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Expression of RHOGTPase regulators in human myometrium.

O'Brien M, Flynn D, Mullins B, Morrison JJ, Smith TJ - Reprod. Biol. Endocrinol. (2008)

Bottom Line: RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form.RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity.Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland. margaret.obrien@nuigalway.ie

ABSTRACT

Background: RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs), RHOGTPase-activating proteins (RHOGAPs) and RHO guanine nucleotide inhibitors (RHOGDIs). RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.

Methods: We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells.

Results: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state.

Conclusion: This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us to further our knowledge of RHOGTPase regulation in human myometrium during pregnancy and labour.

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A summary of the fold changes for each gene at labour (n = 7) in comparison to the non-labouring state (n = 7) in human myometrium, normalised to β-Actin, from real-time fluorescence RT-PCR analyses. Fold change at labour is plotted against gene name: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN, respectively. An asterisk indicates P < 0.05.
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Figure 3: A summary of the fold changes for each gene at labour (n = 7) in comparison to the non-labouring state (n = 7) in human myometrium, normalised to β-Actin, from real-time fluorescence RT-PCR analyses. Fold change at labour is plotted against gene name: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN, respectively. An asterisk indicates P < 0.05.

Mentions: Relative quantitative expression analysis was then performed on the seven genes ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN by real-time RT-PCR. In order to minimise any undue experimental error from sources such as pipetting inaccuracies, analysis of each gene was performed in triplicate. RT-PCR product specificity was confirmed using melting curve analysis. Amplification curve crossing points were determined for each gene generated within the initial phase of exponential amplification, per 0.5 μg total RNA in the tissues studied. The mean Ct values for each transcript normalised to β-Actin (per 0.5 μg total RNA), were then averaged and values determined for both labouring (PL, n = 7) and non-labouring myometrium (PNL, n = 7). The mean β-Actin normalised Ct values for PL and PNL ± SEM, respectively for the 7 genes were: ARHGEF1 26.77 ± 0.23, 26.79 ± 0.6; ARHGEF11 33 ± 0.44, 33.58 ± 0.94, ARHGEF12 29.55 ± 0.48, 29.95 ± 0.67, ARHGAP5 27 ± 0.51, 27.46 ± 0.54, ARHGAP24 28.9 ± 0.19, 29.82 ± 0.25, ARHGDIA 27.08 ± 0.46, 27.29 ± 0.67 and MSN 29.09 ± 0.41, 28.5 ± 0.75 which is graphically represented in Figure 2. Relative fold changes were then calculated using the difference in the Ct values (x) between the PL and PNL myometrium for each transcript, Relative fold change = 2x. ARHGAP24 showed the greatest fold change increase at labour, by real-time RT-PCR which was significant (1.84-fold increase P = 0.0126) in comparison to the non-labouring at-term myometrium. ARHGEF11, ARHGEF12, ARHGAP5, ARHGDIA and ARHGEF1 showed slight increases in mRNA expression at labour, none of which were significant. A slight decrease in MSN expression at labour was also observed, which was not significant. A summary of the fold changes observed using real-time fluorescence RT-PCR is presented in Figure 3.


Expression of RHOGTPase regulators in human myometrium.

O'Brien M, Flynn D, Mullins B, Morrison JJ, Smith TJ - Reprod. Biol. Endocrinol. (2008)

A summary of the fold changes for each gene at labour (n = 7) in comparison to the non-labouring state (n = 7) in human myometrium, normalised to β-Actin, from real-time fluorescence RT-PCR analyses. Fold change at labour is plotted against gene name: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN, respectively. An asterisk indicates P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254629&req=5

Figure 3: A summary of the fold changes for each gene at labour (n = 7) in comparison to the non-labouring state (n = 7) in human myometrium, normalised to β-Actin, from real-time fluorescence RT-PCR analyses. Fold change at labour is plotted against gene name: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN, respectively. An asterisk indicates P < 0.05.
Mentions: Relative quantitative expression analysis was then performed on the seven genes ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN by real-time RT-PCR. In order to minimise any undue experimental error from sources such as pipetting inaccuracies, analysis of each gene was performed in triplicate. RT-PCR product specificity was confirmed using melting curve analysis. Amplification curve crossing points were determined for each gene generated within the initial phase of exponential amplification, per 0.5 μg total RNA in the tissues studied. The mean Ct values for each transcript normalised to β-Actin (per 0.5 μg total RNA), were then averaged and values determined for both labouring (PL, n = 7) and non-labouring myometrium (PNL, n = 7). The mean β-Actin normalised Ct values for PL and PNL ± SEM, respectively for the 7 genes were: ARHGEF1 26.77 ± 0.23, 26.79 ± 0.6; ARHGEF11 33 ± 0.44, 33.58 ± 0.94, ARHGEF12 29.55 ± 0.48, 29.95 ± 0.67, ARHGAP5 27 ± 0.51, 27.46 ± 0.54, ARHGAP24 28.9 ± 0.19, 29.82 ± 0.25, ARHGDIA 27.08 ± 0.46, 27.29 ± 0.67 and MSN 29.09 ± 0.41, 28.5 ± 0.75 which is graphically represented in Figure 2. Relative fold changes were then calculated using the difference in the Ct values (x) between the PL and PNL myometrium for each transcript, Relative fold change = 2x. ARHGAP24 showed the greatest fold change increase at labour, by real-time RT-PCR which was significant (1.84-fold increase P = 0.0126) in comparison to the non-labouring at-term myometrium. ARHGEF11, ARHGEF12, ARHGAP5, ARHGDIA and ARHGEF1 showed slight increases in mRNA expression at labour, none of which were significant. A slight decrease in MSN expression at labour was also observed, which was not significant. A summary of the fold changes observed using real-time fluorescence RT-PCR is presented in Figure 3.

Bottom Line: RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form.RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity.Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland. margaret.obrien@nuigalway.ie

ABSTRACT

Background: RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs), RHOGTPase-activating proteins (RHOGAPs) and RHO guanine nucleotide inhibitors (RHOGDIs). RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.

Methods: We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells.

Results: ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state.

Conclusion: This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us to further our knowledge of RHOGTPase regulation in human myometrium during pregnancy and labour.

Show MeSH