Stellate Cells Massimo Pinzani, Fabio Marra

Stellate Cells

Massimo Pinzani, Fabio Marra

3

Hepatic stellate cells (HSC) are located in the space of Disse in close contact with hepatocytes and sinu- soidal endothelial cells. In human liver, HSC are dis- posed along the sinusoids with a nucleus-to-nucleus distance of 40 µm, indicating that the sinusoids are equipped with HSC at certain fixed distances [169].

These observations suggest that, although the total number of HSC constitutes a small percentage of the total number of liver cells (approximately 5%–8%), their spatial disposition and spatial extension may be sufficient to cover the entire hepatic sinusoidal mi- crocirculatory network. This cell type has received much attention in the past two decades by reason of its potential involvement in the fibrogenic transfor- mation of liver tissue following chronic injury. Be- cause of their anatomical location, ultrastructural features, and similarities with pericytes regulating blood flow in other organs, HSC have been proposed to function as liver-specific pericytes. Branches of the autonomic nerve fibers coursing through the space of Disse come in contact with HSC [84], and nerve endings containing substance P and vasoac- tive intestinal peptide have been demonstrated in the vicinity of HSC [167]. Other peculiar features that suggest a functional relationship between the autonomic nervous system and HSC are that the ex- pression of N-CAM, a typical central nervous sys- tem adhesion molecule detected in hepatic nerves, and the expression of glial fibrillary acidic protein (GFAP) are restricted, among liver cell types, to HSC [72]. These observations, while reinforcing a poten- tial functional relationship between the autonomic nervous system and HSC, raise a current key issue concerning the origin of this cell type, previously considered to be of myogenic origin because of the expression of desmin and smooth muscle α-actin ( α-SMA). Along these lines, activated HSC express nestin, a class VI intermediate filament protein originally identified as a marker for neural stem cells [112]. Remarkably, the expression of this cell marker appears to be restricted to HSC and peri- cytes of brain parenchyma vessels, among all organ- specific pericytes.

The most evident ultrastructural feature of HSC in normal adult liver is the presence of cytoplasmic lipid droplets ranging in diameter from 1 to 2 µm (i.e., "fat-storing cells" or "lipocytes") [169]. These lipid droplets are involved in the hepatic storage of retinyl esters, reflecting the key role of HSC in the metabolism and storage of retinoids [9].

Among other cell types potentially involved in the abnormal progressive deposition of fibrillar ex- tracellular matrix (ECM), HSC have received much attention, also because of the possibility of isolating them from liver tissue with a relatively high purity.

Consequently, most of the present knowledge on the cell and molecular biology of hepatic fibrosis de- rives from in vitro studies employing culture-acti- vated HSC isolated from rat, mouse or human liver.

Regardless, it is now evident that distinct ECM-pro-

ducing cells, each with a distinct localization and a

characteristic immunohistochemical and/or elec-

tron microscopic phenotype, are likely to contribute

to liver fibrosis [18]. These include: fibroblasts and

myofibroblasts of the portal tract, smooth muscle

cells localized in vessel walls, and myofibroblasts

localized around the centrolobular vein. It is also

evident that the relative participation of these dif-

ferent cell types is dependent on the development of

distinct patterns of fibrosis. It is likely that all these

different ECM-producing cell types undergo a proc-

ess of activation in conditions of chronic liver dam-

age or, anyhow, in conditions in which the physi-

ological homeostasis of the tissue is chronically per-

turbed. For the reasons previously mentioned, the

process of HSC activation has been the object of sev-

eral studies and consistent information is available

at present. Following prolonged culture on plastic,

HSC undergo a process of activation from the qui-

escent "storing" phenotype to the highly prolifera-

tive "myofibroblast-like" phenotype (Fig. 3.1). This

process is still regarded as similar to that occurring

in liver tissue following chronic damage, although

this assumption likely represents an oversimplifica-

tion. The activated phenotype is characterized by a

dramatically increased synthesis of collagen types

I and III, which appears predominant over the syn-

thesis of collagen type IV (I>III>>IV) and other ECM components. Studies performed in recent years have emphasized some important aspects po- tentially related to the initiation of HSC activation.

A first important element concerns the disruption of the normal ECM pattern that follows liver tissue injury and acute inflammation. A perturbation in the composition of the normal hepatic ECM and/or of the cell–cell relationship between epithelial and mesenchymal cells present in liver tissue, typical of some cholestatic disorders (i.e., those character- ized by bile duct proliferation and lobular invasion), could also be considered a potent stimulus for the activation and proliferation of HSC, as well as other ECM-producing cells. Indeed, loss of adhesion with the various elements constituting the basal mem- brane-like ECM of the space of Disse is likely to de- termine a marked increase of the proliferative and synthetic properties of HSC. This issue is becoming more and more important with the demonstration that the movement, shape and proliferation of cells are greatly influenced by the cooperation of ECM components and cell adhesion molecules.

Several soluble factors, including growth fac- tors, cytokines, chemokines and oxidative stress products, play a role in the activation of HSC (Ta- ble 3.1) [44, 127]. It must be stressed that, although most studies address the role and the biochemical features of individual factors, these mediators do

not work alone but rather in a complex network of interactions with their cellular targets and the ECM.

Thus, the response to single agonists on cultured HSC does not completely reflect the complexity of the in vivo situation. In the past decade, there has been significant elucidation of the cascades of intra- cellular signals downstream of cytokine-receptor binding. In addition, extensive modifications in the composition and organization of the ECM, and par- allel changes in the expression and function of cell membrane molecules have been identified.

The different cytokines can be grouped accord- ing to their class of receptors, which tend to gener- ate similar intracellular signals within each group:

(a) factors promoting HSC proliferation, migration, and survival (polypeptide growth factor receptors);

(b) factors promoting fibrillar ECM accumulation, and particularly transforming growth factor (TGF)- β1 (TGF-β receptor superfamily); (c) factors with a prevalent contractile effect on HSC, such as endothe- lins, angiotensin-II, vasopressin, and thrombin, although all these agents may also promote HSC proliferation (seven transmembrane domain recep- tors); (d) receptors for chemokines and pro-inflam- matory cytokines. An important determinant of the biologic response to these ligands is the interaction between these substances and the rapidly evolving ECM microenvironment, where they bind and can be stored.

