REPRODUCTIONREVIEW

RHO regulation of contraction in the human uterus

J Lartey and A Lo´pez Bernal1 Medical School, Institute of Cellular Medicine, University of Newcastle, 3rd Floor, William Leech Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK and 1Division of Obstetrics and Gynaecology, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Clinical Sciences South Bristol, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK Correspondence should be addressed to J Lartey; Email: [email protected]

Abstract

The state of contraction in smooth muscle cells of the human uterus is dependent on the interaction of activated forms of actin and . Ras homology (RHO) are small monomeric GTP-binding proteins that regulate actin polymerisation and myosin phosphorylation in smooth muscle cells. Their action is determined by their level of expression, GTP-bound state, intracellular localisation and phosphorylated status. Agonist activated RHO proteins bind to effector kinases such as RHO kinase (ROCK) and diaphanous proteins (DIAPH) to regulate smooth muscle contraction by two mechanisms: ROCK activates smooth muscle myosin either by direct phosphorylation at Ser19/Thr18 or through inhibition of myosin phosphatase which is a trimeric protein regulated by ROCK and by other protein kinases. Actin-polymerising proteins such as DIAPH homolog 1 increase filamentous actin assembly to enhance acto-myosin cross bridge formation and contraction. This review explores recent advances in RHO protein signalling in human myometrium and proposes areas of further research to investigate the involvement of these proteins in the regulation of uterine contractility in pregnancy and labour. Reproduction (2009) 138 407–424

Introduction can only be described by analogy with evidence obtained in other systems. Our aim is to explain the The purpose of this paper is to provide an overview of relevance of RHO proteins in the control of myometrial the role of small GTP-binding proteins in myometrial smooth muscle contractility, especially in relation to the function. The past decade has seen a rapid expansion onset of labour. Although this approach can only provide in our knowledge of Ras homology (RHO)-related a narrow perspective in the ever expanding field of proteins in many areas of biology including angiogen- small , we believe it is a framework of current esis, vascular contractility, cytoskeletal dynamics, understanding and a stimulus for research on this endosomal trafficking, neuronal growth, stem cell multifunctional family of proteins for scientists and proliferation, chemotaxis and inflammation, and there clinicians interested in reproduction. are excellent reviews covering these areas (Bishop & Premature birth is the most important cause of Hall 2000, Somlyo & Somlyo 2000, Heasman perinatal death and long term morbidity in the world & Ridley 2008). Moreover, RHO GTPases are promis- today. Although an increasing proportion of premature ing cellular targets for new anticancer drugs and other therapeutic agents for the management of births are induced secondary to pre-existing fetal and asthma (Kume 2008), hypertension (Oka et al. 2008, maternal conditions, over half of these deliveries are due Ponnuchamy & Khalil 2009), and CNS disorders, from to the spontaneous onset of uterine contractions stroke to Alzehimer’s disease (Kubo & Yamashita 2007, (spontaneous preterm labour). So far, our ability to Shin et al. 2008). successfully treat women whose pregnancies are com- The fundamental role of RHO proteins in multiple cell plicated by preterm labour is hampered by a lack of signalling pathways is well established and this is an area knowledge of the endocrine and biochemical factors of rewarding research opportunities; however, it remains that initiate human parturition. relatively unexplored in reproductive tissues. In this The human uterus is a smooth muscle organ which review, we have focused on RHO proteins that have undergoes considerable distension during gestation been described in uterine tissues, but in some instances without expelling its contents. It is not clear the experimental data are scarce and their possible role what regulates this state of relative quiescence.

q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-09-0160 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 07:02:34AM via free access 408 J Lartey and A Lo´pez Bernal

During labour, it contracts in a regular and coordinated fashion to forcibly expel the fetus. Contraction in smooth muscle tissues is regulated by two key enzymes: calcium–calmodulin-dependent myosin light chain kinase (MYLK) which phosphorylates the regulatory 20 kDa myosin light chain (MYL) to generate increases in tension and contraction, and a trimeric protein phosphatase called myosin phosphatase (MLCP) which induces a state of relaxation through dephosphorylation of activated MYL (Somlyo & Somlyo 1994, Word 1995). Therefore, the force of contraction and MYL phosphorylation in the human uterus during labour is determined by the equilibrium between MYLK and MLCP. The mechanisms that regulate these two enzymes in the human uterus remain poorly understood.

The RHO family Figure 1 Dendritic tree representation of the human RHO family of GTP binding proteins. This tree is based on the alignment of RHO RHO proteins are small monomeric GTP-binding protein domains using Clustal W alogorithm and includes RHO, RAC, proteins that regulate acto-myosin interactions in smooth CDC42, ARF and RND proteins and new constitutively active GTPase muscle cells. Their function is primarily determined by deficient proteins RRAD and GEM. Protein sequences were their level of expression, state of phosphorylation, obtained from UniProt (2008) and tree viewed using Jalview editor subcellular localisation and binding of GTP. At a (Clamp et al. 2004). 2C constant level of intracellular calcium ([Ca ]i), acti- vated GTP-bound RHOA binds to RHO kinase (ROCK) Structure and regulation of RHO proteins which inhibits the regulatory binding subunit of myosin These small monomeric GTP-binding proteins usually phosphatase and prevents dephosphorylation of MYL. have between 190 and 250 residues and measure The increase in MYL phosphorylation and contraction at w20–30 kDa in size. All RHO proteins have conserved 2C 2C a constant [Ca ]i is termed ‘Ca sensitisation’ residues at Gly14, Thr19, Phe30 and Gln63 which are (Somlyo & Somlyo 1994). The purpose of this review is required for GTP binding and hydrolysis. Their core to examine the mechanisms that determine RHO protein structure includes an effector domain; four separate function and outline how these proteins regulate uterine nucleotide binding regions which span the length of the contractility during pregnancy and in labour. core structure; a hypervariable region and a CAAX box RHO GTPs belong to the RAS super family of GTP motif, see Fig. 2. The effector domain changes confor- binding proteins. They function as molecular switches mation between the GTP- and GDP-bound states. which regulate a wide variety of cellular functions RHO proteins have intrinsically low levels of GTPase ranging from membrane trafficking, transcription, activity and cycle between an active GTP-bound state cell growth, actin polymerisation, stress fiber formation and an inactive GDP-bound state. RHO activity is and smooth muscle contraction. determined by three classes of regulators: guanine The mammalian RHO family is made-up of at least ten nucleotide exchange factors (ARHGEFs); GTPase activat- different proteins namely RHO gene family member A (RHOA); RHO gene family member B (RHOB); RHO ing proteins (ARHGAPs) and guanine nucleotide gene family member C (RHOC); RHO family GTPase dissociation inhibitors (ARHGDIAs). ARHGEFs activate 1–3 (–3); RAS-related protein RAC1–2; cell the exchange of GTP for GDP (Van Aelst & D’Souza- division cycle 42 GTP-binding protein (CDC42); RHO Schorey 1997). ARHGAPs reduce the levels of active gene family member D (RHOD); RHO gene family GTP-bound RHO by accelerating the rate of GTP hydro- member F in filopodia (RIF) also known as RHOF; RHO lysis to yield GDP (Moon & Zheng 2003). ARHGDIAs gene family member Q (RHOQ) also known as TC10 extract prenylated RHO proteins from membranes and and ADP-ribosylation factors (ARF1 and ARF6). Human sequester them in the cytosol by shielding their prenyl RHOA, RHOB, RHOC and RHOD are located on groups (Michaelson et al. 2001). The effect of these regu- 3p21.3, 2p24, 1p13.1 and 11q14.3 latory proteins on the GTPase cycle is outlined in Fig. 3. respectively. RAC1, CDC42 and their close RHO The complex interaction between these regulatory family relation RHOG are found at 7p22, 1p36.1 and proteins imply that ARHGDI levels relative to ARHGAP 11p15.5–p15.4 respectively. A dendritic tree represen- and ARHGEFs levels may determine RHO activity in tation of the human family of RHO GTP binding proteins uterine smooth muscle tissue during gestation. To this is outlined in Fig. 1. end, we used immunoblotting to quantify myometrial

