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Gregory A Michelotti,
Guanhua Xie,
Marzena Swiderska,
Steve S Choi,
Gamze Karaca,
Leandi Krüger,
Richard Premont,
Liu Yang,
Wing-Kin Syn,
Daniel Metzger,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: When regenerative processes cannot keep pace with cell death, functional epithelia are replaced by scar. Scarring is characterized by both excessive accumulation of fibrous matrix and persistent outgrowth of cell types that accumulate transiently during successful wound healing, including myofibroblasts (MFs) and progenitors. This suggests that signaling that normally directs these cells to repair injured epithelia is deregulated. To evaluate this possibility, we examined liver repair during different types of liver injury after Smoothened (SMO), an obligate intermediate in the Hedgehog (Hh) signaling pathway, was conditionally deleted in cells expressing the MF-associated gene, αSMA. Surprisingly, blocking canonical Hh signaling in MFs not only inhibited liver fibrosis but also prevented accumulation of liver progenitors. Hh-sensitive, hepatic stellate cells (HSCs) were identified as the source of both MFs and progenitors by lineage-tracing studies in 3 other strains of mice, coupled with analysis of highly pure HSC preparations using flow cytometry, immunofluorescence confocal microscopy, RT-PCR, and in situ hybridization. The results identify SMO as a master regulator of hepatic epithelial regeneration based on its ability to promote mesenchymal-to-epithelial transitions in a subpopulation of HSC-derived MFs with features of multipotent progenitors.
The Journal of clinical investigation 04/2013; · 15.39 Impact Factor
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Isaac S Chan,
Cynthia D Guy,
Yuping Chen,
Jiuyi Lu,
Marzena Swiderska, Gregory A Michelotti,
Gamze F Karaca,
Guanhua Xie,
Leandi Kruger,
Wing-Kin Syn,
Blair R Anderson,
Thiago A Pereira,
Steve S Choi,
Albert S Baldwin,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) typically develop in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts (MF). Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent MF in hepatocarcinogenesis is unknown. Here we used human HCC samples, a mouse HCC model, and hepatoma cell/MF co-cultures to examine the hypothesis that Hh signaling modulates MF metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce HH-ligands to stimulate glycolysis in neighboring MF, resulting in release of MF-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCC.
Cancer Research 10/2012; · 7.86 Impact Factor
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Yuping Chen,
Steve S Choi, Gregory A Michelotti,
Isaac S Chan,
Marzena Swiderska-Syn,
Gamze F Karaca,
Guanhua Xie,
Cynthia A Moylan,
Francesca Garibaldi,
Richard Premont,
Hagir B Suliman,
Claude A Piantadosi,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism.
Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity.
Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh.
Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.
Gastroenterology 08/2012; 143(5):1319-1329.e11. · 11.68 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Although α1AR antagonists have been used for more than two decades to treat lower urinary tract symptoms (LUTS), we have little understanding
of the mechanistic basis of their efficacy and their role in the development of LUTS. It is clear that α1ARs play a critical
role in bladder dysfunction and recent data suggest that α1AR subtype switching may play a key role in this pathophysiology, providing support for use of α1dAR-selective antagonists in treating irritative symptoms. This review seeks to summarize current levels of understanding in
this field and discusses new concepts that suggest increased levels of complexity involving cross-talk in multiple receptor
systems. Effective therapeutic modalities likely will involve increased subtype selective α1AR antagonists and other pharmacodynamic factors.
Current Prostate Reports 04/2012; 2(2):78-86.
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Wing-Kin Syn,
Kolade M Agboola,
Marzena Swiderska, Gregory A Michelotti,
Evaggelia Liaskou,
Herbert Pang,
Guanhua Xie,
George Philips,
Isaac S Chan,
Gamze F Karaca,
Thiago de Almeida Pereira,
Yuping Chen,
Zhiyong Mi,
Paul C Kuo,
Steve S Choi,
Cynthia D Guy,
Manal F Abdelmalek,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells.
The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans.
When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18-/- and CD1d-/- mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity.
Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.
Gut 03/2012; 61(9):1323-9. · 10.11 Impact Factor
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[show abstract]
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ABSTRACT: OBJECTIVE: Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. DESIGN: Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. RESULTS: Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from Smo(loxP/loxP) transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. CONCLUSIONS: LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.
