used.19, 20 One hundred nanograms plasmid DNA per 6-well culture plate was co-introduced with pSV40-β-gal (Promega, Mannheim, Germany) for transfection efficiency normalization,21 using Fugene6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturers' instructions. One day after transfection, cells were serum-starved for 16 hours followed by stimulation with 1 ng/mL TGF-β1 or 10 ng/mL BMP-2 for 8 hours. Cell lysis and luciferase assays were carried out using a luciferase assay system (Promega).

Knock-Down of Endogenous Id1 and ALK1.

Cells were infected with recombinant adenoviruses encoding small interfering RNA for Id1 or ALK1. The sequence encoding short interfering RNA (siRNA) for Id1, CGTCCTGCTCTACGACATC (ACC# M31885, NT 254-272), was first cloned in pSUPER, and the BamHI/XhoI insert was subcloned into pENTRY1A vector (Invitrogen Corp., Carlsbad, CA), recombinated by a clonase reaction in pAD/DEST (Invitrogen), and adenoviruses were generated according to the manufacturers' protocol. The adenoviral construct encoding interfering RNA for ALK1 was previously described.22 Lysates and immunostainings were performed 48 hours after virus infection.

Results

Smad7 Interferes With Id1 Expression in Activated HSCs.

We have previously shown that expression of the TGF-β antagonist Smad7 inhibits activation of primary cultured HSCs and experimentally induced liver fibrosis in rats.7 To identify Smad7-dependent gene responses, HSCs were infected at day 2 after seeding with an adenovirus driving ectopic Smad7 expression, and total RNA was purified 2 days later, when the stress response caused by viral infection was no longer predominant. Transcript profiles were generated in comparison to Ad-LacZ–infected cells using the Affymetrix rat genome RG-U34 microarray (∼8.800 probe sets). A total of 377 genes were found to be differentially regulated by Smad7 with a change of at least twofold (P < .001). Among these, 230 were identified with informative annotations. The affected genes were subsequently clustered into seven categories according to their biological characteristics and functions (manuscript in preparation). Among the most intensely regulated genes was a recently identified TGF-β/BMP-2 target gene,23 Id1, whose known functions include cell cycle regulation or inhibition of epithelial to mesenchymal transition.24, 25 Smad7-dependent downregulation of Id1 mRNA levels was confirmed by real-time PCR analysis and showed a decrease of message levels by 70% to 80% (Fig. 1A). Id1 protein expression during transdifferentiation was completely undetectable after overexpression of Smad7 (Fig. 1B).

Figure 1.

Smad7 abrogates Id1 expression during transdifferentiation of HSCs. HSCs were infected with adenoviruses (AdLacZ, AdSmad7) as indicated (50 MOI) at day 2 after plating. (A) Total RNA was analyzed by SYBR green I–based real-time PCR with rat Id1 specific primers as indicated in Materials and Methods. Three different preparations of rat HSCs were investigated independently and in triplicate. (B) Cell lysates for total protein were prepared at day 4. Western blot analysis was performed using anti Id1 antiserum. As loading control, the same blot was probed with an anti–β-actin antibody. HSC, hepatic stellate cells.

Ectopic Id1 Expression Enhances Activation of HSCs and Abrogates Inhibitory Effects of Smad7.

To assess the effect of Id1 on the HSC phenotype and on Smad7 activities, adenoviruses carrying cDNA for either Id1 or Smad7 were introduced into 2-day-old HSCs. Ectopic expression of Id1 was confirmed by Western blot analysis (Fig. 2A). Morphological changes were monitored by phase contrast and fluorescence microscopy until day 7 of culture (Fig. 2B–C). As expected from previous studies, Smad7 expressing HSCs remained in a quiescent stage with compact spherical shape and sustained retinoid storage. In contrast, HSCs expressing Id1 alone or together with Smad7 showed characteristic transdifferentiation-dependent morphological changes that include loss of fat droplets and flattened appearance with spreading and cytoplasmic extensions representing myofibroblasts (MFBs). In comparison with untreated (not shown) or Ad-LacZ–infected controls, Id1 overexpressing cells were more progressed in the transdifferentiation process. Because α-SMA fiber formation is one typical feature of transdifferentiation, which is potently abrogated by Smad7, the influence of ectopic Id1 expression on α-SMA fiber formation was examined. HSCs were cultured on glass coverslips, infected with adenoviruses expressing Id1 or Smad7, and cultured until day 7, when α-SMA staining was performed. Ectopic expression of Id1 eliminated the effect of co-expressed Smad7 and resulted in polymerization of α-SMA into fiber structures (Fig. 2C). These data imply that ectopic Id1 expression suffices to overcome inhibitory Smad7 effects on HSC transdifferentiation.

