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Laurence J Miller - One of the best experts on this subject based on the ideXlab platform.
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Secretin Receptor Dimerization. Prototypic of Class B GPCR Behavior
G-Protein-Coupled Receptor Dimers, 2017Co-Authors: Kaleeckal G. Harikumar, Laurence J MillerAbstract:The Secretin Receptor is a class B1 GPCR that has been demonstrated to associate with itself, with structurally-related Receptors, and even with select Receptors from another class of GPCRs. The predominant complex formed with itself represents a symmetrical homo-dimer, along the lipid face of transmembrane segment four. This exists constitutively, arriving at the plasma membrane in this form after biosynthesis and being unaffected by agonist binding. There is evidence to suggest that this state of the Receptor is functionally important, contributing to its high affinity binding of natural agonist and the high potency of responses to Secretin. It also explains the negative cooperativity responsible for the reduced affinity binding and biological activity of agonist occupation of the second protomer comprising this complex. These themes appear to be consistent among other family members, and most members of the class B GPCR family can not only form homo-dimers, but also associate with each other to form hetero-complexes. Such complexes can explain agonist-induced cross-internalization of the associated Receptors, providing an important mechanism for Receptor regulation. It will be important to explore the physiologic implications of these processes, and to examine whether they are important in vivo.
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Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the Secretin Receptor
The Biochemical journal, 2017Co-Authors: Richard J. Ward, Laurence J Miller, Kaleeckal G. Harikumar, John D. Pediani, Graeme MilliganAbstract:Previous studies have indicated that the G protein-coupled Secretin Receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that Receptor dimerization is critical for high potency signalling by Secretin. However, whether all of the Receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the Secretin Receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor Receptor is predominantly monomeric in the absence of ligand and whilst wild type Receptor was rapidly converted to a dimeric form by ligand, a mutated form of this Receptor remained monomeric. Equivalent studies showed that at moderate expression levels the Secretin Receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate induced up-regulation of the Receptor resulted in a shift from monomeric towards oligomeric organization. By contrast, a form of the Secretin Receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the Secretin Receptor-interacting G protein Gαs did not alter Receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the Receptor polypeptide, whilst short term treatment with Secretin had no effect on organization of the wild type Receptor but increased the dimeric proportion of the mutated Receptor variant.
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The orthosteric agonist-binding pocket in the prototypic class B G-protein-coupled Secretin Receptor.
Biochemical Society transactions, 2013Co-Authors: Laurence J Miller, Maoqing DongAbstract:Class B GPCRs (G-protein-coupled Receptors) share heptahelical topology and G-protein binding with other superfamily members, yet have unique structures and modes of activation. Natural ligands for these Receptors are moderate-length peptides with C-terminal α-helices. NMR and crystal structures of the peptide-bound disulfide-bonded Receptor N-terminal domains demonstrate that these helices occupy a conserved groove; however, the details of this interaction vary from one Receptor to another. In this review, we focus on the prototypic Secretin Receptor and use extensive intrinsic photoaffinity labelling, structure–activity series, alanine-replacement mutagenesis and fluorescence analysis to define the molecular basis for this interaction. Additionally, experimental validation of predictions coming from in silico molecular modelling has provided a basis for enhancement of binding affinity. Such insights will be useful in the rational development of drugs acting at this important group of targets.
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Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of Secretin Receptor antagonists
Journal of Computer-Aided Molecular Design, 2012Co-Authors: Maoqing Dong, Laurence J Miller, Andrew J. BordnerAbstract:Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the Secretin Receptor, the prototypical member of class B G protein-coupled Receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the Secretin Receptor bound to an N-terminal truncated Secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the Receptor N-terminal domain. Amino acid replacements predicted to most enhance Receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on Receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the Receptor extracellular domain contribute to their higher binding affinities.
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Ligand binding and activation of the Secretin Receptor, a prototypic family B G protein-coupled Receptor.
