TGF-β and the Smad Pathwayin Liver Fibrogenesis

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TGF- β and the Smad Pathway in Liver Fibrogenesis

Axel M. Gressner, Steven Dooley, Ralf Weiskirchen

12

12.1 The Pluripotency of TGF- β in Liver Pathobiology

Transforming growth factors- β (TGF-βs) are multi- functional polypeptide growth factors, which gov- ern fundamental cellular functions, tissue homeos- tasis, tumor suppression, development and immune responses [17]. They are produced by the majority of nucleated cells. In liver, each non-parenchymal cell type (hepatic stellate cell [HSC], Kupffer cell, sinusoidal endothelial cell) is capable of producing TGF- β with different expression profiles regarding the three isoforms, β1, β2 and β3, which are about 80% homologous to each other but reside on differ- ent chromosomes [10]. Although clearly detectable in hepatocytes, the parenchymal liver cells are un- likely to synthesize and secrete substantial amounts of this cytokine [77, 80]. It is suggested that an up- take mechanism might exist on hepatocytes for cir- culating TGF- β.

12.1.1

Synthesis and Processing of TGF-

β

Biologically active TGF- β is a disulfide-linked homodimeric peptide with a molecular mass of 25 kDa, capable of sequential binding to three TGF- β receptors (TβRs) [23, 55]. Before doing so, TGF-β undergoes extensive post-translational modifica- tions, which start intracellularly by furin-catalyzed endoproteolytic cleavage of the proform (100 kDa) between two arginine residues at positions 278 and 279 into a large N-terminal fraction, designated la- tency-associated peptide (75 kDa, LAP), and a small C-terminal fragment, i.e., the mature, 25-kDa TGF- β homodimer (Fig. 12.1). Both fractions remain non-covalently attached to each other and before se- cretion, LAP is linked by disulfide bridges with one of the four isoforms of the large (molecular mass 125–160 kDa) latent TGF- β-binding protein (LTBP), which shows a 30% amino acid sequence homology

to the matrix proteins fibrillin-1 and -2 [57, 58, 60].

Rat and human liver cells secrete a large latent TGF-

β complex with a molecular mass >225 kDa [14],

which is supposed to be initially fixed to components

of the extracellular matrix (ECM, e.g., fibronectin)

via LTBP in a transglutaminase-dependent reaction

[37, 69]. Thus, connective tissue serves as a reservoir

for (latent) TGF- β, which can be released by sequen-

tial proteolytic degradation of LTBP, involving the

plasminogen/plasmin/plasminogen activator sys-

tem [50, 74, 82]. Subsequent diffusion of the small

remnant LAP-TGF- β aggregate to the surface of the

target cell enables the binding of this complex by

mannose-6-phosphate (M6P) residues of LAP to the

cation-independent M6P-IGF-II receptor [36], where

the final activation of latent TGF- β by the action of

cell surface-associated and microenvironmental

proteases (e.g., calpains, cathepsin G, matrix-metal-

loproteinases-2 and -9, mast cell chymase, plasmin)

takes place. However, it is likely that the important

regulatory step of extracellular TGF- β activation

from the large latent TGF- β complex is much more

complicated and involves several, complementary

or even competitive pathways, since thrombospon-

din-1 [19, 64, 85], specific integrins ( α

v

β6) [63], and

reactive oxygen species (ROS) [5] have been impli-

cated. Once generated, active TGF- β can be antago-

nized by a number of binding molecules, which are

synthesized by hepatocytes (e.g., α

2

-macroglobulin)

and HSC (e.g., proteoglycans such as biglycan, deco-

rin) [11, 18]. Since the expression of proteoglycans is

stimulated by TGF- β, a negative feedback loop might

operate. Proteolytic shedding of the type III TGF- β

receptor (betaglycan) from the cell surface and of

endoglin (CD 105), a high affinity TGF- β-binding

site in vascular endothelial cell, might be additional

pathways to inactivate the ligand by preventing its

binding to the signal-generating serine-threonine-

kinase receptors T βRI and TβRII. Thus, there are

multiple pathways that can control TGF- β bioactiv-

ity in the tissue and it is clear from numerous exper-

imental studies that post-translational regulation

is likely to be more important than transcriptional

control of this important cytokine.