3.1

Polypeptide Growth Factor Receptors

Platelet-derived growth factor (PDGF), a dimer of two polypeptide chains referred to as A- and B- chain, is the most potent mitogen for cultured HSC isolated from rat, mouse, or human liver [117, 123, 125]. Of the three possible dimeric forms of PDGF (-AA, -AB, and -BB), PDGF-BB is most potent in stimulating HSC growth/chemotaxis and the rela- tive intracellular signaling, in agreement with a predominant expression of PDGF receptor β (or type B) subunits compared to PDGF receptor α (or type A) subunits in activated HSC [123]. Impor- tantly, co-distribution of PDGF with cells express- ing PDGF receptor subunits has been demonstrated following both acute and chronic liver tissue dam- age [121, 124], thereby confirming an active role of this growth factor in liver repair and fibrosis. In ad- dition, PDGF is pro-fibrogenic in conditions where inflammation is less evident such as experimental cholestatic liver injury [58, 70]. In cholestasis, PDGF synthesis and release is sustained by proliferating

Fig. 3.1. Activation and phenotypical modulation of hepatic stellate cells (HSC). Activation of HSC and transition to the so- called “myofibroblast-like” phenotype is associated with remark- able changes in their biology. Activated HSC become highly pro- liferative, motile and contractile. In addition, they are responsible for the deposition of increasing amounts of fibrillar extracellular matrix associated with a reduced capability towards its degrada- tion and remodeling. Activated HSC are also responsible for the synthesis and secretion of several pro-inflammatory mediators, including chemokines, thus leading to further amplification of the inflammatory process

bile duct cells. Recent work has shown that PDGF, in addition to inducing proliferation and chemoat- traction of HSC toward bile ducts, is able to mediate the myofibroblastic conversion of peribiliary ECM- producing cells distinct from HSC [71].

Because of the relevance of this polypeptide growth factor in stellate cell growth and chemotaxis, considerable effort has been invested in understand- ing the intracellular signaling events elicited by the interaction of PDGF with its receptor. Recently, two additional PDGF polypeptide chains were discov- ered, namely PDGF-C and PDGF-D. The discovery of two additional ligands for the two PDGF recep- tors suggests that PDGF-mediated signaling is more complex than previously anticipated [88]. However, no data are currently available concerning HSC.

Figure 3.2 illustrates the major signaling path- ways elicited by the interaction of PDGF with its membrane receptors. This response is the net result of the activation of both positive and negative in- tracellular signals, and, accordingly, each pathway leading to a specific effect is often provided with an intrinsic autoregulation. PDGF receptors, which

have intrinsic tyrosine kinase activity, dimerize and become auto-phosphorylated on tyrosine residues upon binding to their ligand [22, 24, 116]. Associa- tion of the PDGF receptor with the adapter protein Grb2 leads to recruitment of the exchange factor mSos with the consequent activation of Ras. This event is followed by the sequential activation of Raf-1, MEK and extracellular-signal-regulated ki- nase (ERK) [104, 105]. Nuclear translocation of ERK is associated with the phosphorylation of several transcription factors, including Elk-1 and SAP, and represents an absolute requirement for triggering a proliferative response [113]. In cultured human HSC, there is activation of the ERK pathway fol- lowed by increased expression of c-fos in response to PDGF [97, 98, 102]. The activation of this pathway is necessary for PDGF-induced cell proliferation and, accordingly, the pharmacological blockade of signaling molecules upstream of ERK (e.g. MEK) leads to a dose-dependent inhibition of cell growth.

This observation is supported by the reduction in the downstream activation of the proto-oncogene

Table 3.1. Growth factors, cytokines and other soluble factors affecting HSC biology

Injured hepatocytes

T lymphocytes Mononuclear/

Kupffer

Sinusoidal endothelium

Platelets Serum Autocrine

ROI TNF-α PDGF-AB PDGF-BB PDGF-AB Thrombin MCP-1

Reactive aldehydes

IFN-γ bFGF bFGF EGF/

TGF-α

A-II PDGF

IGF-I TGF-β VEGF TGF-β AVP TGF-β

VEGF TNF-α IL-1 TX VEGF

IL-1 TGF-β IGF-I ET-1

PGs IGF-I VEGF

ROI PGs

NO ET-1 ROI

Different cellular sources are responsible for the release of factors affecting HSC activation and their pro-fibrogenic properties once activated. A-II angiotensin II, EGF epidermal growth factor, ET-1 endothelin-1, bFGF basic fibroblast growth factor, IFN-γ interferon-γ, IGF-I insulin-like growth factor-I, IL-1 interleukin-1, MCP-1 monocyte chemotactic protein-1, NO nitric oxide, PDGF platelet-derived growth factor, PGs prostaglandins, ROI reactive oxygen species, TGF-α transforming growth factor-α, TGF-β transforming growth factor-β1, TNF-α tumor necrosis factor-α, TX thromboxane A2, VEGF vascular endothelial growth factor.

follows the inhibition of PDGF-induced ERK acti- vation [102]. Inhibition of ERK is associated with a reduction of STAT-1 activation induced by PDGF, although the potential role of the crosstalk occur- ring between ERK and STAT-1 in PDGF-induced cell growth is presently unclear. Work by Reeves and coworkers indicates that PDGF-induced generation of the lipid second messenger phosphatidic acid (PA) contributes to ERK activation in rat HSC, an action attributed to a positive interaction of PA with signaling molecules upstream of ERK [126].

ERK activation in rat HSC occurs following in vivo liver injury induced by the acute administration of CCl

[102]. In this model, increased ERK activity temporally precedes HSC proliferation and peaks at 48 h after the administration of the toxin. Remark- ably, this time point is associated with maximal availability of PDGF in acutely injured liver tissue [121], and precedes HSC proliferation, which begins at 48 h and peaks at 72 h after liver damage.

Studies that employ other drugs to interfere with pathways upstream of ERK provide additional evidence for a key role of ERK in mediating PDGF- induced cell growth in HSC. Preincubation of HSC with drugs increasing intracellular cAMP levels such as pentoxifylline, a phosphodiesterase inhibi- tor, leads to a remarkable reduction in the PDGF-

induced ERK phosphorylation and activity, c-fos expression, and mitogenesis [94]. Other examples of upstream ERK antagonists are compounds that increase prostaglandin (PG) E

synthesis, which act via an increase in intracellular cAMP [40, 93, 174].

Interestingly, stimulation of HSC with PDGF leads to increased synthesis and release of PGE

. This causes in turn an autocrine increase in intracellular cAMP leading to a self-limitation of PDGF mitogen- ic potential [40, 93]. Increased levels of intracellular cAMP may inhibit PDGF-induced cell growth with two main mechanisms: (a) inhibition of Raf kinase, an upstream activator of ERK, occurring through phosphorylation of Raf-1 by cAMP-activated protein kinase A (PKA) [59], and (b) inhibition of STAT-1 activation [69].