Figure 2.

Id1 expression enhances activation of HSCs and is required for α-SMA fiber formation. HSCs were infected with adenoviruses (AdLacZ, AdSmad7) as indicated at day 2 after plating (50 MOI) on glass coverslips. Representative images were taken on day 7 of culture. (A) Titration of virus and corresponding ectopic Id1 protein expression was monitored by Western analysis. Expression of ERK1/2 was determined to assure equal loading. (B) Morphological changes were monitored by phase contrast microscopy after virus infections as indicated. (C) Immunofluorescence staining for α-SMA (right). Nuclei were stained with 4,6-diamidino-2-phenylindole (left). Staining was documented by fluorescence microscopy with an LSM 510 laser scanning microscope; original magnification, 200×. HSC, hepatic stellate cell; MOI, multiplicities of infection; α-SMA, alpha smooth muscle actin.

RNA Interference-Mediated Knock-Down of Id1 Expression Inhibits α-SMA Fiber Formation.

To conversely examine the effect of depleted Id1 expression on in vitro transdifferentiation, siRNA targeting Id1 expression was designed and introduced by means of adenoviral infection. Efficacy of Id1 knock down was demonstrated by immunoblotting, showing approximately 90% down-regulated Id1 protein levels at 100 MOI (Fig. 3A). α-SMA fiber formation was strongly enhanced by Id1 overexpression and almost absent from Id1-depleted cells (Fig. 3C), whereas total expression of α-SMA was not reduced (Fig. 3B). Other typical morphological changes, such as loss of fat droplets and cell spreading, were not blocked by Id1 knock-down.

Figure 3.

Overexpression of Id1 enhances and knock-down of Id1 abrogates α-SMA fibers. HSCs were infected with adenoviruses (AdLacZ, AdId1, AdsiId1) as indicated. (A) Titration of Adsi-Id1 effectively down-regulates Id1 in Western blot analysis. β-actin was assayed as loading control. (B) Total α-SMA levels at day 7 are unchanged by Id1 knock-down or overexpression. (C) Representative immunofluorescent images from day 7 of cell culture (see Fig. 2C); original magnification 100×. For each image a cut-out is shown below at higher magnification (approximately 2×). HSC, hepatic stellate cell; α-SMA, alpha smooth muscle actin.

TGF-β Induces Expression of Id1 in HSCs Through the ALK1 But Not the ALK5 Pathway.

ALK1 was recently identified as TGF-β type I receptor in endothelial cells.23 In this cell type, TGF-β regulates epithelial cell function via balancing ALK5 and ALK1 signaling, and Id1 was found as target for the ALK1 specific response. Therefore, we examined the TGF-β type I receptor expression pattern in HSCs by real-time PCR analysis. ALK1, ALK3, and ALK5 mRNA are constantly expressed in transdifferentiating HSCs (Fig. 4A). Furthermore, expression of the two known TGF-β type III receptors, betaglycan and endoglin, was identified (Fig. 4B). Interestingly, endoglin mRNA and protein increase initially during culture activation and remain at elevated levels at least up to day 8 (Fig. 4B–C).

Figure 4.

TGF-β type I and III receptor expression in transdifferentiating HSCs. Total RNA samples were processed by real-time RT-PCR. ALK1, 3, and 5 (A) as well as betaglycan and endoglin (B) mRNA expression was investigated by LightCycler technology. [cp] = crossing points. (C) Western blot analysis of endoglin expression. TGF-β, transforming growth factor beta; HSC, hepatic stellate cells; RT-PCR, reverse transcription polymerase chain reaction.

Stimulation of primary HSCs and cirrhotic fat storing cells (CFSC), a cell line representing activated HSCs, with 5 ng/mL TGF-β1 strongly but transiently induced Id1 protein accumulation within 1 hour (Fig. 5A). TGF-β1 induced both Smad2/3 and Smad1/5/8 pathways, as shown by phosphorylation of Smad2/Smad1 (Fig. 5A). To identify the pathway leading to Id1 expression, we used ectopic expression of constitutively active receptors. caALK5 selectively induced Smad2 phosphorylation and did not increase Id1 expression, whereas caALK1 and caALK3, which is the BMP-2 receptor, both phosphorylated Smad1 and not Smad2 and induced Id1 accumulation (Fig. 5B). Exactly the same result was obtained for CFSC (Fig. 5B). To further confirm that TGF-β upregulates Id1 expression via activation of ALK1, siRNAs targeting ALK1 expression were designed, and a dose-dependent knock-down effect was proven and confirmed in ALK1 overexpressing CFSC (Fig. 6A). Accordingly, AdsiALK1 infection dose dependently decreased Smad1 phosphorylation and Id1 accumulation in TGF-β–treated CFSC (Fig. 6B). Finally, inhibition of ALK5 activity in HSCs using a chemical compound (SB431542) reduced Smad2 phosphorylation, but did not inhibit Smad1 phosphorylation, further confirming ALK1 as the responsible receptor (Fig. 5C). Interestingly, blocking endogenous ALK5 activity increases basal phospho-Smad1, possibly by enhancing autocrine ALK1 activity.