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12.1.2

Pathobiology of TGF-

β in Liver

The pathobiological relevance of TGF- β for the liver is due to an array of effects on hepatocellular prolif- eration and liver regeneration, on induction of pa- renchymal cell apoptosis, on immune surveillance, and finally on hepatic fibrogenesis (Fig. 12.2).

Together with activin A, another member of the TGF- β superfamily, TGF-β is a strong inhibitor of liver cell proliferation and, thus, a negative regulator of liver regeneration and liver cell mass. It may serve as the terminator (stop signal) of the replicative re-

sponse of liver tissue to partial hepatectomy. TGF- β and activin are proposed to be major determinants of the homeostasis of normal liver cell mass.

The cytokine was also recognized as an impor- tant inducer of rapid apoptosis of hepatocytes [42, 70], which might be initiated and mediated by dif- ferent signals linked to suppression of phosphor- ylation of the retinoblastoma gene product pRb, to the induction of p53 and/or bax, to the increase of ROS accompanied by reduction of intracellular glu- tathione, and induction of cytosolic transglutami- nase [86]. We could show that TGF- β secreted by transdifferentiated HSC (myofibroblasts, MFB) can induce hepatocellular apoptosis after activation in a

Fig. 12.1. Schematic pres- entation of TGF-β processing inside and outside the cell.

LAP latency-associated pep- tide, LTBP latent TGF-β-binding protein

Fig. 12.2. Synopsis of the pathobiochemical effects of TGF-β on liver

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paracrine manner [41]. Dexamethasone, phenobar- bital, bacterial lipopolysaccharide, peroxisome pro- liferators (nafenopin) and epidermal growth factor (EGF) are among other molecules that can inhibit TGF- β-initiated apoptosis of hepatocytes. It is likely that apoptosis of hepatocytes observed in various liver diseases, including viral hepatitis, primary bil- iary cirrhosis and alcoholic liver diseases is at least partially mediated by the overexpression of TGF- β under these pathobiochemical conditions.

TGF- β has complex effects on T-lymphocytes, because it acts not only directly on this cell type, but also indirectly by regulating the function of antigen-presenting cells [44]. It is an important im- munosuppressant, but recently TGF- β was recog- nized also as an anti-apoptotic survival factor for T-lymphocytes [17]. The net result of TGF- β effects on T-lymphocytes depends on their status of differ- entiation and on the cytokine milieu. With respect to liver diseases, TGF- β is potentially involved in the mechanism of autoimmune liver diseases [7], which, after consolidation of these data, would provide a novel therapeutic strategy focused on this cytokine [44, 45].

TGF- β plays an outstanding role in the pathogen- esis of liver fibrosis (fibrogenesis) due to its multiple effects on HSC activation and transdifferentiation, stimulation of matrix protein expression (collagens, proteoglycans, structural glycoproteins), enhance- ment of hyaluronan synthesis, depression of matrix metalloproteinases, increase of tissue inhibitors of metalloproteinases (TIMPs), and autoinduction of the cytokine [10, 13, 43]. Hepatic stellate cells are not only an important source but also the predomi-

nant target cell of TGF- β, which is recognized in numerous experiments as a profibrogenic master cytokine [43]. Consequently, eradication or antago- nism of TGF- β is a rational antifibrotic therapeutic approach, which proved successful under various experimental conditions [30, 40, 96, 103]. Due to a more detailed understanding of the intracellular Smad-dependent signaling pathways of TGF- β and their crosstalk with other signal cascades, an inhibi- tory strategy of this cytokine is no longer limited to ligand inactivation, but can also use intracellular mediators of the TGF- β signaling pathway, which will be discussed in the following sections.

