RESUMO
The role of cannabinoid receptor I (CBR-1) in the induction of decidualization was examined using decidual fibroblasts and human endometrial stromal cells as model systems. Decidual fibroblasts decidualized in vitro for 3 and 6 days in the presence of the CBR-1 agonist R(+)-WIN 55,212-2 mesylate (WIN, 0.1-10 microM) expressed less of the decidualization-specific markers prolactin, CBR-1, forkhead (FKHR), TIMP-3, laminin, endometrial bleeding associated factor (EBAF), decorin and insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels compared to control cells. The maximal decrease for each transcript was in the range of 50-99%. In contrast, cells exposed to the CBR-1 inhibitor AM-251 (1 microM) expressed about two-fold higher levels of the decidualization-specific marker gene mRNAs. The WIN-exposed cells showed a marked decrease in intracellular cAMP levels and a progressive, concentration-dependent increase in DNA fragmentation (TUNEL assay) and caspase 3 levels during decidualization compared to control cells. These studies strongly suggest that activation of CBR-1 inhibits human decidualization and stimulates apoptosis by a cAMP-dependent mechanism.
Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , Decídua/fisiologia , Endométrio/fisiologia , Fibroblastos/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Células Estromais/fisiologia , Benzoxazinas , Bloqueadores dos Canais de Cálcio/farmacologia , Canabinoides/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Humanos , Marcação In Situ das Extremidades Cortadas , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Laminina/genética , Laminina/metabolismo , Fatores de Determinação Direita-Esquerda , Morfolinas/farmacologia , Naftalenos/farmacologia , Piperidinas/farmacologia , Prolactina/genética , Prolactina/metabolismo , Pirazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/genética , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The importance of the transcription factor TWIST1 for uterine decidualization was examined in human uterine fibroblast (HUF) cells decidualized in vitro with medroxyprogesterone, estradiol (E2), and prostaglandin E2. TWIST1 mRNA levels increased by 6.0- to 6.8-fold during the first 1-2 d of decidualization and remained above predecidualization levels for up to 15 d. Pretreatment of HUF cells with a TWIST1 small interfering RNA (siRNA) for 3 d before the induction of decidualization resulted in less morphologic differentiation than HUF cells pretreated with a nonsilencing control RNA. In addition, the cells pretreated with TWIST1 siRNA expressed 75-95% less IGF binding protein 1, LEFTY2, fibromodulin, laminin, and several other mRNA during decidualization, including the mRNA for the transcription factors forkhead box protein O1 and v-ets-erythroblastosis virus E26, both of which were previously shown to be critical for the induction of decidualization. The HUF cells pretreated with the TWIST1 siRNA also underwent less apoptosis during decidualization than the control cells, as evidenced by a 20% decrease in DNA fragmentation (terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling assay) and a 43-48% decrease in caspase 3, BCL2-associated X protein, and TNF receptor superfamily member 6 mRNA levels. Although the knockdown of TWIST1 expression markedly attenuated the induction of decidualization, overexpression of TWIST1 alone was insufficient to induce the decidualization of HUF cells. Taken together, these findings strongly implicate an essential role for TWIST1 in the initiation of human decidualization and uterine stromal cell apoptosis that occurs upstream of the induction of forkhead box protein O1 and v-ets-erythroblastosis virus E26 mRNA.
Assuntos
Decídua/metabolismo , Fibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Células Estromais/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Decídua/efeitos dos fármacos , Dinoprostona/farmacologia , Estradiol/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Medroxiprogesterona/farmacologia , Proteínas Nucleares/genética , Placenta/efeitos dos fármacos , Gravidez , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Interferente Pequeno , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Proteína 1 Relacionada a Twist/genéticaRESUMO
CONTEXT: Parathyroid hormone-like hormone (PTHLH) is abundantly expressed by human endometrial stromal cells during decidualization. However, the role for PTHLH in the decidualization process is unknown. OBJECTIVE: To examine the effects of PTHLH on the induction and maintenance of decidualization of human uterine fibroblast (HUF) cells in vitro. DESIGN: HUF cells were treated with a PTHLH siRNA or a PTHLH receptor antagonist (bPTH(7-34)) before or after decidualization with medroxyprogesterone acetate (MPA), estradiol (E(2)), and prostaglandin E(2) (PGE(2)). Decidualization was monitored by immunocytochemistry and the induction of decidualization-specific marker genes, including IGFBP-1, prolactin, lefty, and transcription factor FOXO1. RESULTS: HUF cells decidualized after pretreatment with a PTHLH siRNA showed greater morphologic changes of decidualization, greater IGFBP-1 protein, and two- to threefold more IGFBP-1, prolactin, lefty, and FOXO1 mRNAs than cells pretreated with a nonsilencing RNA. The PTHLH siRNA pretreated cells also had 31% less DNA fragmentation (TUNEL assay) and 30-35% less caspase 3 levels during decidualization than cells pretreated treated with nonsilencing RNA. Treatment of HUF cells with PTHLH siRNA or bPTH(7-34) at 9 d after the induction of decidualization also resulted in 2.1- to 3.2-fold greater IGFBP-1, prolactin, lefty, and FOXO1 mRNA levels than that noted in control cells treated with nonsilencing RNA. CONCLUSIONS: These finding strongly suggest that PTHLH represses the induction of human decidualization, stimulates stromal cell apoptosis, and limits the extent of uterine stromal cell differentiation. Because PTHLH and its receptor are expressed by HUF cells and placental cells, the inhibitory effect of PTHLH on decidualization appears to be due, at least in part, to an autocrine/paracrine mechanism.
Assuntos
Decídua/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Útero/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Caspase 3/análise , Caspase 3/biossíntese , Células Cultivadas , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Marcadores Genéticos , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fatores de Determinação Direita-Esquerda/biossíntese , Comunicação Parácrina/efeitos dos fármacos , Prolactina/biossíntese , RNA/biossíntese , RNA/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/citologiaRESUMO
The aim of this study was to examine whether the transcription factor ETS1 plays a critical role in the regulation of human decidualization. Decidual fibroblast cells were decidualized in vitro by treatment with medroxyprogesterone, estradiol (E(2)) and dibutyryl cyclic AMP or prostaglandin E(2) in the absence or presence of an ETS1 antisense oligonucleotide (oligo) that blocks the translation of ETS1 mRNA. Control experiments were performed using a control oligo that did not affect ETS1 expression and the induction of specific marker genes for decidualization. The ETS1 antisense oligo markedly inhibited ETS1 protein expression and significantly inhibited downstream targets of ETS1 action. On day 6 of culture, the decidualized fibroblast cells that had been exposed to the ETS1 antisense oligo contained 40-90% less mRNAs for prolactin, insulin growth factor binding protein 1 (IGFBP-1) and other decidualization-specific markers (laminin, tissue inhibitor of metalloproteinase-3 [TIMP3], endometrial bleeding associated factor [EBAF] and decorin) than those of control cells that had not been exposed to the ETS1 antisense oligo. GAPDH mRNA levels, which do not change during decidualization, were unaffected by either the ETS1 antisense or the control oligo. The cells decidualized in the presence of the ETS1 antisense oligo also released significantly less prolactin, EBAF and IGFBP-1 protein, determined by western blot analyses, than the control cells. Taken together, these findings strongly suggest that ETS1 plays a critical role in the induction of human decidualization.