Gut-enriched Kruppel-like Factor Research Articles

GKLF, a transcriptional activator of Txnip, drives microglia activation in kainic acid-induced murine models of epileptic seizures.

Neuroinflammation is a major component of epilepsy. Gut-enriched Kruppel-like factor (GKLF), a transcription factor of Kruppel-like factor family, has been reported to promote microglia activation and mediate neuroinflammation. However, the role of GKLF in epilepsy remains poorly characterized. This study focused on the function of GKLF in neuron loss and neuroinflammation in epilepsy and the molecular mechanism underlying microglia activation induced by GKLF upon lipopolysaccharides (LPS) treatment. An experimental epileptic model was induced by an intraperitoneal injection of 25mg/kg kainic acid (KA). Lentivirus vectors (Lv) carrying Gklf CDS or short hairpin RNA targeting Gklf (shGKLF) was injected into the hippocampus, resulting in Gklf overexpression or knockdown in the hippocampus. BV-2 cells were co-infected with Lv-shGKLF or/and Lv carrying thioredoxin interacting protein (Txnip) CDS for 48h and treated with 1μg/mL LPS for 24h. Results showed that GKLF enhanced KA-induced neuronal loss, pro-inflammatory cytokine secretion, activation of NOD-like receptor protein-3 (NLRP3) inflammasomes and microglia, and TXNIP expression in the hippocampus. GKLF inhibition showed negative effects on LPS-induced microglia activation, as evidenced by reduced pro-inflammatory cytokine secretion and activation of NLRP3 inflammasomes. GKLF bound to Txnip promoter and increased TXNIP expression in LPS-activated microglia. Interestingly, Txnip overexpression reversed the inhibitory effect of Gklf knockdown on microglia activation. These findings indicated that GKLF was involved in microglia activation via TXNIP. This study demonstrates the underlying mechanism of GKLF in the pathogenesis of epilepsy and uncovers that GKLF inhibition may be a therapeutic strategy for epilepsy treatment.

Mechanism of KLF4 in the development and progression of hepatocellular carcinoma

The pathogenesis of hepatocellular carcinoma is complex and has not been fully clarified. KLF4, also known as gut-enriched Kruppel-like factor (GKLF), plays the dual regulatory role of tumor suppressor and tumor promoter. KLF4 plays an anti-cancer role in liver cancer. This article introduces the biological function and regulatory mechanism of KLF4 in the progression of liver cancer, and research on the mechanism of action of KLF4 in liver cancer is expected to provide new ideas for targeted therapy and prognosis monitoring of liver cancer.

Transcription factors and epigenetic modulation: its therapeutic implication in chronic kidney disease.

Recently emerging evidence has shown that epigenetic mechanisms are involved in initiation and progression of various diseases, including kidney diseases. In the present article, we review the current data regarding the role of epigenetic modulation in chronic kidney disease (CKD) and kidney fibrosis, including DNA methylation and histone modification. Especially we focused on the role of transcription factors in epigenetic modulation and the possibility of therapeutic target of CKD. We have recently reported that transcription factor Kruppel-like factor 4 (also known as gut-enriched Kruppel-like factor) is expressed in kidney podocytes (visceral epithelial cells) and modulates podocyte phenotype by gene-selective epigenetic control. Targeting transcription factors for epigenetic modification may be a good candidate for remission and regression of CKD. It is necessary for the therapy of CKD with an epigenetic-based approach to investigate organ-, tissue-, or gene-specific treatment methods for reduction of side effects.

Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake

Pancreatic acinar cells (PAC) obtain thiamin from the circulation via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3, respectively). Chronic alcohol exposure of PAC inhibits thiamin uptake, and, on the basis of in vitro studies, this inhibition appears to be transcriptionally mediated. The aim of this study was to confirm the involvement of a transcriptional mechanism in mediating the chronic alcohol effect in in vivo settings and to delineate the molecular mechanisms involved. Using transgenic mice carrying full-length SLC19A2 and SLC19A3 promoters, we found that chronic alcohol feeding led to a significant reduction in the activity of SLC19A2 and SLC19A3 promoters (as well as in thiamin uptake and expression of THTR-1 and -2). Similar findings were seen in 266-6 cells chronically exposed to alcohol in vitro. In the latter studies, the alcohol inhibitory effect was found to be mediated via the minimal SLC19A2 and SLC19A3 promoters and involved the cis-regulatory elements stimulating protein 1 (SP1)/gut-enriched Kruppel-like factor and SP1-GG-box and SP1/GC, respectively. Chronic alcohol exposure of PAC also led to a significant reduction in the expression of the SP1 transcription factor, which upon correction (via expression) led to the prevention of alcohol inhibitory effects on not only the activity of SLC19A2 and SLC19A3 promoters but also on the expression of THTR-1 and -2 mRNA and thiamin uptake. These results demonstrate that the inhibitory effect of chronic alcohol exposure on physiological/molecular parameters of thiamin uptake by PAC is mediated via specific cis-regulatory elements in SLC19A2 and SLC19A3 minimal promoters.

Association between gut-enriched Kruppel-like factor and prognosis of patients with gastric cancer

To explore the expression of gut-enriched Kruppel-like factor 4(KLF4) in gastric cancer, and its association with prognosis. Surgical specimens were collected from 264 patients undergoing radical surgery between 2004 and 2009 in the Affiliated Qianfoshan Hospital, Shandong University. KLF4 mRNA level of specimens was detected by real-time PCR. KLF4 protein expression was measured by immunohistochemistry on tissue microarray, which contained primary gastric cancer, corresponding para-cancerous tissue, and paired lymph node metastases. Real-time PCR revealed that mRNA level of KLF4 was down-regulated in gastric cancer compared with paired normal gastric mucosa. Immunohistochemistry on tissue microarray showed gastric cancer tissues had significantly lower KLF4 levels compared with paired normal gastric tissues. By univariate and multivariate analysis, KLF4 was a significant predictor of survival and recurrence. KLF4 expression is significantly down-regulated in gastric cancer, and is an independent predictor of survival and recurrence.

Effects of transfected gut-enriched Kriippel-like factor on growth of human gastric carcinoma cell line SGC-7901 and its xenograft in nude mice

Objective To investigate the antitumour effects of transfected gut-enriched Kruppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice. Key words: Gut-enriched Kruppel-like transcription factors; Transfection; Stomach neoplasms; Cell line, tumor; Mice, nude; Neoplasm transplantation

Interleukin-18 Suppresses Adiponectin Expression in 3T3-L1 Adipocytes via a Novel Signal Transduction Pathway Involving ERK1/2-dependent NFATc4 Phosphorylation

An inverse correlation between the pro-inflammatory cytokine interleukin-18 and the anti-atherogenic adipokine adiponectin has been reported in the chronic pathological conditions obesity, insulin resistance, coronary artery disease, and metabolic syndrome. We investigated whether this relationship is coincidental or has a causal basis. Here we show that interleukin-18 (IL-18) suppresses adiponectin transcription, mRNA expression, and secretion by 3T3-L1 adipocytes. IL-18 suppresses adiponectin promoter-reporter activity, an effect reversed by deletion or mutation of the NFATc4 core DNA-binding site. IL-18 induces NFATc4 phosphorylation (Ser(676)), nuclear translocation, and in vivo DNA binding. IL-18 induces ERK1/2 phosphorylation and enzyme activity, and pretreatment with the MEK inhibitor U0126, ERK1/2 inhibitor PD98059, or small interference RNA targeted to ERK1/2 attenuates ERK1/2 activation and NFATc4 phosphorylation. Finally, inhibition of ERK1/2 or NFATc4 knockdown reverses IL-18-mediated adiponectin suppression. In contrast to its inhibitory effects on adiponectin expression, IL-18 potently stimulates PAI-1 secretion. These data demonstrate for the first time that IL-18 selectively suppresses adiponectin expression via ERK1/2-dependent NFATc4 activation and suggest that the inverse relationship observed between IL-18 and adiponectin in various chronic pathological conditions is causally related. Thus, targeting IL-18 expression may enhance adiponectin expression and mitigate disease progression.

Cell Growth Inhibition by Okadaic Acid Involves Gut-Enriched Kruppel-like Factor–Mediated Enhanced Expression of c-Myc

Human breast cancer (HBC) cell growth suppression by okadaic acid (OA) was previously found to involve elevated expression of oncogenes c-myc and c-fos and apoptosis. Since, c-Myc influences diverse pathways of cell growth, we hypothesized that elevated levels of c-Myc are involved in HBC growth suppression. Here, we investigated whether induction of c-Myc by OA or protein synthesis inhibitor cycloheximide contributed to HBC growth inhibition and the mechanisms involved. OA, cycloheximide, or the chemotherapeutic drug Taxol suppressed HBC cell growth. However, OA or cycloheximide treatments over 6 or 10 h, respectively, induced c-Myc expression. Depletion of c-Myc, on the other hand, resulted in enhanced HBC cell viabilities when exposed to OA or cycloheximide, but not by Taxol. OA induced c-myc transcription by targeting an 80-bp region from positions -11 to +70, relative to the P1 transcription start of mouse c-myc promoter. Gel mobility shift assays revealed binding of HBC cell nuclear proteins to the OA-responsive c-myc promoter fragment, whereas binding of one complex was elevated in the case of the OA-treated or cycloheximide-treated HBC cell nuclear extracts. Database search revealed presence of a consensus sequence for zinc finger protein gut-enriched Kruppel-like factor (GKLF) in OA-responsive region of the c-myc promoter. Mutation of GKLF consensus sequences abrogated OA responsiveness of the c-myc promoter, and OA treatments caused enhanced expression of GKLF in HBC cells. Thus, OA-dependent attenuation of HBC growth is accomplished, in part, by zinc finger transcription factor GKLF-mediated enhanced transcription of c-myc.

Genetic and epigenetic analysis of the KLF4 gene in gastric cancer

KLF4, which is also known as the gut-enriched Kruppel-like factor, plays important roles during the proliferation and differentiation of gastrointestinal epithelial cells. A loss of KLF4 expression has been observed in human tumors, particularly in the gastrointestinal tract. In this study, the molecular basis of the KLF4 inactivation in gastric cancer was investigated by analyzing the somatic mutation, the allelic loss with two microsatellite markers, D9S53 and D9S105, and hypermethylation of the KLF4 gene in 47 gastric adenomas and 81 gastric adenocarcinomas. Mutational analysis revealed one mutation of the KLF4 gene in a diffuse-type advanced gastric adenocarcinoma, but not in the gastric adenoma. This mutation was a somatic missense mutation, GGG-->AGG (Gly-->Arg) at codon 107 in exon 3, which encodes a transcriptional activation domain of the protein. An allelic loss was found in 7 (22.6%) of the 31 informative gastric adenoma cases and 15 (31.3%) of the 48 informative cancer cases at one or both markers. In addition, promoter hypermethylation of the KLF4 gene was observed in only two gastric cancers. These results suggest that genetic and epigenetic alterations of the KLF4 gene might play a minor role in gastric carcinogenesis.