Figure 5.

(A) TGF-β induces Id1 expression and Smad1/2 phosphorylation in HSCs. At day 4, after 16 hours' serum starvation (0.5% FCS), HSCs were stimulated with 5 ng/mL TGF-β1. Cells were lysed after 1, 2, and 6 hours, and analyzed for Id1, phospho-Smad1/2, total Smad1/2, and β-actin by Western blot. (B) Id1 induction is mediated by ALK1 or ALK3 but not ALK5. Cell lysates from HSCs (4d) and CFSC expressing constitutively active ALK1, 3, or 5 after adenoviral infection were analyzed by Western blot for Id1, phospho-Smad1/2, and total Smad1/2. (C) In HSCs, phosphorylation of Smads1 and 2 was investigated by Western blot after stimulation with TGF-β (5 ng/mL, 1 hour) in the presence or absence of 5 μmol/L SB431542 and after addition of the inhibitor alone. TGF-β, transforming growth factor beta; HSC, hepatic stellate cell.

Figure 6.

siRNA knock-down of ALK1 expression reduces Smad1 phosphorylation and Id1 expression (A) To demonstrate efficient siRNA knock-down for ALK1, CFSC were co-infected with AdcaALK1 (containing an HA-tag) and AdsiALK1. By Western blotting using an antibody against HA, a dose-dependent knock-down of ALK1 is shown. (B) TGF-β–stimulated CFSC (5 ng/mL, 1 hour) were pretreated with increasing amounts of AdsiALK1. Phospho-Smad1, total Smad1, Id1, and β-actin were analyzed by Western blot. siRNA, small interfering RNA.

The presented data indicate that in HSCs, TGF-β signaling via ALK5 activates the Smad2/3 pathway, whereas signaling via ALK1 leads to phosphorylation of Smad1. Furthermore, TGF-β/ALK1/Smad1 but not TGF-β/ALK5/Smad2/3 pathways stimulate Id1 expression in HSCs.

To investigate TGF-β–dependent induction of Id1 at the transcriptional level, HSCs were infected with AdBRE-Luc, containing a cassette of BMP-response elements from the mouse Id1 promoter, and CFSC were transiently transfected with a mouse Id1-promoter luciferase reporter construct (−1231/+88),19 followed by stimulation with TGF-β1 or BMP-2. Comparable results were obtained in both experimental setups, showing induction of luciferase activity with TGF-β1 and BMP-2 (Fig. 7).

Figure 7.

TGF-β and BMP-2 activate an Id1-promoter BMP response element in HSCs and CFSC. HSCs were infected with an adenovirus carrying a cassette of BMP-response elements from the mouse Id1 promoter, whereas CFSC were transiently transfected with an Id1-promoter reporter plasmid. Cells were then stimulated with 1 ng/mL TGF-β1 or 10 ng/mL BMP-2 for 14 hours, and luciferase activity was measured. Each assay was performed in quadruplicates and repeated in three independent experiments. Mean value of one representative experiment ± SD is shown. TGF-β, transforming growth factor beta; BMP, bone morphogenetic proteins.

Smad1 Phosphorylation and Id1 Expression Increase With Severity of Bile Duct Ligation–Induced Liver Damage.

To assess a profibrogenic role for Id1 in vivo, liver tissue samples from bile duct–ligated (BDL) rats were immunostained against Id1 (Fig. 8). Specimen obtained 1 week after BDL displayed no signal for Id1 (data not shown), whereas, 2 to 5 weeks after BDL, the amount of positively stained nuclei was increased in fibrotic areas. In parallel with Id1 expression, nuclear staining for phospho-Smad1 occurred (Fig. 8). Staining of tissue samples from control animals indicated no detectable Id1 expression. These data support the hypothesis that Id1 induction via a Smad1-dependent pathway might play a role in liver fibrogenesis.

Figure 8.