Gut 02/2012; · 10.11 Impact Factor
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George M Philips,
Isaac S Chan,
Marzena Swiderska,
Vanessa T Schroder,
Cynthia Guy,
Gamze F Karaca,
Cynthia Moylan,
Talaignair Venkatraman,
Sebastian Feuerlein,
Wing-Kin Syn,
Youngmi Jung,
Rafal P Witek,
Steve Choi, Gregory A Michelotti,
Fatima Rangwala,
Elmar Merkle,
Christopher Lascola,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50-60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2(-/-) mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.
Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2(-/-) mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.
Unlike controls, Mdr2(-/-) mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.
Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.
PLoS ONE 01/2011; 6(9):e23943. · 4.09 Impact Factor
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ABSTRACT: Trans-differentiation of quiescent hepatic stellate cells (Q-HSCs), which exhibit epithelial and adipocytic features, into myofibroblastic-HSC (MF-HSCs) is a key event in liver fibrosis. Culture models demonstrated that Hedgehog (Hh) pathway activation is required for transition of epithelioid/adipocytic Q-HSCs into MF-HSCs. Hh signaling inhibits adiposity and promotes epithelial-to-mesenchymal transitions (EMTs). Leptin (anti-adipogenic, pro-EMT factor) promotes HSC trans-differentiation and liver fibrosis, suggesting that the pathways may interact to modulate cell fate. This study aimed to determine whether leptin activates Hh signaling and whether this is required for the fibrogenic effects of leptin. Cultures of primary HSCs from lean and fa/fa rats with an inherited ObRb defect were examined. Inhibitors of PI3K/Akt, JAK/STAT, and Hh signaling were used to delineate how ObRb activation influenced Hh signaling and HSC trans-differentiation. Fibrogenesis was compared in wild type and db/db mice (impaired ObRb function) to assess the profibrotic role of leptin. The results demonstrate that leptin-ObR interactions activate Hh signaling with the latter necessary to promote trans-differentiation. Leptin-related increases in Hh signaling required ObR induction of PI3K/Akt, which was sufficient for leptin to repress the epithelioid/adipocytic program. Leptin-mediated induction of JAK/STAT was required for mesenchymal gene expression. Leptin-ObRb interactions were not necessary for HSC trans-differentiation to occur in vitro or in vivo but are important because liver fibrogenesis was attenuated in db/db mice. These findings reveal that leptin activates Hh signaling to alter gene expression programs that control cell fate and have important implications for liver fibrosis and other leptin-regulated processes involving EMTs, including development, obesity, and cancer metastasis.
Journal of Biological Chemistry 11/2010; 285(47):36551-60. · 4.77 Impact Factor
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Wing-Kin Syn,
Steve S Choi,
Evaggelia Liaskou,
Gamze F Karaca,
Kolade M Agboola,
Ye Htun Oo,
Zhiyong Mi,
Thiago A Pereira,
Marzena Zdanowicz,
Padmini Malladi, [......],
Syamal D Bhattacharya,
Vanessa Teaberry,
Alessia Omenetti,
Manal F Abdelmalek,
Cynthia D Guy,
David H Adams,
Paul C Kuo, Gregory A Michelotti,
Peter F Whitington,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc(+/-) ) (overly active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc(+/-) mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). CONCLUSION: OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH.
Hepatology 09/2010; 53(1):106-15. · 11.66 Impact Factor
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Steve S Choi,
Alessia Omenetti,
Rafal P Witek,
Cynthia A Moylan,
Wing-Kin Syn,
Youngmi Jung,
Liu Yang,
Debra L Sudan,
Jason K Sicklick, Gregory A Michelotti,
Marcos Rojkind,
Anna Mae Diehl
[show abstract]
[hide abstract]
ABSTRACT: Myofibroblastic hepatic stellate cells (MF-HSC) are derived from quiescent hepatic stellate cells (Q-HSC). Q-HSC express certain epithelial cell markers and have been reported to form junctional complexes similar to epithelial cells. We have shown that Hedgehog (Hh) signaling plays a key role in HSC growth. Because Hh ligands regulate epithelial-to-mesenchymal transition (EMT), we determined whether Q-HSC express EMT markers and then assessed whether these markers change as Q-HSC transition into MF-HSC and whether the process is modulated by Hh signaling. Q-HSC were isolated from healthy livers and cultured to promote myofibroblastic transition. Changes in mRNA and protein expression of epithelial and mesenchymal markers, Hh ligands, and target genes were monitored in HSC treated with and without cyclopamine (an Hh inhibitor). Studies were repeated in primary human HSC and clonally derived HSC from a cirrhotic rat. Q-HSC activation in vitro (culture) and in vivo (CCl(4)-induced cirrhosis) resulted in decreased expression of Hh-interacting protein (Hhip, an Hh antagonist), the EMT inhibitors bone morphogenic protein (BMP-7) and inhibitor of differentiation (Id2), the adherens junction component E-cadherin, and epithelial keratins 7 and 19 and increased expression of Gli2 (an Hh target gene) and mesenchymal markers, including the mesenchyme-associated transcription factors Lhx2 and Msx2, the myofibroblast marker alpha-smooth muscle actin, and matrix molecules such as collagen. Cyclopamine reverted myofibroblastic transition, reducing mesenchymal gene expression while increasing epithelial markers in rodent and human HSC. We conclude that Hh signaling plays a key role in transition of Q-HSC into MF-HSC. Our findings suggest that Q-HSC are capable of transitioning between epithelial and mesenchymal fates.
