Transcriptional Regulation in Hepatic Stellate Cells

• ABSTRACT
• REGULATION OF GENE EXPRESSION BY TRANSCRIPTION FACTORS
• CHROMATIN REMODELING ENZYMES AND TRANSCRIPTION
• PARADIGMS OF TRANSCRIPTIONAL REGULATION IN HEPATIC STELLATE CELLS
• CCAAT/Enhancer-Binding Proteins
• C/EBPS INDUCED BY OXIDATIVE STRESS LEAD TO TRANSCRIPTIONAL UPREGULATION OF THE α₁(I) COLLAGEN GENE
• C/EBPS INDUCED BY TUMOR NECROSIS FACTOR-α LEAD TO TRANSCRIPTIONAL REPRESSION OF THE α₁(I) COLLAGEN GENE
• c-Myb and Cyclic AMP Response Element Binding Protein (CREB)
• Nuclear Factor κB
• NF-κB IN STELLATE CELLS
• ACTIVITIES OF NF-κB INDUCED BY CYTOKINES
• Peroxisome Proliferator-Activated Receptors (PPARs)
• Kruppel-like Zinc Finger Factors
• KRUPPEL-LIKE FACTOR 6: POTENTIAL ROLE IN HSC ACTIVATION
• CONCLUSIONS AND FUTURE DIRECTIONS
• ABBREVIATIONS
• REFERENCES

ABSTRACT

Modulation of gene expression through altered transcription regulates stellate cell behavior in normal liver and following hepatic injury. Transcription factors are generally classified according to conserved motifs within either the activation- or DNA- binding domains of the molecules. Transcriptional activity in stellate cells represents a delicate fine tuning of multiple inputs. Activities of these transcription factors are modified by their intracellular localization, rate and pathway of degradation, oligomerization, and interactions with heterologous factors and chromatin, as well as by posttranslational modifications, including phosphorylation, glycosylation, and acetylation. General paradigms of transcriptional control are increasingly being validated in hepatic stellate cells, particularly involving the transcription factors CCAAT/enhancer-binding proteins, c-myb, CREB, nuclear factor κB, peroxisome proliferator-activated receptor, and Kruppel-like zinc finger factors. Although there are no simple rules that govern mechanisms of transcriptional regulation in stellate cells, continued advances will yield new insights into their role in normal liver homeostasis and in the response to injury.

With the importance of stellate cell activation in hepatic fibrosis clearly established, investigators are increasingly exploring fundamental features of the cell's biology. Among these, modulation of gene expression through altered transcription is a critical level of regulation. This review will focus on advances in our understanding of mechanisms regulating gene transcription in stellate cells, and the characterization of transcription factors controlling stellate cell behavior in normal and injured liver.

The fundamental features of stellate cell activation, or transdifferentiation, have been well described in this issue of Seminars in Liver Disease and previous reviews[1] [2] and are summarized only briefly here. In normal liver, hepatic stellate cells (HSCs) store retinoids (vitamin A) within cytoplasmic lipid droplets. Upon liver injury, HSCs activate (transdifferentiate) into a myofibroblast-like phenotype characterized by expression of α-smooth muscle actin (α-SMA) and loss of retinoid. Activation of stellate cells has been documented in animal models and human liver disease, and can also be recapitulated by growth in culture, where primary quiescent HSCs are activated on a plastic substratrum. The temporal sequence of molecular and cellular events during stellate cell activation has been characterized both in vivo and in culture.[3] [4] [5] For example, during culture activation, α-SMA is detected at days 3-5 of primary culture.[6] Activated HSCs are highly migratory or chemotactic, and remodel the extracellular matrix by secreting matrix metalloproteinases and depositing type I collagen (see articles by Schuppan and Geerts in this issue). In addition, HSCs respond to a number of paracrine and autocrine cytokines elicited during liver injury. These cytokines as well as cellular oxidative stress have profound effects on both collagen production and cellular proliferation (see article by Pinzani in this issue).

REGULATION OF GENE EXPRESSION BY TRANSCRIPTION FACTORS

Most genes in eukaryotes are regulated in part by controlling their synthesis of messenger RNA (mRNA) in a process known as transcription. The recognized modes of regulating transcription continue to expand in complexity, and extend far beyond the initial, simple paradigm whereby a single transcription factor binds to a cognate element in a gene promoter to either promote or repress mRNA expression (see Goodrich,[7] Calkhoven,[8] and Lemon[9] for general reviews of transcriptional regulation). Rather, gene expression is exquisitely fine tuned through the summation of a complex of transcription factors acting as activators and repressors simultaneously, as well through alterations in chromatin structure and charge.

