National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland.
Reprod Biol Endocrinol. 2010 May 28;8:55. doi: 10.1186/1477-7827-8-55.
Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated.
mRNA and protein were isolated from human myometrium and the myometrial smooth muscle cell line hTERT-HM; and real-time fluorescence RT-PCR, western blotting and fluorescence microscopy performed. The effects of beta-Estradiol and bacterial lipopolysaccharide (LPS) on hTERT-HM gene expression were evaluated by western blotting.
We have reported for the first time the expression and processing of ghrelin, GHS-R1, GOAT and PC1/3 expression in human myometrium, and also the down-regulation of ghrelin mRNA and protein expression during labour. Furthermore, GHS-R1 protein expression significantly decreased at labour. Myometrial GOAT expression significantly increased during term non-labouring pregnancy in comparison to both non-pregnant and labouring myometrium. Mature PC1/3 protein expression was significantly decreased at term pregnancy and labour in comparison to non-pregnant myometrium. Ghrelin, GHS-R1, GOAT and PC1/3 mRNA and protein expression was also detected in the hTERT-HM cells. Ghrelin protein expression decreased upon LPS treatment in these cells while beta-Estradiol treatment increased GHS-R1 expression.
Ghrelin processing occurred in the human myometrium at term pregnancy and in the non-pregnant state. GOAT expression which increased during term non-labouring pregnancy demonstrating a similar expression pattern to prepro-ghrelin and GHS-R1, decreased at labour, signifying possible myometrial ghrelin acylation. Moreover, the presence of PC1/3 may contribute to pro-ghrelin processing. These results along with the previous in vitro data suggest that myometrially-produced and processed ghrelin plays a significant autocrine or paracrine role in the maintenance of relaxation in this tissue during pregnancy. Furthermore, the significant uterine modulators LPS and beta-Estradiol are involved in the regulation of ghrelin and ghrelin receptor expression respectively, in the human myometrium.
Ghrelin 是一种 28 个氨基酸的辛酰化肽，主要在胃中合成。它刺激生长激素释放，促进食物摄入，并表现出许多其他不同的作用。我们的研究小组之前已经确定 Ghrelin 抑制了体外的人类收缩性。因此，本研究的目的是研究 Ghrelin、其受体、生长激素促分泌素受体 1 型（GHS-R1）、催化 Ghrelin 辛酰化的 Ghrelin O-酰基转移酶（GOAT）、负责前 Ghrelin 加工的前激素转化酶 1/3（PC1/3）在人类分娩前妊娠、分娩中和非妊娠状态下的子宫肌中表达。还研究了培养的子宫平滑肌细胞中 Ghrelin 和 Ghrelin 受体表达的调节。
从人子宫肌和子宫平滑肌细胞系 hTERT-HM 中分离出 mRNA 和蛋白质；并进行实时荧光 RT-PCR、Western 印迹和荧光显微镜检查。通过 Western 印迹评估β-雌二醇和细菌脂多糖（LPS）对 hTERT-HM 基因表达的影响。
我们首次报道了 Ghrelin、GHS-R1、GOAT 和 PC1/3 在人子宫肌中的表达和加工，以及 Ghrelin mRNA 和蛋白表达在分娩过程中的下调。此外，GHS-R1 蛋白表达在分娩时明显下降。与非妊娠和分娩的子宫肌相比，足月非临产妊娠期间 GOAT 表达明显增加。与非妊娠子宫肌相比，成熟的 PC1/3 蛋白表达在足月妊娠和分娩时显著降低。Ghrelin、GHS-R1、GOAT 和 PC1/3 的 mRNA 和蛋白表达也在 hTERT-HM 细胞中检测到。LPS 处理后 Ghrelin 蛋白表达下降，而β-雌二醇处理增加了 GHS-R1 的表达。
Ghrelin 在足月妊娠和非妊娠状态下在人子宫肌中加工。GOAT 表达在足月非临产妊娠期间增加，与前 Ghrelin 和 GHS-R1 表达模式相似，在分娩时下降，表明可能存在子宫肌 Ghrelin 酰化。此外，PC1/3 的存在可能有助于前 Ghrelin 的加工。这些结果以及之前的体外数据表明，在妊娠期间，子宫肌产生和加工的 Ghrelin 在维持该组织松弛方面发挥着重要的自分泌或旁分泌作用。此外，显著的子宫调节剂 LPS 和β-雌二醇分别参与 Ghrelin 和 Ghrelin 受体在人子宫肌中的表达调节。
