Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 &mgr;M whereas the concentration in plasma is considerably less [1]. The SPP concentration in serum is 0.5 &mgr;M, approximately two-fold higher than the plasma concentration [1]. The lipids are present during tissue injury and promote cellular processes involved in wound repair [2]. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.     

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.     

Dual effects of lysophosphatidic acid on human airway smooth muscle cell proliferation and survival

Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2-20 microM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10-100 microM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+ LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 microM LPA stimulated HASM cell mitogenesis. However, with 100 microM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3':5'-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+ free and Ca2+ containing medium. After 24 h, a 100 microM LPA solution in Ca2+ free medium contained markedly greater bioactivity than a 100 microM LPA solution made in Ca2+ containing medium. In summary, formation of Ca2+ LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+ free but not in Ca2+ containing medium induce apoptosis of HASM cells.     

Ligand density and integrin repertoire regulate cellular response to LPA

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance α5β1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for α4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of α4β1-expressing CHOα4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOα4 and human A375 melanoma cells in an α4β1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for α4β1. LPA signaling combined with α4β1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.     

Phospholipase D stimulation is required for sphingosine-1-phosphate activation of actin stress fibre assembly in human airway epithelial cells.

In human airway epithelial cells, sphingosine-1-phosphate (SPP) and lysophosphatidic acid (LPA) stimulated the production of phosphatidic acid (PA), which was inhibited by the primary alcohol butan-1-ol, but not by the inactive butan-2-ol, clearly indicating phospholipase D (PLD) involvement. Both SPP and LPA stimulated actin stress fibre formation, which was also butan-2-ol-insensitive and inhibited by butan-1-ol. SPP-induced PLD activation and cytoskeletal remodelling were insensitive to brefeldin A and toxin B from Clostridium difficile, which conversely blocked the effect of LPA, suggesting that the monomeric GTPases ADP ribosylation factor (ARF) and Rho are involved in LPA, but not in SPP responses. Pertussis toxin inhibited SPP- but not LPA-induced effects. PLD activation and stress fibre formation by both lysolipids were abolished by the tyrosine kinase inhibitor genistein. Addition of PA to cells caused a massive stress fibre assembly. In conclusion, PLD is one of the signalling components linking SPP-receptor activation to assembly of actin stress fibres.     

LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6), showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1) being 10-fold more potent than acyl-LPA(18:1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.     

Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors

Lysophosphatidic acid (LPA) exerts multiple biological functions through G protein-coupled receptors (EDG2/LPA(1), EDG4/LPA(2), and EDG7/LPA(3)) and is present in serum where it is associated with albumin. In this study we examined LPA activity in various biological fluids by measuring the LPA-induced increase in the intracellular concentration of calcium ion in three types of Sf9 insect cells, each expressing one of the LPA receptors. Using this system, we found that EDG2 and EDG4, but not EDG7, were activated strongly by addition of incubated plasma. By contrast, LPA detected in seminal plasma, which contains a low concentration of albumin, selectively activated EDG7. After LPA in these samples was extracted and reconstituted, it activated all three receptors. We also found that serum albumin readily inhibits the activation of EDG7 but not the activation of EDG2 or EDG4. In addition, plasma from Nagase analbuminemic rats but not plasma from control Sprague-Dawley rats was found to strongly activate EDG7, although the plasma of these two types of rats contained equal amounts of LPA and activated both EDG2 and EDG4. The present study shows that serum albumin can negatively regulate EDG7 but not EDG2 or EDG4, and suggests that protein factors are present in seminal plasma and deliver LPA efficiently to EDG7 but not to EDG2 or EDG4.     

Structure-activity analysis of the effects of lysophosphatidic acid on platelet aggregation.

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate or LPA) is a phospholipid mediator displaying numerous and widespread biological activities and thought to act via G-protein-coupled receptors. Here we have studied the effects on human platelets of a number of LPA analogues, including two enantiomers of both N-palmitoyl-(L)-serine-3-phosphate ((L) and (D)NAPS for N-acyl-phosphoserine) and 2-(R)-N-palmitoyl-norleucinol-1-phosphate ((R) and (S)PNPA), cyclic analogues of 1-acyl-sn-glycero-3-phosphate (cPA) and of 1-O-hexadecyl-sn-glycero-3-phosphate (cAGP), sphingosine-1-phosphate (SPP), as well as two palmitoyl derivatives of dioxazaphosphocanes bearing either a P-H or a P-OH bond (DOXP-H and DOXP-OH, respectively). Nine of these compounds induced platelet aggregation with the following order of potency: SPP < cAGP < DOXP-OH < (L)NAPS = (D)NAPS < (R)PNPA = (S)PNPA < LPA < AGP, EC50 varying between 9.8 nM and 8.3 microM. Two of these compounds (SPP and cAGP) appeared as weak agonists inducing platelet aggregation to only 33% and 41%, respectively, of the maximal response attained with LPA and other analogues. In cross-desensitization experiments, all of these compounds specifically inhibited LPA-induced aggregation, suggesting that they were all acting on the same receptor(s). In contrast, cPA and DOXP-H did not trigger platelet aggregation but instead specifically inhibited the effects of LPA in a concentration-dependent manner. The inhibitory action of cPA did not vary with the acyl chain length or the presence of a double bond and did not involve an increase in cAMP. These data thus confirm the lack of stereospecificity of platelet LPA receptor(s). In addition, since the order of potency of some analogues is different from that described in other cells, our results suggest that platelets contain (a) pharmacologically distinct receptor(s) whose molecular identity still remains to be established. Finally, this unique series of compounds might be used for further characterization of other endogenous or recombinant LPA receptors.     

Specific receptor subtype mediation of LPA-induced dual effects in cardiac fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid messenger with diverse effects mediated via receptors LPA1, LPA2 and LPA3. Our previous study revealed that serum LPA level is elevated after myocardial infarction (MI). However, very little is known about the effects of LPA on cardiac fibroblasts (CFs) that play a crucial role in left ventricular remodeling after MI. Here we demonstrated that LPA dose-dependently induced proliferation and collagen synthesis with the maximum stimulation at 10 microM that was preferentially mediated by LPA3. LPA also dose-dependently induced apoptotic cell death, as estimated by MTT assay, hoechst staining, TUNEL and flow cytometric analysis, with an IC(50) of 50 microM. