Biomedical subjects

Scott L Friedman

Publications and source records attributed to Scott L Friedman.

At least 37 records · Page 2Linked to original sources

PubMed2005

Regulation of Kruppel-like factor 6 tumor suppressor activity by acetylation.

Krüppel-like factor 6 (KLF6) is a zinc finger transcription factor and tumor suppressor that is inactivated in a number of human cancers by mutation, allelic loss, and/or promoter methylation. A key mechanism of growth inhibition by wild-type KLF6 is through p53-independent up-regulation of p21(WAF1/cip1) (CDKN1A), which is abrogated in several tumor-derived mutants. Here we show by chromatin immunoprecipitation that transactivation of p21(WAF1/cip1) by KLF6 occurs through its direct recruitment to the p21(WAF1/cip1) promoter and requires acetylation by histone acetyltransferase activity of either cyclic AMP-responsive element binding protein-binding protein or p300/CBP-associated factor. Direct lysine acetylation of KLF6 peptides can be shown by mass spectrometry. A single lysine-to-arginine point mutation (K209R) derived from prostate cancer reduces acetylation of KLF6 and abrogates its capacity to up-regulate endogenous p21(WAF1/cip1) and reduce cell proliferation. These data indicate that acetylation may regulate KLF6 function, and its loss in some tumor-derived mutants could contribute to its failure to suppress growth in prostate cancer.

Acetylation↗

PubMed2005

Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10.

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.

Administration, Oral↗

PubMed2005

BMP-7 opposes TGF-beta1-mediated collagen induction in mouse pulmonary myofibroblasts through Id2.

Mesenchymal cells, primarily fibroblasts and myofibroblasts, are the principal matrix-producing cells during pulmonary fibrogenesis. Transforming growth factor (TGF)-beta signaling plays an important role in stimulating the expression of type I collagen of these cells. Bone morphogenetic protein (BMP)-7, a member of the TGF-beta superfamily, has been reported to oppose the fibrogenic activity of TGF-beta1. Here, we have addressed the effects of BMP-7 on the fibrogenic activity of pulmonary myofibroblasts. We first established cell lines from the lungs of transgenic mice harboring the COL1A2 upstream sequence fused to luciferase. They displayed a spindle shape and expressed vimentin and alpha-smooth muscle actin, but not E-cadherin. COL1A2 promoter activity was dose dependently induced by TGF-beta1, which was further augmented by adenoviral overexpression of Smad3, but was downregulated by Smad7. Under the identical condition, adenoviral overexpression of BMP-7 attenuated the TGF-beta1-dependent COL1A2 promoter activity. By immunocytochemistry, the ectopic expression of BMP-7 led to the nuclear localization of phospho-Smad1/5/8 and suppressed that of Smad3. BMP-7 suppressed the expression of mRNAs for COL1A2 and tissue inhibitor of metalloproteinase-2 while increasing those of inhibitors of differentiation (Id) 2 and 3. Ectopic expression of Id2 and Id3 was found to decrease the COL1A2 promoter activity. Finally, BMP-7 and Id2 decreased TGF-beta1-dependent collagen protein secretion. In conclusion, these data demonstrate that BMP-7 antagonizes the TGF-beta1-dependent fibrogenic activity of mouse pulmonary myofibroblastic cells by inducing Id2 and Id3.

Animals↗

PubMed2005

Sex steroids have differential effects on growth and gene expression in primary human prostatic epithelial cell cultures derived from the peripheral versus transition zones.

The majority of human prostate cancers arise from the peripheral zone (PZ). Prostate epithelial stem cells have been localized to the basal epithelial cell compartment. In addition, basal cells have been shown to maintain luminal epithelial cell differentiation and may mediate signals between the stromal and luminal cell compartments. Therefore, the study of adult prostate basal cells derived from different prostate zones may give insights into the mechanisms underlying normal and abnormal prostate growth. We herein compare the basal and sex steroid-stimulated expression and activity of several genes/proteins that are known to be critical in prostate cancer development in primary cultures of basal cells derived from the transition zone (TZ) and PZ of prostatectomy specimens. Our results demonstrate that prostate basal cells derived from the PZ versus TZ are more viable in culture, particularly in response to sex steroid addition. PZ cells exhibit higher telomerase activity and increased expression levels of androgen receptor, the anti-apoptotic protein bcl-2, and the dominant-negative splice variant of Kruppel-like Factor 6. PZ cells have lower basal expression levels of estrogen receptor-beta, the pro-apoptotic protein Bax, and cell-cycle inhibitor proteins (p53, p21(waf1/Cip1)). Finally, we demonstrate divergent responses to sex hormones in the two basal cell populations. The gene expression pattern in the PZ cells may partially explain the predominance of prostate cancer development in this region.

Cell Proliferation↗

PubMed2005

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

Alternative Splicing↗

PubMed2005

Krüppel-like factor-6 promotes preadipocyte differentiation through histone deacetylase 3-dependent repression of DLK1.