12.2 TGF- β Signal Transduction in Liver Cells

12.2.1

The TGF-

β Signaling Network

Binding of TGF- β family members to TGF-β type II receptors triggers heteromerization with and transphosphorylation of type I receptors (Fig. 12.3).

The signal is propagated through phosphorylation of receptor-associated Smads (Smad2 and 3 for TGF- β and activin, Smad1, Smad5 and Smad8 for bone morphogenic protein [BMP]; R-Smads), which oligomerize with the common mediator Smad4 (co- Smad). Upon type I receptor activation, phosphor- ylated R-Smads interact with Smad4 and translocate into the nucleus, where they affect transcription of

Fig. 12.3. Multiple level con- trol of Smad signaling. Smad7 inhibits R-Smad activation.

Cytoplasmic crosstalk induces context-dependent transcriptional responses. Ubiquitous factors provide uniform responses in different cell types. Cell-specific factors dictate differential cell-type- dependent responses for the same Smads. TGIF, Ski, and SnoN are transcriptional co- repressors

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target genes via direct DNA binding or by associa- tion with numerous DNA-binding proteins [72].

Other signaling pathways communicate with Smad-mediated signals, thereby transforming the initially simple core TGF- β/Smad signaling pathway into a much more complex view of cellular regula- tion by TGF- β, which now reflects the multiplicity of potential TGF- β actions. Positive regulators of TGF- β signals include, for example, upstream accessory proteins, such as Smad anchor for receptor activa- tion (SARA), which is involved in Smad2 phospho- rylation and formation of transcriptionally active Smad2–Smad4 heterodimers [95].

A cohort of nuclear Smad-binding proteins was identified, acting as positive or negative regulators of transcription. In general, TGF- β mediates its physiological function via activation of target gene transcription and negatively acting Smad partners are required to prevent the inappropriate activation of TGF- β signaling, or to turn off the pathway fol- lowing normal activation [59].

The first repressors identified were a class of Smad proteins named inhibitory Smads (Smad6, 7) [66, 68, 94]. Smad6 and Smad7 provide their inhibi- tory effects at the cell surface receptors. Addition- ally, nuclear acting repressors were identified, e.g., Ski and SnoN, that snatch away activated complexes containing Smad2, 3, and 4, which results in tran- scriptional repression of Smad-responsive promot- ers [1, 56, 89, 91, 92].

12.2.2

Crosstalk Between TGF-

β Signaling

and Other Pathways in Liver

The analysis of a crosstalk among different signal- ing pathways to specify, enhance, or inhibit TGF- β responses is very important to identify relevant TGF- β effects in a specific cellular context. Signal- ing by interferon- γ (IFN-γ), for example, inhibits TGF- β signaling by direct signal transducers and activators of transcription (STAT)-mediated tran- scriptional induction of Smad7 [99]. In liver, the po- tential of IFN- γ to counteract activation of HSC has been shown [6, 75]. Due to the profibrogenic role of TGF- β, a direct link to the TGF-β pathway was obvi- ous in the liver, and we could identify participation of the above-mentioned pathway in clinically rel- evant beneficial effects of IFN- γ in liver fibrosis .

Recently, mitogen-activated protein kinase (MAPK) and PI3P kinase pathways were identified as direct and/or indirect mediators of TGF- β signal- ing in general [28, 52, 101, 107] and especially in the liver [93, 104].

Involvement of ROS and lipid peroxidation has been demonstrated in activation of HSC and con- tributes to both onset and progression of fibro- sis as induced by alcohol, viruses, iron, or copper overload, cholestasis, and hepatic blood congestion [76]. Expression and synthesis of TGF- β is modu- lated through redox-sensitive reactions, thereby providing a direct connection between TGF- β and ROS. Thus, infection of HSC with an adenovirus encoding a soluble TGF- β type II receptor attenu- ated NADH oxidative activity, ROS generation and lipid peroxidation and prevented HSC activation, at least partially by inhibiting oxidative stress [20]. It is currently not known, if this link between TGF- β signaling and oxidative stress in HSC involves Smad signaling.