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by stimulating the activity of RAC–GEF to increase RAC-dependent cell migration (Katoh et al. 2006). RHOB and RHOD are RHO family members which regulate receptor trafficking by controlling the motility of early endosomes (Murphy et al. 1996, Gampel et al. 1999). Expression of activated RHOD mutants attenuates lysophosphatidic acid (LPA)-induced stress fiber formation in 3T3 fibroblasts and baby hamster kidney cells (Murphy et al. 1996, Matsumoto et al. 1997). This appears to be regulated through the inhibition of RHOA; however, the exact mechanism is not known.

Figure 2 The core structure of RHO GTP binding proteins. This diagram is based on several studies (Adamson et al. 1992, Foster et al. 1996, RND proteins Van Aelst & D’Souza-Schorey 1997, Hall 1998, Mackay & Hall 1998, Bishop & Hall 2000). The available functional and structural data show RND proteins are a subfamily of RAS small GTP-binding that RHO–GTP-binding proteins are made-up of an effector domain, proteins made-up of three members RND1, formerly four separate guanosine phosphate binding regions that span the length known as RHO6; , also known as RHON or of the core structure, a hypervariable region and a CAAX box motif RHO7; and , previously known as RHO8. The (C, Cys; A, aliphatic residue; X, any residue; Ridley 2006). The effector name RND ‘round’ is due to their inhibition of RHOA domain (residues 26–45) changes conformation between the GTP stress fiber formation and induction of ‘rounded’ cell bound and GDP bound states. All RHO proteins have conserved phenotype in Swiss 3T3 fibroblasts. The RND1 gene residues at Gly14, Thr19, Phe30 and Gln93 which are required for GTP binding and hydrolysis. Modification of Gly14 to Val and Gln63 to Leu located in 12q12–q13, was first identified results in activation of GTPase activity. Alteration of Phe30 to Leu by screening a human fetal cDNA library with bovine increases the cycling of the GTPase. Mutation of Thr19 inactivates RHO RND1 as bait (Nobes et al. 1998). The human RND2 and is used as a dominant negative form of RHO. The N-terminus gene is located on chromosome 17q21 and it encodes a region also contains switch 1 (residues 27–40) and switch 2 (residues protein with 227 amino acids. 59–78) regions which change conformation between GTP- and RND3 was first identified by Foster et al. (1996) by GDP-bound states and may facilitate changes in effector region required for binding to downstream targets (Aspenstrom et al. 2004). using rat ARHGAP5 as bait in a yeast two hybrid assay. Covalent modification of conserved residues Asn41, Thr37, Glu63 and Other investigators subsequently described an RND3 Cys90 by bacterial toxins can render RHO protein biologically inactive protein which had 15 extra residues at its N-terminal end (Chardin et al. 1989, Aktories et al. 1992, Genth et al. 2003). The compared to the protein described by Foster et al.(Nobes hypervariable region made-up of residues 173–189 is the region of et al. 1998). Human RND3 is located on chromosome most diversity between individual RHO family members. It may 2q23.3. RND proteins have three guanine-binding contain sites for palmitoylation and a polybasic region which can motifs, two loops and three major residues that determine membrane association. The C terminal CAAX box motif dictates post-translational prenylation, proteolysis and carboxymethy- coordinate magnesium binding in the GTP state. They lation of the prenylated Cys residue. The conserved residue Asn41 have an effector region in common with other RHO within the RHO effector region undergo inhibitory ADP ribosylation by family members but they possess key structural C3 transferases (Nobes et al. 1998, Aktories et al. 2000). differences within the catalytic domain which dis- tinguish them from other RHO proteins. Unlike RHO ARHGDIA and ARHGEF1 expression during pregnancy proteins, RND proteins almost have no GTPase activity and in labour. ARHGDIA levels were invariant in non- and exist in a constitutively active GTP-bound state pregnant and pregnant (labouring and non-labouring) (Foster et al. 1996, Nobes et al. 1998, Wennerberg et al. women (Lartey et al. 2006d ). The presence of several 2003). RND proteins have a CAAX box motif and a ARHGEF and ARHGAP isoforms in human myometrium terminal methionine which implies that they undergo emphasises the need to investigate the involvement of farnesylation. these proteins in uterine function (O’Brien et al. 2008). Other new GTPase deficient, constitutively active, proteins including GTP-binding protein overexpressed in skeletal muscle (GEM) and Ras-related associated with diabetes (RRAD) have now been located on chromo- Other monomeric GTP-binding proteins somes 16q22 and 8q13–q21 respectively, Fig. 1. ARF proteins are monomeric guanine binding proteins that control the internalisation of G-protein-coupled Regulation of RND protein activity receptors (GPCR) such as the oxytocin (OXT) receptor and LH receptor by regulating the availability of adaptor RND protein activity is determined by the levels of proteins like arrestin 2 to the activated receptor and protein expression, prenylation, subcellular localisation ligand-bound complex (Mukherjee et al. 2000). RHOG and state of phosphorylation. RND proteins bind is a CDC42 related protein that acts upstream of RAC to ROCK1 and inhibit RHOA downstream effects www.reproduction-online.org Reproduction (2009) 138 407–424