Transcription begins with initiation, requiring cooperative assembly of both gene-specific and basic transcriptional complexes in specific regions of DNA proximate to the transcriptional initiation site. Transcriptional initiation requires more than just transcription factor binding. After gene-specific transcription factors are bound, a transcriptional preinitiation complex may form by recruitment of the multi-subunit RNA polymerase, together with numerous general transcription factors (GTFs) that comprise the basic transcription machinery. The ability to initiate transcription may be dictated by the ability to recruit the basic transcription machinery.

The DNA regulatory elements of most genes are typically found upstream of the transcriptional initiation site in a region termed a promoter. These regulatory elements are short stretches of DNA sequence to which specific transcription factors are recruited and bound. Promoter regions are distinct in each gene, in that they have a unique array of regulatory elements. Genes also may have other regulatory elements distal to the promoter region in regions called enhancer or silencer elements, to which transcription factors may also bind.

The cell type-specific expression of genes is in part determined by the expression and activation of transcription factors. It is important to recognize that the modes of regulating expression of the same gene product may differ markedly among different cell types. For example, transcriptional regulation of the α₂(I) collagen gene is different between stellate cells and skin fibroblasts, even though the protein product ultimately produced may be identical.[10] This clearly indicates that one must study stellate cells and not other fibrogenic cell types for findings to be directly relevant to liver fibrosis.

The capacity of transcription factors to interact with DNA or other proteins can be significantly influenced by posttranslational modifications. In addition, the nature of the DNA sequence may inform the protein in a way that alters its conformation or capacity to bind other transcription factors or coactivators.[11] Posttranslational modifications may include phosphorylation, sumoylation, prenylation, acetylation, and glycosylation (see Buschmann et al,[12] Lakin and Jackson,[13] Berk,[14] Ito et al[15] Appella and Anderson,[16] Comer and Hart,[17] and references therein for reviews). These modifications can elicit a broad range of biological outcomes, including changes in either intracellular localization, transcriptional activity, targeting for degradation, binding affinity to DNA or other proteins, oligomerization, and half-life. Some of these alterations in transcriptional activity have been demonstrated in hepatic stellate cells, with several examples provided below.

Broadly, transcription factors are classified in families as defined by conserved motifs either in the DNA-binding domain or other regions of the molecule. Motifs within a transcription factor typically include at least a DNA binding domain and activation domain, the latter necessary for interacting with other proteins in assembling a transcriptional complex. For example, zinc finger transcription factors all share a DNA binding motif in which a loop of amino acids, typically containing a fixed number of histidines or cysteines are bound together in a complex by a zinc ion, and their activity can be abrogated by chelators that sequester zinc.[18] These zinc fingers typically bind DNA by intercalating into the major groove of DNA, but they also may be essential for protein-protein interactions.[19] [20] The nature of activation domains within zinc finger proteins can diverge tremendously in their sequence, length, and location within the molecule. Transcription factors also may be defined by their localization or bifunctional nature, for example STATs (signal transducers and activators of transcription). Some of the transcription factor families relevant thus far to stellate cells include helix-loop-helix proteins, winged helix proteins, LIM domain protein (named for Lin-11, Isl-1, and Mec-3 genes), Rel homology domain proteins (NF-κB nuclear hormone receptors, including peroxisome proliferator-activated receptors [PPARs] and retinoic acid receptors [RARs]), CCAAT/enhancer binding proteins (or bZIP domain proteins), and SMADs (Table [1]). It is certain, however, that the number of transcription factors identified thus far in stellate cells represents only a small fraction of those actually regulating stellate cell behavior. Thus, rapid expansion of the list of transcription factors expressed by stellate cells is expected, particularly with the recent completion of the human genome sequencing and expanded use of microarrays.

CHROMATIN REMODELING ENZYMES AND TRANSCRIPTION

The chromatin structure plays a major role in transcriptional regulation. Chromosomal DNA is not free, but bound to complexes of histone proteins. The basic structural unit of chromatin is the nucleosome, composed roughly of 146 base pairs of DNA wrapped tightly around a disc-shaped core of histone proteins. The higher ordered structure of chromatin can be described as a string of linked nucleosomes that are further folded into a compact and condensed structure. A number of transcriptional coactivators and corepressors have recently been identified that are capable of remodeling chromatin, resulting in the enhancement or repression of transcription (for review see Fry[21] and the article by Geerts in this issue).

