Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice.

UNLABELLED: Alcoholic hepatitis (AH) is a distinct spectrum of alcoholic liver disease (ALD) with intense neutrophilic (polymorphonuclear; PMN) inflammation and high mortality. Although a recent study implicates osteopontin (SPP1) in AH, SPP1 is also shown to have protective effects on experimental ALD. To address this unsettled question, we examined the effects of SPP1 deficiency in male mice given 40% calories derived from ad libitum consumption of the Western diet high in cholesterol and saturated fat and the rest from intragastric feeding of alcohol diet without or with weekly alcohol binge. Weekly binge in this new hybrid feeding model shifts chronic ASH with macrophage inflammation and perisinusoidal and pericellular fibrosis to AH in 57% (15 of 26) of mice, accompanied by inductions of chemokines (Spp1, Cxcl1, and interleukin [Il]-17a), progenitor genes (Cd133, Cd24, Nanog, and epithelial cell adhesion molecule), PMN infiltration, and clinical features of AH, such as hypoalbuminemia, bilirubinemia, and splenomegaly. SPP1 deficiency does not reduce AH incidence and inductions of progenitor and fibrogenic genes, but rather enhances the Il-17a induction and PMN infiltration in some mice. Furthermore, in the absence of SPP1, chronic ASH mice without weekly binge begin to develop AH. CONCLUSION: These results suggest that SPP1 has a protective, rather than causal, role for experimental AH reproduced in our model.

Hepatic stellate cell-derived delta-like homolog 1 (DLK1) protein in liver regeneration.

Hepatic stellate cells (HSCs) undergo myofibroblastic activation in liver fibrosis and regeneration. This phenotypic switch is mechanistically similar to dedifferentiation of adipocytes as such the necdin-Wnt pathway causes epigenetic repression of the master adipogenic gene Pparγ, to activate HSCs. Now we report that delta-like 1 homolog (DLK1) is expressed selectively in HSCs in the adult rodent liver and induced in liver fibrosis and regeneration. Dlk1 knockdown in activated HSCs, causes suppression of necdin and Wnt, epigenetic derepression of Pparγ, and morphologic and functional reversal to quiescent cells. Hepatic Dlk1 expression is induced 40-fold at 24 h after partial hepatectomy (PH) in mice. HSCs and hepatocytes (HCs) isolated from the regenerating liver show Dlk1 induction in both cell types. In HC and HSC co-culture, increased proliferation and Dlk1 expression by HCs from PH are abrogated with anti-DLK1 antibody (Ab). Dlk1 and Wnt10b expression by Sham HCs are increased by co-culture with PH HSCs, and these effects are abolished with anti-DLK Ab. A tail vein injection of anti-DLK1 Ab at 6 h after PH reduces early HC proliferation and liver growth, accompanied by decreased Wnt10b, nonphosphorylated ß-catenin, p-ß-catenin (Ser-552), cyclins (cyclin D and cyclin A), cyclin-dependent kinases (CDK4, and CDK1/2), p-ERK1/2, and p-AKT. In the mouse developing liver, HSC precursors and HSCs express high levels of Dlk1, concomitant with Dlk1 expression by hepatoblasts. These results suggest novel roles of HSC-derived DLK1 in activating HSCs via epigenetic Pparγ repression and participating in liver regeneration and development in a manner involving the mesenchymal-epithelial interaction.

Morphogens and hepatic stellate cell fate regulation in chronic liver disease.

Hepatic stellate cells (HSC) are the liver mesenchymal cell type which responds to hepatocellular damage and participates in wound healing. Although HSC myofibroblastic trans-differentiation (activation) is implicated in excessive extracellular matrix deposition, molecular understanding of this phenotypic switch from the viewpoint of cell fate regulation is limited. Recent studies demonstrate the roles of anti-adipogenic morphogens (Wnt, Necdin, Shh) in epigenetic repression of the HSC differentiation gene Pparγ as a causal event in HSC activation. These morphogens have positive cross-interactions which converge to epigenetic repression of Pparγ involving the methyl-CpG binding protein MeCP2. However, these morphogens expressed by activated HSC may also participate in cross-talk between HSC and hepatoblasts/hepatocytes to support liver regeneration, and their aberrant regulation may contribute to liver tumorigenesis. Implications of HSC-derived morphogens in these possibilities are discussed.

Epigenetic cell fate regulation of hepatic stellate cells.