AJP Gastrointestinal and Liver Physiology 10/2009; 297(6):G1093-106. · 3.43 Impact Factor
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Langtian Yuan,
Jin-Soo Seo,
Nam Sook Kang,
Shahar Keinan,
Sarah E Steele, Gregory A Michelotti,
William C Wetsel,
David N Beratan,
Young-Dae Gong,
Tong H Lee,
Jiyong Hong
[show abstract]
[hide abstract]
ABSTRACT: From a screen of small molecule libraries to identify potential therapeutics for psychostimulant abuse, 3-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-ones were shown to be isoform-selective PKC-zeta inhibitors.
Molecular BioSystems 10/2009; 5(9):927-30. · 3.53 Impact Factor
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ABSTRACT: Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein-GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.
Journal of Neurochemistry 12/2008; 108(1):147-57. · 4.06 Impact Factor
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ABSTRACT: The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.
Journal of Biological Chemistry 03/2008; 283(5):2973-85. · 4.77 Impact Factor
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ABSTRACT: A growing body of evidence implicates alpha1-adrenergic receptors (alpha1ARs) as potent regulators of growth pathways. The three alpha1AR subtypes (alpha1aAR, alpha1bAR, alpha1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell-specific regulation of the human alpha1dAR subtype, we cloned and characterized the alpha1dAR promoter region in two human cellular models that display disparate levels of endogenous alpha1dAR expression (SK-N-MC and DU145). Results reveal that alpha1dAR basal expression is regulated by Sp1-dependent binding of two promoter-proximal GC boxes, the mutation of which attenuates alpha1dAR promoter activity 10-fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with alpha1dAR promoter methylation-dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation-dependent disruption of Sp1 binding in a cell-specific manner resulting in repression of basal alpha1dAR expression.
The FASEB Journal 08/2007; 21(9):1979-93. · 5.71 Impact Factor
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ABSTRACT: Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously.
Journal of Biological Chemistry 10/2005; 280(36):31368-77. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Cardiac surgery with cardiopulmonary bypass induces both systemic and local inflammatory responses implicated in the pathogenesis of myocardial dysfunction. Multifactorial perioperative sources of myocardial injury complicate understanding of the molecular mechanisms involved. By using microarray technology, this study examines myocardial gene expression responses to cardiopulmonary bypass in the absence of cardioplegic arrest and ischemia-reperfusion injury.
We used a unique rat model of cardiopulmonary bypass in which sternotomy, direct operations on the heart, aortic crossclamping, and cardioplegic arrest were not performed. Hearts from 6 animals randomized to either 90 minutes of cardiopulmonary bypass or sham control animals were used to perform cDNA microarray analyses of 2343 genes. Real-time quantitative polymerase chain reaction was used to confirm the microarray results for a subset of genes.
Compared with sham-operated control animals, myocardium from animals undergoing cardiopulmonary bypass revealed 42 differentially expressed genes. Upregulated genes include the transcription activator nuclear factor kappaB, adhesion molecules (vascular cell adhesion molecule 1 and P-selectin), and interleukin 6 receptor subunits; downregulated genes include transforming growth factor beta receptor 2, tissue inhibitor of metalloproteinase 3, and mitogen-activated protein kinase 1. Distinct proinflammatory gene cascades were confirmed by means of category overrepresentation analysis.
This study represents an initial report on the use of microarray technology to elucidate cardiac transcriptional programs in response to cardiopulmonary bypass-specific injury in vivo. These preliminary findings, combined with future functional genomic studies superimposing ischemia and reperfusion and other inflammatory stimuli, should improve our understanding of the molecular regulatory networks involved in myocardial responses to injury and aid in the development of novel cardioprotective and perfusion strategies.