British Journal of Pharmacology, 2012Co-Authors: Laurence J Miller, Maoqing Dong, Kaleeckal G. HarikumarAbstract:The Secretin Receptor is a prototypic member of family B G protein-coupled Receptors that binds and responds to a linear 27-residue peptide natural ligand. The carboxyl-terminal region of this peptide assumes a helical conformation that occupies the peptide-binding cleft within the structurally complex disulphide-bonded amino-terminal domain of this Receptor. The amino terminus of Secretin is directed toward the core helical bundle domain of this Receptor that seems to be structurally distinct from the analogous region of family A G protein-coupled Receptors. This amino-terminal region of Secretin is critical for its biological activity, to stimulate Gs coupling and the agonist-induced cAMP response. While the natural peptide ligand is known to span the two key Receptor domains, with multiple residue-residue approximation constraints well established, the orientation of the Receptor amino terminus relative to the Receptor core helical bundle domain is still unclear. Fluorescence studies have established that the mid-region and carboxyl-terminal end of Secretin are protected by the Receptor peptide-binding cleft and the amino terminus of Secretin is most exposed to the aqueous milieu as it is directed toward the Receptor core, with the mid-region of the peptide becoming more exposed upon Receptor activation. Like other family B peptide hormone Receptors, the Secretin Receptor is constitutively present in a structurally specific homo-dimeric complex built around the lipid-exposed face of transmembrane segment four. This complex is important for facilitating G protein association and achieving the high affinity state of this Receptor. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
Billy K. C. Chow - One of the best experts on this subject based on the ideXlab platform.
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Origin of Secretin Receptor Precedes the Advent of Tetrapoda: Evidence on the Separated Origins of
2016Co-Authors: Janice K. V. Tam, Jessica Y S Chu, Kwan-wa Lau, Leo T. O. Lee, Alain Fournier, Hubert Vaudry, Billy K. C. ChowAbstract:At present, Secretin and its Receptor have only been identified in mammals, and the origin of this ligand-Receptor pair in early vertebrates is unclear. In addition, the elusive similarities of Secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of Secretin Receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) Secretins, we provide evidence that the Secretin ligand-Receptor pair has already diverged and become highly specific by the emergence of tetrapods. The Secretin Receptor-like sequence cloned from lungfish indicates that the Secretin Receptor was descended from a VPAC-like Receptor prior the advent of sarcopterygians. To clarify the controversial relationship of Secretin and orexin, orexin type-2 Receptor was cloned from X. laevis. We demonstrated that, in frog, Secretin and orexin could activate their mutual Receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the Secretin/glucagon superfamily, Secretin was found to be the only peptide that could activate the orexin Receptor. We therefore hypothesize that Secretin and orexin are of different ancestral origin
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Secretin Receptor knockout mice are resistant to high fat diet induced obesity and exhibit impaired intestinal lipid absorption
The FASEB Journal, 2014Co-Authors: Revathi Sekar, Billy K. C. ChowAbstract:Secretin, a classical gastrointestinal hormone released from S cells in response to acid and dietary lipid, regulates pleiotropic physiological functions, such as exocrine pancreatic secretion and gastric motility. Subsequent to recently proposed revisit on Secretin's metabolic effects, we have confirmed lipolytic actions of Secretin during starvation and discovered a hormone-sensitive lipase-mediated mechanistic pathway behind. In this study, a 12 wk high-fat diet (HFD) feeding to Secretin Receptor-knockout (SCTR(-/-)) mice and their wild-type (SCTR(+/+)) littermates revealed that, despite similar food intake, SCTR(-/-) mice gained significantly less weight (SCTR(+/+): 49.6±0.9 g; SCTR(-/-): 44.7±1.4 g; P<0.05) and exhibited lower body fat content. These SCTR(-/-) mice have corresponding alleviated HFD-associated hyperleptinemia and improved glucose/insulin tolerance. Further analyses indicate that SCTR(-/-) have impaired intestinal fatty acid absorption while having similar energy expenditure and locomotor activity. Reduced fat absorption in the intestine is further supported by lowered postprandial triglyceride concentrations in circulation in SCTR(-/-) mice. In jejunal cells, transcript and protein levels of a key fat absorption regulator, cluster of differentiation 36 (CD36), was reduced in knockout mice, while transcript of Cd36 and fatty-acid uptake in isolated enterocytes was stimulated by Secretin. Based on our findings, a novel positive feedback pathway involving Secretin and CD36 to enhance intestinal lipid absorption is being proposed.