Phosphatidylinositol 3-kinase (PI 3-K), another molecule that is recruited by the activated PDGF re- ceptor, is comprised of an 85-kDa regulatory subu- nit, equipped with two SH-2 domains, and a cata- lytic 110-kDa subunit [104]. PDGF stimulation leads to the association of PI 3-K with the activated recep- tor, and to tyrosine phosphorylation of p85 but not of p110. The downstream effectors of PI 3-K activa- tion are only partially known and include protein kinase C ζ, ribosomal S6 kinase, and protein kinase B (c-Akt). Nevertheless, this pathway is sufficient

Fig. 3.2. Major intracellular signaling pathways induced by platelet-derived growth factor (PDGF) and crosstalk with in- tegrin receptors. Ras plays a key role in the crosstalk between PDGF receptor and focal adhesion kinase (FAK) in human HSC.

Integrin dimerization leads to the phosphorylation of FAK and

to the consequent formation of a functional FAK/c-Src complex.

This complex, through interaction with the signaling molecule Shc, leads to an enhanced GTP exchange on Ras, thus facilitating the activation of downstream effectors

to transduce PDGF-dependent mitogenic signals [168] and to be necessary for cell chemotaxis [74].

Thus, in human HSC cultures, PI 3-K activation is necessary for both mitogenesis and chemotaxis in- duced by PDGF [99]. The in vivo relevance of this finding is suggested by the recruitment of the p85 subunits by the PDGF receptor and activation of PI 3-K following acute CCl4-induced liver damage in the rat. Wortmannin, a fungal metabolite that binds and non-competitively inhibits PI 3-K, induces a dose-dependent inhibition of PDGF-BB-induced PI 3-K activation in HSC with a maximal effect at 100 nM. This concentration, which does not affect either PDGF receptor autophosphorylation or the physical association between the PI 3-K p85 subunit and the receptor, virtually abolishes PDGF-induced mitogenesis and chemotaxis in HSC, indicating a functional involvement of this pathway. Similar observations have been made with other PI 3-K in- hibitors such as LY294002 [51]. In addition, PI 3-K is involved in the activation of the Ras-ERK pathway in human HSC, although it is not strictly necessary, since both wortmannin and LY294002 inhibit ERK activation only by 40%–50% [51, 99]. Therefore, in HSC, PI 3-K regulates PDGF-related mitogenesis and cell migration by pathways that are at least in part independent of ERK activation.

In addition to the involvement in cell growth and migration, growth factor-induced PI 3-K acti- vation may contribute to the downstream signaling that regulates cell survival. Current evidence sug- gests that the “survival” or anti-apoptotic action of PI 3-K is mediated by the activation of c-Akt (also referred to as protein kinase B [PKB]), a signaling protein whose activity is regulated by several up- stream events, and particularly the generation of phosphoinositides by PI 3-K [29]. PDGF and insu- lin-like growth factor-I (IGF-I) provide an example of two contrasting paradigms of action. In human HSC, PDGF induces a tenfold increase in DNA syn- thesis and cell migration, whereas the effect of IGF-I is in general one fifth of that of PDGF. Regardless, PDGF and IGF-I are equipotent in the activation of the Ras/ERK and the PI 3-K pathways, at least at early time points (10–15 min) after stimulation [51].

In addition, IGF-I acts as a survival rather than a mitogenic growth factor in this cell type. Gentilini and coworkers have shown that in human activated HSC, IGF-I can activate the c-Akt pathway and its downstream targets regulating cell survival [54].

Importantly, in these experiments, activation of the c-Akt pathway is a PI 3-K-dependent event that is reversed by PI 3-K inhibitors.

In general, early signaling events (i.e., observed within 5–20 min after growth factor stimulation) may be responsible for one or more biologic effects

occurring after several hours. However, it is increas- ingly evident that the biologic effect of a growth fac- tor may be dependent on the activation of one or more intracellular signals occurring with a cyclic and/or reiterated pattern between the interaction of the growth factor and its receptor (early signaling) and the completion of the biologic effect (interme- diate and late signaling). This modality has been extensively elucidated for the relationship between Ras/ERK activation and the progression of the cell cycle induced by PDGF [83] and likely applies to other pathways such as PI 3-K [82]. In particular, this holds true for human HSC, where PDGF trig- gers a biphasic activation of ERK, with a late peak at 15–24 h after addition of the stimulus [10].

In addition to specific intracellular signaling pathways that involve protein phosphorylation, PDGF signaling relies also on changes in [Ca

]

and pH. In particular, in HSC and other cells, sus- tained changes in [Ca

]

and intracellular pH are necessary for the correct articulation of pathways involving protein phosphorylation. The mitogenic potential of different PDGF dimeric forms is pro- portional to their effects on [Ca

]

in activated rat and human HSC [118, 123]. The increase in [Ca

]

induced by PDGF in HSC is characterized by two main components: (a): a consistent and transient in- crease (peak increase), due to calcium release from intracellular stores following the activation of PLC γ and the consequent PIP

hydrolysis, and (b) a lower but longer-lasting increase (plateau phase) due to an influx from the external medium. Induction of replicative competence by PDGF is dependent on the maintenance of sustained increase in [Ca

]

due to calcium entry rather than from the release from in- tracellular stores [138, 172]. Extracellular calcium entry induced by PDGF was originally ascribed to the opening of low-threshold voltage-gated calcium channels consistent with “T” type designation [90].

Subsequently, this channel has been better charac- terized and defined as a PDGF-receptor-operated non-selective cation channel controlled by the tyro- sine kinase activity of the PDGF-R and, particularly, by the activation of Ras through Grb2-Sos [16].

Stimulation with PDGF increases the activity of

the Na

/H

exchanger in rat or human HSC with

consequent sustained changes in intracellular pH

[33–35]. This increased activity appears to occur

through calcium-calmodulin and protein kinase C-

dependent pathways. Inhibition of the activity of the

Na

/H

exchanger by pretreatment with amiloride

inhibits PDGF-induced mitogenesis, thus indicat-

ing that changes in intracellular pH induced by this

growth factor are essential for its full biologic activ-

ity [8]. In addition, PDGF-induced Na

/H

exchang-

er activity is linked to the activation of PI 3-K, and

is blocked by preincubation with PI 3-K inhibitors.

Furthermore, inhibition of the Na

/H

exchanger leads to the interruption of downstream signaling events essential for growth factor-mediated cy- toskeletal reorganization such as PDGF-induced fo- cal adhesion kinase (FAK) phosphorylation [13].

Several observations indicate that a complex interplay may occur among a PDGF signaling and products of oxidative stress in conditions charac- terized by chronic inflammation typical of liver disease. In this context, the relationship between the activation of PDGF receptors and the action of reactive aldehydes (HAKs) appears to be of particu- lar interest. HAKs, and particularly 4-hydroxy-2,3- nonenal (HNE), exert direct pro-fibrogenic effects via an upregulation of the procollagen type I gene.

