RESUMEN
The purpose of this study was to determine the role of programmed cell death (apoptosis) in the disappearance of keratocytes beneath an epithelial debridement wound in the cornea and to investigate a potential role of interleukin-1 (IL-1) in induction of apoptosis in stromal fibroblasts in vitro and keratocytes in vivo. Keratocyte and stromal fibroblast cell morphology was examined in wounded and unwounded mouse corneas using transmission electron microscopy. Nuclear DNA fragmentation was detected with the TUNEL assay for 3'-hydroxyl DNA ends. The effect of IL-1 on keratocytes in vivo was determined by microinjection of IL-1 alpha into the central corneal stroma via a limbal entry site. The in vitro effects of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were determined with primary cultures of human corneal stromal and dermal fibroblasts. Cell shrinkage, blebbing with formation of membrane bound bodies, condensation and fragmentation of the chromatin, and DNA fragmentation, consistent with apoptosis were detected in anterior stromal keratocytes after epithelial scrape wounds. Thus, disappearance of keratocytes from the underlying stroma following epithelial debridement is mediated by apoptosis. Microinjection of IL-1 alpha into the central stroma of the mouse cornea caused a redistribution of keratocytes in the stroma via apoptosis and, possibly, negative chemotaxis. IL-1 alpha and IL-1 beta induced apoptosis in corneal stromal and dermal fibroblasts in vitro. The epithelial/endothelial-stromal IL-1 system may mediate corneal tissue organization and responses to mechanical- and pathogen-induced injury through induction of keratocyte apoptosis. Keratocyte apoptosis is likely an initiating event in wound healing following corneal surgery. We hypothesize that derangement's in this system may have a role in the pathogenesis of keratoconus and other diseases of the cornea.
Asunto(s)
Apoptosis , Lesiones de la Cornea , Interleucina-1/farmacología , Cicatrización de Heridas/fisiología , Animales , Apoptosis/efectos de los fármacos , Recuento de Células , Células Cultivadas , Córnea/efectos de los fármacos , Córnea/ultraestructura , ADN/análisis , Epitelio/lesiones , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Factores de TiempoRESUMEN
We sought to determine whether the hepatocyte growth factor/scatter factor (HGF/SF)- and keratinocyte growth factor-receptor systems were expressed in normal breast cells, breast carcinoma cell lines, normal breast tissues, and breast cancer tissues. Reverse transcriptase-polymerase chain reaction and hot blotting were used to detect HGF, HGF/SF (met) receptor, KGF, and KGF receptor mRNAs in human mammary epithelial (HME) and stromal (HMS) cells. We also examined breast carcinoma (MDA-MB-157, SCC 38, and SCC 70) and spontaneously immortalized breast epithelial (HMT 3522) cell lines, as well as normal breast and breast carcinoma tissues. PCR products were also confirmed by nucleic acid sequencing. The effects of HGF and KGF, compared to EGF and heparin-binding EGF, on the proliferation of normal human mammary epithelial cells in serum-free defined medium was determined by cell counting. HGF and KGF mRNAs were detected in HMS cells, but not HME cells. KGF receptor mRNA was detected in HME cells, but not HMS cells. HGF/SF receptor mRNA was detected in both HME and HMS cells. mRNAs were also detected in normal breast and breast carcinoma tissues, as well as breast carcinoma and transformed breast epithelial cell lines. Alternative cDNA sequences that are predicted to code for a soluble KGF receptor and a membrane bound, truncated HGF/SF receptor were detected in breast epithelial cells and breast tissues. HGF and KGF maintained viability and stimulated proliferation of HME cells.
Asunto(s)
Mama/metabolismo , Factores de Crecimiento de Fibroblastos , Regulación Neoplásica de la Expresión Génica , Sustancias de Crecimiento/biosíntesis , Factor de Crecimiento de Hepatocito/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/biosíntesis , Adulto , Anciano , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mama/citología , Neoplasias de la Mama/metabolismo , Bovinos , Línea Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero , ADN , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/metabolismo , Femenino , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-met , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/genética , Células del Estroma , Células Tumorales CultivadasRESUMEN
PURPOSE: The purpose of this study was to determine whether messenger RNA coding for hepatocyte growth factor (HGF), HGF receptor (MET), keratinocyte growth factor (KGF), KGF receptor, and fibroblast growth factor (FGF) receptor-2 were produced in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells, as well as ex vivo corneal epithelium, endothelial cells transfected with the SV40 large T antigen, and control embryonic lung fibroblasts. The effects of exogenous HGF and KGF, compared to epidermal growth factor, on the proliferation of first passage corneal cells were also examined. METHODS: Polymerase chain reaction was used to amplify complementary DNA for each modulator from each cell type. Hot blotting was used to demonstrate the specificity of amplification products. Proliferation of first passage corneal epithelial, stromal fibroblast, and endothelial cells in response to varying concentrations of HGF, KGF, and epidermal growth factor was measured. RESULTS: Specific amplification products for messenger RNA coding for each modulator were detected in each corneal cell type, although very low levels of HGF and KGF messenger RNA appeared to be present in corneal epithelial cells relative to stromal fibroblasts and corneal endothelial cells. Amplification products that may have been derived from alternative transcripts were detected for several of the modulators. HGF and KGF stimulated proliferation in a dose-response manner in first passage corneal epithelial and endothelial cells, but not stromal fibroblast cells. CONCLUSIONS: Human corneal epithelial, stromal fibroblasts, and endothelial cells produce messenger RNA coding for HGF and KGF, although low levels appear to be present in the epithelial cells. All three major cell types of the cornea produce messenger RNA coding for HGF receptor, KGF receptor, and FGF receptor-2. The proliferation of human corneal epithelial and endothelial cells, but not stromal fibroblasts, was stimulated by exogenous HGF and KGF. HGF and KGF likely have intracrine, autocrine, and/or paracrine functions in the cornea. Exogenous HGF and KGF may be useful in corneal preservation and for regulating corneal wound healing.