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Secretin Receptor-knockout mice are resistant to high-fat diet-induced obesity and exhibit impaired intestinal lipid absorption
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2014Co-Authors: Revathi Sekar, Billy K. C. ChowAbstract:Secretin, a classical gastrointestinal hormone released from S cells in response to acid and dietary lipid, regulates pleiotropic physiological functions, such as exocrine pancreatic secretion and gastric motility. Subsequent to recently proposed revisit on Secretin's metabolic effects, we have confirmed lipolytic actions of Secretin during starvation and discovered a hormone-sensitive lipase-mediated mechanistic pathway behind. In this study, a 12 wk high-fat diet (HFD) feeding to Secretin Receptor-knockout (SCTR−/−) mice and their wild-type (SCTR+/+) littermates revealed that, despite similar food intake, SCTR−/− mice gained significantly less weight (SCTR+/+: 49.6±0.9 g; SCTR−/−: 44.7±1.4 g; P
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MOLECULAR EVOLUTION OF GPCRS: Secretin/Secretin Receptors
Journal of molecular endocrinology, 2014Co-Authors: Janice K. V. Tam, Leo T. O. Lee, Jun Jin, Billy K. C. ChowAbstract:In mammals, Secretin is a 27-amino acid peptide that was first studied in 1902 by Bayliss and Starling from the extracts of the jejunal mucosa for its ability to stimulate pancreatic secretion. To date, Secretin has only been identified in tetrapods, with the earliest diverged Secretin found in frogs. Despite being the first hormone discovered, Secretin’s evolutionary origin remains enigmatic, it shows moderate sequence identity in nonmammalian tetrapods but is highly conserved in mammals. Current hypotheses suggest that although Secretin has already emerged before the divergence of osteichthyans, it was lost in fish and retained only in land vertebrates. Nevertheless, the cognate Receptor of Secretin has been identified in both actinopterygian fish (zebrafish) and sarcopterygian fish (lungfish). However, the zebrafish Secretin Receptor was shown to be nonbioactive. Based on the present information that the earliest diverged bioactive Secretin Receptor was found in lungfish, and its ability to interact with both vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potently suggested that Secretin Receptor was descended from a VPAC-like Receptor gene before the Actinopterygii‐Sarcopterygii split in the vertebrate lineage. Hence, Secretin and Secretin Receptor have gone through independent evolutionary trajectories despite their concurrent emergence post-2R. A functional Secretin‐Secretin Receptor axis has probably emerged in the amphibians. Although the pleiotropic actions of Secretin are well documented in the literature, only limited information of its physiological functions in nonmammalian tetrapods have been reported. To decipher the structural and functional divergence of Secretin and Secretin Receptor, functional characterization of the ligand‐ Receptor pair in nonmammals would be the next perspective for investigation.
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Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human Secretin Receptor gene
Biochimica et biophysica acta, 2012Co-Authors: Yuan Yuan, Billy K. C. Chow, Vien H. Y. Lee, Leo T. O. LeeAbstract:Abstract In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (− 83 to − 67, relative to ATG) of human Secretin Receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR . Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.
Maoqing Dong - One of the best experts on this subject based on the ideXlab platform.
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Rational development of a high-affinity Secretin Receptor antagonist
Biochemical pharmacology, 2020Co-Authors: Maoqing Dong, Kaleeckal G. Harikumar, Sweta R. Raval, Juliana E. Milburn, Carolyn E. Clark, Rafael Alcala-torano, Juan Carlos Mobarec, Christopher A. Reynolds, Giovanna Ghirlanda, Arthur ChristopoulosAbstract:Abstract The Secretin Receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this Receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu22 with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with Receptor residues Leu31, Val34, and Phe92 that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y10,c[E16,K20],I17,Cha22,R25)sec(6–27), exhibited high binding affinity (4 nM), similar to natural Secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the Receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr5, exhibited partial agonist activity, while further truncation of even a single residue (Phe6) reduced binding affinity. This sec(6–27)-based peptide will be an important new tool for pharmacological and structural studies.
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The orthosteric agonist-binding pocket in the prototypic class B G-protein-coupled Secretin Receptor.