Preadipocyte differentiation occurs during distinct periods of human development and is a key determinant of body mass. Transcriptional events underlying adipogenesis continue to emerge, but the link between chromatin remodeling of specific target loci and preadipocyte differentiation remains elusive. We have identified Krüppel-like factor-6 (KLF6), a recently described tumor suppressor gene, as a repressor of the proto-oncogene Delta-like 1 (Dlk1), a gene encoding a transmembrane protein that inhibits adipocyte differentiation. Forced expression of KLF6 strongly inhibits Dlk1 expression in preadipocytes and NIH 3T3 cells in vivo, whereas down-regulation of KLF6 in 3T3-L1 cells by small interfering RNA prevents adipogenesis. Repression of Dlk1 requires HDAC3 deacetylase activity, which is recruited to the endogenous Dlk1 promoter where it interacts with KLF6. Our studies identify the interaction between HDAC3 and KLF6 as a potential mechanism underlying human adipogenesis, and highlight the role of KLF6 as a multifunctional transcriptional regulator capable of mediating adipocyte differentiation through gene repression.

3T3 Cells↗

PubMed2005

Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu.

Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 microm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and alpha B-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased alpha B-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiments.

Animals↗

PubMed2005

A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.

Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer-associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.

Alleles↗

PubMed2005

Interleukin-6 protects hepatocytes from CCl4-mediated necrosis and apoptosis in mice by reducing MMP-2 expression.

BACKGROUND/AIMS: Interleukin-6 stimulates liver regeneration and promotes hepatoprotection following experimental liver injury, but underlying mechanisms have not been fully characterized. Because studies suggest matrix metalloproteinase-2 (MMP-2) may promote liver injury, we examined whether IL-6 exerted its protective effects via regulation of MMP-2. METHODS: MMP-2 was analyzed in livers of IL-6-/- and IL-6+/+ mice following CCl(4) administration. IL-6-/- mice were pretreated with IL-6 and liver histology and MMP-2 expression were examined after liver injury. IL-6-/- mice were treated with an MMP-2 inhibitor and assessment of injury (histology and serum ALT levels), apoptosis by TUNEL assay, and hepatocyte proliferation by BRDU-labeling was performed. These studies were complemented by analysis of cultured stellate cells. RESULTS: MMP-2 mRNA, protein, and activity was increased in IL-6-/- livers. Restoration of IL-6 signaling in IL-6-/- mice rescued injury and restored MMP-2 expression to wild-type levels. Furthermore, pharmacologic inhibition of MMP-2 decreased hepatocellular injury and apoptosis in IL-6-/- mice. In cultured stellate cells, recombinant IL-6 suppressed endogenous MMP-2 mRNA and protein expression. CONCLUSIONS: IL-6 may be hepatoprotective in acute injury through down-regulation of MMP-2. These findings suggest a role for MMP-2 in amplifying liver injury in vivo.

Acute Disease↗

PubMed2005

Mac the knife? Macrophages- the double-edged sword of hepatic fibrosis.

Progression of hepatic fibrosis requires sustained inflammation leading to activation of stellate cells into a fibrogenic and proliferative cell type, whereas regression is associated with stellate cell apoptosis. The contribution of hepatic macrophages to these events has been largely overlooked. However, a study in this issue of the JCI demonstrates that macrophages play pivotal but divergent roles, favoring ECM accumulation during ongoing injury but enhancing matrix degradation during recovery. These findings underscore the potential importance of hepatic macrophages in regulating both stellate cell biology and ECM degradation during regression of hepatic fibrosis.

Animals↗

PubMed2005

Prevention of rat hepatocarcinogenesis by acyclic retinoid is accompanied by reduction in emergence of both TGF-alpha-expressing oval-like cells and activated hepatic stellate cells.

We investigated the preventive effects of a synthetic acyclic retinoid, NIK-333, on the early and late events of hepatocarcinogenesis in male F344 rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). NIK-333 was administered once a day on consecutive days at a dose of 10, 40, or 80 mg/kg body weight along with the supplementation with 3'-MeDAB-containing diet for 16 wk. Animals from each group were sacrificed at 4 and 16 wk after the commencement of the experiment to determine the effect of NIK-333 on the early and late stages of carcinogenesis, respectively. NIK-333 suppressed the emergence of both oval-like cells expressing transforming growth factor (TGF)-alpha, putative progenitors of hepatocellular carcinoma (HCC), and activated hepatic stellate cells, major matrix-producing cells of the liver, in the early stage and inhibited the incidence of HCC in the late phase. These results suggest that NIK-333 is a promising drug for the chemoprevention of HCC by uniquely suppressing the early events of hepatocarcinogenesis, that is, development of both oval-like cells and fibrogenesis.

Actins↗

PubMed2004

Lhx2-/- mice develop liver fibrosis.

Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2(-/-) mice is disrupted, we analyzed liver development in Lhx2(-/-) embryos in detail. Lhx2(-/-) embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2(-/-) liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.

Adipocytes↗

PubMed2004

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells.