Research in the past three decades has identified key mediators and signaling mechanisms responsible for myofibroblastic transdifferentiation (MTD) of hepatic stellate cells (HSC), the pivotal event in liver fibrogenesis. Yet, fundamental understanding of the MTD from the viewpoint of cell fate or lineage regulation has been elusive. Recent studies using genetic cell fate mapping techniques demonstrate HSC are derived from mesoderm and at least in part via septum transversum and mesothelium. HSC express markers for different cell types derived from multipotent mesenchymal progenitors. A regulatory commonality between differentiation of adipocytes and that of HSC is shown, and a shift from adipogenic to myogenic or neuronal phenotype characterizes HSC MTD. Central to this shift is a loss of expression of the master adipogenic regulator peroxisome proliferator activated receptor-γ (PPAR-γ). Restored expression of PPAR-γ and/or other adipogenic transcription factors reverses myofibroblastic HSC to differentiated cells. In MTD, Pparγ is epigenetically repressed by induction of methyl-CpG binding protein 2 and its enrichment to the promoter and polycomb repressive complex-facilitated histone H3 lysine 27 di/tri-methylation at the 3' exons. Blocking canonical wingless-related MMTV integration site (Wnt) signaling in myofibroblastic HSC with the co-receptor antagonist Dickkopf-1, abrogates these epigenetic mechanisms, restores PPAR-γ expression and HSC differentiation. Necdin, a melanoma antigen family protein, is identified as an upstream mediator for induction of the canonical Wnt10b and consequent Pparγ repression and HSC MTD. The identified morphogen-induced epigenetic regulation of Pparγ and HSC fate may serve as a novel target for manipulation of liver fibrosis and mesenchymal-epithelial interactions in liver regeneration.

The Necdin-Wnt pathway causes epigenetic peroxisome proliferator-activated receptor gamma repression in hepatic stellate cells.

Hepatic stellate cells (HSCs), vitamin A-storing liver pericytes, undergo myofibroblastic trans-differentiation or "activation" to participate in liver wound healing. This cellular process involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Necdin, a melanoma antigen family protein, promotes neuronal and myogenic differentiation while inhibiting adipogenesis. The present study demonstrates that necdin is selectively expressed in HSCs among different liver cell types and induced during their activation in vitro and in vivo. Silencing of necdin with adenovirally expressed shRNA, reverses activated HSCs to quiescent cells in a manner dependent on PPARγ and suppressed canonical Wnt signaling. Promoter analysis, site-directed mutagenesis, and chromatin immunoprecipitation demonstrate that Wnt10b, a canonical Wnt induced in activated HSCs, is a direct target of necdin. Necdin silencing abrogates three epigenetic signatures implicated in repression of PPARγ: increased MeCP2 (methyl CpG binding protein 2) and HP-1α co-repressor recruitments to Pparγ promoter and enhanced H3K27 dimethylation at the exon 5 locus, again in a manner dependent on suppressed canonical Wnt. These epigenetic effects are reproduced by antagonism of canonical Wnt signaling with Dikkopf-1. Our results demonstrate a novel necdin-Wnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPARγ.

MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis.

BACKGROUND & AIMS: Myofibroblast transdifferentiation generates hepatic myofibroblasts, which promote liver fibrogenesis. The peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of this process. We investigated epigenetic regulation of PPARgamma and myofibroblast transdifferentiation. METHODS: Chromatin immunoprecipitation (ChIP) assays assessed the binding of methyl-CpG binding protein 2 (MeCP2) to PPARgamma and chromatin modifications that silence this gene. MeCP2(-/y) mice and an inhibitor (DZNep) of the epigenetic regulatory protein EZH2 were used in the carbon tetrachloride model of liver fibrosis. Liver tissues from mice were assessed by histologic analysis; markers of fibrosis were measured by quantitative polymerase chain reaction (qPCR). Reverse transcription PCR detected changes in expression of the microRNA miR132 and its target, elongated transcripts of MeCP2. Myofibroblasts were transfected with miR132; PPARgamma and MeCP2 expressions were analyzed by qPCR or immunoblotting. RESULTS: Myofibroblast transdifferentiation of hepatic stellate cells is controlled by a combination of MeCP2, EZH2, and miR132 in a relay pathway. The pathway is activated by down-regulation of miR132, releasing the translational block on MeCP2. MeCP2 is recruited to the 5' end of PPARgamma, where it promotes methylation by H3K9 and recruits the transcription repressor HP1alpha. MeCP2 also stimulates expression of EZH2 and methylation of H3K27 to form a repressive chromatin structure in the 3' exons of PPARgamma. Genetic and pharmacologic disruptions of MeCP2 or EZH2 reduced the fibrogenic characteristics of myofibroblasts and attenuated fibrogenesis. CONCLUSIONS: Liver fibrosis is regulated by an epigenetic relay pathway that includes MeCP2, EZH2, and miR132. Reagents that interfere with this pathway might be developed to reduce fibrogenesis in chronic liver disease.