Biochemical Society transactions, 2013Co-Authors: Laurence J Miller, Maoqing DongAbstract:Class B GPCRs (G-protein-coupled Receptors) share heptahelical topology and G-protein binding with other superfamily members, yet have unique structures and modes of activation. Natural ligands for these Receptors are moderate-length peptides with C-terminal α-helices. NMR and crystal structures of the peptide-bound disulfide-bonded Receptor N-terminal domains demonstrate that these helices occupy a conserved groove; however, the details of this interaction vary from one Receptor to another. In this review, we focus on the prototypic Secretin Receptor and use extensive intrinsic photoaffinity labelling, structure–activity series, alanine-replacement mutagenesis and fluorescence analysis to define the molecular basis for this interaction. Additionally, experimental validation of predictions coming from in silico molecular modelling has provided a basis for enhancement of binding affinity. Such insights will be useful in the rational development of drugs acting at this important group of targets.
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Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of Secretin Receptor antagonists
Journal of Computer-Aided Molecular Design, 2012Co-Authors: Maoqing Dong, Laurence J Miller, Andrew J. BordnerAbstract:Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the Secretin Receptor, the prototypical member of class B G protein-coupled Receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the Secretin Receptor bound to an N-terminal truncated Secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the Receptor N-terminal domain. Amino acid replacements predicted to most enhance Receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on Receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the Receptor extracellular domain contribute to their higher binding affinities.
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Ligand binding and activation of the Secretin Receptor, a prototypic family B G protein-coupled Receptor.
British Journal of Pharmacology, 2012Co-Authors: Laurence J Miller, Maoqing Dong, Kaleeckal G. HarikumarAbstract:The Secretin Receptor is a prototypic member of family B G protein-coupled Receptors that binds and responds to a linear 27-residue peptide natural ligand. The carboxyl-terminal region of this peptide assumes a helical conformation that occupies the peptide-binding cleft within the structurally complex disulphide-bonded amino-terminal domain of this Receptor. The amino terminus of Secretin is directed toward the core helical bundle domain of this Receptor that seems to be structurally distinct from the analogous region of family A G protein-coupled Receptors. This amino-terminal region of Secretin is critical for its biological activity, to stimulate Gs coupling and the agonist-induced cAMP response. While the natural peptide ligand is known to span the two key Receptor domains, with multiple residue-residue approximation constraints well established, the orientation of the Receptor amino terminus relative to the Receptor core helical bundle domain is still unclear. Fluorescence studies have established that the mid-region and carboxyl-terminal end of Secretin are protected by the Receptor peptide-binding cleft and the amino terminus of Secretin is most exposed to the aqueous milieu as it is directed toward the Receptor core, with the mid-region of the peptide becoming more exposed upon Receptor activation. Like other family B peptide hormone Receptors, the Secretin Receptor is constitutively present in a structurally specific homo-dimeric complex built around the lipid-exposed face of transmembrane segment four. This complex is important for facilitating G protein association and achieving the high affinity state of this Receptor. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
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Differential determinants for coupling of distinct G proteins with the class B Secretin Receptor.
American journal of physiology. Cell physiology, 2012Co-Authors: Gene L. Garcia, Maoqing Dong, Laurence J MillerAbstract:The Secretin Receptor is a prototypic class B G protein-coupled Receptor that is activated by binding of its natural peptide ligand. The signaling effects of this Receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this Receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and Secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind Secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some Receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.
Fanyin Meng - One of the best experts on this subject based on the ideXlab platform.
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Knockout of Secretin Receptor reduces biliary damage and liver fibrosis in Mdr2^−/− mice by diminishing senescence of cholangiocytes
Laboratory Investigation, 2018Co-Authors: Tianhao Zhou, Fanyin Meng, Julie Venter, Konstantina Kyritsi, Heather Francis, Antonio Franchitto, Domenico Alvaro, Thao K Giang, Marco Marzioni, Paolo OnoriAbstract:In this study the authors demonstrate that the Secretin/Secretin Receptor axis plays a regulatory role in biliary proliferation and liver fibrosis through differential changes in the senescence of cholangiocytes and hepatic stellate cells in a mouse model of primary sclerosing cholangitis. Targeting senescent cholangiocytes by modulation of the Secretin/Secretin Receptor axis may provide a key therapeutic approach in the treatment of cholestatic liver diseases. Secretin Receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2^−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR^−/−, Mdr2^−/−, and SR^−/−/Mdr2^−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2^−/− mice was reduced in SR^−/−/Mdr2^−/− mice. Enhanced senescence levels in cholangiocytes from Mdr2^−/− mice were reversed to normal in SR^−/−/Mdr2^−/− mice. However, senescence was decreased in HSCs from Mdr2^−/− mice but returned to normal values in SR^−/−/Mdr2^−/− mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.