12.2.3

The Role of Smad Activation in HSC during Fibrogenesis

In the normal liver, HSC, which comprise about 5%

of the total number of resident liver cells, are the major storage site for retinoids. Following liver in- jury of any etiology, HSC undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile MFB [9, 10, 15, 29, 31, 32, 38] and TGF- β is a very potent profibrogenic mediator of cellular responses leading to tissue repair, ECM production, growth regulation, and apoptosis [10]. During fibrogenesis, tissue and blood levels of active TGF- β are elevated and overexpression of TGF- β1 in transgenic mice can induce fibrosis. Additionally, experimental fi- brosis can be inhibited with strategies that abrogate TGF- β signaling. These findings, along with the po- tency of TGF- β to upregulate ECM expression and the presence of functional T βR on the surface of HSC, has led to a widely accepted model, in which persistent autocrine stimulation of activated HSC/

MFB by TGF- β is a key mechanism in liver fibro-

genesis [39] (Fig. 12.4). Based on the identification

of downstream events of TGF- β signal transduction

during the past few years, molecular mechanisms

underlying the profibrogenic effects of TGF- β sig-

nal transduction are the subject of intense investi-

gations. Many of these studies were performed with

primary cultured HSC, which were spontaneously

activated by contact to the plastic surface of the cul-

ture well. In this in vitro model of fibrogenesis, HSC

are responsive to TGF- β-dependent Smad2/3 phos-

phorylation during the initial stages of activation,

whereas fully transdifferentiated MFB are insensi-

tive to treatment with TGF- β1 [25]. Thereby, HSC

transduce TGF- β1-dependent signals, which result

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in growth inhibition of the cells and transcription of TGF- β target genes, like Col I and Smad7 (Fig. 12.4).

MFB instead are neither growth inhibited nor do they display activation of TGF- β-dependent tran- scription. T βR-I and TβR-II, as well as Smad2 and Smad4, are expressed in similar amounts in HSC and MFB. TGF- β-dependent stimulation of Smad7 expression was found specifically in HSC and in- creased expression of Smad3 was detected in MFB.

Furthermore, TGF- β1-dependent phosphorylation of Smad2/3, subsequent enhanced nuclear localiza- tion of activated Smad complexes, as well as bind- ing and activation of a strongly responsive TGF- β response element (TRE) were found to be limited to HSC [26]. Ectopic expression of a constitutively active T βR-I in MFB was able to overcome TGF-β insensitivity and to restore the signaling pathway, leading to reactivation of the TRE-driven reporter construct [26]. This indicates that the principal machinery, necessary to transmit TGF- β signals, is functional in MFB. Furthermore, the results point to the availability of T βR at the surface of the cells as a cause for TGF- β insensitivity, a model that was confirmed by the finding that ligand binding to the cell surface receptors is diminished in MFB. Possi- ble reasons for this have also been published, e.g., reduced expression of T βR-II [81] and/or cell sur- face availability of expressed receptors [25].

In the liver, injury by a variety of means results in a rapid induction of TGF- β synthesis, predominant- ly in HSC (Fig. 12.4), consistent with a ubiquitous role for TGF- β in wound healing. Concomitant with increased TGF- β production, HSC increase produc- tion of collagen, and it is suggested that unbalanced TGF- β activity during wound repair could lead to damaging fibrotic responses and scarring. Howev- er, because TGF- β suppresses the cellular immune response, it is considered a potent anti-inflamma- tory cytokine and may also be anti-fibrogenic under some circumstances. Therefore, in addition to al-

tered cytokine production, changes in the multiplic- ity of components that control the TGF- β signaling pathway may underlie the onset of the pathological condition.