44 KRUPPEL-LIKE FACTOR 4 REGULATES ENDOTHELIAL INFLAMMATION.

<h3>Background</h3> The vascular endothelium plays a critical role in vascular homeostasis. A limited proinflammatory, prothrombotic state is appropriate in the context of infection or wound healing, but sustained endothelial activation leads to deleterious conditions such as atherosclerosis and pathologic thrombosis. Inflammatory cytokines and nonlaminar blood flow induce endothelial dysfunction and confer a proadhesive and prothrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factors (KLFs) are a subclass of the zinc-finger family of transcription factors. Previous studies demonstrate that KLF proteins typically regulate critical aspects of cellular differentiation and tissue development. Studies from our laboratory and others demonstrate an emerging role for this family of transcriptional regulators in vascular biology. KLF4/GKLF (gut-enriched Kruppel-like factor) was first identified as being highly expressed in epithelial cells, and subsequent work has verified a critical role in skin and intestinal development. KLF4 has also been identified in endothelial cells, yet its function in vessel biology has yet to be elucidated. <h3>Methods and Results</h3> Immunohistochemical analysis of mouse and human vascular tissues demonstrates the expression, in vivo, of KLF4 in human and mouse endothelial cells. Northern blot analysis of total mRNA harvested from primary endothelial cell lines derived from various human arterial vascular beds (aorta, pulmonary artery, and umbilical artery) shows expression of endothelial KLF4 in a variety of arterial and venous cell lines. Furthermore, we demonstrate that endothelial KLF4 is induced by proinflammatory stimuli and shear stress. Overexpression of KLF4 induces expression of multiple anti-inflammatory and antithrombotic factors, including eNOS and thrombomodulin, and inhibits basal and cytokine-mediated expression of a diverse set of proinflammatory factors, including MCP-1, RANTES, CRP, PAI-1, IL-6, and IL-8. The significance of endogenous expression of KLF4 on target genes was assessed in experiments using siRNA-mediated knockdown of KLF4. These experiments demonstrate that KLF4 depletion leads to enhancement of TNF-α-induced VCAM-1 and tissue factor expression. In addition to the determination of target genes, we have verified the functional importance of KLF4 by demonstrating that KLF4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. As a first step toward understanding the molecular basis of KLF4-mediated regulation of endothelial target genes, we assessed the effect of KLF4 on target gene promoters. KLF4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. <h3>Conclusion</h3> Inflammatory cytokines and the biomechanical effects of laminar shear stress are the most potent effectors of endothelial homeostasis identified to date. Perturbation of endothelial function by proinflammatory cytokines or nonlaminar flow has been documented in most vascular disease states, including vasculitis, aneurysm formation, pathologic thrombosis, and atherosclerosis. The observations described above implicate KLF4 as a novel regulator of endothelial activation in response to proinflammatory stimuli. A greater understanding of KLF4 function is thus of considerable scientific and potentially therapeutic interest.

Molecular mechanisms involved in the adaptive regulation of human intestinal biotin uptake: a study of the hSMVT system

Biotin, a water-soluble micronutrient, is vital for cellular functions, including growth and development. The human intestine utilizes the human sodium-dependent multivitamin transporter (hSMVT) for biotin uptake. Evidence exists showing that the intestinal biotin uptake process is adaptively regulated during biotin deficiency. Nothing, however, is known about molecular mechanism(s) involved during this adaptive regulation. This study compared two human-derived intestinal epithelial cell lines (HuTu-80 and Caco-2) during biotin-deficient or biotin-sufficient states and with an approach that assessed carrier-mediated biotin uptake, hSMVT protein and RNA levels, RNA stability, and hSMVT promoter activity. The results showed that during biotin deficiency, a significant and specific upregulation in carrier-mediated biotin uptake occurred in both human intestinal epithelial cell lines and that this increase was associated with an induction in protein and mRNA levels of hSMVT. The increase in mRNA levels was not due to an increase in RNA stability but was associated with an increase in activity of the hSMVT promoter in transfected human intestinal cells. Using promoter deletion constructs and mutational analysis in transiently transfected HuTu-80 and Caco-2 cells, a biotin deficiency-responsive region was mapped to a 103-bp area within the hSMVT promoter that contains gut-enriched Kruppel-like factor (GKLF) sites that confer the response to biotin deficiency. These results confirm that human intestinal biotin uptake is adaptively regulated and provide novel evidence demonstrating that the upregulation is not mediated via changes in hSMVT RNA stability but rather is due to transcriptional regulatory mechanism(s) that likely involve GKLF sites in the hSMVT promoter.

Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer

Abnormalities in the expression of DMBT1 (deleted in malignant brain tumors 1) have been implicated in the development of esophageal, gastric and colorectal cancers of the alimentary tract, but the underlying mechanism remains unclear. In the present study, using the gastric cell line AGS, we identified two intracellular signaling molecules protein kinase C (PKC) and extracellular signal-related kinase (ERK). They mediated both the phorbol myristate acetate (PMA) downregulation of DMBT1 expression and the initiation of cell differentiation, which was measured by cell cycle withdrawal and the induction of the tissue-specific marker trefoil factor 1 (TFF1). A time-course study showed that following the PMA activation of ERK kinase, the induction of TFF1 and the reduction of DMBT1 were detected at the same time point. We then demonstrated a minimal level of DMBT1 in proliferating AGS cells seeded at low density, where ERK activity was high. Reduction of ERK activity, either by an ERK inhibitor PD98059 or by high-density seeding, significantly reduced AGS cell growth judged by CFSE labeling. This cellular effect was elicited by cyclin D/p21 (Cip/Waf1) and G(0)/G(1) arrest, and was accompanied by a marked increase in DMBT1-expressing cells. Finally, we showed that siRNA directed against DMBT1 had no effect on the induction of a cell growth arrest marker, gut-enriched Kruppel-like factor (GKLF), but reduced the PMA induction of TFF1. Along with its upregulation coinciding with G(0)/G(1) arrest, and its attenuation in differentiated cells, these results suggest that the transient induction of DMBT1 is apparently specific at an early stage of gastric epithelial differentiation-like process, when it may play a role in cell fate decision. Consistent with such a potential function, we detected frequent abnormalities of the DMBT1 expression in the specimens of human gastric adenocarcinoma.

The Leukocyte Integrin Gene CD11d Is Repressed by Gut-enriched Kruppel-like Factor 4 in Myeloid Cells

The myeloid-specific leukocyte integrin CD11d encodes the alphaD subunit for the alphaDbeta2 receptor. A yeast one-hybrid screen showed that a longer isoform of gut-enriched Kruppel-like factor 4 (GKLF) we term GKLFa interacts with the CD11d promoter. Purified GST-GKLFa protein was shown to bind within the -61 to -44 region that overlaps a binding site for the CD11d transcriptional activators Sp1 and transforming growth factor beta-inducible early gene-1 (TIEG1). Transfection of GKLF/GKLFa in myeloid cells reduced CD11d promoter activity, whereas, down-regulation of GKLF/GKLFa with small interfering RNAs led to up-regulation of CD11d expression. Differentiation of myeloid cells with phorbol ester led to activation of the CD11d promoter and reduced occupancy of the promoter by GKLF/GKLFa but an increased occupancy by TIEG1 in vivo. Binding of GKLF/GKLFa, Sp1, and TIEG1 to the CD11d promoter in vivo is dependent on their zinc finger DNA binding domains. GKLFa physically associates with the histone deacetylases (HDAC) 1 and 2, and both HDACs are bound to the CD11d promoter in vivo but released after exposure of myeloid cells to phorbol ester suggesting that GKLF/GKLFa recruits HDACs to effect repression.

52. Gut Enriched Kruppel-Like Factor (GKLF) Inhibits Differentiation and Induces Self-Renewal of Murine Embryonic Stem Cells

