Wouter H. Moolenaar

The emerging role of lysophosphatidic acid in cancer

The bioactive phospholipid lysophosphatidic acid (LPA) stimulates cell proliferation, migration and survival by acting on its cognate G-protein-coupled receptors. Aberrant LPA production, receptor expression and signalling probably contribute to cancer initiation, progression and metastasis. The recent identification of ecto-enzymes that mediate the production and degradation of LPA, as well as the development of receptor-selective analogues, indicate mechanisms by which LPA production or action could be modulated for cancer therapy.

Lysophosphatidate-induced cell proliferation: Identification and dissection of signaling pathways mediated by G proteins

Lysophosphatidate (LPA), the simplest natural phospholipid, is highly mitogenic for quiescent fibroblasts. LPA-induced cell proliferation is not dependent on other mitogens and is blocked by pertussis toxin. LPA initiates at least three separate signaling cascades: activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis with subsequent Ca2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing the first two responses but fails to activate Gi and to stimulate DNA synthesis. Our data suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that the phosphoinositide hydrolysis-protein kinase C pathway is neither required nor sufficient, by itself, for mitogenesis. The results further suggest that LPA or LPA-like phospholipids may have a novel role in G protein-mediated signal transduction.

The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein

Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes [3-5], although the underlying mechanisms are poorly understood as cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signalling motifs. We have used a yeast two-hybrid protein interaction screen to identify proteins that bind to the carboxy-terminal tail of Cx43 and thereby isolated the zona occludens-1 (ZO-1) protein. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain [6-9]. The interaction of Cx43 with ZO-1 occurred through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associated with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy revealed that endogenous Cx43 and ZO-1 colocalised at gap junctions. We suggest that ZO-1 serves to recruit signalling proteins into Cx43-based gap junctions.

Lysophosphatidic acid: G-protein signalling and cellular responses

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates a specific G protein coupled receptor to evoke multiple cellular responses. Recent work has identified two cDNAs encoding putative LPA receptors, various LPA-like agonists that act on distinct receptors, and new pathways that link the receptor(s) to such diverse events as Ras signalling, cytoskeletal remodelling and membrane depolarization.

International Union of Pharmacology. XXXIV. Lysophospholipid Receptor Nomenclature

The lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are now recognized as important extracellular signaling molecules. These lipid mediators are pleiotropic; among the most common cellular responses are mitogenesis, cell survival (anti-apoptosis), inhibition of adenylyl cyclase and calcium mobilization. Physiologic events associated with these mediators include platelet aggregation, vasopressor activity, wound healing, immune modulation, and angiogenesis. Many of the actions of LPA and S1P are mediated through a set of eight G protein-coupled receptors. Five of these are S1P-prefering while the remaining three are LPA receptors. These receptors are expressed widely and in aggregate signal through a variety of heterotrimeric G proteins. The lysophospholipid receptor family is referred to commonly as the “Edg” group (e.g., Edg-1, Edg-2, etc.). Herein, the molecular pharmacology of the lysophospholipid receptors is reviewed briefly, and a rational nomenclature for LPA and S1P receptors that is consistent with the International Union of Pharmacology guidelines is proposed.

Autotaxin, a Secreted Lysophospholipase D, Is Essential for Blood Vessel Formation during Development

ABSTRACT Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the Gα13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Gα13.

Detecting cAMP‐induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP–Epac–YFP fusion construct. When expressed in mammalian cells, CFP–Epac–YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP‐raising agents, whereas it fully recovers after addition of cAMP‐lowering agonists. Thus, by undergoing a cAMP‐induced conformational change, CFP–Epac–YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)‐based sensor, Epac‐based cAMP probes show an extended dynamic range and a better signal‐to‐noise ratio; furthermore, as a single polypeptide, CFP–Epac–YFP does not suffer from the technical problems encountered with multisubunit PKA‐based sensors. These properties make Epac‐based FRET probes the preferred indicators for monitoring cAMP levels in vivo.

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine‐1‐phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E‐115 neuronal cells, S1P mimics the G protein‐coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho‐dependent contraction of the actin cytoskeleton. S1P is approximately 100‐fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross‐desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho‐regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein‐coupled receptors to mediate similar actions.

TRPM7, a novel regulator of actomyosin contractility and cell adhesion

Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, α‐kinases control actomyosin relaxation, a similar role for mammalian α‐kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an α‐kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca2+‐ and kinase‐dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase‐dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase‐dependent and ‐independent pathways on actomyosin contractility.

Gap junction protein connexin-43 interacts directly with microtubules

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.