This effect is mediated through a peculiar signal pathway based on activation and nuclear transloca- tion of c-Jun NH

-terminal kinases (JNKs), upreg- ulation of c-jun and increased AP-1 binding [115].

HNE, as well as other HAKs at the same low concen- trations, induce procollagen type I synthesis, and abolish PDGF-BB mitogenic signaling in human HSC [137]. This occurs through specific inhibition of the intrinsic tyrosine kinase activity associated with the PDGF- β receptor subunit, while autophos- phorylation of other receptors, such as PDGF- α re- ceptor or EGF receptor is not affected.

Established "angiogenic" growth factors such as basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF) exert important biologic effects on HSC. Both bFGF and VEGF play a central role in angiogenesis in health and disease and in all conditions characterized by chronic wound healing. When compared to other mitogenic and chemoattractant growth factors for HSC, bFGF is second only to PDGF-BB in potency [41, 117]. In addition, the potential in vivo relevance of bFGF in hepatic fibrogenesis has been clearly de- lineated in animal models [110, 176]. No studies have examined the expression of bFGF receptors and the relative intracellular signaling in HSC. However, it is conceivable that activation of HSC is associated with an increased expression of receptors for this growth factor and studies addressing this specific issue are awaited, especially in view of recent ad- vances in bFGF signaling [32].

The VEGF family of ligands and receptors has been the focus of attention in vascular biology for more than a decade [27]. Activation of HSC is as- sociated with an increased expression of VEGF and VEGF receptors, and VEGF induces cell growth in this cell type [1, 88]. However, in later stages of ac- tivation, expression of VEGFR1 progressively in- creases, whereas VEGFR2 expression decreases. At this later stage of activation, stimulation with VEGF

induces an attenuation of the contractile properties of HSC and of α-SMA expression [106]. In agree- ment with the available background information on VEGF intracellular signaling, stimulation of activat- ed HSC with this growth factor induces activation of the ERK and PI 3-K pathways [163]. Importantly, several stimuli potentially relevant during chronic liver injury, including hypoxia, nitric oxide, and oxidative stress-related conditions, can upregulate the VEGFR1 [2, 25].

3.2

TGF- β Receptor Superfamily

Expression of TGF- β is markedly increased in ani- mal models of liver fibrosis and in patients with chronic liver disease [20, 111]. Overexpression of TGF- β is associated with increased deposition of matrix in the target tissue, and neutralization of the biologic activity of this cytokine ameliorates ex- perimental liver fibrosis [53, 148]. These data clearly establish a role for TGF- β in mediating the develop- ment of fibrosis during chronic liver injury. TGF- β is the most potent stimulus for production of fibril- lar and non-fibrillar matrix by HSC, and it also in- duces qualitative changes in the matrix by differen- tially stimulating its components [43]. In addition, TGF- β has effects on matrix degradation, which are characterized by mixed actions on matrix metallo- proteinases, and by inhibition of tissue inhibitors of metalloproteinases (TIMPs) and plasminogen acti- vator inhibitor (PAI).

Quiescent HSC are poorly responsive to TGF- β

when maintained in suspension, a condition that

prevents transition to an activated state [42]. Ac-

cordingly, affinity labeling studies have demon-

strated small amounts of T βRIII in quiescent cells,

while all three TGF- β receptors are present after

activation. Nevertheless, in non-activated cells,

T βRII expression could be demonstrated by west-

ern blotting, and mRNA transcripts for T βRII and

T βRIII (betaglycan) were actually greater than af-

ter activation on uncoated plastic [42]. Decreased

mRNA levels for T βRII in activated HSC have been

confirmed by Roulot et al. [145], suggesting that the

ratio between type II and type I receptors may be

critical to mediate differentially the biologic effects

of TGF- β, including matrix synthesis. The biologi-

cal significance of reduced expression of T βRIII in

activated versus quiescent HSC remains to be es-

tablished. Similar to other TGF- β-responsive cells,

HSC show phosphorylation of Smad2 and Smad3

upon exposure to this cytokine [36]. Interestingly,

compared to early cultured HSC, fully activated my-

ofibroblast-like cells show reduced Smad activation, and this finding is associated with lower efficacy of the cytokine in inducing biologic actions in fully ac- tivated cells [36]. However, these changes are likely to be limited to fully activated, myofibroblast-like cells, because binding of TGF- β to its receptors, as evaluated by affinity binding, is not reduced in HSC isolated 48 or 72 h after acute CCl

intoxication in the rat [79]. Interaction between activated Smad proteins and other factors may be relevant for the cell specificity of TGF- β actions. Sp1 binding to the promoter of the α2(I) collagen gene mediates the in- creased expression by TGF- β in HSC [76], while in hepatocytes Sp3 binds the same element, but with little transactivating activity. In HSC, but not in hepatocytes, activated Smad3 physically interacts with Sp1, thus providing a link between TGF- β sign- aling and matrix upregulation in specific cell types [78]. Moreover, constitutive phosphorylation and nuclear localization of Smad3 was found in a clone of HSC exhibiting high levels of collagen and PAI- 1 expression together with poor response to TGF- β, confirming the relevance of this pathway for the upregulation of extracellular matrix production in HSC [77].

Besides Smads, other signaling pathways are activated by TGF- β in HSC, with potential involve- ment in ECM synthesis and other activities. TGF- β1 induces activation of the ERK pathway through se- quential activation of Ras, Raf-1 and MEK [133]. In- terestingly, in the absence of TGF- β, the members of this pathway have divergent actions on collagen gene expression. While dominant-negative ERK, which inhibits ERK signaling, reduced procollagen α1(I) transcription, dominant-negative raf increased col- lagen reporter gene expression. These results indi- cate that activation of ras exerts negative signals on collagen transcription via a Raf-dependent, ERK-in- dependent pathway, while ERK positively modulates expression of the collagen gene [30].

Other Smad-independent signaling pathways contribute to the biologic effects of TGF- β in HSC, in particular the exposure of the cells to reactive oxygen intermediates. TGF- β induction of procol- lagen α1(I) mRNA is mimicked by the addition of H

O

, and is prevented by the addition of the anti- oxidant PDTC, or of catalase to cultured HSC. These effects are mediated, at least in part, by activation of the C/EBP β transcription factor, which binds to a cis-acting element of the promoter [48]. It has been proposed that glutathione levels in HSC en- able the cells to discriminate between exogenously produced H

O

, and therefore oxidative stress, and H

O

generated as a signaling molecule such as in response to TGF- β [31]. In particular, exposure to TGF- β would be associated with high intracellular

levels of H

O

, which could be removed efficiently by catalase, yielding high levels of glutathione and allowing autocrine secretion of TGF- β. On the con- trary, exogenously added H

O

, simulating a condi- tion of oxidative stress, generates low intracellular levels of H

O

, which is catabolized by glutathione peroxidase, resulting in low glutathione levels and interruption of a TGF- β autocrine pathway [31].

