Expression of RHOGTPase regulators in human myometrium.
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.


CONCLUSIONS
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.
O'Brien
Margaret
M
National Centre for Biomedical and Engineering Science, Orbsen Building, National University of Ireland Galway, University Road, Galway, Ireland. margaret.obrien@nuigalway.ie
Flynn
David
D
Mullins
Brian
B
Morrison
John J
JJ
Smith
Terry J
TJ
D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D002585:Cesarean Section; D003199:Computer Systems; D005260:Female; D005453:Fluorescence; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007044:Hysterectomy; D066298:In Vitro Techniques; D007743:Labor, Obstetric / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009215:Myometrium / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569638:ARHGDIA protein, human; D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D008840:Microfilament Proteins; D012333:RNA, Messenger; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C071058:moesin; D020741:rho GTP-Binding Proteins