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. 2010 May 28:8:55.
doi: 10.1186/1477-7827-8-55.

Ghrelin in the human myometrium

Margaret O'Brien  1 , Padraig Earley, John J Morrison, Terry J Smith
Affiliations

Ghrelin in the human myometrium

Margaret O'Brien et al. Reprod Biol Endocrinol. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
Graphical representation of real-time fluorescence RT-PCR results of β-Actin-normalised relative ghrelin mRNA quantity plotted against myometrial pregnancy state. NP (n = 3), PNL (n = 3), PL (n = 3) ± SEM (indicated by the error bars). * indicates a significance value of P < 0.05 between NP v PL and PNL v PL.
Figure 2
Figure 2
A representative western blot of ghrelin (sc anti-rabbit antibody) protein expression in human myometrium. NP (n = 4), PNL (n = 4), PL (n = 5) and HeLa cells (H). (i) Prepro-ghrelin and (ii) pro-ghrelin and (iii) mature ghrelin bands are indicated. The corresponding β-Actin (ACTB) western blot is presented underneath each blot. Molecular weights are indicated in kDa. Quantitative densitometric analysis of each ghrelin isoform is presented below the relevant blot(s) (i-iii), with β-Actin normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars). * indicates a significance value of P < 0.05. Significance values indicated are compared to NP tissue expression.
Figure 3
Figure 3
A representative western blot of ghrelin (sc anti-goat antibody) protein expression in human myometrium. NP (n = 4), PNL (n = 4) and PL (n = 5) (i) prepro-ghrelin and (ii) pro-ghrelin and (iii) mature ghrelin bands are indicated. The corresponding β-Actin (ACTB) western blot is presented underneath each blot. Molecular weights are indicated in kDa. Quantitative densitometric analysis of each ghrelin isoform is presented below the relevant blot(s) (i-ii), with β-Actin-normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars). * indicates a significance value of P < 0.05. Significance values indicated are compared to NP tissue expression.
Figure 4
Figure 4
A representative Western blot of GHS-R1 protein expression in human myometrial tissue samples. PL (n = 4), PNL (n = 4) and NP (n = 5), and HeLa cells (H). The corresponding ACTB protein expression is presented underneath. Molecular weights are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin-normalised densitometric units for GHS-R1 plotted against pregnancy state ± SEM (indicated with error bars). * indicates a significance value of P < 0.05.
Figure 5
Figure 5
Representative Western blots of GOAT protein expression in human myometrium. (a) PL (n = 5), PNL (n = 4), NP (n = 5), (b) HeLa cells (H) (n = 1) and myometrial PL (n = 3). The corresponding ACTB western blots are presented underneath each western blot. Molecular weights (M) are indicated in kDa. Quantitative densitometric analysis of each protein band indicated a(i-iii) is presented, with β-Actin-normalised densitometric units for GOAT plotted against pregnancy state ± SEM (indicated with error bars). ** indicates a significance value of P < 0.01, *** indicates a significance value of P < 0.001.
Figure 6
Figure 6
A representative Western blot of PC1/3 protein expression in human myometrium. NP (n = 4), PNL (n = 4), PL (n = 5) and HeLa cells (H). The corresponding ACTB western blots are presented underneath. Molecular weights (M) are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin-normalised densitometric units for PC1/3 plotted against pregnancy state ± SEM (indicated with error bars). * indicates a significance value of P < 0.05, compared to the NP tissue.
Figure 7
Figure 7
Representative Western blot of prepro-ghrelin protein expression in LPS (50 ng/ml) treated hTERT-HM cells compared to control (C) untreated cells. The corresponding ACTB western blot is presented underneath the western blot. Molecular weights are indicated in kDa. Quantitative densitometric analysis of this western blot is presented, with β-Actin-normalised densitometric units for prepro-ghrelin plotted against treatment ± SEM (indicated with error bars). * indicates a significance value of P < 0.05, ** indicates a significance value of P < 0.01.
Figure 8
Figure 8
Representative Western blot of GHS-R1 protein expression of β-Estradiol 10-8M (BE) treated hTERT-HM cells compared to control (C) untreated cells. The corresponding ACTB western blot is presented underneath the western blot. Molecular weights are indicated in kDa. Quantitative densitometric analysis of this western blot is presented, with β-Actin-normalised densitometric units for prepro-ghrelin plotted against treatment ± SEM (indicated with error bars). ** indicates a significance value of P < 0.01.
Figure 9
Figure 9
Representative Western blots of (i and ii) GOAT protein expression (iii) PC1/3 expression in hTERT-HM cells and in HeLa cells (H). The corresponding ACTB western blot is presented underneath. Molecular weights are indicated in kDa.
Figure 10
Figure 10
Representative immunofluorescence images of ghrelin localisation in human myometrial smooth muscle cells (hTERT-HM). The FITC negative secondary antibody control is presented (iii) and scale bars are included.
Figure 11
Figure 11
Representative immunofluorescence images of GHS-R1 localisation in human myometrial smooth muscle cells (hTERT-HM). The FITC negative secondary antibody control is presented (iv) and scale bars are included.
Figure 12
Figure 12
Representative immunofluorescence images of GOAT localisation in human myometrial smooth muscle cells (hTERT-HM). The FITC negative secondary antibody control is presented (ii) and a scale bar is included.
Figure 13
Figure 13
Representative immunofluorescence images of PC1/3 localisation in human myometrial smooth muscle cells (hTERT-HM). The FITC negative secondary antibody control is presented (iii).
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