TNF-alpha represses transcription of human Bone Morphogenetic Protein-4 in lung epithelial cells.

Bone Morphogenetic Proteins are key signaling molecules in vertebrate development. Little is known about Bmp gene regulation in any organ. In Drosophila, the Bmp gene, dpp is regulated by Dorsal, the invertebrate homologue of Rel-NF-kB. In this study we examined whether TNF-alpha, which activates NF-kB, can regulate Bmp4 gene expression. TNF-alpha reduced Bmp4 mRNA in lung adenocarcinoma A549 cells and repressed transcriptional activity of the human Bmp4 promoter in a dose-dependent manner. Similar repression was observed when the Bmp4 promoter was co-transfected with a p65 (RelA) expression vector in the absence of TNF-alpha treatment, suggesting that RelA mediates the effect of TNF-alpha. In support of this finding, the repressor effect of TNF-alpha on Bmp4 was abrogated by a co-transfected dominant negative mutant of IkB (S32A/S36A). The human Bmp4 promoter contains 3 putative consensus binding sites for NF-kB. Surprisingly, only one of the latter binding sites was capable of binding NF-kB. Repressor effect of NF-kB was not dependent on any of the three binding sites, but localized to a 122 bp fragment which bound both RelA and SP1. SP1 stimulated transcription, whereas increasing doses of RelA opposed this effect. In vivo, TNF-alpha inhibited branching morphogenesis and LacZ gene expression in Bmp4-lacz transgenic lungs. These data support a model in which TNF-alpha-induced RelA interacts with SP1 to bring about transcriptional repression of Bmp4 gene. These findings provide a mechanistic paradigm for interactions between mediators of inflammation and morphogenesis with relevant implications for normal lung development and pathogenesis of disease.

Physical and functional interactions between homeodomain NKX2.1 and winged helix/forkhead FOXA1 in lung epithelial cells.

NKX2.1 is a homeodomain transcription factor that controls development of the brain, lung, and thyroid. In the lung, Nkx2.1 is expressed in a proximo-distal gradient and activates specific genes in phenotypically distinct epithelial cells located along this axis. The mechanisms by which NKX2.1 controls its target genes may involve interactions with other transcription factors. We examined whether NKX2.1 interacts with members of the winged-helix/forkhead family of FOXA transcription factors to regulate two spatially and cell type-specific genes, SpC and Ccsp. The results show that NKX2.1 interacts physically and functionally with FOXA1. The nature of the interaction is inhibitory and occurs through the NKX2.1 homeodomain in a DNA-independent manner. On SpC, which lacks a FOXA1 binding site, FOXA1 attenuates NKX2.1-dependent transcription. Inhibition of FOXA1 by small interfering RNA increased SpC mRNA, demonstrating the in vivo relevance of this finding. In contrast, FOXA1 and NKX2.1 additively activate transcription from Ccsp, which includes both NKX2.1 and FOXA1 binding sites. In electrophoretic mobility shift assays, the GST-FOXA1 fusion protein interferes with the formation of NKX2.1 transcriptional complexes by potentially masking the latter's homeodomain DNA binding function. These findings suggest a novel mode of selective gene regulation by proximo-distal gradient distribution of and functional interactions between forkhead and homeodomain transcription factors.

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy.

OBJECT: Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. METHODS: A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. CONCLUSIONS: Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

NKX2.1 regulates transcription of the gene for human bone morphogenetic protein-4 in lung epithelial cells.

