RESUMO
Serotonin signaling plays key roles in augmentation of pancreatic ß-cell function during pregnancy. Increased expression of tryptophan hydroxylase 1 (Tph1), a rate-limiting enzyme for serotonin synthesis by lactogenic hormones, is involved in this phenomenon. To investigate its mechanisms, we here performed 5'-RACE and identified ß-cell-specific transcription initiation sites for Tph1. Prolactin enhanced the expression of mRNA containing these exons; however, reporter gene plasmids containing the proximal 5'-flanking region of these exons did not show prolactin responsiveness in MIN6 cells. Prolactin-induced Tph1 expression was inhibited by a Jak2 inhibitor and was partially inhibited by an MEK1/2 or PI3K inhibitor. Therefore, we analyzed interferon γ-activated sequences (GAS) and found GAS-A about 9-kbp upstream of the transcription start site. The reporter gene plasmid containing the GAS-A region linked to a heterologous promoter showed increased promoter activity by prolactin, which was inhibited by the forced expression of a dominant-negative mutant form of Stat5A and a Jak2 inhibitor. Chromatin immunoprecipitation analysis showed that prolactin treatment augmented Stat5 binding to the GAS-A region in MIN6 cells, as well as in isolated mouse islets, and that Stat5 recognized the GAS-A region in pregnant mouse islets. In addition, the transactivation activity of Stat5 was enhanced by prolactin through the Erk and PI3K pathways in MIN6 cells. Finally, serotonin expression was attenuated in islets of ß-cell-specific Stat5-deficient mice compared with that of control littermates during pregnancy. Our findings suggest that prolactin-induced Tph1 expression is mediated by the activation of Jak2/Stat5, Erk, and PI3K pathways in ß cells.
Assuntos
Células Secretoras de Insulina/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Animais , Linhagem Celular Tumoral , Éxons/genética , Feminino , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Prolactina/genética , Prolactina/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Serotonina/genética , Serotonina/metabolismo , Transdução de Sinais/genética , Transativadores/genética , Transativadores/metabolismo , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Gênica/genéticaRESUMO
A microstructure-based hydrogel was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic ß-cells. To effectively form homogeneous cell clusters in vitro, we made cell-containing hydrogel membrane constructs with an adapted grid structure based on a hexagonal micropattern. Homogeneous cell clusters (average diameter: 83.6 ± 14.2 µm) of pancreatic insulinoma (MIN6) cells were spontaneously generated in the floating hydrogel membrane constructs, including a hexagonal grid structure (size of cavity: 100 µm, interval between cavities: 30 µm). Interestingly, 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to each hexagonal cavity of the hydrogel grid structure. The fate and insulin secretion of homogeneous cell clusters in the hydrogel grid structure were also assessed. The results of these designable hydrogel-cell membrane constructs suggest that facultative in vitro ß-cell proliferation and maintenance can be applied to biofunctional assessments.
Assuntos
Materiais Biomiméticos , Técnicas de Cultura de Células , Proliferação de Células , Hidrogéis/química , Células Secretoras de Insulina , Membranas Artificiais , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , CamundongosRESUMO
Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.