A family of transcriptional coactivators termed HATs for their inherent histone acetyl transferase enzymatic activity has been identified. These coactivators can covalently modify by acetylation lysine residues within the amino-terminal domains of nucleosomal histones. Multiple acetylations by HATs are thought to enhance transcription by destabilizing the higher order folding and condensation of chromatin. In contrast to HATs, a family of transcriptional corepressors termed histone deacetylases (HDACs) repress transcription through their opposing enzymatic activity that can remove acetyl groups from nucleosomal histones.

HATs and HDACs can recognize and bind to transcription factors and other cofactors in the transcriptional preinitiation complex. Typically, multiple HAT family members or multiple HDAC family members are recruited to the complex. Therefore, HAT coactivator enrichment of a preinitiation complex leads to the enhanced transcription of a gene, whereas HDAC corepressor enrichment leads to repression. Still another level of transcriptional regulation by HATs and HDACs is their release from preinitiation complexes. So conversely, HAT coactivator release leads to transcriptional repression, whereas HDAC corepressor release leads to gene transcriptional activation. Therefore, a transcription factor can function either as an activator or repressor depending on its modulation by the chromatin remodeling cofactors, HATs, and HDACs.

What then determines cofactor recruitment or release of either HATs or HDACs at a given transcriptional preinitiation site? Cellular responses to stimuli result in transcription factor and cofactor modifications such as the phosphorylation and dephosphorylation of specific amino acid residues within the proteins. These modifications alter the conformation of proteins leading to either the exposure or concealment of potential interaction sites between transcription factors and the chromatin remodeling cofactors. Therefore, gene activation or inactivation by transcriptional regulation is highly sensitive to stimuli that alter the acetylation or phosphorylation status of chromatin or DNA-binding proteins.

PARADIGMS OF TRANSCRIPTIONAL REGULATION IN HEPATIC STELLATE CELLS

Exploration of transcriptional regulation in stellate cells has focused on the role of transcription factors in cellular activation accompanying liver injury and fibrosis. Key stimuli eliciting changes in transcriptional activity have been identified. In liver injury, for example, oxidative stress from hepatocytes and inflammatory cells or cellular fibronectin from sinusoidal endothelial cells can initiate the activation of stellate cells. Following initial activation, the stellate cell is responsive to a range of extracellular stimuli, particularly cytokines, which can modulate the expression of structural genes, including α₁(I) collagen.[22] Recent studies also have underscored the potential of activated stellate cells to revert to a quiescent state or to undergo programmed cell death, a process termed apoptosis (see article by Iredale in this issue).

In the following sections are examples in which specific transcriptional events in stellate cells have been elucidated. This is not intended as a comprehensive list, but rather to display the broad range of regulatory mechanisms and factors that contribute to the cell's unique and highly plastic phenotype.

CCAAT/Enhancer-Binding Proteins

A role for CCAAT/enhancer-binding proteins (C/EBPs) in modulating transcriptional activity of the α₁(I) collagen gene in stellate cells has recently been demonstrated.[23] [24] [25] The C/EBPs comprise a family of transcription factors that are critical for cellular differentiation in a variety of tissues (reviewed by Lekstrom-Himes and Xanthopoulos[26]) and also have roles in the regulation of hepatocyte regeneration (reviewed by Diehl[27]). In stellate cells, C/EBPδ and two isoforms of C/EBPβ modulate the transcriptional activity of the α₁(I) collagen gene.

The prototypic C/EBP is a modular protein consisting of an activation domain, a dimerization bZIP region, and a DNA-binding domain. All family members have the conserved dimerization domain, required for DNA binding, through which they form homo- and heterodimers with other family members. C/EBPs are least conserved in their activation domains and vary from strong activators to dominant negative repressors. Thus, it is thought that dimerization of different C/EBPs precisely modulates transcriptional activity of target genes.

C/EBPS INDUCED BY OXIDATIVE STRESS LEAD TO TRANSCRIPTIONAL UPREGULATION OF THE α₁(I) COLLAGEN GENE

Numerous studies have implicated oxidative stress and transforming growth factor β (TGF-β) in liver fibrosis, activation of HSCs, and increased type I collagen expression.[28] [29] Ethanol induces oxidative stress through its metabolism via the microsomal P450-dependent system, which consequently generates free radicals.[30] [31] Ethanol is also metabolized by alcohol dehydrogenase, and its metabolite acetaldehyde is modestly fibrogenic.[32]

Recent reports now demonstrate a shared mechanism involving C/EBPs that accounts for upregulation of the α₁(I) collagen gene by either TGF-β or acetaldehyde.[23] [25] Both compounds induce oxidative stress in stellate cells, which is mediated by increased accumulation of hydrogen peroxide (H₂O₂). This in turn leads to DNA-binding activity of C/EBPs at the α₁(I) collagen promoter and its transactivation. Direct H2O₂ treatment of HSCs elicits similar results. In contrast, catalase, an H₂O₂ enzyme scavenger, abrogates the TGF-β or acetaldehyde-induced C/EBP DNA-binding activity and α1(I) collagen gene upregulation. The cis-acting regulatory element responsive to TGF-β or acetaldehyde has been localized to the α₁(I) collagen promoter (-378 to -344).