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Figure 3 The RHO GTPase cycle. (1) RHO guanine dissociation inhibitors (ARHGDI) extract prenylated RHO proteins from plasma membrane and sequester the GDP-bound RHO protein in an inactive complex within the cytosol. ARHGDIs shield the RHO protein prenyl groups and prevent them from entering the GTPase cycle (Michaelson et al. 2001, DerMardirossian & Bokoch 2005). ARHGDIs only inhibit GPD dissociation of prenylated RHOA proteins suggesting that post-translational modification is required for ARHGDI inhibition (Hori et al. 1991, Adamson et al. 1992). ARHGDIs also inhibit nucleotide exchange by interacting with RHO switch regions 1 and 2 and competitively restricting RHO access to ARHGAPs and ARHGEFs (Li & Zheng 1997, Hoffman et al. 2000). (PRKA) phosphorylation of membrane-bound GTP–RHOA causes a ARHGDI induced translocation into the cytosol where it forms an inactive complex with ARHGDI (Lang et al. 1996). (2) ERM (ezrin, radixin and moesin) proteins bind to ARHGDI and release the inactive RHO–GDP from ARHGDI enabling it to enter the GTPase cycle (Takahashi et al. 1997). (3) The GTPase cycle. RHO–GTP-binding proteins cycle between two conformations: an active GTP-bound state catalysed by RHO guanine nucleotide exchange factors (ARHGEFs) and an inactive GDP-bound state which occurs as a result of GTP hydrolysis catalysed by RHO GTPase activation proteins (ARHGAPs). (4) ARHGEFs enhance the exchange of GTP for GDP (Van Aelst & D’Souza-Schorey 1997, Schmidt & Hall 2002). Cells usually have up to 10-fold higher levels of GTP compared to GDP and therefore the exchange of GTP for GDP is limited by the release of GDP which can be inhibited by ARHGDIs. GEF catalysed nucleotide exchange occurs in a two-stage reaction. ARHGEFs initially bind with low affinity to GDP-bound RHO and catalyse the release of GDP. This leads to the formation of a higher affinity intermediate which spontaneously binds to GTP (Cherfils & Chardin 1999, Ridley 2006). ARHGEFs are therefore distinct from other RHO regulating proteins because they preferentially bind to nucleotide depleted forms rather than GDP- or GTP-bound forms (Hart et al. 1996, Meller et al. 2002). ARHGEFs are directed to specific subcellular locations by interaction via their PDZ domains with other PDZ containing target proteins. Some ARHGEFs require this additional reaction for activation. In some instances, ARHGEF–PDZ interaction may be regulated by phosphorylation (e.g. by focal adhesion kinase (FAK) and kalirin7 (KALRN7; Penzes et al. 2003). (5) ARHGAPs are characterised by a 150-amino acid RHOGAP domain which is made-up of nine a helices and a highly conserved catalytic arginine residue finger (Gamblin & Smerdon 1998). The RHOGAP domain interacts with the GTP binding core made-up of switch 1, switch 2 and the P-loop and induces a conformational change in the RHO–GTPase active site (Moon & Zheng 2003). ARHGAPs facilitate GTP hydrolysis by allowing an arginine residue to enter and stabilise the GTPase active site (Bishop & Hall 2000). This interaction reduces the energy required for GTP hydrolysis (Li et al. 1997, Nassar et al. 1998). Ligand-activated protein kinases like MAPK phosphorylate and activate ARHGAPs (Roof et al. 1998, Taylor et al. 1998). In some instances, ARHGAP activation is preceded by dimerisation of ARHGAP domains. Activated ARHGAPs increase the intrinsic rate of GTP hydrolysis to generate inactive GDP-bound RHO (Lamarche & Hall 1994). (6) Active GTP-bound RHO translocates to the plasma membrane where it interacts with RHO effectors like ROCK to mediate downstream effects such as stress fiber formation and enhancement of contraction. possibly by preventing RHOA binding to ROCK1 RHOA actin filaments; and an unphosphorylated form (Riento et al. 2003). Thus, the relative levels of RHOA which is found in both cytosolic and membranous and RND3 may determine the overall RHO activity and compartments (Riento et al.2003, 2005). ROCK- function. mediated phosphorylation prolongs the half-life of RND2 is usually cytosolic and preferentially localises RND3 and translocates the protein from the membrane to early endosomes where it binds to vacuolar protein to the cytosol where it disrupts RHOA stress fibers sorting 4A (VPS4A) to regulate endosomal trafficking, (Riento et al. 2005). The resultant RND phosphorylation whereas RND3 also localizes to perinuclear structures requires an activated RHOA/ROCK and a protein kinase like the Golgi (Tanaka et al. 2002). After farnesylation, C (PRKC) pathway (Riento et al. 2005). Platelet-derived RND proteins are equally distributed between plasma growth factor (PDGF) stimulation activates ROCK membrane and the cytosol (Riento et al. 2003). through a PRKC-dependent mechanism to cause It is thought that RND3 exists in two conformations: a phosphorylation of RND3 at Ser7 and Ser11. RND3 phosphorylated cytosolic-bound form with an extended phosphorylation is inhibited by both ROCK and half-life, which is associated with increased disruption of PRKC inhibitors (Riento et al. 2005). PRKC-induced

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Downloaded from Bioscientifica.com at 10/01/2021 07:02:34AM via free access Small G proteins and human labour 411 phosphorylation increases RND activity by prolonging domain binds to the PH and RBD domains to form an the half-life and stability of the phosphorylated protein autoinhibitory closed inactive loop. Binding of active and in some instances C-terminal phosphorylation GTP-bound RHOA to the RBD leads to autopho- causes membrane translocation (Berzat et al. 2005). sphorylation and disruption of the autoinhibitory interaction between the C-terminal PH and RBD domains and the N-terminal kinase domain. The RHO and RND effector interactions activated kinase adopts an open conformation that enables it to bind downstream targets. Structural differences can determine RHO and RND RHO and RND proteins bind to different regions on effector binding. RHO proteins have an ‘insert domain’ ROCK1. RND proteins binding to the N-terminal RND (residues 126–136) which forms a surface loop near the GTP binding site required for GTPase activity. Deletion binding domain made-up of the first 420 amino terminal of the RHOA insert domain makes the protein unstable amino acids including the kinase domain while RHO (Zong et al. 1999). Sequence differences within the insert proteins bind to the C-terminal RBD sited at the end of loop region between RHOA, RHOB and RHOC enables the central coiled-coil domain (Ishizaki et al. 1996). them to selectively bind to effectors like RHO-associated RND proteins optimally bind to RHOA-activated ROCK, protein kinases (ROCK1, ROCK2), Protein kinase N1 suggesting that prior RHOA action is a prerequisite for (PKN1) and the human formin protein diaphanous RND inhibition. The mechanism of RND inhibition of homolog 1 (DIAPH1; Wheeler & Ridley 2004). Other RHOA may be threefold: direct inhibition of the RHOA factors that determine effector specificity include: effector ROCK1 as outlined above, stimulation of i) conformation changes in switch 1 (residues 27–40) ARHGAPs or inhibition of ARHGEFs. Currently, there and switch 2 (residues 59–78) domains between GTP is no evidence that RND proteins are regulated through and GDP-bound states; and ii) coordination of the sequestration by ARHGDIs. interaction between the insert loop and the effector Activated ROCK binds to a number of downstream domain (residues 26–45). These lead to precise changes targets including myosin phosphatase RHO interacting in effector region required for binding to specific protein (Surks et al. 2003, 2005), zip kinase also known downstream targets (Freeman et al. 1996, Aspenstrom as death-associated protein kinase 3 (DAPK3; Hagerty et al. 2004). et al. 2007), integrin linked kinase (ILK; Kiss et al. 2002, ROCKs have four functional domains: an N-terminal Muranyi et al. 2002) and the PRKC inhibitory phospho- kinase domain, a central coiled-coil domain which protein CPI-17 or PPP1R14A (Kitazawa et al. 2000, contains a RHO-binding domain (RBD) and a C-terminal 2003, Eto et al. 2001) to regulate MYL phosphorylation. pleckstrin homology (PH) domain, Fig. 4. The kinase ROCKs regulate actin myofilament assembly by binding to actin nucleators and polymerizers such as DIAPH1, LIM domain kinase 1 and ERM (ezrin, radixin and moesin) proteins. Dysregulation of these actin- binding proteins can result in different disorders. For instance, mutations in DIAPH1 gene have been implicated in non-syndromic deafness (Lynch et al. Figure 4 The structure of RHO associated kinases. The diagram is drawn 1997) and premature ovarian failure (Bione et al. 1998). from information presented in several publications (Ishizaki et al. 1996, We have recently characterised DIAPH1, DIAPH2 and Nakagawa et al. 1996, Riento & Ridley 2003, Loirand et al. 2006). DAPK3 expression in the human uterus during preg- ROCKs are serine/threonine kinases that have four functional domains: an N-terminal kinase domain; a central coiled-coil domain (CC) which nancy and noted interesting changes in expression contains a RHO-binding domain (RBD); a C-terminal pleckstrin during labour (Lartey et al. 2007, Lartey & Lopez Bernal homology (PH) domain; and a cysteine-rich domain (CRD). There is 2009a). It is not certain how these proteins regulate 92% similarity within the N-terminal kinase domains of ROCK1 and uterine smooth muscle MYL phosphorylation and ROCK2 (Nakagawa et al. 1996). The C-terminal coiled-coil and contraction and their function needs to be investigated. pleckstrin domains of the two isoforms are w55 and 66% identical The two ROCK isoforms have their own distinct respectively (Nakagawa et al. 1996). The RBD lies at the C-terminal end functions in the human placenta and in the developing of the coiled-coil domain. The kinase domain binds to the PH and RBD K K domains to form an autoinhibitory closed inactive loop. ROCKs are fetus. ROCK2 deficient (ROCK2 / ) mice usually die activated by GTP–RHO binding to the RBD (Ishizaki et al. 1996), or in utero due to placental dysfunction and intrauterine arachidonic acid (AA) binding to the PH domain (Feng et al. 1999). growth restriction caused by thrombus formation in the Cleavage of the autoinhibitory C-terminal region results in ROCK placenta. ROCK1K/K mice have a different phenotype activation. ROCK1 is cleaved by caspase-3 (Coleman et al. 2001, and usually survive pregnancy and labour to die post- Sebbagh et al. 2001) while ROCK2 is activated by granzyme B cleavage natally from filamentous actin accumulation leading near the CRD domain (Sebbagh et al. 2005). RND proteins inhibit to impairment in the closure of the umbilical vein ROCKs by binding to the RND-binding domain (RND-DB) made-up of the first 420 N-terminal amino acids (Riento et al. 2003). RAD and (Rikitake et al. 2005). Analysis of ROCK2 null mice GEM proteins bind to the CC domain to down regulate ROCK activity suggests that ROCK1 does not compensate for the loss (Ward et al. 2002). Numbers represent amino acid residues. of ROCK2 function (Thumkeo et al. 2003). www.reproduction-online.org Reproduction (2009) 138 407–424

