Limits...
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.

Show MeSH

Related in: MedlinePlus

Representative Western blot of ARHGDIA expression in samples of PL (n = 4), PNL (n = 4), NP (n = 4) human myometrium, and HeLa cells (H). The corresponding ACTB protein expression is presented. Protein bands of interest are indicated with arrows and molecular weights are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2254629&req=5

Figure 7: Representative Western blot of ARHGDIA expression in samples of PL (n = 4), PNL (n = 4), NP (n = 4) human myometrium, and HeLa cells (H). The corresponding ACTB protein expression is presented. Protein bands of interest are indicated with arrows and molecular weights are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars).

Mentions: Protein expression of ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP24 and ARHGDIA by western blotting was demonstrated in human myometrium (Figures 5, 6, 7 and 8). Bands of approximately the correct size of 115, 171, 230, 190, 80 and 27 kDa were obtained for ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24 and ARHGDIA respectively, in the myometrium. ARHGAP24 expression was previously demonstrated in HeLa cells [20] and these were used as a positive control for the western blot experiments in human myometrium. HeLa cell lysate was also used in the other western blots, with expression of ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24 and ARHGDIA protein reported in this cancer cell line. The mean β-Actin normalised densitometric units ± SEM for ARHGAP24 were PL (n = 4) 0.4902 ± 0.095, PNL (n = 4) 0.389 ± 0.044 and NP (n = 4) 0.5751 ± 0.089 (Figure 5). From the densitometric analyses therefore, there was a decrease in ARHGAP24 protein expression at PNL in comparison to NP and there was an increase in protein expression at PL in comparison to PNL.


Expression of RHOGTPase regulators in human myometrium.

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

Representative Western blot of ARHGDIA expression in samples of PL (n = 4), PNL (n = 4), NP (n = 4) human myometrium, and HeLa cells (H). The corresponding ACTB protein expression is presented. Protein bands of interest are indicated with arrows and molecular weights are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Representative Western blot of ARHGDIA expression in samples of PL (n = 4), PNL (n = 4), NP (n = 4) human myometrium, and HeLa cells (H). The corresponding ACTB protein expression is presented. Protein bands of interest are indicated with arrows and molecular weights are indicated in kDa. Quantitative densitometric analysis of the western blot is presented, with β-Actin normalised densitometric units for each protein plotted against pregnancy state ± SEM (indicated with error bars).
Mentions: Protein expression of ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP24 and ARHGDIA by western blotting was demonstrated in human myometrium (Figures 5, 6, 7 and 8). Bands of approximately the correct size of 115, 171, 230, 190, 80 and 27 kDa were obtained for ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24 and ARHGDIA respectively, in the myometrium. ARHGAP24 expression was previously demonstrated in HeLa cells [20] and these were used as a positive control for the western blot experiments in human myometrium. HeLa cell lysate was also used in the other western blots, with expression of ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24 and ARHGDIA protein reported in this cancer cell line. The mean β-Actin normalised densitometric units ± SEM for ARHGAP24 were PL (n = 4) 0.4902 ± 0.095, PNL (n = 4) 0.389 ± 0.044 and NP (n = 4) 0.5751 ± 0.089 (Figure 5). From the densitometric analyses therefore, there was a decrease in ARHGAP24 protein expression at PNL in comparison to NP and there was an increase in protein expression at PL in comparison to PNL.

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
Related in: MedlinePlus