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the Secretin Secretin Receptor axis modulates ductular reaction and liver fibrosis through changes in transforming growth factor β1 mediated biliary senescence
American Journal of Pathology, 2018Co-Authors: Fanyin Meng, Julie Venter, Konstantina Kyritsi, Domenico Alvaro, Tianhao Zhou, Paolo Onori, Romina Mancinelli, Thao Giang, Eugenio GaudioAbstract:Activation of the Secretin (Sct)/Secretin Receptor (SR) axis stimulates ductular reaction and liver fibrosis, which are hallmarks of cholangiopathies. Our aim was to define the role of Sct-regulated cellular senescence, and we demonstrated that both ductular reaction and liver fibrosis are significantly reduced in Sct−/−, SR−/−, and Sct−/−/SR−/− bile duct ligated (BDL) mice compared with BDL wild-type mice. The reduction in hepatic fibrosis in Sct−/−, SR−/−, and Sct−/−/SR−/− BDL mice was accompanied by reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatant, as well as decreased expression of markers of cellular senescence in cholangiocytes in contrast to enhanced cellular senescence in hepatic stellate cells compared with BDL wild-type mice. Secretin directly stimulated the senescence of cholangiocytes and regulated, by a paracrine mechanism, the senescence of hepatic stellate cells and liver fibrosis via modulation of transforming growth factor-β1 biliary secretion. Targeting senescent cholangiocytes may represent a novel therapeutic approach for ameliorating hepatic fibrosis during cholestatic liver injury.
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knockout of Secretin Receptor reduces biliary damage and liver fibrosis in mdr2 mice by diminishing senescence of cholangiocytes
Laboratory Investigation, 2018Co-Authors: Tianhao Zhou, Fanyin Meng, Julie Venter, Konstantina Kyritsi, Heather Francis, Thao Giang, Antonio FranchittoAbstract:Secretin Receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR−/−, Mdr2−/−, and SR−/−/Mdr2−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2−/− mice was reduced in SR−/−/Mdr2−/− mice. Enhanced senescence levels in cholangiocytes from Mdr2−/− mice were reversed to normal in SR−/−/Mdr2−/− mice. However, senescence was decreased in HSCs from Mdr2−/− mice but returned to normal values in SR−/−/Mdr2−/− mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.
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The role of the Secretin/Secretin Receptor axis in inflammatory cholangiocyte communication via extracellular vesicles
Scientific reports, 2017Co-Authors: Keisaku Sato, Shannon Glaser, Fanyin Meng, Julie Venter, Thao Giang, Gianfranco AlpiniAbstract:Small and large intrahepatic bile ducts consist of small and large cholangiocytes, respectively, and these cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, Secretin (SCT) that binds to Secretin Receptor (SR), is a key mediator in cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other cholangiocytes including elevated cytokine production and cell proliferation. Large but not small cholangiocytes show inflammatory responses against large but not small cholangiocyte-derived EVs. Large cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down cholangiocytes fail to induce inflammatory reactions in control large cholangiocytes. This study identifies cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.
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the role of the Secretin Secretin Receptor axis in inflammatory cholangiocyte communication via extracellular vesicles
Scientific Reports, 2017Co-Authors: Keisaku Sato, Shannon Glaser, Fanyin Meng, Julie Venter, Gianfranco Alpini, Thao GiangAbstract:Small and large intrahepatic bile ducts consist of small and large cholangiocytes, respectively, and these cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, Secretin (SCT) that binds to Secretin Receptor (SR), is a key mediator in cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other cholangiocytes including elevated cytokine production and cell proliferation. Large but not small cholangiocytes show inflammatory responses against large but not small cholangiocyte-derived EVs. Large cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down cholangiocytes fail to induce inflammatory reactions in control large cholangiocytes. This study identifies cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.
Julie Venter - One of the best experts on this subject based on the ideXlab platform.
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Secretin/Secretin Receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2019Co-Authors: Lindsey Kennedy, Julie Venter, Heather Francis, Pietro Invernizzi, Marco Carbone, M. Eric Gershwin, Francesca Bernuzzi, Antonio Franchitto, Domenico AlvaroAbstract:Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the Secretin (Sct)/Secretin Receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-β Receptor II (dnTGF-βRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-βRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-βRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-βRII mice, there was increased microRNA-125b/TGF-β1/TGF-β Receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/Secretin Receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.