Journal of Thoracic and Cardiovascular Surgery 09/2005; 130(2):330-9. · 3.41 Impact Factor
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[hide abstract]
ABSTRACT: Alpha1-adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct human alpha1A-adrenoceptor isoforms have been identified from human tissues, including five full-length and 11 truncated versions. An updated scheme for the identification of alpha1A-adrenoceptor splice variants is proposed. Given the established roles of alpha1-adrenoceptors in benign prostatic hyperplasia, myocardial hypertrophy and other cardiovascular disorders, elucidation of the biological significance of the signaling diversity and potential pharmacological roles of alpha1A-adrenoceptor splice variants are important areas of future research.
Trends in Pharmacological Sciences 10/2004; 25(9):449-55. · 10.93 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Although alpha(1)AR antagonists have been used for more than two decades to treat lower urinary tract symptoms (LUTS), we have little understanding of the mechanistic basis of their efficacy and their role in the development of LUTS. It is clear that alpha(1)ARs play a critical role in bladder dysfunction and recent data suggest that alpha(1)AR subtype switching may play a key role in this pathophysiology, providing support for use of alpha(1)(d)AR-selective antagonists in treating irritative symptoms. This review seeks to summarize current levels of understanding in this field and discusses new concepts that suggest increased levels of complexity involving cross-talk in multiple receptor systems. Effective therapeutic modalities likely will involve increased subtype selective alpha(1)AR antagonists and other pharmacodynamic factors.
Current Urology Reports 09/2004; 5(4):258-66.
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[show abstract]
[hide abstract]
ABSTRACT: Recent studies reveal important and distinct roles for cardiac alpha(1a) adrenergic receptors (alpha(1a)ARs). Surprisingly, given their importance in myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as frequent use of rat cardiomyocyte model systems, the rat alpha(1a)AR gene promoter has never been characterized. Therefore, we isolated 3.9 kb of rat alpha(1a)AR 5'-untranslated region and 5'-regulatory sequences and identified multiple transcription initiation sites. One proximal (P1) and several clustered upstream distal promoters (P2, P3, and P4) were delineated. Sequences surrounding both proximal and distal promoters lack typical TATA or CCAAT boxes but contain cis-elements for multiple myocardium-relevant nuclear regulators including Sp1, GATA, and CREB, findings consistent with enhanced cardiac basal alpha(1a)AR expression seen in Northern blots and reporter constructs. Promoter analysis using deletion reporter constructs reveals, in addition to a powerful upstream enhancer, a key region (-558/-542) important in regulating all alpha(1a)AR promoters with hypoxic stress. Gel shift analysis of this 14-bp region confirms a hypoxia-induced shift independent of direct hypoxia-inducible factor binding. Mutational analysis of this sequence identifies a novel 9-bp hypoxia response element, the loss of which severely attenuates hypoxia-mediated repression of alpha(1a)AR transcription. These findings for the alpha(1a) gene should facilitate elucidation of alpha(1)AR-mediated mechanisms involved in distinct myocardial pathologies.
Journal of Biological Chemistry 04/2003; 278(10):8693-705. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Recent studies reveal important and distinct roles for cardiac α1a adrenergic receptors (α1aARs). Surprisingly, given their importance in myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as frequent
use of rat cardiomyocyte model systems, the rat α1aAR gene promoter has never been characterized. Therefore, we isolated 3.9 kb of rat α1aAR 5′-untranslated region and 5′-regulatory sequences and identified multiple transcription initiation sites. One proximal
(P1) and several clustered upstream distal promoters (P2, P3, and P4) were delineated. Sequences surrounding both proximal
and distal promoters lack typical TATA or CCAAT boxes but contain cis-elements for multiple myocardium-relevant nuclear regulators including Sp1, GATA, and CREB, findings consistent with enhanced
cardiac basal α1aAR expression seen in Northern blots and reporter constructs. Promoter analysis using deletion reporter constructs reveals,
in addition to a powerful upstream enhancer, a key region (–558/−542) important in regulating all α1aAR promoters with hypoxic stress. Gel shift analysis of this 14-bp region confirms a hypoxia-induced shift independent of
direct hypoxia-inducible factor binding. Mutational analysis of this sequence identifies a novel 9-bp hypoxia response element,
the loss of which severely attenuates hypoxia-mediated repression of α1aAR transcription. These findings for the α1a gene should facilitate elucidation of α1AR-mediated mechanisms involved in distinct myocardial pathologies.
Journal of Biological Chemistry 03/2003; 278(10):8693-8705. · 4.77 Impact Factor