C/EBPβ is the C/EBP family member involved in upregulation of α₁(I) collagen. However, there are actually two isoforms of C/EBPβ, p35 C/EBPβ and p20 C/EBPβ. The p35 isoform is a prototypic C/EBP, whereas the p20 isoform lacks the activation domain and is perhaps a repressor form of C/EBPβ. The p20 isoform also binds to DNA with higher affinity. The ratio of these two isoforms is thought to modulate transcriptional activity. Both isoforms are induced by the oxidative stress mediated by H₂O₂, TGF-β, or acetaldehyde and can bind to the α₁(I) collagen promoter. Induction of the p35 isoform is far greater than the p20 isoform, and this presumably leads to transcriptional upregulation.

C/EBPS INDUCED BY TUMOR NECROSIS FACTOR-α LEAD TO TRANSCRIPTIONAL REPRESSION OF THE α₁(I) COLLAGEN GENE

In contrast to TGF-β or acetaldehyde, tumor necrosis factor-α (TNF-α) inhibits type I collagen synthesis in stellate cells.[24] Interestingly, a TNF-α responsive element in the α₁(I) collagen promoter colocalizes with the TGF-β and acetaldehyde responsive element. Modulation of C/EBP family members and C/EBPβ isoforms at this cis-acting responsive element accounts for transcriptional repression of the α₁(I) collagen gene.

TNF-α enhances the nuclear concentration and DNA-binding activity of C/EBPδ and both the p20 and p35 isoforms of C/EBPβ. In contrast to TGF-β, acetaldehyde induction of C/EBPβ, the p20 C/EBPβ repressor isoform, is induced more prominently. C/EBPδ, although a prototypic C/EBP, may modulate transcriptional repression through its DNA-binding domain, which contains two proline and four glycine residues that diminish DNA binding affinity.[26]

The mechanism by which the p20 isoform of C/EBPβ is generated is controversial. Alternative translational initiation or limited proteolytic cleavage of the p35 isoform have been suggested.[33] [34] TNF-α is known to induce multiple proteolytic activities, including matrix metalloproteinases[35] and several members of the adamalysin family of proteolytic enzymes.[36] Thus, intracellular proteolytic processing of the p35 to p20 isoform of C/EBPβ is an intriguing possibility that should be explored.

c-Myb and Cyclic AMP Response Element Binding Protein (CREB)

A hallmark of stellate cell activation is the expression of α-SMA.[6] The transcription factor c-myb modulates transcription of the α-SMA gene in stellate cells through its binding to the proximal E box motif in the α-SMA promoter.[37] [38] High levels of c-myb expression and DNA-binding activity are found in activated HSCs but not in quiescent cells. More importantly, the expression of c-myb can be induced in quiescent HSCs in vitro by oxidative stress mediated by the generation of free radicals using either ascorbate/FeSO₄ or malondialdehyde, a product of lipid peroxidation. In addition, HSC proliferation and activation through these inducers of oxidative stress, or with TGF-α or collagen type I substratum, can all be blocked by antioxidants such as d-alpha-tocopherol and butylated hydroxytoluene. These findings emphasize the enlarging importance of oxidative stress as a mediator of stellate cell activation. Expression of c-myb in HSCs in vivo is also stimulated in carbon tetrachloride-induced liver injury[37] and in patients with chronic hepatitis C.[39]

Phosphorylation of the transcription factor CREB at serine 133 (CREB-PSer133) is an important determinant of HSC quiescence by inhibiting the entry of stellate cells into S phase of the cell cycle.[40] The introduction into quiescent cells of a mutated CREB that cannot be phosphorylated at serine 133 (CREB-Ala133) promotes a fivefold increase in cells that enter the S phase, as determined by proliferating cell nuclear antigen staining. However, the mutant CREB-Ala133 cannot promote proliferation when quiescent cells are treated with the antioxidant butylated hydroxytoluene, suggesting that oxidative stress affects the cascade leading to HSC proliferation at a site distal to CREB.

