American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015
Although significant research data exists about the pathophysiology of Nonalcoholic Steatohepatit... more Although significant research data exists about the pathophysiology of Nonalcoholic Steatohepatitis (NASH), finding an efficient treatment regimen for it remains elusive. The present study used Sparstolonin B (SSnB), novel TLR4 antagonist derived from Chinese herb Spaganium stoloniferum as a possible drug to mitigate early inflammation in NASH. This study uses an early steatohepatitic injury model in high fat fed mice with CYP2E1-mediated oxidative stress as a second hit. SSnB was administered for 1 week along with BDCM, an inducer of CYP2E1-mediated oxidative stress. Results showed that SSnB administration attenuated inflammatory morphology and decreased elevation of liver enzyme ALT. Mice administered SSnB also showed decreased mRNA expression of proinflammatory cytokines TNF-α, IFNγ, IL1β and IL23 while protein levels of both TNF-α and IL1β were significantly decreased. SSnB significantly decreased Kupffer cell activation as evidenced by decreases in CD68 and MCP1 mRNA and protein levels with concomitant inhibition of macrophage infiltration in the injured liver. Mechanistically, SSnB decreased TLR4 trafficking to the lipid rafts, a phenomenon described by the colocalization of TLR4 and lipid raft marker flotillin-2 in tissues and immortalized Kupffer cells. Since we have shown previously that NADPH oxidase drives TLR4 trafficking in NASH, we studied the role of SSnB in modulating this pathway. SSnB prevented NADPH oxidase activation in vivo and in vitro as indicated by decreased peroxynitrite formation. In summary the present study reports a novel use of a TLR4 antagonist SSnB in mitigating inflammation in NASH and in parallel showed unique molecular mechanism of decreasing nitrative stress.
Targeted therapies and the consequent adoption of "personalized" oncology have achieved... more Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity…
Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and sec... more Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.
Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous s... more Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO ...
Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain gro... more Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain growth, cancer cells undergo a complex metabolic rearrangement characterized by changes in metabolic pathways involved in energy production and biosynthetic processes. The relevance of the metabolic transformation of cancer cells has been recently included in the updated version of the review "Hallmarks of Cancer", where the dysregulation of cellular metabolism was included as an emerging hallmark. While several lines of evidence suggest that metabolic rewiring is orchestrated by the concerted action of oncogenes and tumor suppressor genes, in some circumstances altered metabolism can play a primary role in oncogenesis. Recently, mutations of cytosolic and mitochondrial enzymes involved in key metabolic pathways have been associated with hereditary and sporadic forms of cancer. Together, these results suggest that aberrant metabolism, once seen just as an epiphenomenon of oncogeni...
Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and sec... more Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were incr...
Alpha1-adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct h... more 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.
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker a... more Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker and a down-stream target in Wnt/β-catenin signaling. In human papillary thyroid cancer (PTC), over activation of Wnt/β-catenin has been associated with tumor aggressiveness. Using established human cell lines (TPC-1, KTC-1, Nthy-ori-3-1), we report LGR5 and R-spondin (RSPO1-3) overexpression in PTC and manipulate LGR5 and Wnt/β-catenin signaling via both pharmacologic and genetic interventions. We test the association of LGR5 tumor expression with markers of PTC aggressiveness using a Discovery Cohort (n = 26 patients) and a Validation Cohort (n = 157 patients). Lastly, we explore the association between LGR5 and the BRAFV600E mutation (n = 33 patients). Our results reveal that LGR5 and its ligand, RSPO, are overexpressed in human PTC, whereby Wnt/β-catenin signaling regulates LGR5 expression and promotes cellular migration. In two separate cohorts of patients, LGR5 and RSPO2 were associated with markers of tumor aggressiveness including: lymph node metastases, vascular invasion, increased tumor size, aggressive histology, advanced AJCC TNM stage, microscopic extra thyroidal extension, capsular invasion, and macroscopic invasion. As a biomarker, LGR5 positivity predicts lymph node metastasis with 95.5% sensitivity (95% CI 88.8%-98.7%) and 61% specificity (95% CI: 48.4%-72.4%) and has a negative predictive value (NPV) of 91.3% (95% CI 79.2%-97.5%) for lymph node metastatic disease. In human PTC, LGR5 is also strongly associated with the BRAFV600E mutation (p = 0.005). We conclude that overexpression of LGR5 is associated with markers of tumor aggressiveness in human PTC. LGR5 may serve as a future potential biomarker for patient risk stratification and loco regional metastases in PTC.
