RESUMO
Studies in both humans and rodents have found that insulin(+) cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin(+) cells arise de novo from ductal epithelium. We have found that insulin(+) cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated insulin(+) cells arise from the same cell of origin. Using a mouse model that develops insulin(+) cell-containing hyperplastic ducts in response to the growth factor TGFalpha, we performed genetic lineage tracing experiments to determine which cells gave rise to both hyperplastic ductal cells and duct-associated insulin(+) cells. We found that hyperplastic ductal cells arose largely from acinar cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin(+) cells adjacent to acinar-derived ductal cells arose from pre-existing insulin(+) cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of endocrine cells to reorganize in association with duct structures.
Assuntos
Ilhotas Pancreáticas/metabolismo , Pâncreas/fisiologia , Adulto , Animais , Diferenciação Celular , Colangiopancreatografia Retrógrada Endoscópica , Células Endócrinas , Células Epiteliais/metabolismo , Epitélio/metabolismo , Humanos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Pâncreas/metabolismo , Pâncreas Exócrino/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Transdução de SinaisRESUMO
BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is produced as a type-I, single-pass transmembrane protein that can be cleaved to release a diffusible peptide. HB-EGF, often overexpressed in damaged or diseased epithelium, is normally expressed in pancreatic islets, but its function is not understood. METHODS: To understand the function of each isoform of HB-EGF, we made transgenes expressing either a constitutively transmembrane or a constitutively secreted protein. RESULTS: The transmembrane isoform was not an inert precursor protein, but a functional molecule, downregulating the glucose-sensing apparatus of pancreatic islets. Conversely, the secreted form of HB-EGF improved islet function, but had severe fibrotic and neoplastic effects on surrounding tissues. Each isoform had a more severe phenotype than that of full-length HB-EGF, even though the full-length protein was efficiently cleaved, thus producing both isoforms, suggesting that a level of regulation was lost by separating the isoforms. CONCLUSIONS: This work demonstrates that islet function depends on the ratio of cleaved to uncleaved HB-EGF and that the transmembrane intermediate, while deleterious to islet function, is necessary to restrict action of soluble HB-EGF away from surrounding tissue.
Assuntos
Intolerância à Glucose/etiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/fisiologia , Pancreatopatias/etiologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiopatologia , Camundongos , Camundongos Transgênicos , Pancreatopatias/metabolismo , Pancreatopatias/patologia , Isoformas de Proteínas/fisiologia , Precursores de Proteínas/fisiologiaRESUMO
The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.
Assuntos
Receptores ErbB/metabolismo , Pâncreas/metabolismo , Pancreatopatias/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células , Quimiotaxia , Modelos Animais de Doenças , Receptores ErbB/genética , Fibrose , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Pâncreas/patologia , Pancreatopatias/genética , Pancreatopatias/patologia , Pancreatopatias/prevenção & controle , Proteínas Recombinantes/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Increased expression of cPLA2 (cytosolic phospholipase A2) has been shown to be the cause of tumorigenesis of NSCLC (non-small-cell lung cancer). Our laboratory has previously demonstrated that oncogenic forms of Ras increase transcription of cPLA2 in normal lung epithelial cells and NSCLC lines through activation of the ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) MAPK (mitogen-activated protein kinase) family. We have also defined a minimal region of the cPLA2 promoter that is critical for this induction. To identify potential transcription factors that bind to this region and regulate expression, a yeast one-hybrid screen was performed with a rat lung cDNA library. Multiple members of the Krüppel family were identified, with LKLF (lung Krüppel-like factor) being isolated a number of times. Overexpression of LKLF in lung epithelial cells or Drosophila SL-2 cells increased cPLA2 promoter activity. Conversely, expression of a dominant negative form of LKLF inhibited induction of cPLA2 promoter activity by oncogenic Ras in normal lung epithelial cells and NSCLC. By electrophoretic mobility-shift assay analysis, it was found that LKLF bound to a GC-rich region of the cPLA2 promoter located between -37 and -30 upstream from the transcription start site. Expression of siRNA (small interfering RNA) directed against LKLF inhibited basal expression of cPLA2 in lung epithelial cells and blocked induction by H-Ras. In NSCLC, siRNA against LKLF co-operated with siRNA against Sp1 (stimulatory protein 1) to inhibit cPLA2 promoter activity. Finally, recombinant LKLF was a substrate for ERKs. These results indicate that LKLF is an important regulator of cPLA2 expression and participates in the induction of this protein, which is critical for increased eicosanoid production associated with lung tumorigenesis.