Bone morphogenetic protein 4, BMP4, plays an important role in the development of various organs including the lungs. Little is known regarding the regulation of Bmp4 gene expression in any organ. In the lung, indirect evidence indicates that NKX2.1, a homeodomain transcriptional factor with a demonstrated role in lung morphogenesis, may be a potential upstream regulator of Bmp4 gene expression. In particular, Bmp4 mRNA is reduced or absent in Nkx2.1(-/-) lungs. The human Bmp4 gene has been reported to include two regions of promoter activity in an embryonal carcinoma cell line, Tera2EC. The hBmp4.1 promoter is located upstream of exon I, whereas the second promoter, hBmp4.2, is localized within intron 1 and upstream of exon II. In the current study, we used a co-transfection assay in lung epithelial cells to examine the response of the two hBmp4 promoters to transcriptional stimulation by NKX2.1. Two DNA sequences were identified on the hBmp4.1 promoter that bind NKX2.1 and serve as functional cis-active NKX2.1-responsive elements. Similarly, NKX2.1 stimulated transcription from the hBmp4.2 promoter through two consensus binding sites localized within 412 nucleotides from the site of transcriptional initiation. Thus, both hBmp4 promoters include specific cis-active elements that bind to and mediate transcriptional regulation by NKX2.1. These findings bear functional implications regarding the regulation of a key signaling molecule by a homeodomain transcriptional regulator of lung epithelial morphogenesis.

Timeless in lung morphogenesis.

The Clock gene, timeless, regulates circadian rhythm in Drosophila, but its vertebrate homolog is critical to embryonic development. Timeless was shown to be involved in murine urethral bud branching morphogenesis. We generated a polyclonal antibody to mouse TIMELESS (mTIM) and studied its distribution and its potential role during lung development, which also requires branching morphogenesis. In the early mouse embryo, TIM was localized to all organs, especially the neural epithelium. In embryonic day (E) 9.5 embryos, TIM was present in both epithelial and mesenchymal cells at the onset of lung morphogenesis. In E15 embryos, TIM decreased in the mesenchyme but remained pronounced in the epithelium of both large and small airways. Later, TIM was localized to a specific subset of epithelial cells with alveolar type 2 phenotype. This finding was verified by immunostaining of isolated alveolar type 2 cells. In the proximal airways, TIM was colocalized with CCSP to nonciliated columnar epithelial cells. Antisense oligonucleotides to mTim specifically inhibited branching morphogenesis of embryonic lungs in explant culture without affecting SpC expression an alveolar type 2 cell marker. In cultured lung cells, expression of TIM is independent of cell cycle and proliferation. These studies indicate that the function of Timeless is highly conserved in organs whose formation requires branching morphogenesis.

Transforming growth factor-beta inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors.

Transforming growth factor-beta (TGF-beta) represses surfactant protein B (Sp-B) gene transcription through a mechanism that remains unknown. A homeodomain and a forkhead transcription factor, NKX2.1 and HNF-3, respectively, are known activators of Sp-B transcription. Because SMADs are the effectors of TGF-beta-induced gene activation, we examined the possibility that gene repression by TGF-beta may also occur through interactions of SMADs with NKX2.1 and HNF-3. We found that lung epithelial carcinoma H441 cells contain SMAD2/3 and -4, which localize to the nucleus in response to TGF-beta treatment. The activity of a transfected Sp-B promoter/reporter construct was reduced in a dose-dependent manner by TGF-beta. Cotransfection with a mutant, constitutively activated form of the Tgf-beta type I receptor repressed Sp-B promoter activity in the absence of TGF-beta ligand. Dominant negative mutants of Smads blocked the repressor activity of TGF-beta. SMAD3, but not SMAD2, mediated the repressor activity of TGF-beta on the Sp-B promoter. Mutations within a 70-base pair domain that includes binding sites for NKX2.1, hepatocyte nuclear factor 3 (HNF-3), or cAMP response element-binding protein (CREB) eliminated SMAD3-dependent repression of Sp-B transcription. Electrophoretic mobility shift analysis showed no evidence for direct binding of SMAD3 to the Sp-B promoter, and a DNA binding mutant of SMAD3 also repressed Sp-B, suggesting that direct DNA binding of SMAD3 may not be required. Using a mammalian two hybrid assay, we found physical and functional interactions between SMAD3 and both NKX2.1 and HNF-3. Also, a glutathione S-transferase-fused SMAD3 directly binds to in vitro synthesized NKX2.1 or HNF-3, demonstrating protein-protein interactions between SMAD3 and the two transcriptional factors. The DNA binding of NKX2.1 to Sp-B promoter was reduced in response to TGF-beta treatment, although expression of Nkx2.1 was not affected. We conclude that SMAD3 interactions with the positive regulators NKX2.1 and HNF-3 underlie the molecular basis for TGF-beta-induced repression of Sp-B gene transcription.