HSC also produce activin and respond to recom- binant activin. Activin A increased mRNA levels for type I collagen, synergistically with TGF- β, thus establishing that activin receptors are functional [155]. The dynamics of activin receptor expression during HSC activation and the specific role of ac- tivin receptor-dependent signaling pathways have not yet been evaluated.

3.3

Seven Transmembrane Domain Receptors

Several factors acting through receptors belonging to the "seven transmembrane domain" family are active in HSC. Due to similarities in biologic actions and signaling these receptors may be divided, for the purpose of this review, into two subgroups, namely receptors for "vasoconstrictors" and chemokine re- ceptors. Endothelins, angiotensin II, vasopressin, and thrombin, although generally referred to as

"vasoconstrictors", promote pro-fibrogenic actions

and are considered to be pleiotropic cytokines when

viewed in the context of the chronic wound-healing

process. Endothelin-1 (ET-1), a potent vasoactive

21-amino-acid peptide secreted by endothelial as

well as other cell types, has been shown to exert a

multifunctional role in a variety of tissues and cells

[153, 154, 175], including the liver. Infusion of ET-1

in the isolated perfused rat liver causes a sustained

and dose-dependent increase in portal pressure

associated with increased glycogenolysis and oxy-

gen consumption [49, 50, 141, 164]. ET-1 stimulates

glycogenolysis, phosphoinositide turnover and re-

petitive, sustained intracellular calcium transients

in isolated rat hepatocytes [48, 150]. Other studies

indicate that ET-1 may also have important interac-

tions with liver non-parenchymal cells. Cultured

sinusoidal endothelial cells isolated from rat liver

have been shown to release ET-1 [136], and preferen-

tial binding sites for ET-1 have been identified, both

in vivo and in vitro [45, 55], on HSC. As previously

mentioned, ET-1 induces a dose-dependent increase

in intracellular free calcium, coupled with cell con-

traction in this cell type. Importantly, activated rat

and human HSC have been shown to express pre-

proET-1 mRNA [66, 125] and to release ET-1 in cell supernatants in response to agonists such as angi- otensin II, PDGF, TGF- β and ET-1 itself [125], thus raising the possibility of a paracrine and autocrine action of ET-1 [140]. ET-1 synthesis in HSC is regu- lated through modulation of endothelin converting enzyme-1 (ECE-1), the enzyme that converts pre- cursor ET-1 to the mature peptide, rather than by modulation of the precursor pre-proET-1 [151]. Re- cent evidence suggests that upregulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins occurs in HSC after liver injury and during activation in vitro [152]. In addition, TGF- β1, stimulates ET-1 production by inducing ECE-1 mRNA stabilization. Overall, it is increasingly evi- dent that the process of HSC activation and pheno- typical modulation is characterized by a close and complex relationship with the ET system. The abil- ity to synthesize and release ET-1 is associated with a progressive shift in the relative predominance of ET

and ET

receptors observed during serial sub- culture: ET

are predominant in the early phases of activation, whereas ET

become increasingly more abundant in “myofibroblast-like” cells [125]. The upregulation of the ET

receptor is prevented by the incubation of HSC with retinoic acid during the ac- tivation process [21], thus confirming that increased expression of this receptor is part of the phenotypi- cal modulation of HSC towards the “myofibroblast- like” phenotype [134]. The shift in the relative ET receptor densities may be directed at differentiating the possible paracrine and autocrine effects of ET-1 on HSC during the activation process. Indeed, when HSC are provided with a majority of ET

receptors (early phases of activation), stimulation with ET-1 causes a dose-dependent increase in cell growth, ERK activity and expression of c-fos. These effects, likely related to the activation of the Ras-ERK path- way, are completely blocked by pretreatment with BQ-123, a specific ET

receptor antagonist [125], and are in agreement with studies performed in oth- er vascular pericytes such as glomerular mesangial cells [171]. Conversely, in later stages of activation, when the number of ET

receptors increases, ET-1 appears to induce a prevalent anti-proliferative ef- fect linked to the activation of this receptor subtype [91]. In this setting the activation of the ET

recep- tor stimulates the production of prostaglandins, leading to an increase in intracellular cAMP, which in turn reduces the activation of both ERK and JNK. In addition, both cAMP and prostaglandins upregulate ET

binding sites, thus suggesting the possibility of a positive feedback regulatory loop.

In addition, recent studies have further defined the action of cAMP on the ET-1 receptor system. Cyclic AMP rapidly desensitizes ET

in activated HSC and

shifts their ET-1 responsiveness from picomolar to nanomolar concentrations with respect to Ca

sig- nals and HSC contraction. ET

desensitization also occurs in response to prostaglandin E

, adenosine, or ET

stimulation [135].

Analogously to what is proposed for ET-1, an- giotensin II, vasopressin, and thrombin, although generally referred to as “vasoconstrictors”, promote pro-fibrogenic actions and are considered to be plei- otropic cytokines when viewed in the context of the chronic wound-healing process. Circulating levels of angiotensin II (A-II), a powerful vasoconstric- tor, are frequently increased in cirrhotic patients, and have been implicated in the circulatory distur- bances typical of this clinical condition. However, A-II, in addition to its action as a vasoconstrictor, is provided with biologic properties potentially rel- evant for the progression of chronic fibrogenic dis- orders. These include increase in cell proliferation and cell hypertrophy, and accordingly A-II may be considered also as a pleiotropic cytokine. In a recent study, Bataller and coworkers [6] have reported that activated human HSC express A-II receptors of the AT1 subtype, and that an increased expression of this type of receptor may represent a feature of HSC activation. Stimulation with A-II elicits a marked dose-dependent increase in [Ca

]

concentration associated with rapid cell contraction, in agree- ment with a previous report [120]. Moreover, A-II stimulates DNA synthesis and cell growth. The in- volvement of AT1 receptors in these effects of A-II is confirmed by their complete abrogation follow- ing preincubation with the AT1 receptor antago- nist losartan. Although the antifibrogenic effects of pharmacological AT1 receptor blockade appear promising in other conditions, i.e., myocardioscle- rosis [89], no information is currently available for hepatic fibrogenesis. Analogous effects on HSC bi- ology have been described for arginine vasopressin (AVP). Human activated HSC express V1 receptors, and stimulation with AVP elicits a dose-dependent increase in intracellular [Ca

]

coupled with cell contraction. Moreover, AVP increases ERK activity, DNA synthesis, and cell growth [5].

The serine protease thrombin (THR) regulates platelet aggregation, endothelial cell activation and other important responses in vascular biology and in acute and chronic wound repair. Although THR is a protease, it acts as a traditional hormone or as a pleiotropic cytokine based on the nature of its re- ceptors, the protease-activated receptors or PARs.

