Margot L. Day

All Three WW Domains of Murine Nedd4 Are Involved in the Regulation of Epithelial Sodium Channels by Intracellular Na

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle’s syndrome where mutations delete or alter the PY motif of either the β or γ subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind α-, β-, and γ-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+channel and of WW domain 1 to an unknown associated protein.

Circadian clockwork genes are expressed in the reproductive tract and conceptus of the early pregnant mouse

Circadian genes are expressed in some peripheral tissues, but the expression status of the female reproductive tract and the conceptus over the preimplantation period is unknown. Oocytes, uterine, oviducal tissues and preimplantation conceptuses from days 1-4 of mouse pregnancy were analysed for transcript presence by reverse transcription polymerase chain reaction. Transcripts encoded by the seven known mammalian canonical circadian genes (Per1-3, Cry1-2, Bmal1 and Clock), plus the mammalian genetic homologue of the Drosophila canonical gene Timeless, were detected in the uteri and oviducts taken from mice on days 1-4 of pregnancy and in unfertilized oocytes. After fertilization, transcripts for Per1, Cry1, Bmal1, Clock and Tim have been detected unambiguously. Transcript levels for each of these five genes fall at the two-cell stage, but are restored rapidly for Per1, Cry1 and Bmal1, presumptively by zygotic gene expression. In contrast, transcripts for Clock and Tim recover more slowly. It is concluded that circadian genes are expressed, and may therefore have a role, during the early development of the mammal.

Egg timers: how is developmental time measured in the early vertebrate embryo?

Eggs and early embryos appear to be programmed to undertake particular developmental decisions at characteristic times, although precisely how these decisions are timed is unknown. We discuss the possible roles and interactions during early vertebrate development of two broad categories of timers: 1) those that involve cyclic or sequential mechanisms, referred to as clocks; and 2) those that require an increase or decrease in some factor to a threshold level for progression of time, referred to as hourglass timers. It is concluded that both clock-like timers linked to various features of the cell cycle and hourglass timers are involved in early developmental timing. The possible involvement of elements of circadian clock timers is also considered. BioEssays 22:57-63, 2000.

Cell cycle regulation of a T-type calcium current in early mouse embryos

Abstract In this paper we report that the amplitude of the T-current changes in a cell cycle-dependent manner in single- and two-cell embryos, being large in unfertilized oocytes and decreasing after fertilization throughout the first cell cycle to a minimum in early M phase, before increasing again during late telophase. This increase can be prevented by arresting the cell cycle in metaphase, but is not prevented by cytochalasin D. During the second cell cycle the T-current remains large until late G2 phase when it decreases. Protein synthesis inhibition has no effect on the changes in T-current amplitude during the cell cycle. This finding, along with the observation that the size of the T-current does not increase until mitosis exit, suggests that cell cycle regulation of the current does not involve the cycling of cyclin-dependent kinase (cdk)1/cyclin B activity. Inhibition of the T-current with pimozide has no effect on cleavage. The cell cycle-dependent changes in T-current amplitude appear to be driven by the same cdk1/cyclin-B-independent clock that we have previously shown to drive cyclic changes in K+ channel activity in early mouse embryos. Thus, multiple ion transport systems in the preimplantation mouse embryo are controlled by this cdk1/cyclin-B-independent clock.

The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na+ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.

A cytoplasmic cell cycle controls the activity of a K+ channel in pre‐implantation mouse embryos

We previously have reported that the activity of a 240 pS K+ channel varies during the cell cycle in pre‐implantation mouse embryos. In the present study, we show that: (i) the cycling of channel activity is not prevented by inhibiting protein synthesis and hence does not involve cyclin‐dependent kinase 1 (cdk1)–cyclin B; and (ii) the cycling of channel activity continues in anucleate zygote fragments with a time course similar to that observed in nucleate fragments. We further demonstrate that: (i) persistent activation of the K+ channel in one‐cell embryos arrested in metaphase requires the maintenance of an active cdk1–cyclin B complex; and (ii) both DNA synthesis inhibition with aphidicolin and DNA damage produced by mitomycin C prevent the down‐regulation of the channel at the start of S phase by a mechanism that requires tyrosine kinase activation. Thus, the 240 pS K+ channel in these cells is controlled by a previously unsuspected cytoplasmic clock that functions independently of the well‐known clock controlling the chromosomal cell cycle, but can interact with it.