Transcription activation and repression of eukaryotic genes are associated with conformational an... more Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsio...
Human C8 is one of five terminal complement components (C5b, C6, C7, C8, C9) that interact to for... more Human C8 is one of five terminal complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex. It is composed of three nonidentical subunits (α, β, γ) which are encoded in separate genes. C8α and C8β are homologous to each other and to C6, C7 and C9. All share common structural motifs that include cysteine-rich
In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the... more In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated wit...
Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem-cell associated ... more Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem-cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with nonalcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced nonalcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog (Shh), but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. YAP+RDC promote scarring, rather than effective regeneration, during NASH.
Initiation and progression of hepatocellular carcinoma (HCC) is intimately associated with a chro... more Initiation and progression of hepatocellular carcinoma (HCC) is intimately associated with a chronically diseased liver tissue. This diseased liver tissue background is a drastically different microenvironment from the healthy liver, especially with regard to immune cell prevalence and presence of mediators of immune function. To better understand the consequences of liver disease on tumor growth and the interplay with its microenvironment, we utilized two standard methods of fibrosis induction and orthotopic implantation of tumors into the inflamed and fibrotic liver to mimic the liver condition in human HCC patients. Compared to non-diseased controls, tumor growth was significantly enhanced under fibrotic conditions. The immune cells that infiltrated the tumors were also drastically different, with decreased numbers of natural killer cells but greatly increased numbers of immune-suppressive CD11b(+) Gr1(hi) myeloid cells in both models of fibrosis. In addition, there were model-sp...
American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015
Although significant research data exists about the pathophysiology of Nonalcoholic Steatohepatit... more Although significant research data exists about the pathophysiology of Nonalcoholic Steatohepatitis (NASH), finding an efficient treatment regimen for it remains elusive. The present study used Sparstolonin B (SSnB), novel TLR4 antagonist derived from Chinese herb Spaganium stoloniferum as a possible drug to mitigate early inflammation in NASH. This study uses an early steatohepatitic injury model in high fat fed mice with CYP2E1-mediated oxidative stress as a second hit. SSnB was administered for 1 week along with BDCM, an inducer of CYP2E1-mediated oxidative stress. Results showed that SSnB administration attenuated inflammatory morphology and decreased elevation of liver enzyme ALT. Mice administered SSnB also showed decreased mRNA expression of proinflammatory cytokines TNF-α, IFNγ, IL1β and IL23 while protein levels of both TNF-α and IL1β were significantly decreased. SSnB significantly decreased Kupffer cell activation as evidenced by decreases in CD68 and MCP1 mRNA and protein levels with concomitant inhibition of macrophage infiltration in the injured liver. Mechanistically, SSnB decreased TLR4 trafficking to the lipid rafts, a phenomenon described by the colocalization of TLR4 and lipid raft marker flotillin-2 in tissues and immortalized Kupffer cells. Since we have shown previously that NADPH oxidase drives TLR4 trafficking in NASH, we studied the role of SSnB in modulating this pathway. SSnB prevented NADPH oxidase activation in vivo and in vitro as indicated by decreased peroxynitrite formation. In summary the present study reports a novel use of a TLR4 antagonist SSnB in mitigating inflammation in NASH and in parallel showed unique molecular mechanism of decreasing nitrative stress.
Targeted therapies and the consequent adoption of "personalized" oncology have achieved... more Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity…
Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and sec... more Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.
Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous s... more Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO ...
Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain gro... more Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain growth, cancer cells undergo a complex metabolic rearrangement characterized by changes in metabolic pathways involved in energy production and biosynthetic processes. The relevance of the metabolic transformation of cancer cells has been recently included in the updated version of the review "Hallmarks of Cancer", where the dysregulation of cellular metabolism was included as an emerging hallmark. While several lines of evidence suggest that metabolic rewiring is orchestrated by the concerted action of oncogenes and tumor suppressor genes, in some circumstances altered metabolism can play a primary role in oncogenesis. Recently, mutations of cytosolic and mitochondrial enzymes involved in key metabolic pathways have been associated with hereditary and sporadic forms of cancer. Together, these results suggest that aberrant metabolism, once seen just as an epiphenomenon of oncogeni...
Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and sec... more Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were incr...
Alpha1-adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct h... more 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.
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker a... more Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker and a down-stream target in Wnt/β-catenin signaling. In human papillary thyroid cancer (PTC), over activation of Wnt/β-catenin has been associated with tumor aggressiveness. Using established human cell lines (TPC-1, KTC-1, Nthy-ori-3-1), we report LGR5 and R-spondin (RSPO1-3) overexpression in PTC and manipulate LGR5 and Wnt/β-catenin signaling via both pharmacologic and genetic interventions. We test the association of LGR5 tumor expression with markers of PTC aggressiveness using a Discovery Cohort (n = 26 patients) and a Validation Cohort (n = 157 patients). Lastly, we explore the association between LGR5 and the BRAFV600E mutation (n = 33 patients). Our results reveal that LGR5 and its ligand, RSPO, are overexpressed in human PTC, whereby Wnt/β-catenin signaling regulates LGR5 expression and promotes cellular migration. In two separate cohorts of patients, LGR5 and RSPO2 were associated with markers of tumor aggressiveness including: lymph node metastases, vascular invasion, increased tumor size, aggressive histology, advanced AJCC TNM stage, microscopic extra thyroidal extension, capsular invasion, and macroscopic invasion. As a biomarker, LGR5 positivity predicts lymph node metastasis with 95.5% sensitivity (95% CI 88.8%-98.7%) and 61% specificity (95% CI: 48.4%-72.4%) and has a negative predictive value (NPV) of 91.3% (95% CI 79.2%-97.5%) for lymph node metastatic disease. In human PTC, LGR5 is also strongly associated with the BRAFV600E mutation (p = 0.005). We conclude that overexpression of LGR5 is associated with markers of tumor aggressiveness in human PTC. LGR5 may serve as a future potential biomarker for patient risk stratification and loco regional metastases in PTC.
Transcription activation and repression of eukaryotic genes are associated with conformational an... more Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsio...
Human C8 is one of five terminal complement components (C5b, C6, C7, C8, C9) that interact to for... more Human C8 is one of five terminal complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex. It is composed of three nonidentical subunits (α, β, γ) which are encoded in separate genes. C8α and C8β are homologous to each other and to C6, C7 and C9. All share common structural motifs that include cysteine-rich
In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the... more In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated wit...
Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem-cell associated ... more Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem-cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with nonalcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced nonalcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog (Shh), but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. YAP+RDC promote scarring, rather than effective regeneration, during NASH.
Initiation and progression of hepatocellular carcinoma (HCC) is intimately associated with a chro... more Initiation and progression of hepatocellular carcinoma (HCC) is intimately associated with a chronically diseased liver tissue. This diseased liver tissue background is a drastically different microenvironment from the healthy liver, especially with regard to immune cell prevalence and presence of mediators of immune function. To better understand the consequences of liver disease on tumor growth and the interplay with its microenvironment, we utilized two standard methods of fibrosis induction and orthotopic implantation of tumors into the inflamed and fibrotic liver to mimic the liver condition in human HCC patients. Compared to non-diseased controls, tumor growth was significantly enhanced under fibrotic conditions. The immune cells that infiltrated the tumors were also drastically different, with decreased numbers of natural killer cells but greatly increased numbers of immune-suppressive CD11b(+) Gr1(hi) myeloid cells in both models of fibrosis. In addition, there were model-sp...
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Papers by Gregory Michelotti