PARs are G-protein-coupled receptors that use a fas- cinating mechanism to convert an extracellular pro- teolytic cleavage event into a transmembrane signal:

these receptors carry their own ligands, which re-

main cryptic until unmasked by receptor cleavage

[26]. Four PARs are known in mouse and human:

human PAR1, PAR3, and PAR4 can be activated by THR, whereas PAR2 is activated by trypsin and tryptase as well as by coagulation factors VIIa and Xa, but not by THR. Studies by Marra and cowork- ers have shown that expression of PAR1 is markedly increased in chronic fibrogenic disorders involving liver. In addition, human HSC express PAR1, and this expression increases during HSC activation [96, 100]. Stimulation of human HSC with THR induces cell contraction [120], proliferation [96], synthesis and release of chemokines such as MCP-1 [100], or platelet activating factor [122]. The signaling mech- anisms specifically regulating THR action in HSC have not been reported thus far.

In addition to ET-1, A-II and thrombin, the po- tential involvement of other vasoconstrictors syn- thesized and released within liver tissue has been suggested. Titos and coworkers [165] have recently reported that in cirrhotic rat liver there is an in- creased synthesis of cysteinyl leukotrienes (LTs). In this context, hepatocytes exhibit the highest abil- ity to generate cysteinyl LTs, which elicit a strong contractile response in activated HSC. These find- ings further reinforce the concept of an imbalance between vasoconstrictor and vasodilator agents within the intrahepatic circulation of cirrhotic liver.

Importantly, the concentration of vasoconstrictors acting on the intrahepatic microvasculature of cir- rhotic liver may increase as a consequence of clini- cal or subclinical events such as infections in the peritoneal cavity, which are clearly associated with a worsening of portal hypertension and with an in- creased incidence of variceal bleeding [57]. Appro- priate use of drugs currently indicated for the treat- ment of portal hypertension [175] should be care- fully reconsidered in light of the current knowledge of the cellular and molecular mechanisms of portal hypertension.

The chemokine system is a major modulator of many critical functions, both in physiologic and pathologic conditions, including inflammation, de- velopment, leukocyte trafficking, angiogenesis and cancer [143]. In HSC, secretion of several chemok- ines belonging to different subclasses regulates re- cruitment of inflammatory cells to sites of damage [101]. These findings demonstrate a first line of in- teraction between the inflammatory and the repara- tive phases of the wound-healing response. Howev- er, chemokines also directly regulate the behavior of cells involved in tissue repair. In fact, glomeru- lar mesangial cells, or smooth muscle cells express chemokine receptors, which mediate functional re- sponses that are relevant for tissue fibrosis [54, 142].

In agreement with these observations, the CC chem- okine monocyte chemoattractant protein-1 (MCP-

1) induces concentration-dependent chemotaxis of HSCs [101]. Interestingly, HSC do not express the re- ceptor CCR2, which mediates the biologic responses of this chemokine in leukocytes. Nevertheless, in- cubation of stellate cells with MCP-1 is associated with a rise in intracellular calcium concentration and activation of intracellular signaling pathways, including protein tyrosine phosphorylation, and activation of PI 3-K, which are necessary to stimu- late cell migration. The receptor(s) responsible for these effects of MCP-1 have not been identified yet, but a possible candidate could be CCR11, which can bind MCP-1 with high affinity [160]. The chemok- ine receptor CXCR3, which binds the ligands IP-10, Mig, and I-TAC, is the first chemokine receptor to be identified in HSC [10]. Its expression increases after a few days in culture on uncoated plastic, and receptor activation is associated with increased cell migration, establishing that CXCR3 is functional in HSC. Interaction of CXCR3 with its ligands leads to activation of the Ras/ERK cascade through a Src- dependent pathway, and to activation of PI 3-K and its downstream kinase Akt. Interestingly, CXCR3 activation increases cell proliferation in glomerular mesangial cells but not in HSC. This discrepancy in the biologic actions elicited by this receptor is ac- companied by a different time course of ERK activa- tion in the two cell types. Indeed, in HSC ERK is ac- tivated only transiently, whereas in mesangial cells a biphasic activation occurs, including a late peak at 15–24 h [10]. The molecular mechanisms underly- ing this different behavior in the two cell types has not yet been elucidated.

Expression of other functional chemokine re- ceptors has been demonstrated recently in cultured HSC. CCR7, a receptor expressed by different T cell subtypes (see above) is also present in activated HSC, where it mediates cell migration and secretion of other chemokines [11]. Similarly, CCR5 has been found on the surface of HSC. Remarkably, HSC also express the CCR5 ligand, CCL5, indicating the exist- ence of an autocrine loop involving these two mol- ecules [158]. Exposure of HSC to recombinant CCL5 resulted in increased DNA synthesis and migration, providing additional evidence for a crosstalk be- tween leukocytes and HSC during liver fibrosis.

Collectively, these data support the view that the

mechanisms regulating leukocyte infiltration and

the persistence of inflammation are also responsible

for migration and proliferation of HSC to the same

sites of liver injury, contributing to the pathogenesis

of tissue repair and fibrogenesis.

3.4

TNF Receptor Superfamily

The receptors for tumor necrosis factor (TNF) be- long to a superfamily that includes several trans- membrane molecules that bind cytokines or other ligands and orphan receptors, and are character- ized by similar, cysteine-rich extracellular domains [4]. This family may be divided into two large sub- groups depending on the presence or absence of a

“death domain”, which allows physical associa- tion with molecules containing the same domain.

TNFR1, Fas, and the p75 receptor for nerve growth factor belong to the group of “death domain” recep- tors, while TNFR2, and CD40 lack this domain [86].

TNFR1 plays a major role in mediating the biologic actions of TNF, because animals lacking this re- ceptor, unlike those lacking TNFR2, are unable to mount inflammatory reactions in response to TNF [144]. TNF activates pathways that regulate gene transcription and inflammation, and others leading to cell death. Binding of TNF induces homotrimeri- zation of TNFR-1, which binds to the death domain- containing protein TRADD [86]. Association of TRADD with RIP and TRAF2 is responsible for the downstream activation of NF- κB and JNK, respec- tively, leading to regulation of gene transcription.

Activation of other members of the mitogen-activat- ed protein kinase (MAPK) family, including ERK1/2 and p38MAPK, is also elicited by TNF. In contrast, cell death is mediated by interaction of TRADD with FADD, which initiates a cascade of proteases, known as caspases, leading to the apoptotic effect. It is im- portant to note that most cells become sensitive to TNF-induced apoptosis only when protein synthesis or RNA transcription is inhibited.

