RESUMO
Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. We and others have previously shown that KLF4 is involved in bone modeling and remodeling but roles played by KLF4 during skeletogenesis are still not fully understood. Here, we show that KLF4 is expressed in the epiphyseal growth plate and articular chondrocytes. Most articular chondrocytes expressed KLF4 in embryos but it localized only in a subset of superficial zone cells in postnatal mice. When KLF4 was overexpressed in chondrocytes in vitro, it severely repressed chondrocytic gene expressions. Global gene expression profiling of KLF4-transduced chondrocytes revealed matrix degrading proteinases of the matrix metalloproteinase and disintegrin and metalloproteinase with thrombospondin-1 domain families within the group of upregulated genes. Proteinase induction by KLF4 was alleviated by Trichostatin A treatment suggesting the possible involvement of epigenetic mechanisms on proteinase induction by KLF4. These results indicate the possible involvement of KLF4 in physiological and pathological aspects during cartilage development and maintenance.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Endopeptidases/biossíntese , Fatores de Transcrição Kruppel-Like/metabolismo , Metaloproteinases da Matriz/biossíntese , Trombospondina 1/biossíntese , Animais , Células Cultivadas , Endopeptidases/genética , Regulação da Expressão Gênica no Desenvolvimento , Ácidos Hidroxâmicos/farmacologia , Fator 4 Semelhante a Kruppel , Masculino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos ICR , Inibidores da Síntese de Proteínas/farmacologia , Trombospondina 1/genéticaRESUMO
Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.
Assuntos
Colo/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Regeneração/genética , Animais , Antineoplásicos Hormonais/farmacologia , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Ciclina B/metabolismo , Ciclina D1/metabolismo , Regulação para Baixo , Células Caliciformes/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Antígeno Ki-67/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Litostatina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite , Celulas de Paneth/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Fatores de Transcrição SOX9/metabolismo , Deleção de Sequência , Transdução de Sinais/genética , Tamoxifeno/farmacologia , Regulação para CimaRESUMO
Gut radiation-induced injury is a concern during treatment of patients with cancer. Krüppel-like factor 4 (KLF4) is expressed in differentiated villous epithelial cells of the small intestine. We previously showed that KLF4 protects cells from apoptosis following γ-irradiation in vitro. We sought to determine whether KLF4 mediates the small intestinal response to γ-irradiation in vivo. Mice with intestinal epithelium-specific deletion of Klf4 (Klf4(ΔIS)) and control (Klf4(fl/fl)) mice were irradiated with total-body γ-radiation. Following irradiation, the Klf4(ΔIS) mice had significantly increased mortality compared with irradiated Klf4(fl/fl) mice. Immunohistochemistry and immunofluorescence staining were used to assess the morphological changes, levels of proliferation, and apoptosis in the intestinal epithelium. At 96 h following irradiation, there was a regenerative response manifested by an expansion of the proliferative zone in both mouse groups, with the control mice having a higher proliferative activity than the Klf4(ΔIS) group. In addition, there was a significant increase in the number of Klf4/Ki67-copositive cells in the irradiated control mice compared with unirradiated mice. Also, the irradiated Klf4(ΔIS) mice had a significantly higher number of crypt cells positive for apoptosis, p53, and p21 compared with irradiated Klf4(fl/fl) mice. Taken together, our data suggest that Klf4 may function as a radioprotective factor against gastrointestinal syndrome in mice following γ-irradiation by inhibiting apoptosis in the acute response to irradiation and contributing to crypt regeneration.
Assuntos
Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Modelos Animais de Doenças , Raios gama , Intestinos/efeitos da radiação , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy. METHODS: To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4(-/-)), we transfected Klf4(-/-)MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4(+/+)) and untransfected or mock-transfected Klf4(-/-)MEFs. RESULTS: We show that overexpression of Klf4 in Klf4(-/-)MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation. CONCLUSION: Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.