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GPCR activation of RHOA/ROCK proteins cause increases in MYL phosphorylation and contraction 2C in smooth muscle cells at a constant [Ca ]i, see Fig. 5. In addition to inhibitory phosphorylation of the GPCR-dependent activation of RHOA is primarily myosin binding subunit, ROCKs directly phosphorylate mediated through the Ga12/13 heterotrimeric proteins. MYL at Ser19/Thr18 (Amano et al. 1996). There is now The ‘active’ heterotrimeric Ga12/13 protein complex evidence that individual pathways can cooperatively binds to ARHGEFs through a regulator of G-protein enhance MYL phosphorylation. signalling (RGS) domain and sets off a downstream cascade by catalysing nucleotide exchange on ARH- GEF1 and ARHGEF12. Thus, ARHGEFs with RGS domains function both as GTPase activating proteins RHO and RND protein regulation of uterine and as effectors for Ga12/13 (Suzuki et al. 2009). Ligand- contractility bound GPCR–Ga13–ARHGEF1 complex has a number The force of contraction and MYL phosphorylation in of downstream actions: i) to stimulate GTP exchange of uterine smooth muscle cells depends on the resultant RHOA, leading to ROCK1 activation; and ii) activation equilibrium between MLCK and MLCP. Agonist acti- of phospholipase D which cleaves phosphatidylcholine vation of the RHOA and subsequently ROCK1 causes to yield phosphatidic acid and diacylglycerol. The latter inhibitory phosphorylation of the myosin-binding sub- stimulates the PRKCB-dependent phosphoprotein unit of MLCP (PPP1R12A). This produces a greater level C PPP1R14A which inhibits the PPP1c catalytic subunit of MYL phosphorylation and tension at a given Ca2 - C of MLCP. Therefore, GPCR receptor activation results in dependent MYLK activity, resulting in Ca2 sensitisa- ROCK and PRKCB-mediated inhibition of MLCP to tion, see Fig. 5.

C Figure 5 RHO proteins, myosin equilibrium and Ca2 sensitisation. Ligand-bound GPCRs regulated smooth muscle phosphorylation by three main pathways. Activated phospholipase C cleaves phosphatidyl inositol phosphates to yield inositol triphosphate (IP3) and diacylglycerol (DAG). Phospholipase D can also be activated to generate DAG from phosphatidylcholine (Ha et al. 1994, Malcolm et al. 1994, Exton 1997, Gong et al. 2C 2C 1997, Zhou et al. 2003). IP3 promotes intracellular Ca release which stimulates Ca -dependent calmodulin to activate myosin light chain kinase (MYLK). Phosphorylation of the regulatory myosin light chains (MYL) by MYLK causes contraction. Diacylglycerol (DAG) stimulates the PRKCB- dependent phosphoprotein PPP1R14A which inhibits the PPP1c catalytic subunit of MLCP to prevent dephosphorylation of activated MYL. The effect of PRKC can be mimicked by other protein kinases such as PKN1 (Hamaguchi et al. 2000). ARHGEF activated RHOA binds to ROCK and allows ROCK inhibitory phosphorylation of Thr696 on the myosin binding subunit PPP1R12A of MLCP. This attenuates the phosphatase activity and leads C to higher levels of activated MYL. Agonists that increase Ca2 –calmodulin dependent contraction can also increase PRKCB–PPP1R14A and 2C 2C RHOA–ROCK mediated MYL phosphorylation in a [Ca ]i-independent manner to cause further increases in contraction known as Ca sensitisation. RND and RHOD proteins inhibit RHOA–ROCK activity. The exact mechanism of RHOD inhibition is not known.