Smad1 phosphorylation and Id1 expression increase with severity of damage in bile duct–ligated rat liver. (A–B) Liver sections from BDL rats were collected at different time points and stained with Sirius red or immunostained for Id1 and phospho-Smad1. Representative histologies are shown in 40× (A) and 400× (B) magnifications. (C) Quantification of phospho-Smad1- or Id1-positive cells was performed by counting 10 randomly selected fields from four different animals. Numbers indicate percentage of stained cells per field. BDL, bile duct ligated.

Discussion

Transdifferentiation of HSCs with loss of retinoid droplets, increased matrix synthesis, and upregulated α-SMA expression is the driving cellular event during liver fibrogenesis,26 and a cohort of in vitro and in vivo data has established TGF-β as key-mediator of pro-fibrogenic effects.2 In the current report, Id1 is identified as a target gene of TGF-β signaling in HSCs. Id1 exhibits functional properties that make it a central mediator of transdifferentiation of this cell type. Furthermore, underlying signaling events are elucidated, namely that TGF-β signaling through ALK1/Smad1, but not ALK5/Smad2/3, triggers Id1 expression in HSCs (summarized in supplementary figure 1 on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).).

Id proteins are important modulators of signaling pathways involved in development, cell cycle progression, and tumorigenesis.11, 27 They act as dominant negative antagonists of the basic helix-loop–helix (bHLH) family of transcription factors, which regulate differentiation of many cell types. Id proteins, among others, associate with Ets transcription factors and the Rb family of tumor suppressor proteins and are downstream targets of TGF-β and BMP signaling.12 Target genes for Id proteins have been identified largely based on the knowledge of promoters activated by bHLH-proteins. In embryo-fibroblasts from wild-type and Id1 knockout mice, a differential gene expression profiling system displayed targets involved in diverse biological functions such as matrix remodeling, intracellular signaling pathways, and angiogenesis.12 In a TGF-β–dependent global expression profiling with fibroblasts, Id1 was found as an early response gene, followed by up-regulation of smooth muscle cell marker genes such as α- and γ-smooth muscle actin, basic calponin, smooth muscle myosin heavy chain, and transgelin.28 In promoter regions of a number of smooth-muscle–specific genes, E-box sequences were found, which represent binding sites for bHLH transcription factors. Thus, our data indicate that upregulation of genes favoring a myofibroblast phenotype represents profibrogenic TGF-β effects in HSCs that are provided by initiation of a cascade involving ALK1 and Smad1 activation and subsequent induction of Id1 expression.

Nevertheless, the function of Id1 in HSCs is poorly understood. We initially identified Id1 as TGF-β target gene in a Smad7-dependent expression array of HSCs. We have previously demonstrated that Smad7 inhibits activation of HSCs and fibrogenesis in vivo.7 Mesenchymal cells have the propensity to transdifferentiate into α-SMA–positive contractile MFBs during tissue repair and fibrogenesis in various organ systems. These cells are highly activated and synthesize ECM proteins and fibrogenic cytokines. MFBs are the predominant fibroblast phenotype present within fibrotic lesions in a number of organs such as liver, lung, kidney, and heart. The cellular conversion of mesenchymal cells to MFBs during development, injury, and disease is mediated by cytokines, with TGF-β being crucial. Nevertheless, molecular details underlying the profibrogenic TGF-β action in injured liver are not fully delineated. Our finding that Id1 overexpression enhances transdifferentiation of HSCs by means of morphological changes and polymerization of α-SMA, together with the observation that knock-down of Id1 expression is blunting activation of HSCs as measured by α-SMA fiber formation, suggests a central role for Id1 in this process. In most cell types, TGF-β binds and activates TβRII/ALK5 receptor complexes, which subsequently leads to phosphorylation of Smad2/3, whereas BMPs have been described to signal via Smad1/5.29 In endothelial cells (ECs), TGF-β signaling is mediated via these two different type I receptors with opposing effects. Activation of ALK5/Smad2/3 leads to inhibition of migration and proliferation, whereas ALK1/Smad1/5 signaling has the converse effects. From these findings, it has been concluded that the activation state of ECs depends on the balance between both pathways.23 Interestingly, although ALK1 directly antagonizes ALK5/Smad signaling in ECs, ALK5 kinase activity is necessary for optimal TGF-β/ALK1 signal transduction.22 Furthermore, TGF-β/ALK1 signaling in ECs leads to Id1 gene expression.22

We recently observed that quiescent HSCs display Smad2/3 activation, SBE-binding activity and stimulation of luciferase activity that is driven by a (CAGA)9-major late promoter–luciferase reporter gene after TGF-β stimulation,5, 30, 31 indicating ALK5 activity. Furthermore, quiescent cells do not migrate and are sensitive to TGF-β–dependent inhibition of DNA synthesis. Inhibition of ALK5/Smad2/3 activation is blunting up-regulation of ECM components, whereas it has no effect on α-SMA expression.31 We show now that inhibition of ALK5 does not prevent Smad1 phosphorylation. We conclude that phosphorylation of Smad1 (with induction of Id1) seems to be indispensable for polymerization of α-SMA. If Smads-5 or -8 are similarly relevant or if these three Smads are even redundant remains to be investigated.