Assuntos
Fibroblastos/metabolismo , Instabilidade Genômica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Aneuploidia , Animais , Linhagem Celular , Proliferação de Células , Centrossomo/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Expressão Gênica , Técnicas de Inativação de Genes , Fator 4 Semelhante a Kruppel , Camundongos , Micronúcleos com Defeito Cromossômico , TransfecçãoRESUMO
The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is expressed in the post-mitotic, differentiated epithelial cells lining the intestinal tract and exhibits a tumor suppressive effect on intestinal tumorigenesis. Here we report a role for KLF4 in maintaining homeostasis of intestinal epithelial cells. Mice with conditional ablation of the Klf4 gene from the intestinal epithelium were viable. However, both the rates of proliferation and migration of epithelial cells were increased in the small intestine of mutant mice. In addition, the brush-border alkaline phosphatase was reduced as was expression of ephrine-B1 in the small intestine, resulting in mispositioning of Paneth cells to the upper crypt region. In the colon of mutant mice, there was a reduction of the differentiation marker, carbonic anhydrase-1, and failure of differentiation of goblet cells. Mechanistically, deletion of Klf4 from the intestine resulted in activation of genes in the Wnt pathway and reduction in expression of genes encoding regulators of differentiation. Taken together, these data provide new insights into the function of KLF4 in regulating postnatal proliferation, migration, differentiation, and positioning of intestinal epithelial cells and demonstrate an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis in vivo.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Homeostase/fisiologia , Mucosa Intestinal/fisiologia , Fatores de Transcrição Kruppel-Like/deficiência , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Bromodesoxiuridina , Anidrase Carbônica I/metabolismo , Movimento Celular , Proliferação de Células , Efrina-B1/metabolismo , Imunofluorescência , Deleção de Genes , Técnicas Histológicas , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. Furthermore, both ß-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 before caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.
Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Anticarcinógenos/uso terapêutico , Dioxóis/uso terapêutico , Genes APC/fisiologia , Isoquinolinas/uso terapêutico , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição 4 , beta Catenina/fisiologiaRESUMO
BACKGROUND & AIMS: Krüppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut. METHODS: Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre;Klf5fl/fl). Morphologic changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction. RESULTS: Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared with controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphologic changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Caudal type homeobox (Cdx) 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to ß-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9. CONCLUSIONS: Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphologic changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.
Assuntos
Colo/metabolismo , Células Epiteliais/metabolismo , Íleo/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Western Blotting , Fator de Transcrição CDX2 , Diferenciação Celular , Movimento Celular , Proliferação de Células , Colo/patologia , Efrinas/metabolismo , Células Epiteliais/patologia , Genótipo , Proteínas de Homeodomínio/metabolismo , Íleo/patologia , Imuno-Histoquímica , Mucosa Intestinal/patologia , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade , Fenótipo , Reação em Cadeia da Polimerase , Receptores da Família Eph/metabolismo , Regeneração , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. METHODS: C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9. RESULTS: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA. CONCLUSIONS: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.
Assuntos
Colite/enzimologia , Colo/enzimologia , Neoplasias do Colo/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Apoptose , Azoximetano , Caspase 3/metabolismo , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dano ao DNA , Sulfato de Dextrana , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Raios gama , Células HCT116 , Humanos , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptor Notch1/antagonistas & inibidores , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Both mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMin mutation or intestine-specific expression of the KRASV12 gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV12 double transgenic mice to develop intestinal tumors. RESULTS: At 12 weeks of age, ApcMin/KRASV12 mice had three times as many intestinal tumors as ApcMin mice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV12/Klf5+/- mice was also statistically significant compared to ApcMin mice alone, with a 75% decrease. Compared with ApcMin/KRASV12, tumors from both ApcMin/KRASV12/Klf5+/- and ApcMin mice were smaller. In addition, tumors from ApcMin mice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMin and ApcMin/KRASV12 mice but were attenuated in ApcMin/KRASV12/Klf5+/- mice. The levels of beta-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV12/Klf5+/- mice when compared to ApcMin/KRASV12 mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV12 mice and modestly reduced in ApcMin/KRASV12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and beta-catenin, irrespective of the mouse genotype from which tumors were derived. CONCLUSIONS: Results of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV12 mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Genes APC , Fatores de Transcrição Kruppel-Like/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
BACKGROUND & AIMS: Chronic inflammation is a risk factor for colon cancer (CC). Lysophosphatidic acid (LPA), a naturally produced phospholipid, mediates multiple effects that are vital to disease process, including inflammation and cancer. The expression of LPA receptor 2 (LPA2) is up-regulated in several types of cancer, including ovarian and colon cancer, but the importance of LPA and LPA2 in the development and progression of CC is unclear. In this study, we sought to determine whether LPA and LPA2 regulate the progression of CC in vivo. METHODS: We examined the potential role of LPA in CC progression by administering LPA to mice heterozygous for the adenomatous polyposis coli (Apc) allele. We determined the loss of LPA2 function in tumorigenesis in the colon by treating mice with genetic deletion of LPA2 (LPA2-/-) with azoxymethane and dextran sulfate sodium. RESULTS: We found that LPA increased tumor incidence in Apc(min/+) mice. LPA2-/- mice showed reduced mucosal damage and fewer tumors than wild-type (WT) mice. Reduced epithelial cell proliferation and decreases in beta-catenin, Krüppel-like factor 5, and cyclooxygenase-2 expression were observed in LPA2-/- mice. Unlike WT mice, induction of monocyte chemoattractant protein-1 and macrophage migration inhibitory factor was significantly attenuated in LPA2-/- mice with reduced infiltration by macrophages. CONCLUSIONS: These results show that LPA is capable of promoting tumorigenesis in the colon. The absence of LPA2 attenuates several effects that contribute to cancer progression in vivo, and, hence, the current study identifies LPA2 as an important modulator of CC.