Assuntos
Citosol/enzimologia , Fosfolipases A/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transativadores/fisiologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Drosophila/química , Drosophila/citologia , Drosophila/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Indução Enzimática/fisiologia , Células Epiteliais/química , Células Epiteliais/metabolismo , Fosfolipases A2 do Grupo IV , Humanos , Fatores de Transcrição Kruppel-Like , Pulmão/citologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosfolipases A2 , Fosforilação , RNA Interferente Pequeno/genética , Transativadores/biossíntese , Transativadores/metabolismo , Transfecção/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mounting evidence implicates stromal fibroblasts in breast carcinoma progression. We have recently shown in three-dimensional coculture experiments that human mammary fibroblasts stimulate the proliferation of T47D breast carcinoma cells and that this activity requires the shedding of the heparan sulfate proteoglycan syndecan-1 (Sdc1) from the fibroblast surface. The goal of this project was to determine the mechanism of Sdc1 ectodomain shedding. The broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 specifically blocked Sdc1-mediated carcinoma cell growth stimulation, pointing toward MMPs as critical enzymes involved in Sdc1 shedding. MMP-2 and membrane type 1 MMP (MT1-MMP) were the predominant MMPs expressed by the mammary fibroblasts. Fibroblast-dependent carcinoma cell growth stimulation in three-dimensional coculture was abolished by MT1-MMP expression silencing with small interfering RNA and restored either by adding recombinant MT1-MMP catalytic domain or by expressing a secreted form of Sdc1 in the fibroblasts. These findings are consistent with a model where fibroblast-derived MT1-MMP cleaves Sdc1 at the fibroblast surface, leading to paracrine growth stimulation of carcinoma cells by Sdc1 ectodomain. The relevance of MT1-MMP in paracrine interactions was further supported by coculture experiments with T47D cells and primary fibroblasts isolated from human breast carcinomas or matched normal breast tissue. Carcinoma-associated fibroblasts stimulated T47D cell proliferation significantly more than normal fibroblasts in three-dimensional coculture. Function-blocking anti-MT1-MMP antibody significantly inhibited the T47D cell growth stimulation in coculture with primary fibroblasts. In summary, these results ascribe a novel role to fibroblast-derived MT1-MMP in stromal-epithelial signaling in breast carcinomas.
Assuntos
Neoplasias da Mama/patologia , Metaloproteinase 14 da Matriz/fisiologia , Sindecana-1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fibroblastos/fisiologia , Humanos , Metaloproteinase 2 da Matriz/fisiologia , Inibidores de Metaloproteinases de Matriz , RNA Interferente Pequeno/farmacologia , Sindecana-1/químicaRESUMO
The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Comunicação Parácrina , Transdução de Sinais , Sindecana-1/metabolismo , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC , Feminino , Fibroblastos/patologia , Inativação Gênica , Humanos , Camundongos , Mutação , Comunicação Parácrina/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Células Estromais/metabolismo , Células Estromais/patologia , Sindecana-1/genéticaRESUMO
There is a significant body of evidence suggesting that enzymes involved in arachidonic acid metabolism and their eicosanoid products play a role in various cancers, having both pro- and antitumorigenic effects. The goal of this study was to further define the role microsomal prostaglandin E synthases (mPGES-1) play in lung tumorigenesis. Transgenic mice were created with targeted over-expression of human mPGES-1 in the alveolar and airway epithelial cells using an SP-C promoter driven construct. Transgene positive (mPGES-1+) mice were shown to significantly over-express functional mPGES-1 in the lung and more specifically in alveolar type II cells. To study the effects of mPGES-1 over-expression in lung tumor formation, mice were exposed to a complete carcinogen protocol with a single injection of urethane or an initiation/promotion model with a single injection of 3-methylcholanthrene (MCA) followed by multiple injections of butylated hydroxytoluene (BHT). mPGES-1+ mice did not show a significant difference in tumor multiplicity or tumor size at 10, 16, 19 or 30 weeks after urethane injection compared with mPGES-1- mice. No significant difference was seen in tumor incidence, multiplicity or size at 19 weeks after treatment with MCA/BHT. Western blots verified that mPGES-1 expression was increased in tumors versus uninvolved tissue of both mPGES-1+ and mPGES-1- mice with overall expression being significantly higher in mPGES-1+ mice. Cyclooxygenase-2 levels were elevated in tumors in both groups. From these studies we conclude that over-expression of mPGES-1 and highly elevated PGE2 production are not sufficient to induce lung tumors.
Assuntos
Dinoprostona/metabolismo , Oxirredutases Intramoleculares/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Western Blotting , Ciclo-Oxigenase 2 , Humanos , Oxirredutases Intramoleculares/genética , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.