RESUMO
Rapid cell proliferation is a hallmark of transit amplifying cells, but the mechanisms of this localized proliferation are not well understood. The Krüppel-like factor family member Klf5 (IKLF; BTEB2) promotes cell proliferation and is highly expressed in squamous epithelia, in regions of active proliferation. Here, using mouse primary esophageal keratinocytes as a model, we identify a critical role for Klf5 in regulating squamous epithelial proliferation via the epidermal growth factor receptor (EGFR), which, like Klf5, is localized to basal cells in squamous epithelia. We show that Klf5 increases proliferation, transcriptionally up-regulates EGFR, and activates MEK/ERK signaling, as indicated by increased phosphorylation of MEK and ERK. By chromatin immunoprecipitation, we demonstrate that Klf5 binds directly to the 5' regulatory region of EGFR. In addition, we show that regulation of proliferation by Klf5 is dependent on EGFR and MEK/ERK signaling, as the proliferative response to Klf5 is blocked by pharmacologic inhibition of EGFR or MEK. Inhibition of EGFR or MEK also decreases Klf5 expression. Thus, Klf5 regulates MEK/ERK signaling via EGFR and is also downstream of MAPK signaling, providing a novel mechanism for signal amplification or suppression and control of proliferation in basal cells.
Assuntos
Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Células Epiteliais/citologia , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Ligação Proteica , TransfecçãoRESUMO
KLF4 and KLF5, members of the KLF family of transcription factors, play key roles in proliferation, differentiation, and carcinogenesis in a number of gastrointestinal tissues. While KLF4 is expressed in differentiating epithelial cells, KLF5 is found in proliferating cells of the gastrointestinal tract, including the esophagus. KLF4 regulates a number of genes vital for esophageal epithelial differentiation, and decreased expression of KLF4 is seen in esophageal squamous cancers. Nonetheless, the roles of KLF4 and KLF5 in esophageal tumor progression are not known. Here, using TE2 cells stably infected with retroviral vectors to express KLF4 or KLF5, we demonstrate that KLF4 and KLF5 are key players in a number of cellular processes critical for esophageal carcinogenesis. TE2 cells, derived from a patient with poorly differentiated esophageal squamous cancer, normally lack KLF4 and KLF5. Expression of KLF5 in TE2 cells inhibits proliferation, and both KLF4 and KLF5 decrease viability after treatment with hydrogen peroxide and increase anoikis. In response to DNA damage from UV irradiation, viability is decreased in KLF5 but not KLF4 infected cells. Both KLF4 and KLF5 upregulate the cdk inhibitor p21(waf1/cip1) following UV irradiation, but the pro-apoptotic protein BAX is markedly induced only by KLF5. Thus KLF4 may preferentially activate DNA repair pathways while KLF5 induces both DNA repair and apoptosis after UV irradiation. Expression of KLF4 or KLF5 in TE2 cells also inhibits invasion, consistent with a role for each in preventing tumor metastasis. In summary, KLF4 and KLF5 regulate esophageal carcinogenesis by affecting proliferation, apoptosis, and invasion.
Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Neoplasias Esofágicas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Invasividade Neoplásica/prevenção & controleRESUMO
Migration of epithelial cells is critical for normal homeostasis in gut and skin, but the factors regulating this process are not completely understood. The zinc finger transcription factor Klf5 (IKLF; BTEB2) is highly expressed in proliferating cells of esophagus, skin, and other organs. We hypothesized that Klf5 regulates keratinocyte migration via the integrin-linked kinase (ILK), which, like Klf5, is localized to basal keratinocytes. We stably transduced mouse primary esophageal keratinocytes to overexpress Klf5 or small interfering RNA against Klf5. Klf5 overexpression in keratinocytes increased migration and correlated directly with ILK expression and activation. ILK expression restored migratory capacity in keratinocytes with suppression of Klf5, whereas ILK small interfering RNA blocked the increased migration resulting from Klf5 overexpression. By chromatin immunoprecipitation, electromobility shift assay, and luciferase reporter assays, we confirmed that ILK was a direct target for Klf5. In addition, Klf5 induced the activation of the ILK targets Cdc42 and myosin light chain, which are critical for cell migration and motility but not Rac1, AKT, or GSK3beta. Overall, these results demonstrate that Klf5 is a key regulator of cell migration via ILK and provide new insight into the regulation of epithelial cell migration.
