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Biomedical subjects

S L Friedman

Publications and source records attributed to S L Friedman.

At least 19 recordsLinked to original sources
PubMed2006

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.

3' Untranslated Regions↗
PubMed2006

Antifibrotic agents for liver disease.

Complications from chronic hepatitis C (HCV) and recurrent HCV post-transplant are responsible for significant morbidity and mortality in the United States and Europe. Current antiviral therapies are at best, effective in up to 50% of patients in the pre-transplant setting, and in the post-transplant setting are associated with more limited efficacy and increased toxicity. With this reduced efficacy of antiviral strategies in the post-transplant setting, new approaches are urgently needed. Substantial progress has been made in understanding the pathogenesis of hepatic fibrosis over the last 20 years, which has yielded potential new therapeutic targets. The prospect of antifibrotic therapies is nearing reality in order to reduce progression to cirrhosis, thereby reducing morbidity, mortality and the need for re-transplantation. Current and evolving approaches primarily target the activated hepatic stellate cells, which are the main source of extracellular matrix, along with related fibrogenic cell types. Key issues yet to be clarified include the optimal duration of antifibrotic therapies, endpoints of clinical trials, indications in clinical practice and whether combination therapies might yield synergistic activity.

Drug Design↗
PubMed2005

Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis.

BACKGROUND: Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells. METHODS AND RESULTS: LX-1 and LX-2 were generated by either SV40 T antigen immortalisation (LX-1) or spontaneous immortalisation in low serum conditions (LX-2). Both lines express alpha smooth muscle actin, vimentin, and glial fibrillary acid protein, as visualised by immunocytochemistry. Similar to primary HSCs, both lines express key receptors regulating hepatic fibrosis, including platelet derived growth factor receptor beta (betaPDGF-R), obese receptor long form (Ob-RL), and discoidin domain receptor 2 (DDR2), and also proteins involved in matrix remodelling; matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-2, and MT1-MMP, as determined by western analyses. LX-2 have reduced expression of TIMP-1. LX-2, but not LX-1, proliferate in response to PDGF. Both lines express mRNAs for alpha1(I) procollagen and HSP47. Transforming growth factor beta1 stimulation increased their alpha1(I) procollagen mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction. LX-2, but not LX-1, cells are highly transfectable. Both lines had a retinoid phenotype typical of stellate cells. Microarray analyses showed strong similarity in gene expression between primary HSCs and either LX-1 (98.4%) or LX-2 (98.7%), with expression of multiple neuronal genes. CONCLUSIONS: LX-1 and LX-2 human HSC lines provide valuable new tools in the study of liver disease. Both lines retain key features of HSCs. Two unique advantages of LX-2 are their viability in serum free media and high transfectability.

Adipocytes↗
PubMed2001

KLF6, a candidate tumor suppressor gene mutated in prostate cancer.

Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.

Alleles↗
PubMed2001

Phosphorylation of retinoid X receptor alpha at serine 260 impairs its metabolism and function in human hepatocellular carcinoma.

Retinoids induce apoptosis and differentiation of hepatocellular carcinoma (HCC) cells and are used clinically in the chemoprevention of HCC. We have shown previously that hepatocarcinogenesis is accompanied by accumulation of full-length retinoid X receptor alpha (RXRalpha), although the underlying mechanisms and biological implications have remained unclear. The present studies were based on the finding that the accumulated full-length RXRalpha was phosphorylated at serine/threonine residues both in all human HCC tissues examined and in human HCC-derived HuH7 cells. Phosphorylation at serine 260 of RXRalpha, a consensus site of mitogen-activated protein kinase, was closely linked to its retarded degradation, low transactivating activity, and the promotion of cancer cell growth. There was no genomic mutation in the RXRalpha gene, and abrogation of phosphorylation by mitogen-activated protein kinase-specific inhibitors restored the degradation of RXRalpha in an RXR ligand-dependent manner. These results suggest that phosphorylation of RXRalpha may interfere with its metabolism and signaling in human HCC, which could lead to growth promotion of these tumors.

Carcinoma, Hepatocellular↗
PubMed2001

Hepatic fibrosis. Pathogenesis and principles of therapy.

There has been great progress made in our understanding of the cellular mechanisms of hepatic fibrosis. The recognition that the hepatic stellate cell, (formerly know as lipocyte, Ito, or fat-storing cell), played a central role in the fibrotic response was key to our understanding. Stellate cells undergo a process known as activation, in response to any insult. Activation is a broad phenotypic response, characterized by distinct functional changes in proliferation, fibrogenesis, contractility, cytokine secretion, and matrix degradation. Insights gained into the molecular regulations of stellate cell activation may lead to new antifibrotic therapies, which may reduce morbidity and mortality in patients with chronic liver injury.