Several reports suggest a prominent role of Smad3 in wound repair. In a model of cutaneous wound healing, Smad3-deficient mice have a re- duced number of monocytes and neutrophils and the amount of TGF- β at the site of injury was dimin- ished, leading to increased keratinocyte prolifera- tion. In contrast, lack of Smad3 does not influence ECM production, which finally results in increased wound healing [4]. In vitro transdifferentiated MFB display increased Smad3 expression in comparison to HSC. Additionally, studies with wild-type and Smad3 heterozygous or Smad3 homozygous knock- out mice reveal that maximum expression of colla- gen type I in activated HSC in vivo and in culture requires Smad3 [83]; the data further indicate that Smad3 is required for TGF- β-dependent growth in- hibition and TGF- β1-mediated formation of Smad- containing DNA-binding complexes in cultured HSC. Interestingly, there is no influence of Smad3 on HSC activation as assessed by α-smooth muscle actin ( α-SMA) expression. A potential profibrogen- ic role of Smad3 is further confirmed by the find- ing that activated HSC lines display a significant amount of constitutively activated Smad3 [8, 47].

This was further delineated in a recent paper from Furukawa et al. [33], who describe a p38-de- pendent phosphorylation of Smad3 in the linker region, which was determined as induced by TGF- β and platelet-derived growth factor (PDGF)-depend- ent signaling, especially in transdifferentiated MFB.

The authors further suggest from their molecular data that phospho-Smad3/Smad4 complexes trans- locate into the nucleus and display DNA-binding activity, however display no transcriptional activity.

Then, after TGF- β type I receptor-dependent activa- tion, Smad2 becomes phosphorylated, participates

Fig. 12.4. TGF-β in HSC transdifferentiation and fibrogenesis. TGF-β induces transdifferentiation of quiescent HSC. Activated HSC produce TGF-β, which acts on surrounding quiescent HSC and hepatocytes. A continu- ous autocrine signal maintains the transdifferentiated phenotype. TGF-β-dependent apoptosis of hepatocytes leads to progression of fibrogenesis

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in the preformed DNA-binding Smad3/Smad4 com- plex and subsequently, target gene transcription becomes activated. This hypothesis, in our view, is quite hypothetic and needs comprehensive further analyses.

TGF- β/Smad signaling was also investigated further by Liu et al. [54], who likewise identified significant changes during transdifferentiation of HSC. Whereas quiescent cells respond nicely to ex- ogenous TGF- β with phosphorylation of Smad2, in transdifferentiated cells Smad3 was identified as the primary mediator of TGF- β signaling. The identified reason for this was a transdifferentiation-depend- ent downregulation of SARA expression required for TGF- β receptor-dependent Smad2 activation.

In contrast to the above-reported finding, Liu et al.

identified a constitutively activated Smad2 instead of Smad3 in transdifferentiated cells.

Furthermore, Shen and co-workers showed transdifferentiation-dependent upregulation of Smad1 expression in HSC, which was reduced by TGF- β signaling, indicating that BMP-initiated pathways may also play a role in fibrogenic proc- esses [87].

Altogether, TGF- β signaling in quiescent HSC and transdifferentiated MFB is different. The chang- es are quite complex and its detailed molecular pathway leading to activation of HSC is still unclear, as are the target genes that are involved. Further delineation of TGF- β signaling will provide further information to dissect this important profibrogenic pathway step by step.

Beside this complexity of alternative possibili- ties to submit TGF- β signals from the cell mem- brane to the nucleus, quantitative differences may also be critical to direct TGF- β effects to adequate target genes that need to be selected from the mul- tiplicity of possible events. Therefore, a completely different result may occur when we compare basal autocrine stimulation of cells with data obtained from treatment with relatively high amounts of the cytokine (e.g., 5 ng/ml, as is mostly used in the sci- entific community). In line with this, the group of D.C. Rockey has recently published data showing that lower concentrations of a soluble TGF- β type II receptor displayed a more significant inhibition of hepatic fibrosis in mice than higher amounts [103].

Furthermore, in human mammary tumor cells, TGF- β-dependent motility was achieved at very low concentrations of a constitutively activated Alk5, resulting in phosphorylation of PI3 kinase-depend- ent Akt1, but not Smad2, which was achieved only at amounts of Alk5 [28].