TNF has many important effects on HSC rel- evant to the pathophysiology of liver fibrosis. TNF participates in the activation process [73], but has an inhibitory effect on expression and synthesis of type I collagen [3] and on proliferation of HSC [47, 73]. Remarkably, TNF is a critical factor for the

“pro-inflammatory” role of HSC, because it upregu- lates expression and secretion of several cytokines and chemokines (for a review see [101]). Unlike other cytokine receptors, quiescent HSC express mRNA transcripts for TNFR1, and TNF efficiently binds to the cell surface [62]. However, the receptor expressed by quiescent cells seems to be only par- tially functional, because exposure of non-activated cells to the ligand does not result in activation of NF- κB, due to the inability to degrade the inhibi- tory protein I κBα in quiescent cells. Accordingly, increased expression of NF- κB-regulated proteins, such as the adhesion molecule ICAM-1, is observed

only in activated cells. Interestingly, the JNK path- way may be activated by TNF both in quiescent and activated HSC, indicating that the block in NF- κB activation in quiescent cells occurs at a post-recep- tor level. Activation of NF- κB plays a pivotal role in mediating the pro-inflammatory effects of TNF on HSC. Interference with NF- κB activation by protea- some degradation inhibitors or an I κB super-re- pressor blocks the expression of several cytokines, chemokines, and adhesion molecules, including in- terleukin-6 (IL-6), MCP-1, CINC, MIP-2, and ICAM- 1 [37, 101]. Interestingly, novel Rel-like proteins may contribute to the NF- κB DNA-binding complex ob- served in activated HSC and upregulated by expo- sure to TNF [38]. NF- κB activation is also required to induce cyclo-oxygenase 2 (COX-2), which medi- ates the growth-inhibitory effect of TNF- α in these cells and contributes to chemokine expression [37, 47]. NF- κB is also an important mediator of cell sur- vival [7], and exposure of HSC to TNF, together with inhibition of NF- κB activation, resulted in apoptosis [85]. However, when used alone, TNF actually pro- tects HSC from apoptosis, via reduction of Fas lig- and expression [147]. On the other hand, activation of stress-activated protein kinases, such as JNK or p38, may be involved in the phenotypic transition from quiescent to activated HSC [132], and in the expression of matrix metalloproteinases [129]. The inhibition of type I collagen expression is mediated by a complex mechanism. Preincubation of HSC with pertussis toxin abolishes the inhibitory effects of TNF on procollagen α1(I) mRNA expression, and ceramide mimics the effects of this cytokine, indi- cating the involvement of a pathway requiring a G protein and sphingomyelin/ceramide [64]. Moreo- ver, several transcription factors and regulatory elements are implicated in the inhibitory effects of TNF, including a tissue-specific regulatory region, increased binding of p20C/EBP β and C/EBPδ, and reduced binding of Sp1 [63, 65, 80].

An interesting signaling crosstalk involving TNF and the peroxisome proliferator-activated receptor- γ (PPAR-γ) has been described recently. PPAR-γ is a nuclear hormone receptor that binds antidiabetic thiazolidinediones and downregulates the activated phenotype of HSC [46, 103, 109, 161]. In HSC ex- posed to TNF, the transcriptional activity of PPAR- γ was reduced, via phosphorylation of Ser(82) medi- ated by activation of ERK and JNK [161]. These data indicate an additional mechanism by which TNF may be implicated in the acquisition of an activated phenotype by HSC.

Expression of Fas (CD95) by HSC was first re-

ported by Saile et al. [146], who showed that this re-

ceptor is upregulated during activation, in parallel

with the appearance of spontaneous apoptosis. Re-

markably, activated HSC also express Fas ligand, in- dicating that HSC possess the two components of the Fas system necessary to induce apoptosis [147]. As indicated above, Fas ligand may be downregulated by either TNF α or TGF-β [148]. In a different study, activation of Fas by soluble ligand or cross-linking antibodies required protein synthesis or transcrip- tion inhibitors to induce apoptosis in activated HSC [56]. The expression of receptors for TRAIL, another molecule related to TNF, has been reported recently in spontaneously immortalized human HSC (LX- 2) [162]. Both TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was detected in these cells, although the expression of TRAIL-R2/DR5 was much higher.

This process was activation-dependent and resulted in sensibility of HSC to TRAIL-mediated apoptosis.

The p75 nerve growth factor receptor also pos- sesses a death domain and binds NGF with low af- finity as compared to TrkA. In activated HSC, p75 was demonstrated in vivo and in culture, and expo- sure to NGF caused apoptosis [166]. Very recently, mRNA for other neurotrophin receptors has been detected in rat HSC [19]. The biologic significance and signaling of these receptors merit further inves- tigation.

3.5

Other Cytokine Receptors

The actions of interleukin-1 (IL-1) on HSC are re- markably similar to those elicited by TNF with re- gard to its effects on pro-inflammatory molecules, and these actions are mediated by activation of NF- κB [101]. Similarly, quiescent HSC express IL-1 re- ceptors, but the activation of NF- κB is restricted to activated cells [62]. IL-1 also has inhibitory effects on procollagen α1(I) expression, which occurs at a post-transcriptional level [3], and stimulates HSC proliferation [108]. However, the net effects of this cytokine are pro-fibrogenic, because administra- tion of IL-1 receptor antagonist reduces matrix dep- osition [95].

Receptors for interferon- γ (IFN-γ) are ubiqui- tous, although no studies have examined their ex- pression during HSC activation. A critical step in IFN- γ signaling is the activation of the latent tran- scription factor STAT-1, which upon dimerization migrates to the nucleus where it regulates transcrip- tion [28]. IFN- γ downregulates activation, matrix synthesis and proliferation of HSC in vitro and in vivo [28, 92, 130, 138, 139]. In addition, particularly if used in combination with other cytokines or LPS, IFN- γ stimulates the expression of inducible NO synthase [138]. However, the molecular mechanisms

underlying these effects have not been studied. IFN- γ upregulates the expression of chemokines such as MCP-1 in HSC [101], and this effect is differentially regulated as compared to other cytokines [136].

Surprisingly, when human HSC are incubated with IFN- γ for prolonged periods and exposed to mi- togens such as serum or PDGF, an increase in cell proliferation is observed, which is dependent on synergistic activation of STAT-1 α [61, 98].

HSC also respond to other cytokines, including IL-10, IL-6, and oncostatin M [60, 87, 170]. IL-10 in- hibits procollagen α(1) expression at the transcrip- tional level, and may have important anti-fibrogenic properties [170]. Expression of the receptor for IL-10 has been shown recently in activated HSC [107].