Nuclear Factor κB

Nuclear factor κB represents a family of inducible transcription factors that are activated by a variety of stimuli, including viral infection, lipopolysaccharide, oxidative stress, TNF-α, and interleukin-1 (IL-1) (reviewed by Verma et al[41]). The NF-κB family is comprised of at least five members that form homo- or heterodimers, including p65 (RelA), p50, p52, RelB, and c-Rel. They are related by their Rel homology domain, which consists of a DNA-binding domain, a dimerization domain, and a nuclear localization domain. Only p65, c-Rel, and RelB contain transcriptional activation domains. Classic transcriptionally active NF-κB is a heterodimer of p65:p50, but NF-κB may consist of a variety of homo- and heterodimers. p50 and p52, which lack activation domains, are generally considered to be repressors when bound to DNA as homodimers.

In most cell types, NF-κB is found in the cytoplasm as an inactive dimer bound to one of the IκB inhibitory proteins (IκBα, IκBβ, or IκBγ) that mask its nuclear localization signal. Following specific signaling, phosphorylation of IκBα at serines 32 and 36 by IκB kinase[42] leads to its ubiquitinylation and consequent degradation by the proteasome. The NF-κB dimer then translocates into the nucleus. NF-κB transcriptional activity is in part due to p65 interaction with the transcriptional coactivator CBP/p300. This NF-κB transcriptional activity can be further enhanced by protein kinase A-mediated phosphorylation of p65 at serine 276, which exposes an additional site for CBP/p300 interaction by weakening a tertiary interaction between N- and C-terminal regions of p65.[43] [44]

NF-κB IN STELLATE CELLS

Although the mechanisms of NF-κB regulation have been extensively studied, the complexity of how it modulates target genes is still unfolding. This provides a cautionary note in trying to develop simplistic paradigms in understanding its regulation in stellate cells. Several studies have compared NF-κB activity between quiescent and activated HSCs.[45] [46] [47] Although NF-κB activity is increased in culture-activated HSCs with more nuclear p50:p65 heterodimers present, it is required neither for proliferation nor for cellular activation.[48] When NF-κB is inactivated by overexpression of the inhibitory IκB protein in quiescent HSCs, there is no effect on α-SMA or α₁(I) collagen gene expression during subsequent HSC activation, indicating that these functions are NF-κB independent. On the other hand, NF-κB activity does play an important role in preventing apoptosis of activated stellate cells.[47] [48] Furthermore, the NF-κB activity in activated HSCs can be stimulated above its basal levels by cytokines, which leads to downregulation of the α₁(I) collagen gene[46] and upregulation of other genes such as IL-6.[49]

During cellular activation of HSCs, NF-κB activity is increased and modulated by IκB family members. IκB-α levels are reduced, leading to translocation of NF-κB dimers into the nucleus.[45] [47] IκB-β levels are transiently reduced but later restored with a hypophosphorylated form that, rather than inhibiting NF-κB activity, shields it from IκBα interaction.[47] Bcl3, a member of the IκB family, which like IκB-β can function as a positive regulator, is also upregulated in activated HSCs. Thus, both IκB-β and Bcl3 may sustain the transcriptional activity of NF-κB in activated HSCs.[47]

The predominant nuclear NF-κB dimer in quiescent HSCs is the p50:p50 homodimer.[46] In activated HSCs, IκB-α levels are reduced and NF-κB dimers p65:p65 and p50:p65 are translocated to the nucleus. These NF-κB dimers have been identified by electrophoretic mobility shift assays using nuclear extracts incubated with a radiolabeled consensus NF-κB DNA-binding site.[47] In activated HSCs, three DNA-binding complexes have been observed and have been assigned as complex 1, 2, or 3 based on their relative electrophoretic mobilities. Complex 1 is composed of p65:p65 homodimers, whereas complex 2 is composed of p65:p50 heterodimers. Surprisingly, complex 3 with the fastest electrophoretic mobility, did not contain any of the known NF-κB members, suggesting that other factors or potentially novel Rel-like factors comprise complex 3. Complex 3 can be formed from HSC nuclear extracts of several species, including mouse, rat, and human. Interestingly, autocrine-derived factors from the activated stellate cells are required to induce and maintain complex 3 formation at basal levels.

