RESUMEN
Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾ 4n DNA content. RNA-sequencing-based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾ 4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer.
Asunto(s)
División Celular/fisiología , Familia de Proteínas EGF/fisiología , Receptores ErbB/metabolismo , Factores de Transcripción Forkhead/fisiología , Anfirregulina , Células Cultivadas , Familia de Proteínas EGF/genética , Familia de Proteínas EGF/metabolismo , Proteína Forkhead Box M1 , Fase G2 , Silenciador del Gen , Humanos , Queratinocitos/metabolismo , LigandosRESUMEN
The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.
Asunto(s)
Colitis/inmunología , Familia de Proteínas EGF/fisiología , Receptores ErbB/fisiología , Inmunidad Innata/fisiología , Inmunidad Mucosa/fisiología , Interleucina-33/fisiología , Linfocitos/inmunología , Anfirregulina , Animales , Colitis/inducido químicamente , Colitis/terapia , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Familia de Proteínas EGF/deficiencia , Familia de Proteínas EGF/uso terapéutico , Epitelio/inmunología , Epitelio/metabolismo , Epitelio/patología , Retroalimentación Fisiológica , Inmunoterapia Adoptiva , Interleucina-33/biosíntesis , Interleucina-33/genética , Interleucina-33/uso terapéutico , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Pulmón/inmunología , Pulmón/patología , Linfocitos/clasificación , Ratones , Ratones Noqueados , Mucinas/biosíntesis , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/uso terapéutico , Transducción de Señal , Organismos Libres de Patógenos EspecíficosRESUMEN
Stem and progenitor cell research is a complex and exciting field which promises curative discoveries in numerous areas including cancer, diabetes, and regenerative medicine. Use of biotic factors or growth factors has played an essential role in the development of stem cell research. These biologically active components have been administered into stem cells either to improve or maintain the stem cell proliferation, or to encourage controlled differentiation into more defined cell types. Small molecules such as 6-Bromoindirubin-3´-oxime (BIO), cardiogenol-C, etc can help stem cell research by controlling or influencing the regulatory changes in a controlled manner and to help understand the mechanisms during stem cell differentiation. Extra cellular matrix (ECM) is another significant biotic factor, which mediates cell and tissue behavior by influencing cell-matrix interactions. Thus, in this review we would like to emphasize significance of various growth factors in stem cell research.
Asunto(s)
Familia de Proteínas EGF/fisiología , Células Madre/fisiología , Animales , Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular , Proliferación Celular , Humanos , Comunicación Paracrina , Medicina Regenerativa , Ingeniería de Tejidos , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
PURPOSE: To study the effects of epidermal growth factor (EGF) on the proliferation and cellular fill of junctional epithelium (JE) and gingival epithelium (GE) by using an in vitro wound model. METHODS: EGFR mRNA was semiquantitatively determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry was performed to measure EGFR expression. JE and GE cells were plated into 6 well tissue culture plates containing 22mm x 26mm sterile glass coverslips. After cells were grown to confluence, a 3mm wide wound was created at the center of the coverslips. The cells were incubated in medium containing EGF at a 20ng/mL concentration. Negative controls were incubated in keratinocyte serum-free medium. At 5, 9 and 12 days following wounding, the coverslips were removed and stained with hematoxylin and eosin (HE) and proliferating cell nuclear antigen (PCNA). Quantification of percentage of cellular wound fill and PCNA positive nuclei was accomplished by using computer assisted histomorphometry.The data were analyzed with SAS6.12 software package. RESULTS: Densitometric scanning indicated that EGFR mRNA expression in GE cells was 1.2-fold higher than that in JE cells. EGFR protein was positive in GE and JE immunocytochemically. At 9 -day post-wounding, GE and JE demonstrated significantly greater proliferative responses to EGF when compared to negative controls (P<0.05). But there were no significant differences in the proliferative responses to EGF between the two cell types (P>0.05). At each time point, EGF stimulated the cellular fill of JE and GE compared with negative controls (P<0.05). However, GE displayed greater cellular fill significantly than JE at day 9 and 12 post-wounding (P<0.05). CONCLUSIONS: EGFR is present in the JE and GE cells. EGF may regulate the cell fill and proliferation of the two cell types in this in vitro model.