Anping Chen

1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation

1,25-Dihydroxyvitamin D3(1,25(OH)2D3) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)2D3 specifically activated protein kinase C-α (PKC-α) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)2D3 activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)2D3 caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1,25(OH)2D3 also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)2D3 also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)2D3 was mediated by ERK and JNK. Using a specific inhibitor of the Ca2+-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-α cDNA, demonstrated that PKC-α mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1,25(OH)2D3-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)2D3 stimulated AP-1 activation in CaCo-2 cells by a PKC-α- and JNK-dependent mechanism leading to increases in cellular differentiation.

The DNA Binding Protein BTEB Mediates Acetaldehyde-Induced, Jun N-Terminal Kinase-Dependent αI(I) Collagen Gene Expression in Rat Hepatic Stellate Cells

ABSTRACT Alcohol-induced cirrhosis results partially from the excessive production of collagen matrix proteins, which, predominantly αI(I) collagen, are produced and secreted by activated hepatic stellate cells (HSC). The accumulation of αI(I) collagen in HSC during cirrhosis is largely due to an increase in αI(I) collagen gene expression. Acetaldehyde, the major active metabolite of alcohol, is known to stimulate αI(I) collagen production in HSC. However, the mechanisms responsible for it remain unknown. The aim of this study was to elucidate the mechanisms by which αI(I) collagen gene expression is induced by acetaldehyde in rat HSC. In the present study, the acetaldehyde response element was located in a distal GC box, previously described as the UV response element, in the promoter of the αI(I) collagen gene (−1484 to −1476). The GC box was predominantly bound by the DNA binding transcription factor BTEB (basic transcription element binding protein), expression of which was acetaldehyde and UV inducible. Blocking BTEB protein expression significantly reduced the steady-state levels of the acetaldehyde-induced αI(I) collagen mRNA, suggesting that BTEB is required for this gene expression. Further studies found that acetaldehyde activated Jun N-terminal kinase (JNK) 1 and 2 and activator protein 1 (AP-1) transactivating activity. Inhibition of JNK activation resulted in the reduction of the acetaldehyde-induced BTEB protein abundance and αI(I) collagen mRNA levels, indicating that the expression of both genes is JNK dependent in HSC. Taken together, these studies demonstrate that BTEB mediates acetaldehyde-induced, JNK-dependent αI(I) collagen gene expression in HSC.

UV Irradiation Activates JNK and Increases αI(I) Collagen Gene Expression in Rat Hepatic Stellate Cells

Hepatic stellate cells (HSCs) become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of αI(I) collagen gene expression is a central pathogenetic step during the development of cirrhosis. Our recent study in rat HSCs (Davis, B. H., Chen, A., and Beno, D. (1996) J. Biol. Chem. 271, 11039–11042) found that ERK1,2 activation might be required for maximal αI(I) collagen gene expression. However, the role of the parallel JNK cascade in regulating αI(I) collagen gene expression was unknown. In this study, we initially found that UV irradiation of HSCs activated JNK but not ERK1,2. Furthermore, UV irradiation increased endogenous α I(I) collagen mRNA abundance and stimulated α I(I) collagen gene transcription in HSCs. The effect of the activation of JNK and Jun on α I(I) collagen gene expression was further evaluated via transfection of chloramphenicol acetyltransferase reporter plasmids with various sizes of truncated 5′ upstream promoter sequence (UPS) of the αI(I) collagen gene. This revealed that dominant negative transcription factor JUN suppressed α I(I) collagen gene transcription in HSCs maintained in media with 20% serum and constitutively activated JUN increased αI(I) collagen gene transcription in HSCs cultured in media with 0.4% serum. UV activated JNK utilized a distal GC box in the 5′-UPS of the collagen gene to regulate gene transcription. This observation was confirmed by site-directed mutagenesis. In co-transfection experiments, the col-chloramphenicol acetyltransferase reporter with a mutagenized GC box was not suppressed by dn-JUN and was not stimulated by activated JUN or by UV irradiation. Southwestern blotting analyses and gel shift assays with basic transcription element-binding protein antiserum suggested that the GC box was bound by basic transcription element-binding protein, a recently described DNA-binding protein. In conclusion, the current study combined with our previous report suggests that ERK1,2 and JNK cascades regulate αI(I) collagen expression in HSCs through different regions of the 5′-UPS of the gene. The distal GC box in the 5′-UPS of the αI(I) collagen gene may play a central role in receiving extracellular signals through the JNK pathway.