In transdifferentiated HSCs, the TGF-β signaling pathway is modulated, and TGF-β has no longer an inhibitory effect on proliferation. Additionally, activated HSCs lack ligand-dependent Smad2/3 phosphorylation or induction of Smad7 gene expression. Furthermore, transdifferentiated HSCs display significant migration and proliferation activities. We hypothesize that in HSCs, a differential usage of TGF-β receptors ALK5 and ALK1 similar to that in ECs exists. This assumption is supported by our findings that (i) ALK1 and ALK5 are expressed in HSCs and (ii) ALK1/Smad1 signaling but not ALK5/Smad2/3 signaling induces Id1 expression in HSCs. Increased expression and phosphorylation of Smad1 during activation of HSCs has been reported previously,32 pointing to a significant role of Smad1/5/8 signaling during transdifferentiation of HSCs. The existence of two TGF-β/Smad pathways in HSCs, via ALK1 and ALK5, enables a careful fine-tuning of TGF-β–induced biological responses. The mechanism controlling signaling activity of ALK5 versus ALK1 in HSCs is not clear and may depend on concentrations of active TGF-β and the amounts of the different receptors present on the cell surface within a given situation. This may trigger the cellular response in opposite directions, as was proposed for ECs.22, 23, 33 Thus, the counteractive interplay between ALK5 and ALK1 provides a more general, intricately regulated TGF-β–controlled switch, which subsequently may change the cellular fate and therefore may be an important prerequisite for HSC activation.

The accessory receptor endoglin also may be involved in TGF-β signaling. Endoglin expression was found to counteract the inhibitory effect of TGF-β on EC migration.34 Ectopic expression of endoglin modulates TGF-β signaling by favoring usage of ALK1.35 Furthermore, ECs lacking endoglin are growth-arrested because TGF-β/ALK1 signaling is reduced and the TGF-β/ALK5 pathway is enhanced, indicating a pivotal role for endoglin in the balance of ALK1 and ALK5 signal transduction.36 In line with this, increased expression levels of endoglin were observed in transdifferentiating HSCs, pointing to a yet unidentified pathway by which endoglin may mediate profibrogenic responses in the liver. This assumption is supported by data showing endoglin exposed at the plasma membrane of activated HSCs.37

Furthermore, preliminary data suggest that, in addition, BMP-2 signaling via ALK3 represents an active pathway in HSCs, which contributes to Smad1 activation and Id1 expression. Whether BMP-2 and TGF-β enhance each other in this respect or whether cross-regulatory mechanisms exist remains to be determined.

In contrast to our findings and the results from ECs, Id proteins can also antagonize a mesenchymal phenotype. Thus, TGF-β–induced transdifferentiation of NMuMG mammary epithelial cells to a fibroblastic phenotype is mediated via E2A proteins. In this cell type, TGF-β decreases Id1 expression, thereby increasing the amount of functional E2A and antagonizing transdifferentiation.24 Another complex regulatory mechanism was described for Id2 and Id3 in the same cell type. Like Id1, both Id proteins display long-term repression by TGF-β and sustained induction by BMP, whereupon a converse regulation of Id genes by different TGF-β family members is critical for proliferative and differentiation responses.25 We conclude that the outcome of Id protein expression in a specific cell type is determined by the function of the diverse interacting bHLH proteins.

In summary, our data identify Id1 as an important regulator of the transdifferentiation process in HSCs downstream of Smad7 and therefore overriding its inhibitory effect on TGF-β or BMP-2 signaling. Experimental evidence links Id1 to fibrogenesis, which may have implications for chronic liver disease. Furthermore, instead of therapeutically targeting TGF-β by Smad7, for example, a more specific intervention may be feasible to revert profibrogenic events, for example, by blocking Id-protein expression.

Acknowledgements

The authors thank T. Taga, Kumamoto University, Japan, for providing the Id1 expressing adenovirus and A. Lux (Fachhochschule, Mannheim, Germany) for providing an endoglin specific antibody; A. Müller, S. Sauer-Lehnen, and U. Haas for excellent technical assistance. E. Wiercinska was a fellow of the Heinz Breuer-Stiftung der Deutschen Gesellschaft für Klinische Chemie und Laboratoriumsmedizin.

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