Humans↗
PubMed2001

Mechanism of retarded liver regeneration in plasminogen activator-deficient mice: impaired activation of hepatocyte growth factor after Fas-mediated massive hepatic apoptosis.

Urokinase-type plasminogen activator (uPA) is implicated in the regulation of hepatic regeneration by activating hepatocyte growth factor (HGF). Here, we investigated its role in the hepatic regeneration after Fas-mediated massive hepatocyte death employing mice deficient in either uPA or its inhibitor, plasminogen activator inhibitor-1 (PAI-1). We measured kinetics of hepatic levels of proliferating cell nuclear antigen (PCNA)-labeling index, plasmin activity, mature HGF, and its phosphorylated receptor, c-Met. In the genetically targeted and wild-type mice, hepatocytes fell into the same extent of apoptosis 6 to 12 hours after an intraperitoneal injection with anti-Fas antibody, as judged from histologic analysis and a histon-DNA enzyme-linked immunosorbent assay (ELISA). In the wild-type mice, mature HGF emerged in the liver 6 hours following anti-Fas injection, and hepatic PCNA-labeling index started to increase following 24 hours and peaked at 48 hours. In the uPA(-/-) mice, emergence of mature HGF was delayed 12 hours and hepatic regeneration peaked at 96 hours. Supplementation with the uPA gene to the uPA(-/-) mice by in vivo lipofection restored hepatic plasmin levels, and improved a delay in the expression of both mature HGF and phosphorylated c-Met, accompanying a normal rate of liver regeneration. In contrast, PAI-1(-/-) mice showed accelerated liver regeneration; mature HGF emerged as early as 3 hours, and PCNA-labeling index increased at 24 hours. This accelerated regeneration was abolished by administration with anti-HGF antibody. These results strongly suggest a physiologic role of uPA in the proteolytic maturation of HGF, and thereby in hepatic regeneration after Fas-mediated massive hepatocyte death.

Animals↗
PubMed2001

Expression and role of Bcl-xL in human hepatocellular carcinomas.

Transformed hepatocytes survive various apoptotic insults during their growth in vivo. However, molecular mechanisms that inhibit apoptosis and support their survival are not well understood. In this study, we investigated the expression and role of Bcl-xL, an antiapoptotic member of the Bcl-2 family, in human hepatocellular carcinoma (HCC). The Bcl-xL protein was expressed in HepG2, Hep3B, and Huh7 human hepatoma cell lines at high levels, but none of these cells expressed Bcl-2. Down-modulation of Bcl-xL by antisense oligonucleotide activated apoptosis in HepG2 cells in response to cellular stresses induced by staurosporine treatment or by serum starvation. Ectopic expression of transcriptionally active p53 alone was not sufficient for the activation of apoptosis in p53-null Hep3B cells, but apoptosis was induced when endogenous Bcl-xL was simultaneously inhibited by antisense oligonucleotide in these cells. Bcl-xL was expressed in all 20 surgically resected human HCC tissues when examined by Western blot analysis and immunohistochemistry, and levels of its expression were higher in a subset of HCC tissues than those of adjacent nontumor liver tissues or normal livers. We conclude that Bcl-xL expressed in human HCC cells inhibits apoptosis produced by various cellular stresses, such as staurosporine treatment, serum starvation, and p53 activation, and may play an important role in their survival.

Apoptosis↗
PubMed2001

Transcriptional regulation in hepatic stellate cells.

Modulation of gene expression through altered transcription regulates stellate cell behavior in normal liver and following hepatic injury. Transcription factors are generally classified according to conserved motifs within either the activation- or DNA- binding domains of the molecules. Transcriptional activity in stellate cells represents a delicate fine tuning of multiple inputs. Activities of these transcription factors are modified by their intracellular localization, rate and pathway of degradation, oligomerization, and interactions with heterologous factors and chromatin, as well as by posttranslational modifications, including phosphorylation, glycosylation, and acetylation. General paradigms of transcriptional control are increasingly being validated in hepatic stellate cells, particularly involving the transcription factors CCAAT/enhancer-binding proteins, c-myb, CREB, nuclear factor kappaB, peroxisome proliferator-activated receptor, and Kruppel-like zinc finger factors. Although there are no simple rules that govern mechanisms of transcriptional regulation in stellate cells, continued advances will yield new insights into their role in normal liver homeostasis and in the response to injury.