12.2.4

TGF-

β Signaling in Hepatocytes

One important point is TGF- β signaling in hepato- cytes. This cell type is highly sensitive for treatment with exogenous TGF- β (Fig. 12.4) and signaling re- sults in functions that regulate the liver mass. TGF- β-dependent inhibition of hepatocyte proliferation is mediated in part by negative regulation of extra- cellular regulated kinase (ERK) 2 and p70 S6 kinase activity [24]. Further, TGF- β1 decreases cyclin A and induces p21 mRNA in primary cultured hepa- tocytes, leading to growth inhibition [90]. Beside its growth inhibitory role, TGF- β induces apoptosis in hepatocytes, thereby representing two important regulatory mechanisms regarding homeostasis of the liver mass during regenerative processes. Exces- sive amounts of TGF- β, as a result of chronic dis- ease, however, lead to a critical disturbance of this homeostasis and nemesis of hepatocytes, thereby promoting outgrowth of MFB and ECM deposition and fibrogenesis. TGF- β-dependent apoptosis of hepatocytes is mediated by p38 MAPK via an indi- rect mechanism. The stress- and cytokine-inducible GADD45 family proteins function as specific acti- vators of MTK1 (also known as MEKK4), a MAP- KKK upstream in the p38 pathway. TGF- β induces expression of GADD45 β Smad-dependently, which then activates the p38 pathway that triggers apopto- sis in hepatocytes [93, 104].

Finally, another important point is the interplay between TGF- β signaling pathways and the expres- sion of virus-encoded proteins or proteins that are induced after infection with hepatitis B or C virus.

In regard to this, it was recently shown that the hepatitis B virus (HBV)-encoded oncoprotein pX enhances transcriptional activity of TGF- β family ligands by stabilizing Smad4-dependent complexes with components of the basic transcriptional ma- chinery, such as TFIIB [53]. These data were, how- ever, obtained with HBV-infected NIH3T3 cells and do not necessarily represent the situation in infected liver.

12.3 Antifibrotic Therapeutic Strategies

Given the prominent role for TGF- β1 in fibrogen-

esis, a number of therapeutic strategies for block-

ing TGF- β function have been advanced, with par-

ticular emphasis on hepatic fibrosis. Simplistically,

based on their modality of action, these approaches

can be grouped into: (i) strategies targeting enzymes

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involved in the proteolytic activation process; (ii) proteins inhibiting receptor binding of TGF- β such as scavengers or antibodies directed against TGF- β;

(iii) inhibitors of TGF- β synthesis and natural an- tagonists of TGF- β downstream signaling; and (iv) pathways that adversely affect the pleiotropic effects of TGF- β (Fig. 12.5).

12.3.1

Strategies Targeting Enzymes Involved in TGF-

β Activation

Transforming growth factor- β is secreted as a latent complex that must be activated before it can bind to its respective receptors. Thus, mechanisms coun- teracting this activation process provide an option for inhibiting the action of TGF- β [21]. In a recent study, Kondou and co-workers demonstrated that a short blocking peptide targeting thrombospondin-1 (TSP-1), a protease necessary for converting TGF- β from the latent precursor to the biologically active form, was able to prevent hepatic damage and fi- brosis in dimethylnitrosamine (DMN)-treated rats

[51]. In turn, the serine protease inhibitor camostat mesilate (also referred to as FOY 305 or CMM) sup- presses the generation of biologically active TGF- β by inhibiting hepatic plasmin activity, thereby pre- venting HSC activation and hepatic fibrosis induced by porcine serum in rats [71].

12.3.2

Strategies Inhibiting Receptor Binding of TGF-

β In recent years, potential gene therapies using solu- ble or truncated T βR-II, targeting circulating TGF-β have been under close investigation. These artificial receptors (herein termed sT βR-II) are carboxy-ter- minal truncated T βR-II or are chimeric proteins, composed of the extracellular domain of T βR-II linked to the Fc portion of an IgG immunoglobulin.

Both are able to form disulfide-bridged dimers exert- ing their effects by competing with the cell-surface receptors for binding TGF- β and recruiting TβR-I.