ACTIVITIES OF NF-κB INDUCED BY CYTOKINES

Tumor necrosis factor-α, a cytokine that downregulates α₁(I) collagen synthesis in HSCs, significantly upregulates both complex 2 (p50:p65) and complex 3.[47] Increased NF-κB activity inhibits α₁(I) collagen gene expression. However, an interesting twist is that NF-κB inhibition is not mediated through the near consensus NF-κB DNA-binding site (-56 to -47) in the α₁(I) collagen promoter, but instead through two potential GC-rich SP1 binding sites (-80 to -130) as demonstrated by loss of activity following mutation of these sites.[46] In addition, another SP1 binding site (+68 to +86) in the 5′ untranslated region of the first exon of the α₁(I) collagen is important for TNF-α-induced downregulation.[50] It is unclear how NF-κB exerts its inhibitory effects through the SP1 sites. NF-κB can physically interact with SP1 in HSCs, which may attenuate the SP1 activity on the α₁(I) collagen promoter.[46] Thus, the basal transcriptional activity of NF-κB is permissive to α₁(I) collagen gene expression, but its further induction by TNF-α then leads to inhibition of α₁(I) collagen gene expression, which is mediated through SP1 sites. These findings illustrate the extraordinary complexity in trying to dissect mechanisms of transcriptional regulation in stellate cells.

NF-κB activity also can be further induced by the cytokine IL-1β. Like TNF-α, this additional induction of NF-κB activity can occur only in activated HSCs, not in quiescent cells.[45] NF-κB activation by TNF-α or IL-1β leads to the expression of intercellular adhesion molecule type 1 (ICAM-1), and macrophage inflammatory protein type 2 (MIP-2), which both play a role in the immune response during tissue repair. Both TNF-α and IL-1β also lead to the expression of IL-6, an important mediator of hepatocyte regeneration. Furthermore, NF-κB activation by the cytokines endothelin-1 (ET-1) or TNF-α upregulates cyclooxygenase-2 (COX-2) expression that is partly inhibitory to cellular proliferation.[51]

Collectively, these findings document a persistent basal level of NF-κB activity in activated HSCs that is sustained by autocrine factors which preserve the cells in a profibrogenic and proliferative state. In contrast, further upregulation of NF-κB activity by various cytokines maintains the activated cells in a less fibrogenic and less proliferative state, expressing proinflammatory molecules ICAM-1, MIP-2, and COX-2 that may be more amenable to tissue repair. Thus, extracellular factors such as cytokines determine the state of NF-κB activation, the extent of which dictates its appropriate physiologic response (Fig. [1]).

Peroxisome Proliferator-Activated Receptors (PPARs)

Recent exciting studies have identified a role of PPARs in stellate cell activation. PPARs are a family of ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily.[52] PPARs are involved in hepatic lipid metabolism[53] and adipocyte differentiation.[54] Three mammalian subtypes have been identified, referred to as PPAR-α, -β, and γ, which are encoded by separate genes. Although PPAR-γ is predominantly expressed in adipose tissue and plays a key role in adipocyte differentiation and lipid metabolism, it is also expressed in other tissues at much reduced levels.[55]

Interestingly, PPAR-γ mRNA is expressed in HSCs of normal rat liver and its expression is greatly reduced in HSCs from cholestatic liver fibrosis induced by bile duct ligation.[56] A decrease in PPAR-γ protein is also detected following culture activation of human HSCs, suggesting that reduced levels of this transcription factor occur during HSC activation/transdifferentiation.[57] Remarkably, the treatment of culture-activated HSCs with PPAR-γ ligands inhibits HSC proliferation and the expression of α-SMA, type I collagen, and monocyte chemotactic protein 1, a potent chemokine secreted by activated HSCs that recruits and activates monocytes and T-lymphocytes.[56] [57] [58] Thus, these studies indicate that treatment with PPAR-γ ligands can reverse key phenotypic features of activated HSCs.

PPAR-γ functions as a heterodimer and requires another member of the nuclear hormone receptor family, 9-cis retinoic acid (9-cisRA) receptor (RXR) in order to be transcriptionally active. A cquisition of the activated phenotype of HSCs has been correlated with reduction of RXR and retinoic acid receptors,[59] as well as with the depletion of retinoids in culture[4] and in vivo.[5] Thus, it is conceivable that vitamin A metabolites and PPAR-γ agonists cooperate in maintaining the quiescent phenotype of HSCs in normal liver.

A surprising finding is that the treatment of activated HSCs with PPAR-γ ligands restores the level of PPAR-γ mRNA to amounts representative of quiescent HSCs.[56] If the potential reversal of the activated phenotype of HSCs by PPAR-γ agonists is due to PPAR-γ transcriptional activity, it must do so as a heterodimer with RXR, and therefore perhaps RXR levels are restored as well. However, RXR levels have not been examined directly. Interestingly, the combinatorial treatment with both PPAR-γ and RXR agonists leads to a more pronounced inhibition on HSC proliferation.[58] These exciting results from culture studies highlight the need for in vivo investigations to determine if PPAR-γ and RXR agonists, synthetic or natural, will be beneficial in the treatment of liver fibrosis (see article by Bataller and Brenner in this issue).