DNA-Binding Proteins↗
PubMed2001

DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.

Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

Animals↗
PubMed2001

Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs.

This study validated a polymerase chain reaction-based method for the detection of a specific bovine mitochondrial gene derived from rendered bovine tissues and admixed with complete animal feed. Four laboratories participated in this effort: one state laboratory and three Food and Drug Administration (FDA) laboratories, including one FDA field laboratory. The protocol used a statistical approach of 90% probability, with a 95% confidence interval for determining acceptable rates of false-positive and false-negative samples. Each participating laboratory analyzed 30 samples of feed each containing 0, 0.125, and 2.0% bovine meat and bone meal (BMBM), for a total of 90 feed samples. The samples were randomized such that the analysts were unaware of the true identity of the test samples. The results demonstrated that all laboratories met the acceptance criteria established for this protocol. The overall rates of false-negative results were 0.83% (1/120) at the level of 0.125% BMBM and 1.67% (2/120) at the level of 2% BMBM. The overall rate of false-negative results for all levels of BMBM was 1.25% (3/240). The rate for false-positive results was 0.83%.

Animal Feed↗
PubMed2001

Hepatology at Mount Sinai: the present and the future.

The Division of Liver Disease at Mount Sinai, now into its fifth decade, has evolved through two remarkable periods in its development and is on the cusp of a third exciting era. The first, extending from the division's creation in 1957 to the retirement in 1988 of its first division chief, Fenton Schaffner, was characterized by brilliant clinical and pathophysiologic insights derived from the unique collaboration of Schaffner, a master clinician, with Hans Popper, a world renowned pathologist widely acknowledged as the father of the modern discipline of hepatology. The second, extending from the appointment in 1988 of Paul D. Berk as Schaffner's successor to the present day, has witnessed enormous growth in the clinical and scientific activities of the division, together with the emergence of a world-class liver transplant program at Mount Sinai. During this recent period, an extensive program of formal clinical research was established; the basic research program then expanded into the areas of hepatic transport, molecular virology, and the cellular and molecular pathogenesis of hepatic fibrosis; and both the clinical and research productivity of the division increased dramatically. A major undertaking, now in its second year, has been the creation of the Center for the Study of Primary Biliary Cirrhosis; Mount Sinai has contributed important advances toward the understanding of this disease. Funding for the Center, from the Artzt Family Foundation Trust, supports a series of interrelated basic studies on the immunology and pathobiology of the disease, as well as creation of a unique clinical database, a serum and tissue bank, and a program of clinical studies. This integration of basic and clinical research in pursuit of new methods of diagnosis and treatment serves as a model for the division's continued leadership role.

Academic Medical Centers↗
PubMed2000

Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2.

The ability of ethanol and arachidonic acid (AA), as inducers of oxidative stress and key factors in alcoholic liver disease, to up-regulate alpha 2 collagen type I (COL1A2) gene expression was studied in a hepatic stellate cell line overexpressing the ethanol-inducible cytochrome P450 2E1 (CYP2E1) (E5 cells). A time- and dose-dependent induction in COL1A2 mRNA by ethanol or AA was observed that was prevented by diallylsulfide, a CYP2E1 inhibitor. Nuclear run-on experiments showed transcriptional activation of the COL1A2 gene by ethanol and AA. Catalase abrogated the increase in COL1A2 mRNA suggesting an H(2)O(2)-dependent mechanism. Cyclooxygenase-2 (COX-2) levels and production of prostaglandin E(2) upon addition of AA were elevated in the E5 cells. Incubation with NS-398, a COX-2 inhibitor, blocked the effect of AA, but not of ethanol, on COL1A2 expression suggesting that CYP2E1 activates COX-2 expression, and the oxidation of AA by COX-2 is responsible for the increase in COL1A2. Activity of a reporter construct driven by -378 base pairs of the proximal promoter region of the COL1A2 gene increased in E5 but not control cells and was further increased by ethanol or AA. These experiments link CYP2E1-dependent oxidative stress to induction of COX-2 and the actions of ethanol and AA on activation of collagen gene expression in hepatic stellate cells.

Allyl Compounds↗
PubMed2000

The Krüppel-like transcriptional factors Zf9 and GKLF coactivate the human keratin 4 promoter and physically interact.

Zf9/CPBP/KLF6 is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines. Additionally, Zf9 localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, Zf9 transactivates the human keratin 4 (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of Zf9 and GKLF/KLF4, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between GKLF and Zf9, a novel finding for Krüppel-like family members.

Cell Line↗
PubMed2000

Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-beta1 in vascular endothelial cells.

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)

Animals↗