RESUMO
The heterodimeric peptide hormone relaxin acts through the novel G-protein coupled receptor LGR7 to elicit the production of cAMP in the human monocyte cell line THP-1. The very small number of receptors on the cell surface, and the lack of response in cell membranes imply the involvement of a cytoplasmic signal amplification process. Here we show that this process comprises a novel and specific tyrosine kinase activity close to the receptor, and involves neither protein kinase A, mitogen-activated protein kinase, nor phosphoinositide-3 kinase activities as major upstream components. Furthermore, this novel involvement of a tyrosine kinase activity is cell-type dependent, being largely absent from LGR7-transfected HEK293T cells, and receptor-dependent; vasoactive intestinal peptide or isoproterenol signalling in the same cells does not require this tyrosine kinase activity.
Assuntos
Fosfotirosina/fisiologia , Relaxina/metabolismo , Linhagem Celular , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Fosfotirosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The heterodimeric peptide hormone relaxin in most cells appears to signal through a G-protein-coupled receptor, LGR7. Whereas in artificial cell systems, made by transfection of receptor-expressing gene constructs into cells normally not presenting the receptor, classic activation of adenylate cyclase appears to be mediated by Gs, in cells naturally expressing the receptor, this type of coupling appears to be very weak. Instead, there is good evidence of other intermediate steps involving cytoplasmic components and tyrosine kinase activity. Part of the complexity of relaxin signaling is also manifest in the variable time course of cAMP production evident in the THP-1 cell line, which appears to depend on passage number and, hence, presumably on differentiation status. It is therefore important to distinguish between immediate early effects, short to mid-term responses, and long-term responses likely the consequences of specific gene upregulation.
Assuntos
Receptores de Peptídeos/metabolismo , Relaxina/metabolismo , Transdução de Sinais , Animais , Humanos , Receptores Acoplados a Proteínas G , Fatores de TempoRESUMO
The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Decídua/fisiologia , Endométrio/fisiologia , Inibidores Enzimáticos/farmacologia , Relaxina/metabolismo , Transdução de Sinais , Células Estromais/fisiologia , Sulfonamidas , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adulto , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Sinergismo Farmacológico , Retroalimentação Fisiológica , Feminino , Expressão Gênica , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Isoenzimas/antagonistas & inibidores , Isoquinolinas/farmacologia , Pessoa de Meia-Idade , Prolactina/genética , Prolactina/metabolismo , RNA Mensageiro/análise , Relaxina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rolipram/farmacologiaRESUMO
BACKGROUND: The receptor for the peptide hormone relaxin has recently been identified as the heptahelical G-protein coupled receptor, LGR7. In order to generate molecular tools with which to characterize both in vivo and in vitro expression of this receptor in human and primate tissues, specific monotypic antibodies have been generated and applied to a preliminary analysis of human and primate female reproductive tissues. METHODS: Three peptide sequences were identified from the proposed open reading frame of the cloned LGR7 receptor gene, representing both extracellular and intracellular domains. Two to three rabbits were immunized for each epitope, and the resulting sera subjected to a systematic validation using cultured cells transiently transfected with a receptor-expressing gene construct, or appropriate control constructs. RESULTS: Human and monkey (marmoset, macaque) endometrium showed consistent and specific immunostaining in the stromal cells close to glands. Staining appeared to be more intense in the luteal phase of the cycle. Weak immunostaining was also evident in the endometrial epithelial cells of the marmoset. A myoma in one patient exhibited strong immunostaining in the circumscribing connective tissue. Uterine expression was supported by RT-PCR results from cultured primary endometrial and myometrial cells. Human breast tissue (healthy and tumors) consistently indicated specific immunostaining in the interstitial connective (stromal) tissue within the glands, but not in epithelial or myoepithelial cells, except in some tumors, where a few epithelial and tumor cells also showed weak epitope expression. CONCLUSIONS: Using validated monotypic antibodies recognizing different epitopes of the LGR7 receptor, and from different immunized animals, and in different primate species, a consistent pattern of LGR7 expression was observed in the stromal (connective tissue) cells of the endometrium and breast, consistent also with the known physiology of the relaxin hormone.