Assuntos
Movimento Celular/fisiologia , Esôfago/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Queratinócitos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Células Cultivadas , Ativação Enzimática/fisiologia , Esôfago/citologia , Queratinócitos/citologia , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Krüppel-like factor 5 (Klf5; also called IKLF or BTEB2), a zinc-finger transcription factor with proproliferative and transforming properties in vitro, is expressed in proliferating cells of gastrointestinal tract epithelia, including in basal cells of the esophagus. Thus, Klf5 is an excellent candidate to regulate esophageal epithelial proliferation in vivo. Nonetheless, the function of Klf5 in esophageal epithelial homeostasis and tumorigenesis in vivo has not previously been determined. Here, we used the ED-L2 promoter of the Epstein-Barr virus to express Klf5 throughout esophageal epithelia. ED-L2/Klf5 transgenic mice were born at the appropriate Mendelian ratio, survived to at least 1 yr of age, and showed no evidence of esophageal dysplasia or cancer. Staining for bromodeoxyuridine (BrdU) demonstrated increased proliferation in the basal layer of ED-L2/Klf5 mice, but no proliferation was seen in suprabasal cells, despite ectopic expression of Klf5 in these cells. Notably, expression of the KLF family member Klf4, which binds the same DNA sequences as Klf5 and which inhibits proliferation and promotes differentiation, was not altered in ED-L2/Klf5 transgenic mice. In primary esophageal keratinocytes that overexpressed Klf5, expression of Klf4 still inhibited proliferation and promoted differentiation, providing a possible mechanism for the persistence of keratinocyte differentiation in ED-L2/Klf5 mice. To identify additional targets for Klf5 in esophageal epithelia, we performed functional genomic analyses and identified a total of 15 differentially expressed genes. In summary, while Klf5 positively regulates proliferation in basal cells, it is not sufficient to maintain proliferation in the esophageal epithelium.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Esôfago/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Esôfago/citologia , Perfilação da Expressão Gênica/métodos , Inibidores de Dissociação do Nucleotídeo Guanina , Herpesvirus Humano 4/genética , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transfecção , Regulação para CimaRESUMO
BACKGROUND & AIMS: The epithelial zinc-finger transcription factor Klf4 (formerly GKLF) regulates cellular proliferation and differentiation in vitro. Klf4 null mice die by postnatal day 1 and show changes in epithelial differentiation of skin and colon. METHODS: We used tissue-specific gene ablation to generate mice lacking Klf4 in their gastric epithelia. Klf4 mutant mice and controls were killed for histology, immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), and serum gastrin levels. Klf4 messenger RNA (mRNA) levels were analyzed in Foxa3-Cdx2 transgenic mice and controls. Human gastric cancers and matched normal tissue were used for qPCR and immunohistochemistry for KLF4. RESULTS: Klf4 mutant mice survive to adulthood and show increased proliferation and altered differentiation of their gastric epithelia. Klf4 mutants also display aberrant expression of acidic mucins and TFF2/SP-positive cells, findings characteristic of premalignant conditions, but no inflammation, intestinal metaplasia, dysplasia, or cancer up to 1 year of age. Expression of KLF4 is nearly absent in human gastric cancer, suggesting that failure to activate KLF4 during normal cellular differentiation may be a common feature of gastric cancers. p21 WAF1/CIP1 is an in vivo target of Klf4, but Klf4 is not a mediator of Cdx2. CONCLUSIONS: Loss of a single genetic factor, Klf4, leads to dramatic changes in the gastric epithelia of mice, and Klf4 is part of a regulatory pathway involving p21 WAF1/CIP1 but not Cdx2. Thus, Klf4 is critical for normal gastric epithelial homeostasis.
Assuntos
Proteínas de Ligação a DNA/genética , Mutação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Estômago/patologia , Fatores de Transcrição/genética , Animais , Fator de Transcrição CDX2 , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/metabolismo , Mucosa Gástrica/patologia , Proteínas de Homeodomínio/metabolismo , Homeostase , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Camundongos , Lesões Pré-Cancerosas/metabolismo , Neoplasias Gástricas/metabolismo , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Fator Trefoil-2RESUMO
Mice lacking the mesenchymal winged helix transcription factor Foxl1 exhibit markedly abnormal small intestinal epithelia and postnatal growth retardation. We investigated whether defects in intestinal nutrient uptake and specific transport processes exist in mice homozygous for a Foxl1 null allele (Foxl1-/-). Foxl1-/- mice and controls on a defined genetic background were weighed regularly and killed at 2, 4, and 12 wk of age. Intestinal uptake studies, quantitative real-time PCR, RNase protection assays, and Western blot analyses were performed. Foxl1-/- mice have dysmorphic small intestinal epithelia and postnatal growth retardation. Foxl1-/- mice demonstrate decreased small intestinal uptake of D-glucose in all age groups studied. Intestinal uptake of D-fructose and two amino acids, L-proline and L-leucine, is not altered. Consistent with these findings, Foxl1-/- mice show decreased levels of the intestinal D-glucose transporter SGLT1. Expression of sucrase-isomaltase, lactase, GLUT2, and Na+-K+ ATPase are not changed. Foxl1-/- mice demonstrate markedly abnormal intestinal epithelia, postnatal growth retardation, and decreased intestinal uptake of D-glucose. The specific effect of Foxl1 on intestinal d-glucose uptake is due to decreased production of SGLT1 protein in the small intestine. Thus we identified, for the first time, a link between a mesenchymal factor, Foxl1, and the regulation of a specific epithelial transport process.
Assuntos
Proteínas de Ligação a DNA/genética , Glucose/farmacocinética , Transtornos do Crescimento/fisiopatologia , Absorção Intestinal/fisiologia , Mucosa Intestinal/anormalidades , Fatores de Transcrição/genética , Animais , Transporte Biológico/fisiologia , Fatores de Transcrição Forkhead , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/anormalidades , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Lactase/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/enzimologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Transportador 1 de Glucose-Sódio , Sacarase/metabolismoRESUMO
Klf4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract and several other organs. In vitro studies have suggested that Klf4 plays an important role in cell proliferation and/or differentiation. Mice homozygous for a null mutation in Klf4 die within 15 hours of birth and show selective perturbation of late-stage differentiation structures in the epidermis, but the function of Klf4 in the gastrointestinal tract has not been investigated. To address this issue, we have generated Klf4(-/-) mice by homologous recombination in embryonic stem cells. In this study, we provide the first in vivo evidence that Klf4 is a goblet cell-specific differentiation factor in the colon. Klf4(-/-) mice exhibit normal cell proliferation and cell death rates in the colon on postnatal day 1. However, Klf4(-/-) mice demonstrate a 90% decrease in the number of goblet cells in the colon, show abnormal expression of the goblet cell-specific marker Muc2 by in situ hybridization, have abnormal staining of the colonic epithelium with Alcian Blue for acidic mucins, and lack normal goblet cell morphology by ultrastructural analysis. All other epithelial cell types are present in the colon of Klf4(-/-) mice. In summary, Klf4 plays a crucial role in colonic epithelial cell differentiation in vivo.