Consequently, these antagonists prevent phosphor- ylation of T βR-I, thereby blocking signal propagation [35]. The efficacy of soluble receptors was shown in

Fig. 12.5. Therapeutic antagonism of TGF-β-mediated liver fibrosis. In injured liver tissue, stimuli that activate latent TGF-β include thrombospondin-1 (TSP-1), plasmin, and reactive oxygen species (ROS). Once activated, TGF-β binds to TβR-II triggering heterodimerization with and transphosphorylation of TβRI. The signal is then propagated through phosphorylation of receptor-associated Smads2/3 and oligomerization with the common

mediator Smad4. Complexes of Smad proteins translocate into the nucleus, where they affect transcription of target genes via direct DNA binding or by association with numerous DNA-binding proteins, resulting in increased matrix protein synthesis (fibrogenesis), cellular activation, and induction of apoptosis. In addition, TGF-β acts via autocrine and paracrine feedback loops to increase TGF-β production

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many experimental models of hepatic fibrogenesis.

These strategies were sufficient to prevent ongoing hepatic fibrosis induced by administration of DMN or by ligature of the common bile duct in rats [35, 73, 97]. Similar results were observed when animals with established fibrosis received the dominant negative receptor [65]. For potential clinical trials, it should be noted that the effect on fibrosis seemed to be greatest at lower concentrations of sT βR-II [103].

In addition, in transgenic mice, overexpression of a dominant negative T βR-II was sufficient to acceler- ate chemically induced hepatocarcinogenesis [48], showing that beyond its involvement in hepatic fi- brogenesis, TGF- β has tumor-suppressor activities in liver. In a subsequent study, Cui and colleagues found that the infection of culture-activated HSC with an adenovirus expressing a sT βR-II resulted in significant suppression of TGF- β1 synthesis, in- hibition of cell spreading, reduced expression of α- smooth muscle actin, decrease of intracellular ROS (i.e., H

₂

O

₂

) and intracellular lipid peroxidation, re- duction of NADH consumption and oxidase activ- ity, thereby demonstrating that sT βR-II blocks the autocrine TGF- β signaling loop [20].

Moreover, proteins such as α2-macroglobulin, decorin and LAP sharing affinity for TGF- β were able to reduce the paracrine and autocrine stimula- tion of HSC in culture [50, 78, 79, 84]. Molecularly, these proteins are believed to bind TGF- β, thereby reducing the overall free concentration of active TGF- β. Comparably, systemic application of anti- TGF- β antiserum and decorin, another TGF-β- binding protein, was able to suppress accumulation of ECM and histological manifestation of prolifera- tive glomerulonephritis [11, 12].

12.3.3

Inhibitors of TGF-

β Synthesis

and Downstream Signaling

Another option to inhibit TGF- β function is to in- terfere with postreceptor signaling. Transient over- expression of Smad7, a natural inhibitory regulator of TGF- β signal transduction, was shown to prevent bleomycin-induced pulmonary fibrosis in mice [67]

and to arrest transdifferentiation of primary-cul- tured HSC and to accelerate liver fibrosis in rats [27].

Interestingly, HSC overexpressing Smad7 displays higher proliferation activity and suppresses col- lagen expression, indicating that this antagonistic member of the SMAD gene family has therapeutic potential [27]. However, constitutive overexpression of Smad7 was shown to cause inflammatory diseases [61] and may have tumorigenic potential. Therefore, more detailed studies are necessary to prove the ef-

ficacy of this intervention directed against TGF- β function.

Moreover, recent reports demonstrate that lefty, a novel member of the TGF- β superfamily, inhibits phosphorylation of Smad2 and events that lie down- stream from R-Smad phosphorylation following activation of the TGF- β receptor [98]. Therefore, expression of lefty may offer a further therapeutic modality for the treatment of liver fibrogenesis.

Alternatively, a direct blockade of TGF- β1 syn- thesis in HSC, by antisense strategies, was shown to lower the overall concentration of TGF- β1 [2].

Moreover, the expression of that antisense mRNA accelerates the production of collagen and α-SMA in hepatic fibrogenesis in rats [3].