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.

Androgens↗

PubMed2004

Suppression of glioblastoma tumorigenicity by the Kruppel-like transcription factor KLF6.

The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14-15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.

Animals↗

PubMed2004

Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1.

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.

CDC2-CDC28 Kinases↗

PubMed2004

Transcriptional activation of the insulin-like growth factor I receptor gene by the Kruppel-like factor 6 (KLF6) tumor suppressor protein: potential interactions between KLF6 and p53.

The IGF system plays an important role in prostate cancer initiation and progression. Most of the biological actions of IGF-I and IGF-II are mediated by activation of the IGF-I receptor (IGF-IR). Evidence accumulated in recent years indicates that acquisition of the malignant phenotype is initially IGF-IR dependent, but progression toward metastatic stages is usually associated with a decrease in IGF-IR levels. The Kruppel-like factor 6 (KLF6) is a zinc finger-containing transcription factor that was shown to be mutated in a significant portion of prostate and other types of cancer. To examine the potential regulation of IGF-IR gene expression by KLF6, we measured KLF6 levels in prostate-derived cell lines displaying different levels of IGF-IR. The results of Western analysis showed that KLF6 levels were higher in nontumorigenic P69 cells expressing high IGF-IR levels than in metastatic M12 cells containing reduced IGF-IR levels. Transient coexpression of wild-type, but not mutated, KLF6 together with an IGF-IR promoter-luciferase reporter plasmid resulted in an approximately 3.4-fold stimulation of IGF-IR promoter activity. Furthermore, KLF6 expression induced a significant increment in endogenous IGF-IR levels. Deletion analysis of the IGF-IR promoter revealed that a cluster of four GC boxes located between nucleotides -399 and -331 mediates a significant portion of the transactivating effect of KLF6. KLF6, although unable to stimulate IGF-IR promoter activity in Sp1-null Drosophila-derived Schneider cells, significantly enhanced the effect of Sp1. To assess the potential interactions between KLF6 and p53 in the regulation of IGF-IR gene expression, transfections were performed in the colorectal cancer cell line HCT116(+/+), which expresses p53, and its HCT116(-/-) derivative, which lacks p53. KLF6 exhibited an enhanced activity in p53-containing, compared with p53-null, cells. In addition, we were able to detect a physical interaction between KLF6 and p53. In summary, we have identified the IGF-IR gene as a novel downstream target for transcription factor KLF6. The regulation of IGF-IR gene expression by KLF6 may have significant implications in terms of cancer initiation and/or progression.

Animals↗

PubMed2004

The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity.

Retinoid X receptor alpha (RXR alpha) is a member of the steroid hormone receptor superfamily. Using yeast two-hybrid screening, beta-galactosidase assays, and pull-down assays, we show that RNF8, a RING finger protein recently isolated as a protein binding to a ubiquitin-conjugating enzyme, binds to RXR alpha through the N-terminal regions of both proteins. In COS7 cells, overexpressed RNF8 colocalized and interacted with RXR alpha in the nucleus, as shown by fluorescence resonance energy transfer. A point mutation of RNF8, Cys-403 to Ser (C403S), which disrupts the RING finger structure, or deletion of the N-terminal region (Delta N) of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXR alpha. Although transient overexpression of RNF8 had little effect on RXR alpha ubiquitination, RNF8 dose-dependently enhanced RXR alpha-mediated transactivation of the RXR-responsive element (RXRE)-bearing gene promoter without the addition of its ligand, 9-cis-retinoic acid (RA), and up-regulated the expression of the genes downstream of RXRE as well as an RA-response element. This transactivation-enhancing activity was not seen with either the C403S point mutant or the Delta N deletion mutant of RNF8. These results suggest a novel function of RNF8 as a regulator of RXR alpha-mediated transcriptional activity through interaction between their respective N-terminal regions.

Animals↗

PubMed2004

Frequent inactivation of the tumor suppressor Kruppel-like factor 6 (KLF6) in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, reflecting incomplete characterization of underlying mechanisms and lack of early detection. Kruppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that is deregulated in multiple cancers through loss of heterozygosity (LOH) and/or inactivating somatic mutation. We analyzed the potential role of the KLF6 tumor suppressor gene in 41 patients who had HCC associated with hepatitis C virus (16 patients), hepatitis B virus (12 patients, one of whom was coinfected with hepatitis C virus), and other etiologies (14 patients) by determining the presence of LOH and mutations. Overall, LOH and/or mutations were present in 20 (49%) of 41 tumors. LOH of the KLF6 gene locus was present in 39% of primary HCCs, and the mutational frequency was 15%. LOH and/or mutations were distributed across all etiologies of HCC evaluated, including patients who did not have cirrhosis. Functionally, wild-type KLF6 decreased cellular proliferation of HepG2 cells, while patient-derived mutants did not. In conclusion, we propose that KLF6 is deregulated by loss and/or mutation in HCC, and its inactivation may contribute to pathogenesis in a significant number of these tumors.

Base Sequence↗