One of the challenges of gene therapy techniques, in addition to the adequate delivery and expression of the desired gene, is the maintenance of stem cell self-renewal during ex vivo manipulations. However, the genes needed for stem cell self-renewal are largely unknown. Homozygous mutant (Shp-2Δ46-110) embryonic stem (ES) cells bearing a targeted exon 3 deletion of the Shp-2 protein tyrosine phosphatase gene exhibit increased sensitivity to Leukemia Inhibitory Factor (LIF) and increased self-renewal potential as assayed by 2° embryoid body frequency upon secondary plating. LIF maintains the pluripotency of ES cells, suggesting that molecules stimulated by LIF are critical for self-renewal. Based on the functional characteristics of the Shp-2Δ46-110 ES cells, we hypothesized that LIF-responsive genes in these cells would be good candidates for molecules vital for stem cell self-renewal. Using a microarray analysis approach, we generated and previously reported a list of LIF-induced genes in Shp-2Δ46-110 ES cells (International Society of Experimental Hematology, 2002, Abstract #326). The two most significantly upregulated genes were Suppressor of Cytokine Signaling-3 (SOCS-3) and Gut Enriched Kruppel-Like Factor (GKLF). In order to assess the capacity of these proteins to affect stem cell self-renewal, each cDNA was introduced into ES cells followed by examination of ES cell differentiation and self-renewal. The FLAG-tagged SOCS-3 and GKLF cDNAs were subcloned into the murine retroviral vector, pMIEG3, in tandem with the enhanced green fluorescent protein (EGFP) cDNA. WT R1 ES cells were transduced and EGFP-positive cells were selected using FACS. Selected cells were subjected to differentiation followed by secondary assays for hemangioblasts, primitive erythroid colonies, and 2° embryoid bodies. We found that ES cells expressing exogenous SOCS-3 (compared to ES cells tranduced with vector alone) had an increased capacity to differentiate to both hemangioblasts (17+/−2.5 v. 6+/−2.4, p = 0.02, student's t test) and to primitive erythroid cells (100+/−5.4 v. 47.6 +/−20.1, p = 0.03, student's t test). There was no difference in the number of 2° embryoid bodies (6.9+/−1.3 v. 5.5+/−0.7, p>0.5). However, in contrast, introduction of GKLF inhibited the differentiation capacity of the ES cells as we observed a trend toward lower numbers of primitive erythroid colonies compared to vector alone (10.6+/−2.1 v. 47.6+/−20.1, p = 0.09, student's t test). Likewise, ES cells expressing exogenous GKLF had a significantly greater capacity to undergo self-renewal as assessed by 2° embryoid body formation compared to cells transduced with vector alone (117.3+/−19.3 v. 5.5+/−0.8, p = 0.0002, student's t test). In both studies, we demonstrated that the exogenously FLAG-tagged molecules were expressed during ES cell differentiation. GKLF is highly expressed in the epithelial crypts of the colon, the site of somatic epithelial stem cells. This previous information along with our data qualifies GKLF as a reasonable candidate for a protein necessary for somatic stem cell self-renewal. We are currently transducing GKLF into hematopoietic stem cells (HSC) followed by competitive transplant assays to determine if GKLF has the capacity to enhance HSC self-renewal.

Kruppel-like Factor 4 (KLF4) Represses Histidine Decarboxylase Gene Expression through an Upstream Sp1 Site and Downstream Gastrin Responsive Elements

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.

Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor.

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

Sp1 and finb interact with BETA2/NeuroD to enhance its transactivation of the secretin promoter

of full length fragment which covers all three gastrin responsive elements were cloned for yeast one hybrid screening. Electrophoretic mobility shift assays (EMSAs) were then used to confirm the positives. To determine the functional importance of the target factor(s), transient transfection and reporter assays were performed by overexpression of the target nuclear factor(s) in the presence of the HDC promoter construct which contains 125bp upstream sequence and 126bp downstream sequence followed by the luciferase gene. Results: Yeast one hybrid screening indicated that gut-enriched Kruppel like factor (GKLF) binds downstream gastrin responsive elements of the HDC promoter. Electrophoretic mobility shift assays confirmed the binding of GKLF with all three downstream gastrin responsive elements. EMSA competition assay also showed the binding of Spl with all three gastrin responsive elements. Furthermore, EMSAs indicated that GKLF binds Spl bound GC-box fragment which is located upstream of the HDC promoter. Overexpression of GKLF and Spl transactivated HDC promoter activity, which was further supported by the repressive effect o f their dominant negative plasmids. Conclusion: GKLF and Sp 1 synergistically regulate HDC promoter activity through binding both upstream GC box and downstream gastrin responsive elements.

