Citations

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 μg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase ( P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 μM), an inhibitor of MEK, which is a kinase upstream from Erk ( P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity ( P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation ( P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.

Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, "Dyn2." Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.

Sp1 is one of the best characterized transcriptional activators. The biological importance of Sp1 is underscored by the fact that several hundreds of genes are thought to be regulated by this protein. However, during the last 5 years, a more extended family of Sp1-like transcription factors has been identified and characterized by the presence of a conserved DNA-binding domain comprising three Krüppel-like zinc fingers. Each distinct family member differs in its ability to regulate transcription, and, as a consequence, to influence cellular processes. Specific activation and repression domains located within the N-terminal regions of these proteins are responsible for these differences by facilitating interactions with various co-activators and co-repressors. The present review primarily focuses on discussing the structural, biochemical and biological functions of the repressor members of this family of transcription factors. The existence of these transcriptional repressors provides a tightly regulated mechanism for silencing a large number of genes that are already known to be activated by Sp1.

Gentamicin is a widely used ototoxic agent. In this study, we shed light on the mechanisms underlying gentamicin-induced hearing loss. More importantly, we demonstrate in vivo and in vitro the effectiveness of a strategy for preventing drug-induced hearing loss using l -carnitine (LCAR), a safe micronutrient that plays a key role in energy metabolism and detoxification [Rebouche, C. J. & Seim, H. (1998) Annu. Rev. Nutr. 18, 39-61]. We show that LCAR prevents changes in hearing threshold and cochlear damage in newborn guinea pigs exposed to gentamicin in utero . Mechanistically, gentamicin-induced apoptosis of auditory cells is mediated by the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) pathway through up-regulation of the proapoptotic factor Harakiri ( Hrk ). Most important, small interfering RNA (siRNA) experiments demonstrate that Hrk up-regulation is crucial for gentamicin-induced apoptosis. LCAR, in contrast, prevents both gentamicin-induced Hrk up-regulation and apoptosis acting by means of c-Jun N-terminal kinase (JNK). Together, these results outline pathways for gentamicin-induced hearing loss and its prevention and assign a key role to Hrk in these processes. Thus, our data offer a conceptual framework for designing clinical trials using a safe micronutrient, LCAR, as a simple preventive strategy for iatrogenically induced ototoxicity.

Abstract Recent studies using glycogen synthase kinase-3β (GSK-3β)–deficient mouse embryonic fibroblasts suggest that GSK-3β positively regulates nuclear factor κB (NFκB)–mediated gene transcription. Because NFκB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3β in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3β and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3β by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3β influences NFκB-mediated gene transcription at a point distal to the Iκ kinase complex, as only ectopic expression of the NFκB subunits p65/p50, but not an Iκ kinase β constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3β inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3β in cancer cell survival and proliferation and suggest GSK-3β as a potential therapeutic target in the treatment of pancreatic cancer.

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo . However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.