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During pregnancy, the myometrial contractile upregulated in mid-pregnant rabbit myometrium and apparatus undergoes several changes that enable it to suggested that the mid-pregnant increase in RND3 adapt to the physiological alterations in size, stretch and expression was associated with inhibition of RHOA C tension due to the growing fetus. These changes during mediated Ca2 sensitization (Cario-Toumaniantz gestation prime the myometrium for the increase in et al. 2003). contractile activity required for normal labour. Although myometrial levels of actin, myosin, calponin and the steady state levels of MYL phosphorylation do not alter Relevance to human myometrium during pregnancy, tissue strips from pregnant women Our data in humans demonstrate that there is an increase generate greater levels of tension at any given level of in myometrial RND2 and RND3 mRNA levels during MYL phosphorylation in comparison with strips from pregnancy, see Fig. 6. The expression of other RHO non-pregnant women (Word et al. 1993, Riley et al. family members, namely RHOA and RHOB, were also 2005). Pregnant myometrium is more sensitive to the increased. We also noted a dramatic increase in RND2 effects of calcium and agonists like OXT than non- and RND3 protein expression in the pregnant groups. pregnant myometrium. In the rat, pregnant myometrium In contrast, RHOA, RHOB and RND1 protein levels did develops greater levels of tension than non-pregnant not alter with pregnancy or labour. myometrium at a given level of MYL phosphorylation after exposure to a range of stimulants including OXT, C prostaglandins (PGE2 and PGF2a), carbachol or high K (Kim et al. 1998). The mechanism of the increased response to contractile agonists in pregnancy is not C completely understood, but it is likely to involve Ca2 sensitization and be multi-factorial in origin. Moreover, other contractile proteins like caldesmon are elevated in pregnant compared to non-pregnant rat myometrium and may mediate this heightened contractile activity (Li et al. 2003). However, treating permeabilised rat myometrial C strips with Ca2 and calyculin-A (a phosphatase inhibitor) produced similar increases in force in non-pregnant and pregnant myometrial strips (Kim et al.1998). This suggests that the increase in force generated in myometrial smooth muscle during pregnancy is due to inhibition of a phosphatase presumably myosin phosphatase. The first physiological experiments which demon- strated the effect of RND protein on contraction in smooth muscle tissues were performed in permeabilised rat ileal muscle strips and showed that co-transfection of C RHOA and RND1 reverses RHOA mediated Ca2 sensitization (Loirand et al. 1999). Treating rat ileal strips with progesterone and oestrogen increased their RND1 membrane-bound content. These changes in RND1 2C Figure 6 RHO and RND mRNA (A) and protein (B) expression in human expression led to a reduction in agonist-induced Ca myometrium. RHOA, RHOB, RND2 and RND3 mRNA expression in sensitization. The action of RND1 was independent of myometrial samples from non-pregnant (NP) and pregnant not in 2C [Ca ]i and did not affect the calcium-force response labour (P) women were measured by quantitative real-time PCR curve or the force of contraction generated by the myosin (Lartey et al. 2007). The amounts of RHO and RND mRNA were phosphatase inhibitor calyculin-A. Subsequently, other normalised to the amount of RNA polymerase II (RPII) endogenous Z investigators suggested that the increase in steroid control gene mRNA expression within each sample (n 5 for each). Whole tissue homogenates from the samples above were immuno- hormones during gestation causes a time-dependent blotted with RHO and RND antibodies. All the samples were analysed increase in Rnd1 mRNA expression in rat uterus (Kim in duplicate and normalised to individual bands and plotted in et al. 2003). They noted that Rnd1 mRNA levels fell to a logarithmic scale within allotted patient groups. One-way ANOVA pre-pregnancy levels at day 1 post partum. In line with was used to assess differences between the means of the three groups. previous observations they suggested that the increase in Myometrial RHO and RND mRNA expressions were elevated in Rnd1 mRNA expression may function to inhibit RHOA pregnant relative to non-pregnant groups. This is sharply contrasted by C Ca2 sensitization and maintain uterine quiescence the immunoblotting analysis which only demonstrated significant pregnancy related increases in RND2 and RND3 protein expressions. required during gestation. Pacaud’s group used cDNA RHOA, RHOB and RND1 levels were invariant with pregnancy. micro array, real-time PCR and immunoblotting to show The histograms are means and the vertical bars represent S.E.M. that RND3 mRNA and protein expression was (*P!0.01), Student’s t-test. www.reproduction-online.org Reproduction (2009) 138 407–424

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C RHO and RND function in human myometrium may phasic agonist- and K -stimulated contractions be determined by their levels of expression, state of (Kupittayanant et al. 2001). This has led some workers C phosphorylation, intracellular localisation and the to conclude that RHOA-induced Ca2 -independent activity levels of effector molecules like ROCK1 (Bishop contractions primarily regulate the strength of tonic & Hall 2000). The overarching hypothesis is that an contractions and may not play a significant role in increase in RHOA and ROCK1 expression may account augmenting the phasic activity of myometrial tissue in for the increased contractile activity at the end of labour (Kupittayanant et al. 2001). gestation and during labour (Niiro et al. 1997, Moore et al. 2000, Moran et al. 2002, Tahara et al. 2002). The potential regulatory role for RHO proteins in Coupling of RHO activation, membrane uterine smooth muscle contraction was first highlighted by demonstrating an increase in Rock1 and Rock2 translocation and myometrial contraction mRNA expression in rat myometrium during pregnancy RHO proteins shuttle between an active GTP mem- (Niiro et al. 1997). Other investigations subsequently brane-bound form and an inactive GDP cytosol and reported ROCK1 and ROCK2 mRNA and protein presumably ARHGDI sequestered form. Several investi- expression in pregnant and non-pregnant human gators have now linked RHO-mediated increases in myometrium (Moore et al. 2000, Moran et al. 2002, tension and MYL phosphorylation to changes in Friel et al. 2005, Riley et al. 2005). Myometrial RHOA, membrane-bound levels and GTP-bound state of RHO. ROCK1 and ROCK2 levels in women are invariant Exposure of myometrial tissue to stimulatory GPCR among non-pregnant, pregnant not in labour, spon- agonists, e.g. OXT, carbachol, activates RHOA and C taneous labour and spontaneous preterm labour groups ROCK to produce Ca2 -independent increases in (Lartey et al. 2006d ). tension (Taggart et al. 1999, Kupittayanant et al. 2001, Myometrial ROCK activity was elevated after treat- Tahara et al. 2002, 2005, Woodcock et al. 2004). LPA is a ment with the caspase-3 inhibitor Z-DEVD-FMK which classical RHO agonist that increases MYL phosphoryl- prevents cleavage of ‘active’ p160ROCK1 to the ation in myometrial tissue strips (Moore et al. 2000). ‘inactive’ p130ROCK1 (Moore et al. 2002). Chronic Recent experiments demonstrating that LPA increases thromboxane receptor stimulation with U46619 GTP–RHOA levels in treated human myometrial smooth increased p160ROCK1 expression and these changes cells suggest that LPA-induced MYL phosphorylation were reversed by pretreatment with SQ29458, a may in part be due to RHOA activation (Lartey et al. 2007). thromboxane receptor antagonist (Moore et al. 2002). These experiments highlighted the potential role of Agonist activation of RHOA enhances ROCK activity by agonist-induced RHO activation and MYL phosphoryl- increasing the stability of the p160ROCK1 (Moore & ation in human uterine smooth muscle contraction. Lopez Bernal 2003). One of the key experiments linking GPCR receptor There is evidence of other RHO effectors in human stimulation to protein translocation in uterine smooth C myometrium. A constitutively active p34 protein frag- muscle cells demonstrated that carbachol-induced Ca2 ment of the RHO effector p21 protein activated kinase 2 sensitisation resulted in PRKCB, RHOA and ROCK (PAK2) is elevated in human myometrium during translocation to the plasma membrane (Taggart et al. pregnancy (Moore et al. 2000). The expression of PKN 1999). Carbachol stimulated increases in the force of (previously known as PRKC-like protein; Mukai & Ono contraction and MYL phosphorylation at a constant 2C 1994) and its downstream target PPP1R14A are both [Ca ]i were reversed by the ROCK inhibitor Y27632. elevated in human myometrium during pregnancy (Ozaki The same authors showed that contractile agonists like et al. 2003, Lartey et al. 2007). The human formin pro- carbachol and OXT cause membrane translocation of tein DIAPH1 expression is upregulated in spontaneous RHOA in rat myometrial cells (Oh et al. 2003). labour whole tissue homogenates (Lartey et al. 2007). Our experiments have demonstrated increases in RHOA, RHOG, RND2 and RND3 membrane-bound proteins in pregnant myometrium relative to non- Tonic versus phasic contractions pregnant myometrium (Lartey et al. 2006c). This suggests Smooth muscle tissues like the uterus and the gut that the changes in myometrial cell shape, size and contract in two ways: ‘phasic’ contractions which are function during pregnancy are reflected by alterations transient, and ‘tonic’ more sustained contractions in RHO protein activation. We observed increases in (Murthy et al. 2003). During labour the uterus contracts RHOA membrane translocation in human myometrial in a predominantly phasic manner but also requires tissue strips after OXT and carbachol treatment under periods of sustained tension (i.e. tonic contraction) in both tension free and isometric conditions (Lartey & between the phasic contractions which occur approxi- Lo´pez Bernal 2008a, 2008b, 2008c). Curiously, both mately every 2 min. In vitro experiments with human OXT and carbachol also produced marked increases in uterine muscle strips showed that ROCK inhibition RND1 and RND2 membrane translocation (Lartey & with Y27632 was maximal during tonic rather than Lopez Bernal 2008a).