Kruppel-like Zinc Finger Factors

Members of the Kruppel-like family of transcription factors have been identified in activated HSCs. This family of transcription factors shares homology with the zinc finger DNA-binding motif of the Drosophila melanogaster segmentation protein, Kruppel. The zinc fingers of this family contain a single zinc atom that is tetrahedrally coordinated by two cysteine and two histidine residues, referred to as a C₂H2 zinc finger. Multiple fingers are contiguously aligned that fit into the major groove of DNA. In addition to the conserved amino acid sequence in the zinc finger, these proteins share a conserved seven-amino acid interfinger spacer, TGEKP(Y/F)X. The zinc fingers bind to either GC-rich boxes or CACCC elements of promoters. The regions outside the zinc fingers are usually unique. Studies to date have shown that Kruppel-like family members play key roles in a diverse range of biological processes, including cell growth, differentiation, embryogenesis, and tumorigenesis.[60]

KRUPPEL-LIKE FACTOR 6: POTENTIAL ROLE IN HSC ACTIVATION

We have reported that the Kruppel-like factor KLF6 (formerly Zf9/CPBP/GBF) is rapidly induced during HSC activation in vivo as an immediate-early gene.[61] The level of KLF6 protein increases significantly in HSC within only 3 hours of CCl₄-induced liver injury. This rapid induction of KLF6 is also observed in culture-induced activation of HSCs. Similarly, in an adipocyte differentiation model using 3T3-L1 cells, KLF6 is also rapidly induced when cells are stimulated to differentiate.[62] [63] These observations have led to the speculation that KLF6 may have a role in regulating cellular differentiation.

Our laboratory has identified a number of putative transcriptional targets of KLF6 that could contribute to the fibrogenic activity of stellate cells, including collagen α₁(I),[61] TGF-β1 and its signaling receptors,[64] and urokinase-type plasminogen activator,[65] which may activate latent TGF-β1. In view of its ubiquitous distribution, however, the targets of KLF6 are likely to differ among tissues and at different stages of stellate cell activation or quiescence. Moreover, most of the putative targets in stellate cells have been identified by transiently transfecting KLF6 with the appropriate promoter fragment as a reporter gene. This type of analysis is somewhat artificial because reporter genes lack a physiologic chromatin context, and thus the findings must be confirmed by analyzing the endogenous promoter and its protein product. So far, urokinase plasminogen activator is the only fibrosis-related target in which upregulation of the endogenous gene has been verified in stellate cells.

Another candidate target of KLF6 as identified by promoter reporter gene analysis is the leukotriene C₄ (LTC4) synthase gene.[66] Although leukotriene C₄ is expressed predominantly by inflammatory immune cells, activated HSCs produce significant quantities of LTC₄.[67] In the asthmatic lung, LTC₄ provokes smooth muscle contraction and recruits leukocytes.[68] Studies have suggested that HSC production of LTC₄ promotes HSC proliferation[67] and α₁(I) collagen gene expression in vitro.[69] KLF6 upregulation of the endogenous LTC₄ synthase gene in stellate cells awaits verification.

The Kruppel-like family member BTEB/KLF9 (basic transcription element binding protein/Kruppel-like factor 9) is associated with matrix remodeling in HSCs. Acetaldehyde, a stimulant of oxidative stress, induces KLF9 protein expression and its DNA-binding activity to a distal GC-rich box element (-1484 to -1476) in the rat α₁(I) collagen promoter, resulting in the upregulation of collagen synthesis.[70] Although other roles for KLF9 activity in HSCs have yet to be explored, the thyroid hormone T3 can modulate KLF9 expression in neuronal cells of the developing nervous system.[71] HSCs are thought to be developmentally derived from neural origins, and they retain expression of neural markers such as RhoN, glial fibrillar acidic protein, nestin, and neurotrophin receptors. The recent finding that HSCs undergo apoptosis in response to nerve growth factor (see article by Iredale in this issue) and the demonstration that thyroid hormones promote hepatocyte regeneration[72] invite us to explore if there are thyroid hormone responses in stellate cells.

SP1 and SP3 are Kruppel-like family members that also play a role in oxidative stress mediated upregulation of the α₁(I) collagen gene.[73] [74] SP1 DNA-binding activity is greatly increased in activated HSCs compared with quiescent cells. Iron-induced oxidative stress in activated HSCs increases SP1 and SP3 protein levels and their DNA-binding activity to the murine α₁(I) collagen promoter. Antioxidant pretreatment of cells blocks both the increase in SP1 protein and upregulation of collagen.

