ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies

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.

A multi-stage genome-wide association study of uterine fibroids in African Americans

“…CYTH4 is most highly expressed in whole blood and spleen based on observations in GTEx and only expressed at low levels in all other measured tissues. CYTH4 expression has also been observed to be low in myometrial tissue postpartum (Kanamarlapudi et al 2012 ). The top signal from the GPGE analysis also identified predicted reduced CYTH4 in thyroid tissue as associated with fibroid risk, supporting a potential biological role for this gene.…”

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate

“…Previous investigations of cytohesins in the prostate were limited to two studies without regarding BPH or smooth muscle contraction (64,66). Recently, cytohesin expression was detected in the human myometrium; however, no contraction experiments were performed in that study (34). So far, no other attempts have been made to address cytohesins in smooth muscle cells, e.g., vascular smooth muscle cells.…”

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

“…The ARF6-HA/pXS, ARF1-HA/pXS, ARF6 T27N -HA/pXS, and ARF1 T31N -HA/pXS constructs were used to express ARF proteins with a C-terminal hemagglutinin (HA) epitope tag in mammalian cells ( 24 ). ARF6 T44N -HA/pXS, GST-GGA3 protein binding domain (PBD; amino acids 1–316), FLAG-EFA6A, and GFP-EFA6A constructs have been described previously ( 25 – 28 ). EFA6C cDNA was obtained by PCR amplification of mammalian gene collection clone 45998, digested with EcoRI (sense) and SalI (antisense), and cloned into the same sites of pEGFP-C2 (Clontech) to obtain GFP-EFA6C construct.…”