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The RND2 results are quite remarkable as this protein in spontaneous labour and preterm labour. RND2 was only confined to the cytosolic fraction of unstimu- and RND3 levels were markedly upregulated relative lated tissue strips in all pregnant samples used in our to RHOA levels during gestation (Fig. 7). This increase study. At first glance, these findings are contrary to the in RND protein expression correlated with a reduction concurrent increases in RHOA translocation and pre- of ROCK1 inhibitory phosphorylation of PPP1R12A sumed activation observed in the stimulated uterine subunit in the pregnant tissues examined (Lartey et al. strips. However, it is noted that RND2 can function both 2006d). These findings are consistent with the effects of as a RHOA antagonist and agonist (Tanaka et al. 2006a). RND proteins in rat and rabbit myometrium and imply RND2 is usually cytosolic but is recruited to a membrane that, in humans, these proteins may regulate the compartment presumed to be early endosomes by an relative quiescence of the uterus during gestation in effector protein called VPS4A. Therefore, the differences order to allow the fetus to reach an appropriate in RND membrane translocation caused by OXT may be maturity prior to normal labour. The lack of changes as a consequence of its involvement in GPCR receptor/ in RND to RHOA ratios in labouring myometrium vesicular trafficking (Tanaka et al. 2002). More work is suggests that other mechanisms may be important at required to understand RHO and RND functions in this interface. For instance, downregulation in the myometrial cells during pregnancy and in labour. expression of RHOD, a known RHOA inhibitor, may A summary of RND interacting proteins and their account for the threefold increase in GTP-bound mechanisms of actions are presented in Table 1. RHOA levels in labour, see Fig. 7 (Lartey et al. There is evidence that agonist stimulation of other 2006b, 2007). RHOD inhibits other RHO–GTP-binding GPCRs like the corticotrophin-releasing hormone (CRH) proteins. For example, it binds to the cytoplasmic RBD type 2 receptor also results in MYL phosphorylation and domain of the semaphorin receptors Plexin-A1 and contraction in human myometrial smooth muscle cells. Plexin-B1, preventing their dimerisation and activation Urocortin (a 38 kDa CRH neuropeptide) stimulates by RND1 possibly through competitive binding myometrial cell CRHR2 to increase MYL phosphoryl- (Zanata et al. 2002, Tong et al. 2007). The myometrial ation via a variety of mechanisms which include RHOA RHOA–RND–RHOD interactions in human labour activation and RHO membrane translocation. This was require further investigation. confirmed by RHO inhibitors like C3 exotoxin and RHOB and RHOD are endosomal GTP-binding Y27623 which reversed MYL phosphorylation and proteins which regulate actin vesicular dynamics and RHOA translocation. In addition, urocortin mediated GPCR trafficking (Murphy et al. 1996) and are expressed MYL phosphorylation was abolished by the PRKCB in pregnant myometrial tissue, see Fig. 8.Weare inhibitor bisindolylmaleimide and the MAPK1 inhibitor currently investigating their role in human chorionic U0126 suggesting that these additional pathways also gonadotropin/LH and OXT receptor desensitisation. regulate myometrial cell MYL phosphorylation and We have noted some interesting changes in RHOA contraction (Karteris et al. 2004). and RND protein function during abnormal dysfunc- Physiological uterotones such as OXT and PGF2a tional labour, defined as spontaneous labour with C enhance myometrial sensitivity to Ca2 through a ‘secondary arrest’ not treated with OXT. RHOA RHOA–ROCK-dependent mechanism (Woodcock et al. membrane levels increased during gestation and in 2004, 2006). We have mapped the effects of OXT and normal labour at term but were reduced in the carbachol on RHO protein targets downstream of dysfunctional labour group (J Lartey & A Lo´pez Bernal, ROCK1. OXT-induced tension and contraction in unpublished observations). This represents attenuation in human myometrial strips under isometric conditions are RHOA activation in dysfunctional labour relative to associated with concurrent increases in PPP1R12A- spontaneous labour. OXT stimulation increases both Thr696 and MYL-Ser19/Thr18 phosphorylation (Lartey RHOA and RND2 membrane translocation therefore it &Lo´pez Bernal 2008a, 2008b, 2008c). Pregnancy is plausible to suggest that the use of OXT will correct the associated with a marked upregulation in the expression abnormal RHO function in this subgroup. of native and activated forms of the PRKCB-dependent ARF–GTP-binding proteins regulate GPCR receptor inhibitory phospho-protein PPP1R14A (Ozaki et al. desensitization (Mukherjee et al. 2000). ARF–GEFs 2003, Lartey et al. 2007). We have shown that OXT can catalyse the nucleotide exchange on the ARF–GDP- induce changes in PPP1R14A–Thr38 phosphorylation in bound GPCR complex allowing the receptor to inter- human uterine smooth muscle cells (Lartey et al. 2007). nalize (Salvador et al. 2001). Recent experiments from our laboratory have found changes in ARF6 and PSCD3 Involvement of RHO proteins in uterine quiescence (an ARF6 GEF) mRNA and protein expression in preterm myometrium from pregnancies complicated by pre- and abnormal labour eclampsia and intrauterine growth restriction, suggesting We examined a panel of over ten RHO–GTP-binding for the first time that different mechanisms may regulate proteins to determine if changes in RHO protein receptor trafficking during normal and complicated expression were associated with the contractile activity pregnancies (Lartey & Lopez Bernal 2008b). www.reproduction-online.org Reproduction (2009) 138 407–424

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Table 1 Known RND2 interacting proteins.

Kinase Effects and function References Serine/threonine kinases RHOA associated kinase (ROCK) RND proteins inhibit RHOA activity through competitive inhibition of Fujisawa et al. (1996) and ROCK1. RND3 and RHOA bind to different regions on ROCK1. RND Riento et al. (2003, 2005) proteins bind to the N-terminal domain (1–76) while RHO proteins bind to the C-terminal region (934–1015) of ROCK1. The minimum region required for RND inhibition (1–420) includes the RND-binding domain, a kinase domain (76–338) and a further region (338–420). RND1 inhibition of ROCK1 may be dependent of prior activation by GTP– RHOA. RND3 binds optimally to caspase activated or truncated versions of activated ROCK1 ARHGAPs and ARHGEFs ARHGAP5 (or p190RHOGAP) RND3 was initially identified as a binding partner to ARHGAP5, which has Foster et al. (1996), an N-terminal GTP-binding domain, a middle RND-binding region Hansen et al. (2000) and (382–1229) and C-terminal RHOGAP domain. The RND3–RHOGAP- Wennerberg et al. (2003) binding motif include residues 16–93 which contain the phosphate- binding loop, switch 1 and 2 regions. RND1 and RND3 enhance GTPase activity by targeting ARHGAP5 to the plasma membrane where it reduces GTP–RHOA levels and RHOA stress fiber formation Conversely, ROCK1 phosphorylates ARHGAP5 at Ser1150 to reverse Mori et al. (2009) RND1 binding and attenuate GTPase activity

UBX domain protein 11 UBXN11 was the first protein to be identified that specifically interacts with Katoh et al. (2002) (UBXN11) or Socius RND proteins. Transfection of activated RND3 proteins into COS7 cells targets UBXN11 in the plasma membrane to cause actin stress fiber formation and cell rounding. No downstream effects were observed when RND2 and RND3 were co-transfected with UBXN11, suggesting that it is a RND1-specific effector mediating RHOA/ROCK inhibition