Assuntos
Colite/complicações , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Azoximetano , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Neoplasias do Colo/etiologia , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Progressão da Doença , Genes APC , Fatores de Transcrição Kruppel-Like/metabolismo , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , beta Catenina/metabolismoRESUMO
The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor-suppressive role for KLF4 is shown by the recent finding that Klf4 haploinsufficiency in Apc(Min/+) mice promotes intestinal tumorigenesis. Studies also show that KLF4 is required for the terminal differentiation of goblet cells in the mouse intestine. The Notch signaling pathway suppresses goblet cell formation and is up-regulated in intestinal tumors. Here, we investigated the relationship between Notch signaling and KLF4 expression in intestinal epithelial cells. The rate of proliferation of HT29 human colon cancer cells was reduced when treated with the gamma-secretase inhibitor dibenzazepine to inhibit Notch signaling or small interfering RNA directed against Notch. KLF4 levels were increased in dibenzazepine-treated or Notch small interfering RNA-treated cells. Conversely, overexpression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate. Treatment of Apc(Min/+) mice with dibenzazepine resulted in a 50% reduction in the number of intestinal adenomas compared with the vehicle-treated group (P < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from dibenzazepine-treated Apc(Min/+) mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki-67 staining when compared with those from vehicle-treated mice. Results of these studies indicate that Notch signaling suppresses KLF4 expression in intestinal tumors and colorectal cancer cells. Inhibition of Notch signaling increases KLF4 expression and goblet cell differentiation and reduces proliferation and tumor formation. KLF4 is therefore a potential mediator for the antitumor effect of Notch inhibitors such as dibenzazepine.
Assuntos
Adenoma/fisiopatologia , Neoplasias Colorretais/fisiopatologia , Células Caliciformes/fisiologia , Mucosa Intestinal/citologia , Fatores de Transcrição Kruppel-Like/genética , Receptor Notch1/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Divisão Celular/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dibenzazepinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Células Caliciformes/citologia , Células HT29 , Humanos , Mucosa Intestinal/fisiologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND & AIMS: Krüppel-like factor 5 (KLF5) is a zinc finger-transcription factor that regulates cell proliferation. Oncogenic KRAS mutations are commonly found in colorectal cancers. We aimed to determine whether KLF5 mediates KRAS functions during intestinal tumorigenesis. METHODS: The effects of KLF5 on proliferation and transformation were examined in IEC-6 intestinal epithelial cells stably transfected with inducible KRAS(V12G). KLF5 expression was examined in intestinal tumors derived from transgenic mice expressing KRAS(V12G) under villin promoter and in human colorectal cancers with mutated KRAS. RESULTS: Induction of KRAS(V12G) in IEC-6 cells resulted in increased expression of KLF5, accompanied by increased rates of proliferation and anchorage-independent growth. Inhibition of KLF5 expression by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors or KLF5-specific small interfering RNA reduced proliferation and anchorage-independent growth despite KRAS(V12G) induction. Human colorectal cancer cell lines with mutated KRAS contained high levels of KLF5 and reduction of KLF5 by MEK inhibitors or KLF5 small interfering RNA also led to reduced proliferation and transformation. In vivo, both intestinal tumors derived from mice transgenic for villin-KRAS(V12G) and human primary colorectal cancers with mutated KRAS contained high levels of KLF5 and increased staining of the proliferative marker Ki67. CONCLUSIONS: Elevated levels of KLF5 protein are strongly correlated with activating KRAS mutations in intestinal tumors in vitro and in vivo. Inhibition of KLF5 expression in tumor cells resulted in significantly reduced rates of proliferation and transforming activities. We conclude that KLF5 is an important mediator of oncogenic KRAS transforming functions during intestinal tumorigenesis.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/etiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/patologia , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas ras/fisiologia , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas p21(ras)RESUMO