Transactivation of Gut-enriched Kruppel-like factor by butyrate in human colonic cancer cells

Pancreatic cancer has an abysmal prognosis because of late diagnosis and lack response to available therapeutics. Therefore, it is important to identify risk factors and to be able to prevent and detect this cancer in an early, non-invasive stage. Pancreanc lntraepithdial Neoplasias (PanINs) are precursor lesions which could be an ideal target for chemoprevention. COX-2 is up-regulated in PanlNs. We recently reported that 5-1ipoxygenase (5-LOX) is 0verexpressed in resected human pancreatic cancers and that the 5-LOX pathway is even more important than COX-2 in this disease. However, whether the 5-LOX pathway is activated early or only in late disease'and whether it might be a target for chemoprevention Ls not known. Therefore, we investigated the expression of 5-LOX in PanlN lesions in the human pancreas as well as two pancreatic cancer models. Immunocytochemistry for 5-LOX ,a~s undertaken in human surgical specimens from 10 pancreatic adenocarcinomas, 9 cases 0fchronic pancreatitis and I0 normal pancreatic tissues from multi-organ donors, immunoslaining was also carried out in pancreatic specimens of two models of pancreatic adenocarcinoma: carcinogen (BOP)-treated hamsters and Ek-Kras transgenic mice (Kras mutant), as wdl as normal harhster and mouse pancreatic tissues. In human and hamster tissues, 5l.OX was constitutively expressed at a modest level in the cytoplasm of acinar ceils. Intense 5-LOX staining was evident in the nuclear envelope of cancer cells and all grades of PanlNs 0/9/10 human pancreatic adenocareinoma and 7/9 chronic pancreatitis tissues. In contrast, 5-LOX expression was only detected in a small proportion of ductaI cells (0-75%) in l0 normal human pancreatic tissues. Similarly, PanlNs in BOP-treated hamsters and EL-Kras mice showed positive staining, whereas normal ductal cells were completely negative. This study demonstrates that 5-LOX ts up-regulated in human PanINs and early lesions of pancreatic cancer in two different animal models. These findings provide even more evidence that 5-LOX plays a key role in the development of pancreatic cancer. Furthermore, like COX-2, this lipoxygenase pathway is an attractive target for the prevention of this devastating disease 5-LOX and COX-2 inhibitors should eventually be used for chemoprevention in patients at risk for developing pancreatic cancer

Redox-sensitive vascular smooth muscle cell proliferation is mediated by GKLF and Id3 in vitro and in vivo.

Reactive oxygen species such as superoxide and hydroxyl radicals have been implicated in the pathogenic growth of various cell types. The molecular mechanisms involved in redox-sensitive cell growth control are poorly understood. Stimulation of cultured vascular smooth muscle cells (VSMC) with xanthin/xanthin oxidase (X/XO) increases proliferation, whereas stimulation with hydrogen peroxide and Fe3+NTA (H-Fe) causes growth arrest of VSMC. Differential Display led to the identification of two novel, differentially regulated redox-sensitive genes. The dominant negative helix-loop-helix protein Id3 is induced by X/XO and down-regulated by H-Fe. The transcription factor gut-enriched Kruppel-like factor (GKLF) is induced by H-Fe but not by X/XO. Induction of GKLF and inhibition of Id3 via transfection experiments leads to growth arrest, whereas overexpression of Id3 and inhibition of GKLF cause cell growth. Id3 down-regulation is induced via binding of GKLF to the Id3 promotor and concomitantly reduced Id3 gene transcription rate. GKLF induction by H-Fe is mediated through hydroxyl radicals, p38MAP kinase-, calcium-, and protein synthesis-dependent pathways. Id3 is induced by X/XO via superoxide, calcium, p38, and p42/44 MAP kinase. GKLF induces and Id3 depresses expression of p21WAF1/Cip1, p27KIP1, p53. Induction of Id3 is accomplished by angiotensin II via superoxide release. A vascular injury mouse model revealed that Id3 is overexpressed in proliferating vascular tissue in vivo. These findings reveal novel mechanisms of redox-controlled cellular proliferation involving GKLF and Id3 that may have general implications for our understanding of vascular and nonvascular growth control.

Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor (GKLF/KLF4) and Sp1

Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation. Laminin gamma1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos. Laminin gamma1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1-GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin gamma1 chain when laminin is needed to regulate cell differentiation.