Abstract
BackgroundARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 contains very low intrinsic GTP binding activity and depends on GEFs (guanine nucleotide exchange factors) such as CYTH3 (cytohesin 3) to bind GTP. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues.MethodsThe expression of ARF6, ARF1, and CYTH1-4 was investigated by measuring mRNA (using RT-PCR) and protein levels (using immunoblotting) in samples of myometrium obtained from non-pregnant women, and women with normal pregnancies, before or after the spontaneous onset of labour. We also analysed myometrial samples from women with spontaneous preterm labour and from women with complicated pregnancies requiring emergency preterm delivery. The GST)-effector pull down assay was used to study the presence of active ARF6 and ARF1 in all myometrial extracts.ResultsARF6, ARF1 and CYTH3 but not CYTH1, CYTH2 and CYTH4 were expressed in all samples and the levels did not change with pregnancy or labour. However, ARF6 and CYTH3 but not ARF1 levels were significantly reduced in complicated pregnancies. The alterations in the expression of ARF6 and its GEF in human myometrium indicate a potential involvement of this signalling system in modulating the response of myometrial smooth muscle in complicated pregnancies. The levels of ARF6-GTP or ARF1-GTP did not change with pregnancy or labour but ARF6-GTP levels were significantly decreased in women with severe complications of pregnancy.ConclusionsWe have demonstrated a functional ARF6 system in human myometrium and a correlation between ARF6 level and activity in uterine and abnormal pregnancy.
Highlights
Preterm birth, defined as delivery before 37 weeks of gestation, is one of the major causes of perinatal mortality and disability in surviving infants [1,2]
We investigated first the expression of ARF1, ARF6, CYTH1, CYTH2, CYTH3 and CYTH4 mRNAs in myometrium by RTPCR (Figure 1)
Human placental cDNA, where the expression of ARF6 and CYTH3 mRNAs has been previously reported, MDAMB-231 breast cancer cells cDNA and plasmid carrying ARF1, ARF6, CYTH1, CYTH2, CYTH3 or CYTH4 cDNA were used as positive controls along with water and –RT cDNA as negative controls in the RT-PCR analysis [23,26,27,28]
Summary
Preterm birth, defined as delivery before 37 weeks of gestation, is one of the major causes of perinatal mortality and disability in surviving infants [1,2]. There is a need to improve our understanding of the intracellular molecular events involved in the maintenance of uterine quiescence during pregnancy, and in initiating successful parturition at term, in order to establish the basis for the development of new therapeutic strategies to prevent preterm birth. Smooth muscle contractility depends on the interaction between polymerised actin filaments and activated regulatory myosin light chains (MLCs). An increase in intracellular calcium (Ca2+)i concentration driven by membrane depolarization through action potentials or generated by stimulatory GPCR activates calmodulin dependent. ARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues
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