CONCLUSIONS AND FUTURE DIRECTIONS

It is too simplistic based on current knowledge to derive simple rules summarizing mechanisms of transcriptional regulation in stellate cells. The full range of complexity in regulating gene expression in all tissues is equally relevant to stellate cells, and to date few mechanisms have been uncovered in this cell type that are totally unique (a notable exception is the presence of an apparently novel stellate cell-specific response element in the TIMP-1 promoter; see article by Benyon and Arthur in this issue). Thus, the output of gene expression in stellate cells, as in all tissues, is a highly integrated readout of hundreds if not thousands of cooperating and competing signals. Efforts will increasingly analyze these many simultaneous and sequential events at different stages during stellate cell quiescence, activation, and apoptosis using gene arrays and proteomics. These findings will assist in formulating a hierarchy of gene expression in stellate cells, an effort that has barely begun. More refined methods of analyzing gene expression in vivo and in systems in which chromatin maintains a more native conformation are anticipated. In addition, genetic models will be developed in which expression of specific factors is enhanced or abrogated in stellate cells to define mediators critical to their behavior. These efforts are likely to uncover attractive targets for inhibiting stellate cell activation in treating hepatic fibrosis. Equally important, they will reveal new insights into the role of stellate cells in normal liver function and homeostasis, with implications for controlling metabolic, regenerative, and neoplastic pathways in normal and diseased liver.

ABBREVIATIONS

C/EBP CCAAT/enhancer-binding protein

COX-2 cyclooxygenase-2

GTF general transcription factor

HAT histone acetyl transferase

HDAC histone deacetylase

HSC hepatic stellate cell

ICAM-1 intercellular adhesion molecule type 1

IL-1 interleukin-1

LTC₄ leukotriene C4

mRNA messenger RNA

NF-κB nuclear factor κB

α-SMA α-smooth muscle actin

STATs signal transducers and activators of

transcription

TGF-β transforming growth factor-β

TNF-α tumor necrosis factor-α

REFERENCES

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REFERENCES

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 3 Bissell D M. Lipocyte activation and hepatic fibrosis.  Gastroenterology . 1992;  102 1803-1805
  PubMedSearch in Google Scholar
• 4 Friedman S L, Wei S, Blaner W S. Retinol release by activated rat hepatic lipocytes: regulation by Kupffer cell-conditioned medium and PDGF.  Am J Physiol . 1993;  264 947-952
  PubMedSearch in Google Scholar
• 5 Tsukamoto H, Cheng S, Blaner W S. Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation.  Am J Physiol . 1996;  270 G581-586
  PubMedSearch in Google Scholar
• 6 Rockey D C, Boyles J K, Gabbiani G, Friedman S L. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture.  J Submicrosc Cytol Pathol . 1992;  24 193-203
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  PubMedSearch in Google Scholar
• 9 Lemon B, Tjian R. Orchestrated response: a symphony of transcription factors for gene control.  Genes Dev . 2000;  14 2551-2569
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• 10 Inagaki Y, Mamura M, Kanamaru Y. Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells.  J Cell Physiol . 2001;  187 117-123
  CrossrefPubMedSearch in Google Scholar
• 11 Lefstin J A, Yamamoto K R. Allosteric effects of DNA on transcriptional regulators.  Nature . 1998;  392 885-888
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 14 Berk A J. Regulation of eukaryotic transcription factors by post-translational modification.  Biochim Biophys Acta . 1989;  1009 103-109
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• 15 Ito A, Lai C H, Zhao X. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2.  EMBO J . 2001;  20 1331-1340
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• 17 Comer F I, Hart G W. O-GlcNAc and the control of gene expression.  Biochim Biophys Acta . 1999;  1473 161-171
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 37 Lee K S, Buck M, Houglum K, Chojkier M. Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.  J Clin Invest . 1995;  96 2461-2468
  PubMedSearch in Google Scholar
• 38 Buck M, Kim D J, Houglum K, Hassanein T, Chojkier M. c-Myb modulates transcription of the alpha-smooth muscle actin gene in activated hepatic stellate cells.  Am J Physiol Gastrointest Liver Physiol . 2000;  278 G321-328
  PubMedSearch in Google Scholar
• 39 Houglum K, Venkataramani A, Lyche K, Chojkier M. A pilot study of the effects of d-alpha-tocopherol on hepatic stellate cell activation in chronic hepatitis C.  Gastroenterology . 1997;  113 1069-1073
  PubMedSearch in Google Scholar
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