12.3.4

Adverse Cytokines, Scavengers of ROS, Herbal Components, ACE Blockers

There are a number of cytokines that have adverse effects on TGF- β, e.g., transduction of the hepatocyte growth factor (HGF) gene suppresses the increase of TGF- β1, inhibits fibrogenesis and hepatocyte apop- tosis, and generates a complete resolution of fibrosis in the cirrhotic liver [96]. A deletion variant of HGF was previously shown effectively to downregulate mRNA expression of procollagens and TGF- β1 and to inhibit HSC activation in vivo [102]. Furthermore, it is well established that the administration of IFN- α in patients with chronic hepatitis also results in sustained clinical responses with normalization of hepatic TGF- β1 mRNA expression levels, which do not differ from the expression in untreated nor- mal control patients [16]. BMP-7 is another potent antagonist of TGF- β. Although there are presently no reports available describing the administration of this member of the transforming growth factor superfamily during hepatic fibrogenesis, there is unique evidence that this protein counteracts TGF- β1-induced renal fibrogenic diseases [62, 106].

The rationale for the use of antioxidants is the

finding that oxidative stress is associated with in-

creased collagen production, resembling the bio-

logical effects of TGF- β1. Treatment with TGF-β in-

creases production of H

₂

O

₂

in activated HSC, where-

as addition of H

₂

O

₂

induces production and secre-

tion of TGF- β by these cells [22] and sequestering

TGF- β leads to a significant decrease in generation

of ROS [20]. As one consequence, TGF- β-mediated

accumulation of H

₂

O

₂

was shown to result in activa-

tion and binding of a C/enhancer-binding protein

(EBP)- β-containing transcriptional complex to the

α1(I) collagen gene promoter [34]. Thus, antioxi-

dants may have therapeutic impact in chronic liver

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injury by interfering with oxidative signaling cas- cades, in which TGF- β plays a key role. In line with these findings, it is evident that antioxidants such as α-tocopherol (vitamin E), resveratrol, quercetin, and N-acetylcysteine function as fibrosuppressants, at least in part, by inhibition of TGF- β signaling [46, 49]. Also the antifibrotic mechanism of diverse herbal compounds (e.g., Sho-saiko-to) may be based on inhibition of oxidative stress, involving baicalin and baicalein as active ROS scavenging components [88]. In line, other herbal medicines, such as Salvia

miltiorrhiza (Dan-shen), were shown to reduce ex-

perimentally induced hepatic fibrosis in animal models and to suppress expression of TGF- β1 [100].

The drugs perindopril and candesartan are an- tagonists of the renin–angiotensin system by block- ing the angiotensin-converting enzyme (ACE) or the angiotensin-II type 1 receptor (AT

₁

-R). In recent in- vestigations, it was shown that both drugs suppress expression of TGF- β1 and induce cell proliferation in activated HSC and may therefore provide an ef- fective new strategy for treatment of patients with chronic liver disease and fibrosis [105].

In summary, many of the discussed approaches are highly effective in blocking TGF- β in experi- mental models of ongoing or persisting fibrosis.

However, their efficacy and safety in human liver fibrosis remain unknown.

Selected Reading

Gressner AM, Weiskirchen R, Breitkopf K et al. Roles of TGF-β in hepatic fibrosis. Front Biosci 2002;7:D793–D807. (This report highlights the recent findings on TGF-β signaling and therapeutic interventions in hepatic fibrosis. Furthermore, the review gives a brief summary of the potential of circulating TGF-β as a diagnostic tool.)

Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J 2004;18:816–827. (This review discusses the current state of knowledge concerning interactions among the profibrotic proteins TGF-β, CTGF, ED-A fibronectin and the antifibrotic proteins tumor necrosis factor-α and γ-interferon.)

Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 2000;342:1350–

1358. (This report summarizes the mechanisms by which TGF-β mediates its cellular functions, focusing on its role in disease, particularly diseases in which genetic mutations in the TGF-β pathway have been documented.)

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