We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH(2)-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53(+/+) (p53-wt), p53(-/-) (p53-null), p21(-/-) (p21-null), and BAX(-/-) (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Noscapina/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Alelos , Apoptose/fisiologia , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Fase G2/efeitos dos fármacos , Células HCT116 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that the expression of KLF4 was activated by the colorectal cancer tumor suppressor adenomatous polyposis coli (APC) and that KLF4 repressed the Wnt/beta-catenin pathway. Here, we examined whether KLF4 plays a role in modulating intestinal tumorigenesis by comparing the tumor burdens in mice heterozygous for the Apc(Min) allele (Apc(Min/+)) and those heterozygous for both the Apc(Min) and Klf4 alleles (Klf4(+/-)/Apc(Min/+)). Between 10 and 20 weeks of age, Klf4(+/-)/Apc(Min/+) mice developed, on average, 59% more intestinal adenomas than Apc(Min/+) mice (P < 0.0001). Immunohistochemical staining showed that Klf4 protein levels were lower in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with wild-type, Klf4(+/-), or Apc(Min/+) mice. In contrast, the levels of beta-catenin and cyclin D1 were higher in the normal-appearing intestinal tissues of Klf4(+/-)/Apc(Min/+) mice compared with the other three genotypes. Klf4 levels were further decreased in adenomas from both Apc(Min/+) and Klf4(+/-)/Apc(Min/+) mice compared with their corresponding normal-appearing tissues. Reverse transcription-PCR showed an inverse correlation between adenoma size and Klf4 mRNA levels in both Klf4(+/-)/Apc(Min/+) and Apc(Min/+) mice. There was also a progressive loss of heterozygosity of the wild-type Apc allele in adenomas with increasing size from Klf4(+/-)/Apc(Min/+) and Apc(Min/+) mice. Results from this study show that KLF4 plays an important role in promoting the development of intestinal adenomas in the presence of Apc(Min) mutation.
Assuntos
Polipose Adenomatosa do Colo/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Intestinais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclina D , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Técnicas Imunoenzimáticas , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Pólipos Intestinais/genética , Pólipos Intestinais/metabolismo , Pólipos Intestinais/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , beta Catenina/metabolismoRESUMO
AIM: Colorectal cancer (CRC) is the third leading cancer-related cause of death due to its propensity to metastasize. Epithelial-mesenchymal transition (EMT) is a multistep process important for invasion and metastasis of CRC. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor highly expressed in differentiated cells of the intestinal epithelium. KLF4 has been shown to play a tumor suppressor role during CRC tumorigenesis - its loss accelerates development and progression of cancer. The present study examined the relationship between KLF4 and markers of EMT in CRC. METHODS: Immunofluorescence staining for KLF4 and EMT markers was performed on archived patient samples after colorectal cancer resection and on colonic tissues of mice with colitis-associated cancer. RESULTS: We found that KLF4 expression is lost in tumor sections obtained from CRC patients and in those of mouse colon following azoxymethane and dextran sodium sulfate (AOM/DSS) treatment when compared to their respective normal appearing mucosa. Importantly, in CRC patient tumor sections, we observed a negative correlation between KLF4 levels and mesenchymal markers including TWIST, ß-catenin, claudin-1, N-cadherin, and vimentin. Similarly, in tumor tissues from AOM/DSS-treated mice, KLF4 levels were negatively correlated with mesenchymal markers including SNAI2, ß-catenin, and vimentin and positively correlated with the epithelial marker E-cadherin. CONCLUSION: These findings suggest that the loss of KLF4 expression is a potentially significant indicator of EMT in CRC.
RESUMO
Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell growth, proliferation, and differentiation. Since its discovery in 1996, KLF4 has been gaining a lot of attention, particularly after it was shown in 2006 as one of four factors involved in the induction of pluripotent stem cells (iPSCs). Here we review the current knowledge about the different functions and roles of KLF4 in various tissue and organ systems.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Animais , Sequência de Bases , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Homologia de Sequência de AminoácidosRESUMO
Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by gamma-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following gamma-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53-/-) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following gamma-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53-/- cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53+/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, gamma-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following gamma-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.
