Johnny Loke

Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses

In a recent study, a single nucleotide polymorphism (SNP) of the Toll‐like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild‐type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4−/− mice and a human HSC line (LX‐2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hyporesponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS‐induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein‐1, interleukin‐6), down‐regulation of bone morphogenic protein and the activin membrane‐bound inhibitor expression (an inhibitory transforming growth factor β pseudoreceptor), and activation of a nuclear factor κB (NF‐κB)–responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF‐κB, extracellular signal‐regulated kinase (ERK), and phosphatidylinositol 3‐kinase were greatly increased in HSCs from either TLR4−/− or myeloid differentiation factor 88−/− (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; increased apoptosis in these lines was accompanied by decreased phospho‐ERK and Bcl‐2. Conclusion: TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4‐mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression. (HEPATOLOGY 2009.)

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3′-ends for gene annotation.

Mutations in MAP3K1 Cause 46,XY Disorders of Sex Development and Implicate a Common Signal Transduction Pathway in Human Testis Determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell–Derived Factor-1α–Mediated Stellate Cell Activation

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell–derived factor‐1α (SDF‐1α), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF‐1α/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF‐1α is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture‐induced activation. Treatment of stellate cells with recombinant SDF‐1α increases expression of α‐smooth muscle actin and collagen I and stimulates a dose‐dependent increase in HSC proliferation. Inhibitor studies suggest that SDF‐1α/CXCR4‐dependent extracellular signal‐regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. Conclusion: HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF‐1α is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal‐regulated kinase 1/2 and phosphoinositide 3‐kinase pathways mediate SDF‐1α–induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches. (HEPATOLOGY 2009.)

Ras Promotes Growth by Alternative Splicing-Mediated Inactivation of the KLF6 Tumor Suppressor in Hepatocellular Carcinoma

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown. METHODS In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2. RESULTS In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation. CONCLUSIONS Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.

Downregulation of Hepatic Stellate Cell Activation by Retinol and Palmitate Mediated by Adipose Differentiation-Related Protein (ADRP)

Hepatic stellate cells (HSCs) store retinoids and triacylglycerols in cytoplasmic lipid droplets. Two prominent features of HSC activation in liver fibrosis are loss of lipid droplets along with increase of α‐smooth muscle actin (α‐SMA), but the link between these responses and HSC activation remains elusive. In non‐adipose cells, adipose differentiation‐related protein (ADRP) coats lipid droplets and regulates their formation and lipolysis; however its function in HSCs is unknown. Here, we observed, in human liver sections or primary HSC culture, ADRP localization to lipid droplets of HSCs, and reduced staining coincident with loss of lipid droplets in liver fibrosis and in culture‐activated HSCs, consistent with HSC activation. In the LX‐2 human immortalized HSCs, with scant lipid droplets and features of activated HSCs, we found that the upregulation of ADRP mRNA by palmitate is potentiated by retinol, accompanied by increased ADRP protein, generation of retinyl palmitate, and lipid droplet formation. ADRP induction also led to decreased expression of α‐SMA mRNA and its protein, while ADRP knockdown with small interfering RNA (siRNA) normalized α‐SMA expression. Furthermore, ADRP induction by retinol and palmitate resulted in decreased expression of collagen I and matrix metalloproteinase‐2 mRNA, fibrogenic genes associated with activated HSCs, while increasing matrix metalloproteinase‐1 mRNA; ADRP knockdown with siRNA reversed these changes. Tissue inhibitor of metalloproteinase‐1 was not affected. Thus, ADRP upregulation mediated by retinol and palmitate promotes downregulation of HSC activation and is functionally linked to the expression of fibrogenic genes. J. Cell. Physiol. 223:648–657, 2010.