The Antioxidant (–)-Epigallocatechin-3-gallate Inhibits Rat Hepatic Stellate Cell Proliferation in Vitro by Blocking the Tyrosine Phosphorylation and Reducing the Gene Expression of Platelet-derived Growth Factor-β Receptor

During hepatic fibrogenesis, quiescent hepatic stellate cells (HSC) become active and trans-differentiate into myofibroblast-like cells. This process coincides with an increase in cell proliferation, loss of stored vitamin A droplets, and excessive production and deposition of extracellular matrix components. HSC activation is coupled with the sequential expression of cytokine receptors, including platelet-derived growth factor-β receptor (PDGF-βR). Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays critical roles in activation of HSC during hepatic fibrogenesis. We have recently demonstrated that the antioxidant (–)-epigallocatechin gallate (EGCG), a major component in green tea extracts, significantly inhibited the proliferation of passaged HSC. The aim of the present study is to elucidate the underlying mechanisms. Since PDGF is a potent mitogen for HSC and mediates the early proliferative response, it was hypothesized that EGCG might inhibit HSC proliferation by interfering with the PDGF signal transduction. In this report, we demonstrated that EGCG, in two steps, significantly and effectively inhibited the proliferation of primary and passaged HSC. The polyphenolic compound initiated its inhibitory action by rapidly blocking the phosphorylation of tyrosines in PDGF-βR elicited by PDGF in serum. This action was short lived, persisting for a few hours. In addition, this antioxidant inhibited the gene expression of PDGF-βR by blocking the activation of transcription factors activator protein-1 and NF-κB, which were required for the gene transcription. The latter action remained effective for no less than 48 hours. These results provided a novel insight into the mechanisms by which EGCG inhibits HSC growth. The inhibitory effect of the natural antioxidant, its long history of beverage consumption without adverse health effects, and higher potent antioxidant capability make it a good candidate for therapeutic treatment and prevention of hepatic fibrosis.

E-box-binding Repressor Is Down-regulated in Hepatic Stellate Cells during Up-regulation of Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Expression in Early Hepatic Fibrogenesis

Hepatic stellate cells become activated during the early stages of hepatic injury associated with fibrogenesis. The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGFIIR) plays an important role in early fibrogenesis by participating in the activation of latent transforming growth factor-β, a potent inducer of the matrix proteins in activated stellate cells that define the fibrotic phenotype. In this study we examined hepatic stellate cell regulation of M6P/IGFIIR expression and found that M6P/IGFIIR mRNA transcript levels increased in stellate cells from rats exposed to carbon tetrachloride (CCl4), a potent fibrogenic stimulant. Two E-boxes residing in the proximal promoter of M6P/IGFIIR were found to each bind a novel 75-kDa transcription factor (P75) in quiescent stellate cells of normal livers. This E-box binding was down-regulated as an early response in stellate cells exposed to CCl4, coinciding with increased M6P/IGFIIR transcript levels. Mutagenized E-boxes in M6P/IGFIIR promoter-chloramphenicol acetyltransferase (CAT) reporter constructs produced a substantial increase in reporter expression when compared with the corresponding native promoter-CAT construct when transfected in culture-activated stellate cells, suggesting P75’s role as a repressor. The results indicate P75’s participation in the regulation of M6P/IGFIIR transcription in hepatic stellate cells during fibrogenesis.