Bernard H. Davis

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.

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.

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.

Prostaglandin E Suppresses Hepatic Fibrosis: Section I. The In Vivo Approach; Section II. The In Vitro Approach.

Recent studies suggest that prostaglandin E may have the ability to suppress cytokine responsiveness. We examined the effects of prostaglandin E administration on several parameters of the acute and chronic hepatic injury induced by bile duct ligation. Enisoprost, a prostaglandin E1 analog was found to suppress early hepatic and Ito cell type I collagen gene expression without diminishing the induction of the fibrogenic cytokine, transforming growth factor β. Overall hepatic inflammation and cell proliferation were not altered, suggesting that prostaglandin E acts distal to the initial injurious event(s). During the later phases, drug administration reduced total collagen accumulation as well as type I collagen periductular infiltration associated with early nodule formation.Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo coincident with the de novo expression of Ito cell platelet-derived growth factor β (PDGFβ) receptor messenger RNA. PDGF-induced mitogenesis was studied in cultured rat hepatic Ito cells which resemble the myofibroblast associated with liver injury. Pretreatment with prostaglandin E markedly suppressed the PDGF response in a dose-dependent fashion. The PDGF-induced cascade was studied ± PGE to determine the level of regulation which induced the observed suppression. PGE caused no apparent diminution in the abundance of the surface PDGFβ receptor nor its subsequent activation and tyrosine phosphorylation following PDGF stimulation. The cytoplasmic “secondary messengers” mitogen-activated protein kinase pp42–44 and raf kinase appeared to be comparably induced and therefore unaffected by PGE. Raf perinuclear translocation was also intact, and comparable degrees of nuclear egr, fos, and jun expression occurred. Because other studies have suggested that many of these features of the PDGF cascade may be causally and sequentially linked, the data collectively suggest that the dominant PGE mitogenic suppressive effect resides at a Raf-MAP parallel pathway or at a nuclear level distal to the induction of these early growth response genes.