Mutations in MAP3K1 tilt the balance from SOX9/FGF9 to WNT/β-catenin signaling

In-frame missense and splicing mutations (resulting in a 2 amino acid insertion or a 34 amino acid deletion) dispersed through the MAP3K1 gene tilt the balance from the male to female sex-determining pathway, resulting in 46,XY disorder of sex development. These MAP3K1 mutations mediate this balance by enhancing WNT/β-catenin/FOXL2 expression and β-catenin activity and by reducing SOX9/FGF9/FGFR2/SRY expression. These effects are mediated at multiple levels involving MAP3K1 interaction with protein co-factors and phosphorylation of downstream targets. In transformed B-lymphoblastoid cell lines and NT2/D1 cells transfected with wild-type or mutant MAP3K1 cDNAs under control of the constitutive CMV promoter, these mutations increased binding of RHOA, MAP3K4, FRAT1 and AXIN1 and increased phosphorylation of p38 and ERK1/2. Overexpressing RHOA or reducing expression of MAP3K4 in NT2/D1 cells produced phenocopies of the MAP3K1 mutations. Using siRNA knockdown of RHOA or overexpressing MAP3K4 in NT2/D1 cells produced anti-phenocopies. Interestingly, the effects of the MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. Although MAP3K1 is not usually required for testis determination, mutations in this gene can disrupt normal development through the gains of function demonstrated in this study.

A DDX5 S480A Polymorphism Is Associated with Increased Transcription of Fibrogenic Genes in Hepatic Stellate Cells

We recently identified a missense single nucleotide polymorphism (SNP) in DDX5 (rs1140409, p.S480A) that enhances the risk of developing cirrhosis. DDX5 is an ATP-dependent RNA helicase and transcriptional modulator. We hypothesized that the activity of DDX5 in regulating fibrogenic gene transcription in hepatic stellate cells (HSCs) is altered by the S480A SNP. To test this, we employed two approaches: 1) transient overexpression of DDX5 cDNA or siRNA knockdown of endogenous DDX5, with replacement by either DDX5 wild type (WT) or SNP cDNA, or 2) stable expression of exogenous DDX5 WT and SNP in HSC lines. WT DDX5 mRNA in HSCs was inversely correlated with gene expression for α2(I) collagen, tissue inhibitor of metalloproteinase-1, and transforming growth factor-β1. Stable DDX5 SNP-expressing cells had higher basal and transforming growth factor-β1-stimulated expression and enhanced promoter activities of fibrogenic genes. DDX5 variant-expressing cells also had higher Smad3 and AP-1-responsive reporter activities. In a one-hybrid GAL4 system, co-expression of the DDX5 SNP variant with chimeras of GAL4 DNA binding domain linked to JunD or Sp1 displayed higher transactivation of a GAL4-responsive reporter than that of DDX5 WT. Increased fibrogenic gene expression in DDX5 SNP-expressing cells was associated with reduced recruitment of DDX5 homodimers to responsive promoters, but there was no difference in the recruitment of the co-repressor HDAC1 (histone deacetylase 1). These data suggest that DDX5 is a repressor of fibrogenic genes in HSCs through interaction with transcriptional complexes. The enhanced fibrogenic activity of the DDX5 risk variant is linked to a reduced repressive function toward these target genes.

Rapidly screening variants of uncertain significance in the MAP3K1 gene for phenotypic effects

Loke J, Ostrer H. Rapidly screening variants of uncertain significance in the MAP3K1 gene for phenotypic effects.

Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway

Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway disrupt binding of the encoded proteins, transport into the nucleus and initiation of homologous recombination, thereby increasing cancer risk [Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88, 265-275, Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J., Weber, B.L., Ashley, T., Livingston, D.M. et al. (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2, 317-328]. To meet the challenge of correct classification, flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in DSB repair genes disrupted the binding of BRCA1 to BARD1, PALB2, BRCA2 and FANCD2, phosphorylation of p53 or BRCA1 nuclear localization in response to DNA damage caused by diepoxybutane, mitomycin C and bleomycin. Lymphoblastoid cells from individuals with BRCA1 pathogenic mutations, benign variants, and variants of uncertain significance or with known BRCA2, FANCC or NBN mutations were tested. Mutations in BRCA1 decreased nuclear localization of BRCA1 in response to individual or combination drug treatment. Mutations in BRCA1 reduced binding to co-factors, PALB2 and FANCD2 and decreased phosphorylation of p53. Mutations in BRCA2, FANCC and NBN decreased nuclear localization of BRCA1 in response to drug treatment, cofactors binding and p53 phosphorylation. Unsupervised cluster analysis of all and as few as two assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk, fully sequenced controls and variants of uncertain significance (VUS). Thus, two FVA assays distinguish BRCA1 mutations and phenocopies from benign variants and categorize most VUS as benign. Mutations in other DSB repair pathway genes produce molecular phenocopies. FVA assays may represent an adjunct to sequencing for categorizing VUS or may represent a stand-alone measure for assessing breast cancer risk.