Recently, the role of leptin as a mediator of liver fibrogenesis has been demonstrated by different groups. HSC express the short form (ObRa) of the leptin receptor, while the expression of the long form (ObRb), which more effectively transduces leptin's signals, is debated [75, 149]. Recently, the expression and signaling of leptin receptors has been reported in LX-2 cells. These cells express the long form of the receptor, which is responsible for activation of protein tyrosine kinases of the Jak family and ac- tivation of Stat3 and the MAPK cascade [14]. These pathways are ultimately responsible for increased TIMP-1 expression and the resulting pro-fibrogenic effect.

3.6

Cooperation Between Growth Factor Receptor and Integrin Signaling

Binding of cells to the ECM is mediated, at least in

part, by cell surface receptors belonging to the in-

tegrin family. In addition to mediating cell adhe-

sion, integrins make transmembrane connections

to the cytoskeleton and activate many intracellular

signaling pathways. Integrins are heterodimeric

transmembrane proteins that consist of an α and a

β subunit. At present, 8 β and 18 α subunits and 24

possible distinct integrin dimers have been identi-

fied. Combination in dimers leads to the formation

of integrin receptors with defined specificity for dif-

ferent ECM ligands. Expression of different dimers

reflects the attitude of a given cell type to interact

with a specific matrix microenvironment and this

interaction has profound consequences for the bi-

ology of the cell type, including its responsiveness

to soluble factors. In addition to their roles in ad-

hesion to ECM ligands or counter-receptors on ad-

jacent cells, integrins serve as transmembrane me-

chanical links from those extracellular contacts to the cytoskeleton inside cells. For most integrins the linkage is to the actin-based microfilament system, which integrins also regulate and modulate [68].

In part related to the integrin-mediated assembly of cytoskeletal linkages, ligation of integrins also triggers a large variety of signal transduction events that serve to modulate many aspects of cell behavior including proliferation, survival/apoptosis, shape, polarity, motility, gene expression, and differentia- tion. Protein phosphorylation is one of the earliest events detected in response to integrin stimulation, and in particular tyrosine phosphorylation is a com- mon response to integrin engagement in many cell types [12, 13]. Several protein tyrosine kinases have been implicated in integrin-related signaling events.

These signal transduction pathways are complex, like those emanating from receptors for soluble factors (e.g., G protein-coupled and kinase recep- tors). Indeed, many integrin-stimulated pathways are very similar to those triggered by growth factor receptors and are intimately coupled with them. In fact, many cellular responses to soluble growth fac- tors, such as EGF, PDGF, LPA, and thrombin, etc., are dependent on the cell being adherent to a sub- strate via integrins. That is the essence of anchorage dependence of cell survival and proliferation and integrins lie at the basis of these phenomena. Along these lines, studies have begun to address the mul- tiple potential interactions of cells with the micro- environment. The major advances reflect elucida- tion of the fine tuning occurring upon cell adhesion and the consequent cytoskeletal organization. Key elements in these responses are: (a) the specificity of integrin receptors and their downstream signal- ing; (b) the crosstalk between integrin and cytokine signaling; (c) the relationship between the above mechanisms and the organization and tension of the cytoskeleton.

Activated HSC express several integrin β

-associ- ated α subunits. A particularly high expression has been demonstrated for α

β

and α

β

[15]. Focal ad- hesion kinase (FAK) plays a central role in integrin- mediated signal transduction [156, 157]. In addition to its activation by integrins, FAK is also activated by several growth factors. FAK phosphorylation is stimulated by mitogenic neuropeptides such as bombesin, vasopressin, and by PDGF and ET-1. PI 3-K co-immunoprecipitates with tyrosine-phospho- rylated FAK in response to cell adhesion. Tyrosine phosphorylated PLC γ may be a transducer molecule in integrin-mediated signaling pathways. Adhesion of human HSC to ECM proteins does not result in PLC γ tyrosine phosphorylation [157]. Nevertheless, adhesion of HSC induces interactions between PLC γ and cellular proteins undergoing tyrosine phospho-

rylation, one of which has been identified as FAK, suggesting that adhesion of HSC is followed by re- cruitment of PLC γ to phosphorylated FAK. Since PLC γ also physically associates with the PDGF re- ceptor, there may be crosstalk between this receptor and proteins of the focal adhesion complex. Indeed, stimulation of HSC with PDGF-BB leads to cluster- ing of PDGF- β receptor subunits in areas possibly corresponding to focal adhesion complexes. Along these lines, upon autophosphorylation, PDGF re- ceptors are co-distributed with FAK, thus suggest- ing a potential functional crosstalk between these signaling molecules [15]. In addition, experimental evidence indicates that Ras plays a key role in the crosstalk between PDGF receptor and FAK in hu- man HSC [16] (Fig. 3.2).

In a recent study we evaluated the influence of cell adhesion on the major intracellular signaling pathways elicited by PDGF in activated human HSC [17]. PDGF signaling was investigated in an experi- mental condition characterized by lack of cell adhe- sion for different intervals of time. Prolonged lack of cell adhesion resulted in the abrogation of both basal and PDGF-induced FAK tyrosine phospho- rylation. In these conditions ERK/MAPK activity correlated with FAK phosphorylation. Stimulation with PDGF was able to stimulate Ras-GTP loading only in adherent cells. The ability of PDGF to induce PI 3-K activity was abrogated in cells maintained in suspension. The activation of PKB/Akt was only marginally affected by the lack of cell adhesion. We then evaluated the association of FAK with c-Src.

This association was found to be cell adhesion de- pendent and it did not appear to be dependent on phosphorylated FAK. These changes in PDGF-in- duced intracellular signaling were associated with a remarkable reduction of PDGF proliferative po- tential in non-adherent cells, although no marked differences in the apoptotic rate were observed. In summary, these results suggest that cell adhesion differentially regulates major signaling pathways activated by PDGF in human HSC.

Therefore, it appears that multiple receptor sys-

tems can synergize with integrins to regulate bio-

logical phenomena such as cell proliferation, and

cell motility, and the signaling proteins activated

by these synergistic agents are common to different

receptor pathways. However, more recent advances

indicate that cell adhesion and the consequent sig-

naling events are necessary but not sufficient to

provide a complete control of cell functions, and

in particular their response to growth factors and

cytokines. Indeed, a modern concept, defined in its

complexity with the term "cellular tensegrity archi-

tecture" [67], implies that in the presence of growth

factor stimulation and adequate adhesion to the

substratum, the cell is unable to progress into the cell cycle if restricted in its spreading. This view is supported by several observations indicating that lack of cytoskeletal tension, obtainable only with full cell spreading, is equivalent to cytoskeletal dis- ruption and leads to the inability to enter the cell cycle. This general key mechanism appears relevant for the regulation of the molecular signaling cas- cades operating in the context of the structural and mechanical complexity of living tissue and their pathophysiological alterations, including hepatic fi- brogenesis.

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