Growth factor receptor-bound GRB7 is a member of the GRB1/10/14 family which contains a Src Vayssiere et al. (2000) protein 7 (GRB7) homology 2 (SH2) domain, a central pleckstrin homology domain and an N-terminal proline rich SH3 binding motif. The RND1 switch 2 and guanine nucleotide exchange region binds to the SH2 domain in GRB7 and the resultant complex is translocated to the membrane. Therefore, GRB7 acts as an adaptor protein which targets RND1 to the plasma membrane where it stimulates GTPase activity towards GTP RHOA

Rapostilin (Apostle of RND2) Rapostilin is a RND2-specific effector which is homologous to a human Jalink et al. (1994), formin binding protein and a RHO–CDC42 effector protein called CIP4. Chan et al. (1996) It has two functional domains: an N-terminal CIP4 domain and a SH3 and Fujita et al. (2002) domain at the C-terminal. GTP-bound RND2 binds to Rapostilin SH3 domain to reverse RHOA induced changes in cell morphology and function

Plexins – A1, B1 and D1 RND1 binds to plexin-B1 increasing its GTPase activity which opposes Rohm et al. (2000), Zanata RHOA to cause cell contraction in COS7 cells. Active RND1 binds to et al. (2002), Oinuma plexin-A1 and increases its GAP activity to cause actin rearrangements. et al. (2003, 2006) and RND2 binds to plexin-D1 and inhibits it functions Uesugi et al. (2009) Zanata et al. (2002)

Pragmin (Pragma of RND2) RND GTP-binding proteins mediate almost all of their intracellular effects Tanaka et al. (2006a) by inhibition of the RHOA–ROCK1 pathway. However, in some instances RND proteins can act as activators of RHOA. Activated RND2 binds to pragmin which activates RHOA to cause cell contraction

Fibroblasts growth factor receptor RND protein function may also be regulated through sequestration by Harada et al. (2005) associated substrate 2-a and b specific binding proteins. RND1 normally binds to FRS2a and FRS2b (FRS2a and FRS2b) which are docking proteins of fibroblast growth factor (FGF) receptors. FRS2b bound RND1 has no effect on RHOA

Male germ cell RAC GTPase RACGAP1 is a human ARHGAP which is highly expressed in male germ Naud et al. (2003) activating protein (MgcRACGAP) cells in spermatocytes and spermatids. RND2 and RND3 bind to the or RACGAP1 GAP domain of RACGAP1 and mediate RHOA inhibition by increasing GTPase activity

Vacuolar protein sorting 4A VPS4A belongs to a family of which couple the chemical energy Tanaka et al. (2002) (VPS4A) obtained from ATP hydrolysis to the mechanical force of myosin. VPS4A recruits RND2 to early endosomes where it regulates endosomal sorting during vesicular trafficking

GEM interacting protein (GMIP) GMIP is an ARHGAP with a cysteine-rich domain and a RHOGAP domain Aresta et al. (2002) in its C-terminal half. It inhibits RHOA by accelerating RHOA GTPAse activity and downregulates many processes dependent of the RHO pathway

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p300 acetyltransferase, increasing histone acetyl- transferase activity and gene transcription (Tanaka et al. 2006b). ROCK1 co-localizes with centromeres (Chevrier et al. 2002) and ROCK2 is found in the cleavage furrow in late mitosis (Inada et al. 1999). Progesterone and oestrogen regulate the nuclear localisation of some kinases including cyclin-dependent kinases (cdc4; Tong & Pollard 1999) and adaptor molecules like CT10 regulator of kinase in uterine smooth muscle and epithelial cells (Nautiyal et al. 2004). ROCK also induces nuclear translocation of MAPK3/1 (ERK1/2) in smooth muscle cells (Liu et al. 2004). ROCK2 regulates PDGF- induced nuclear localization of serum response factor during differentiation of proepicardial cells into coronary smooth muscle cells (Lu et al. 2001). Growth factors can induce ROCK nuclear translocation. Transforming growth factor-b translocates ROCK2 into the nucleus where it inhibits CDC24 phosphatase to regulate cell cycle progression (Bhowmick et al. 2003). Nuclear localised ROCK2 has kinase activity against PPP1R12A and sensitivity to Y27632 (Narumiya et al. 2000). The Figure 7 RHO and RND protein expressions during pregnancy and nuclear localisation of both proteins during pregnancy labour. (A) Whole tissue homogenates from five individual donors were suggests a role in myometrial cell division and regulation immunoblotted with RHOA, RHOD and RND antibodies and levels of RHO and RND proteins were estimated by densitometry. RND and of gene expression. However, more investigation is RHOD levels were normalised to individual RHOA levels before required to determine whether this change in ROCK plotting in the allotted groups. One-way ANOVA analysis demon- protein function has any effect of PPP1R12A activity and strated that there were significant differences between the individual myofilament sensitivity to physiological uterotones like group means (*P!0.05, **P!0.001). RND/RHOA ratios were elevated OXT and PGF2a in labour. in pregnant not in labour (NIL) and spontaneous labour (SL) PKN1, the first RHOA effector to be discovered (Mukai myometrium compared to non-pregnant (NP) myometrium. We also observed a down regulation of RHOD/RHOA ratios in spontaneous et al. 1994, Palmer et al. 1994) was also present in our labour. (B) A GTP pull-down assay (Lartey et al. 2007) was performed myometrial samples. Its expression was upregulated in using equal amounts of protein from each sample. The GTP-bound all the pregnant groups compared to non-pregnant RHO fraction was normalised to total RHOA content. GTP RHOA groups. PKN1 expression was mirrored by a similar levels in spontaneous labour group were elevated relative to the non- increase in one of its downstream targets, namely pregnant and pregnant not in labour groups. phosphoThr38–PPP1R14A. This represents another C mechanism of Ca2 sensitization in pregnant myome- Novel RHO effector mechanisms during pregnancy trial tissue. Thus, the PKN1–PPP1R14A and the RHOA– and in labour ROCK–RND pathways may converge on MLCP at PPP1c and PPP1R12A sites respectively and crucially can We have outlined the complex interaction of kinases function independently of each other, see Fig. 5. downstream of the RHOA–ROCK complex in regulating We have found that the expression of other RHO smooth muscle MYL phosphorylation. To date, six RHO effectors such as DIAPH1 and DIAPH2 (Watanabe et al. effectors, ROCK1, ROCK2, DIAPH1, DIAPH2, PAK2 and 1997, 1999) is altered with human labour (Lartey et al. PKN1 have been identified in human myometrium 2007). DIAPH1 and DIAPH2 are human formin proteins (Lartey et al. 2007). ROCKs are the best characterized involved in actin reorganization and have been linked to RHO family proteins in smooth muscle cells. Although RHOB and RHOD formation of endosomal vesicles ROCK levels are not altered with pregnancy or labour, (Murphy et al. 1996, Gasman et al. 2003). The role of GTP–RHOA levels are elevated in labour and it appears actin polymerisation in uterine myofilament remodelling that the changes upstream of ROCK control MYL during gestation and in priming the contractile apparatus phosphorylation and contraction in labour. Preliminary for labour is not well understood and is an area of investigations have revealed increases in ROCK1 and considerable research interest. ROCK2 nuclear localization in myometrial and inter- Protein kinases other than ROCK can inhibit MLCP stitial uterine cells myometrial tissue sections during activity by phosphorylating PPP1R12A at Thr696. labour (Lartey & Lopez Bernal 2009b). There are The candidates kinases include PAK (Takizawa et al. previous reports of nuclear localisation of ROCK 2002), distrophia-myotonica protein kinase (Muranyi proteins in other tissues and cells lines. ROCK2 localises et al. 2001), ILK (Muranyi et al. 2002), and DAPK3 to the nucleus in HeLa cells, where it phosphorylates (MacDonald et al. 2001a). We used immunoblotting www.reproduction-online.org Reproduction (2009) 138 407–424