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Secretin Secretin Receptor signaling mediates biliary damage and liver fibrosis in early stage primary biliary cholangitis
The FASEB Journal, 2019Co-Authors: Julie Venter, Lindsey Kennedy, Heather Francis, Pietro Invernizzi, Marco Carbone, Eric M Gershwin, Francesca BernuzziAbstract:Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the Secretin (Sct)/Secretin Receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-β Receptor II (dnTGF-βRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-βRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-βRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-βRII mice, there was increased microRNA-125b/TGF-β1/TGF-β Receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/Secretin Receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.
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Knockout of Secretin Receptor reduces biliary damage and liver fibrosis in Mdr2^−/− mice by diminishing senescence of cholangiocytes
Laboratory Investigation, 2018Co-Authors: Tianhao Zhou, Fanyin Meng, Julie Venter, Konstantina Kyritsi, Heather Francis, Antonio Franchitto, Domenico Alvaro, Thao K Giang, Marco Marzioni, Paolo OnoriAbstract:In this study the authors demonstrate that the Secretin/Secretin Receptor axis plays a regulatory role in biliary proliferation and liver fibrosis through differential changes in the senescence of cholangiocytes and hepatic stellate cells in a mouse model of primary sclerosing cholangitis. Targeting senescent cholangiocytes by modulation of the Secretin/Secretin Receptor axis may provide a key therapeutic approach in the treatment of cholestatic liver diseases. Secretin Receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2^−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR^−/−, Mdr2^−/−, and SR^−/−/Mdr2^−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2^−/− mice was reduced in SR^−/−/Mdr2^−/− mice. Enhanced senescence levels in cholangiocytes from Mdr2^−/− mice were reversed to normal in SR^−/−/Mdr2^−/− mice. However, senescence was decreased in HSCs from Mdr2^−/− mice but returned to normal values in SR^−/−/Mdr2^−/− mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.
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the Secretin Secretin Receptor axis modulates ductular reaction and liver fibrosis through changes in transforming growth factor β1 mediated biliary senescence
American Journal of Pathology, 2018Co-Authors: Fanyin Meng, Julie Venter, Konstantina Kyritsi, Domenico Alvaro, Tianhao Zhou, Paolo Onori, Romina Mancinelli, Thao Giang, Eugenio GaudioAbstract:Activation of the Secretin (Sct)/Secretin Receptor (SR) axis stimulates ductular reaction and liver fibrosis, which are hallmarks of cholangiopathies. Our aim was to define the role of Sct-regulated cellular senescence, and we demonstrated that both ductular reaction and liver fibrosis are significantly reduced in Sct−/−, SR−/−, and Sct−/−/SR−/− bile duct ligated (BDL) mice compared with BDL wild-type mice. The reduction in hepatic fibrosis in Sct−/−, SR−/−, and Sct−/−/SR−/− BDL mice was accompanied by reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatant, as well as decreased expression of markers of cellular senescence in cholangiocytes in contrast to enhanced cellular senescence in hepatic stellate cells compared with BDL wild-type mice. Secretin directly stimulated the senescence of cholangiocytes and regulated, by a paracrine mechanism, the senescence of hepatic stellate cells and liver fibrosis via modulation of transforming growth factor-β1 biliary secretion. Targeting senescent cholangiocytes may represent a novel therapeutic approach for ameliorating hepatic fibrosis during cholestatic liver injury.
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knockout of Secretin Receptor reduces biliary damage and liver fibrosis in mdr2 mice by diminishing senescence of cholangiocytes
Laboratory Investigation, 2018Co-Authors: Tianhao Zhou, Fanyin Meng, Julie Venter, Konstantina Kyritsi, Heather Francis, Thao Giang, Antonio FranchittoAbstract:Secretin Receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR−/−, Mdr2−/−, and SR−/−/Mdr2−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2−/− mice was reduced in SR−/−/Mdr2−/− mice. Enhanced senescence levels in cholangiocytes from Mdr2−/− mice were reversed to normal in SR−/−/Mdr2−/− mice. However, senescence was decreased in HSCs from Mdr2−/− mice but returned to normal values in SR−/−/Mdr2−/− mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.