Assuntos
Centrossomo/fisiologia , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/fisiologia , Raios gama , Fatores de Transcrição/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo , Ciclina E/genética , Deleção de Genes , Genes p53 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , RNA/genética , TransfecçãoRESUMO
In vitro experiments on adult Schistosoma mansoni worms have shown the existence of mutual attraction between males and females mediated by factor(s) released by both sexes. Males were attracted to female-released factor(s), which was found in <30 kDa fractions. A combination of bioassays, size fractionations, chromatographic and chemical and enzymatic treatments were used to identify female-released factor(s) that attracted males. Boiling reduced the attraction of males towards female incubate. Male-attractant factor(s) was not in the female vomitus, and TLC showed the absence of lipids and carbohydrates in the female incubate. Male-attractant factor(s) was found to be sensitive to proteases and to be hydrophilic in nature. Using RP-HPLC, a 3260 Da peptide released by S. mansoni adult females that plays a role in attracting the males was identified, purified and partially characterized. A partial amino acid sequence
Assuntos
Peptídeos/química , Schistosoma mansoni/química , Atrativos Sexuais/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Peptídeos/fisiologia , Schistosoma mansoni/fisiologia , Atrativos Sexuais/isolamento & purificação , Atrativos Sexuais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF) is known to exhibit checkpoint function during the G1/S and G2/M transitions of the cell cycle. The mechanism by which KLF4 exerts these effects is not fully established. Here we investigated the expression profile of KLF4 in an inducible system over a time course of 24 h. Using oligonucleotide microarrays, we determined that the fold changes relative to control in expression levels of KLF4 exhibited a time-dependent increase from 3- to 20-fold between 4 and 24 h following KLF4 induction. During this period and among a group of 473 cell cycle regulatory genes examined, 96 were positively correlated and 86 were negatively correlated to KLF4's expression profile. Examples of upregulated cell cycle genes include those encoding tumor suppressors such as MCC and FHIT, and cell cycle inhibitors such as CHES1 and CHEK1. Examples of downregulated genes include those that promote the cell cycle including several cyclins and those required for DNA replication. Unexpectedly, several groups of genes involved in macromolecular synthesis, including protein biosynthesis, transcription, and cholesterol biosynthesis, were also significantly inhibited by KLF4. Thus, KLF4 exerts a global inhibitory effect on macromolecular biosynthesis that is beyond its established role as a cell cycle inhibitor.
Assuntos
Ciclo Celular/genética , Perfilação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Transcrição Gênica , Linhagem Celular Tumoral , Colesterol/biossíntese , Neoplasias do Colo , Regulação da Expressão Gênica , Humanos , Cinética , Fator 4 Semelhante a Kruppel , Análise de Sequência com Séries de Oligonucleotídeos , TransfecçãoRESUMO
UNLABELLED: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently downregulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the intestinal tract and plays an important role in DNA damage-repair mechanisms. Here, the in vivo effects of Klf4 deletion were examined from the mouse intestinal epithelium (Klf4(ΔIS)) in a genetic or pharmacological setting of colonic tumorigenesis:Apc(Min/âº) mutation or carcinogen treatment with azoxymethane (AOM), respectively.Klf4 (ΔIS)/Apc (Min/âº) mice developed significantly more colonic adenomas with 100% penetrance as compared with Apc(Min/âº) mice with intact Klf4 (Klf4(fl/fl)/Apc (Min/âº)). The colonic epithelium of Klf4 (ΔIS)/Apc (Min/âº)mice showed increased mTOR pathway activity, together with dysregulated epigenetic mechanism as indicated by altered expression of HDAC1 and p300. Colonic adenomas from both genotypes stained positive for γH2AX, indicating DNA double-strand breaks. InKlf4 (ΔIS)/Apc (Min/+) mice, this was associated with reduced nonhomologous end joining (NHEJ) repair and homologous recombination repair (HRR) mechanisms as indicated by reduced Ku70 and Rad51 staining, respectively. In a separate model, following treatment with AOM, Klf4 (ΔIS) mice developed significantly more colonic tumors than Klf4 (fl/fl) mice, with more Klf4 (ΔIS) mice harboring K-Rasmutations than Klf4 (fl/fl)mice. Compared with AOM-treated Klf4 (fl/fl)mice, adenomas of treated Klf4 (ΔIS) mice had suppressed NHEJ and HRR mechanisms, as indicated by reduced Ku70 and Rad51 staining. This study highlights the important role of KLF4 in suppressing the development of colonic neoplasia under different tumor-promoting conditions. IMPLICATIONS: The study demonstrates that KLF4 plays a significant role in the pathogenesis of colorectal neoplasia.