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Figure 8 Localisation of endosomal RHO proteins RHOB and RHOD in myometrial tissue and cultured myometrial cells. RHO protein (red) and actin phalloidin (green) staining of myometrial fibers (!40 magnification) and myometrial cells in primary culture (!63 magnification). The tissue was obtained from pregnant not in labour women. Nuclear proteins were detected with DAPI (blue). RHOB and RHOD images are presented in panels A and B respectively. and immunohistochemistry to characterize DAPK3 like Y32885 inhibit other related kinases like PRK1 and expression in our human tissue samples. Our preliminary PRK2 at concentrations required for ROCK inhibition findings indicate that DAPK3 may undergo differential (Davies et al. 2000). This multi kinase inhibition is also signalling and activation during labour (J Lartey & observed with another ROCK inhibitor compound HA ALo´pez Bernal, unpublished observations). DAPK3 may 1077 (AT877 or fasudil hydrochloride; Amano et al. 1999, C cause Ca2 -independent contraction by either phos- Niggli 1999). Therefore, the potential of ROCK inhibitors phorylation of the PPP1R12A subunit of myosin phos- to relax the uterus may be limited due to their lack of phatase at Thr696 (MacDonald et al. 2001a, Niiro & Ikebe kinase specificity. Further research is required on the 2001, Borman et al. 2002), activation of PPP1R14A by effect of ROCK inhibitors on myometrial tissue during phosphorylation at Thr38 (MacDonald et al. 2001b)or agonist-induced and spontaneous contractions in normal direct phosphorylation of MYL at Ser19 and Thr18 and preterm labour. Furthermore, we need to determine (Murata-Hori et al. 1999). the transcription factors and ERE/PREs that control the remarkable increases in RHO and RND protein expressions and activity during pregnancy and labour. Pharmacological regulation of the RHO pathway We also need to elucidate whether oestrogen stimulation or a functional progesterone withdrawal, or both, can C ROCK inhibition by pyridine derivatives produce changes in uterine smooth muscle Ca2 Physiological uterotones such as PGF2a and OXTactivate sensitization in women as demonstrated in some animal C GPCRs to promote Ca2 release and entry into the species as there can be quite marked changes in RHO C cells, promoting Ca2 –calmodulin-dependent MYLK protein expression and function among species. activation. Moreover, these agonists generate increases Moreover, steroids may induce changes in the activity C in tension at a constant activity of intracellular Ca2 .We of signalling enzymes that result in post-translational have shown that myometrial GTP–RHOA and ROCK modification or phosphorylation of the proteins to activity are elevated during spontaneous preterm labour precipitate labour. There are predicted putative oestro- (Lartey et al. 2007). Thus, it is conceivable that inhibition gen and progesterone response elements of human RND of ROCK activity may lead to attenuation of the tension genes, see Fig. 9. that may be associated with spontaneous preterm labour. Several compounds have been manufactured to inhibit Farnesyltransferase and geranylgeranyltransferase ROCK function. The most widely used N-(4-pyridyl)-4- regulation of RHO protein function (1-aminoethyl) cyclohexane carboxamide Y27632 is a highly potent, cell-permeable pyridine derivative which Farnesyltransferases and geranylgeranyltransferases acts as an ATP-competitive inhibitor with equal potency catalyse the irreversible addition of farnesyl for RAS- against ROCK1 and ROCK2 (Uehata et al. 1997). Y27632 related associated with diabetes or RND proteins; or inhibits agonist-induced phosphorylation of myosin geranylgeranyl for RHO, RAS, RAC, RAP and CDC42. phosphatase and MYL to disrupt RHOA mediated stress Pharmacological inhibition by farnesyltransferase fibers and cause smooth muscle relaxation (Uehata et al. inhibitors (FTIs) and geranylgeranyltransferase inhibitors 1997). However, Y27632 and other similar compounds (GGTIs) prevent isoprenoid modification and block

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Figure 9 Predicted putative EREs and PREs in RND gene promoter regions. Promoter database search was performed using Transcriptional Regulatory Element Database, Zhang Laboratory (http://rulai.cshl.edu/TRED). the lipid-mediated association of RAS and RHO proteins U46619 induced contraction in myometrial and pla- with the plasma membrane (Sebti & Hamilton 2000). cental arteries in normal term pregnancies (Wareing Other small molecule inhibitors such as farnesyl et al. 2005). We have quantified the expression on RHO thiosalicylic acid compete with the isoprenoid attach- proteins during normal and abnormal labour (Lartey ment of small GTPases to membrane binding sites et al.2006a). Analysis of paired placentae and (Smalley & Eisen 2003, Goldberg & Kloog 2006). myometrium from individual samples shows that However, these RHO inhibitors can have other diverse placental RHO protein levels were up to threefold effects on RHO protein function. For instance, FTIs and higher than corresponding myometrial levels, see GGTIs stimulate RHOB transcription leading to Fig. 10. RND3 proteins were differentially expressed increases in RHOB expression. FTIs activate transcrip- in primary, secondary and tertiary human placental tion by a number of mechanisms including histone trophoblasts (Lartey et al. 2006a). RND3 regulation deacetylase 1 dissociation, histone acetyltransferase association and histone acetylation of the RHOB promoter (Delarue et al. 2007). The upregulation of RND protein expression during gestation may function to inhibit RHOA activity thereby maintaining uterine quiescence. Therefore, theuseofRNDproteins inhibitors (FTIs) at appropriate gestations may induce labour in conditions such as pre-eclampsia and intrau- terine growth restriction which necessitate delivery of the fetus.

RHO proteins and uteroplacental vascular disease Figure 10 RHO GTPase expression in human myometrial and placental RHO proteins have been implicated in a range of tissue samples. Paired myometrial (MYO) and placental (PLAC) tissue vascular smooth muscle disorders including hyperten- homogenates from five different spontaneous labour donors were tion, arterioscleriosis and also directly in ischaemic immunoblotted with RHOA, RHOB RHOD and ARF6 antibodies and visualized with ECL. Individual RHO protein bands were myocardial damage. ROCK inhibitors have been used normalised to their own tubulin bands. The normalised RHO in animal models of cardiovascular diseases with varying densitometry values were plotted on a logarithmic scale within allotted degrees of success. The ROCK inhibitor Y27632 reduces groups (*P!0.05, **P!0.01). www.reproduction-online.org Reproduction (2009) 138 407–424

Downloaded from Bioscientifica.com at 10/01/2021 07:02:34AM via free access 420 J Lartey and A Lo´pez Bernal of normal placental development and its role in References uterovascular diseases and placental insufficiency need Adamson P, Marshall CJ, Hall A & Tilbrook PA 1992 Post-translational to be explored. Interestingly, although RND1, RND2 and modifications of p21rho proteins. Journal of Biological Chemistry 267 RND3 mRNAs were expressed in the placental samples 20033–20038. examined, we did not find any evidence of RND1 or Van Aelst L & D’Souza-Schorey C 1997 Rho GTPases and signaling RND2 proteins. This suggests differences in trans- networks. Genes and Development 11 2295–2322. Aktories K, Mohr C & Koch G 1992 Clostridium botulinum C3 criptional or translational regulation of RND proteins ADP-ribosyltransferase. Current Topics in Microbiology and between the human placenta and myometrium. Immunology 175 115–131. 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