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The Role of ARF6 in Biliary Atresia.

Ningappa M, So J, Glessner J, Ashokkumar C, Ranganathan S, Min J, Higgs BW, Sun Q, Haberman K, Schmitt L, Vilarinho S, Mistry PK, Vockley G, Dhawan A, Gittes GK, Hakonarson H, Jaffe R, Subramaniam S, Shin D, Sindhi R - PLoS ONE (2015)

Bottom Line: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66).In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos.Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.

ABSTRACT

Background & aims: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

No MeSH data available.


Related in: MedlinePlus

EGFR signaling regulates biliary morphogenesis.(A) Whole-mount in situ hybridization image showing egfra expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by Tp1:GFP expression. Tg(Tp1:GFP) embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of Tp1:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by Tp1:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.
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pone.0138381.g004: EGFR signaling regulates biliary morphogenesis.(A) Whole-mount in situ hybridization image showing egfra expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by Tp1:GFP expression. Tg(Tp1:GFP) embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of Tp1:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by Tp1:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.

Mentions: Because EGFR signaling activates ARF6 and induces breast cancer invasion, we tested whether impaired EGFR signaling could reproduce the intrahepatic biliary defects observed in arf6-ATG MO-injected larvae [50]. The expression of egfra, a zebrafish orthologue of human EGFR, was clearly observed in the liver at 72 hpf (Fig 4A). Compared with DMSO-treated controls, larvae treated with 4 μM AG1478, a potent EGFR inhibitor [51], from 48 hpf onwards demonstrated 1) reduced BEC filopodial length (Fig 4B), bile canaliculi length (Fig 4C), and PED-6 accumulation in the gallbladder (Fig 4E), 2) enhanced clustering of BECs (Fig 4D) without differences in proliferation at 75 hpf (S7C Fig). Therefore, EGFR-Arf6 signaling pathway may regulate intrahepatic biliary morphogenesis.


The Role of ARF6 in Biliary Atresia.

Ningappa M, So J, Glessner J, Ashokkumar C, Ranganathan S, Min J, Higgs BW, Sun Q, Haberman K, Schmitt L, Vilarinho S, Mistry PK, Vockley G, Dhawan A, Gittes GK, Hakonarson H, Jaffe R, Subramaniam S, Shin D, Sindhi R - PLoS ONE (2015)

EGFR signaling regulates biliary morphogenesis.(A) Whole-mount in situ hybridization image showing egfra expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by Tp1:GFP expression. Tg(Tp1:GFP) embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of Tp1:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by Tp1:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.
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Related In: Results  -  Collection

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pone.0138381.g004: EGFR signaling regulates biliary morphogenesis.(A) Whole-mount in situ hybridization image showing egfra expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by Tp1:GFP expression. Tg(Tp1:GFP) embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of Tp1:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by Tp1:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.
Mentions: Because EGFR signaling activates ARF6 and induces breast cancer invasion, we tested whether impaired EGFR signaling could reproduce the intrahepatic biliary defects observed in arf6-ATG MO-injected larvae [50]. The expression of egfra, a zebrafish orthologue of human EGFR, was clearly observed in the liver at 72 hpf (Fig 4A). Compared with DMSO-treated controls, larvae treated with 4 μM AG1478, a potent EGFR inhibitor [51], from 48 hpf onwards demonstrated 1) reduced BEC filopodial length (Fig 4B), bile canaliculi length (Fig 4C), and PED-6 accumulation in the gallbladder (Fig 4E), 2) enhanced clustering of BECs (Fig 4D) without differences in proliferation at 75 hpf (S7C Fig). Therefore, EGFR-Arf6 signaling pathway may regulate intrahepatic biliary morphogenesis.

Bottom Line: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66).In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos.Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Hillman Center for Pediatric Transplantation of the Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center (UPMC), Pittsburgh, PA, 15224, United States of America.

ABSTRACT

Background & aims: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

No MeSH data available.


Related in: MedlinePlus