Integrin β3 in rat blastocysts and epithelial cells is essential for implantation in vitro: studies with Ishikawa cells and small interfering RNA transfection

BACKGROUND Integrins are involved in the process of embryo-endometrium interaction during implantation. We investigated the localization of integrin β3 in the rat blastocyst and Ishikawa cells using an in vitro co-culture model of implantation. METHODS Zona pellucida-free rat blastocysts were co-cultured with the Ishikawa cells (endometrial adenocarcinoma cell line) to observe the attachment between the embryo and endometrium. Immunofluorescence staining was used to investigate the localization of integrin β3 in rat embryos at different stages of development (each n= 3 embryos) and at the embryo/endometrium interface, observed by confocal microscopy. The Ishikawa cells were transfected with integrin β3 small interfering RNA (siRNA) for 48 h and then co-cultured with Day 5 rat blastocysts to observe the effect on attachment. RESULTS Integrin β3 staining in the rat embryos increased at the blastocyst stage being highly concentrated in the cytoplasm of trophoblast cells (n= 9 embryos). Integrin β3 was localized on the apical surface of the Ishikawa cells (n= 3 experiments). However, integrin β3 relocated to the apical membrane of trophoblast cells in response to attachment to Ishikawa cells (n= 6 embryos). Moreover, when Ishikawa cells were transfected with integrin β3 siRNA, blastocyst attachment was significantly reduced compared with those transfected with control siRNA (16.7 versus 92.3%, respectively, P< 0.05). CONCLUSIONS Integrin β3, localized apically in the blastocyst and the Ishikawa cells, is important during initial attachment of the blastocyst to endometrial cells. This study provides further evidence of the importance of integrins during implantation and may aid in elucidating the molecular mechanism of implantation failure and infertility in women.

β1 and β3 integrins disassemble from basal focal adhesions and β3 integrin is later localised to the apical plasma membrane of rat uterine luminal epithelial cells at the time of implantation

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely β(1) and β(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The β(1) and β(3) integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, β(1) and β(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, β(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in β(1) and β(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, β(1) and β(3) integrin disassembly from focal adhesions and the increase in β(3) integrin apically are key components of hormonally regulated endometrial receptivity.

Ligand-Activated Signal Transduction in the 2-Cell Embryo

Abstract Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (ω-agatoxin; ω-conotoxin MVIIC), N-type (ω-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the α1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (±) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each others responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

The activity of the H+-monocarboxylate cotransporter during pre-implantation development in the mouse

Abstract We have reported previously that cytosolic pH (pHi) in the mouse 2-cell conceptus is controlled by a H+-monocarboxylate cotransporter (MCT) that is sensitive to cinnamates and p-chloromercuriphenylsulfonate. In the present study we have used measurement of pHi with BCECF to characterize the changes in MCT activity during pre-implantation development. We found that the resting pHi in bicarbonate-free conditions increased significantly from the unfertilized oocyte to the 2-cell stage, but thereafter remained constant. There was no evidence for changes in MCT activity during the cell cycle, but MCT activity was found to increase during development. Using RT-PCR we demonstrated that mRNA encoding MCT isoforms 1, 2 and 3 is present throughout pre-implantation development. The inhibitor of MCT1, p-chloromercuribenzoic acid, completely abolished the effect of extracellular l-lactate on pHi suggesting that MCT1, and not MCT2, plays a functional role in pHi regulation in mouse conceptuses, while the role of MCT3 remains unclear. We further found that removal of glucose from the culture medium, which has previously been shown to stimulate pyruvate uptake by blastocysts, had no effect on the activity of the MCT. These findings suggest that the changes in pyruvate uptake that have been observed following compaction are not due to changes in the activity of the MCT. These findings indicate the presence of MCTs during early embryonic development.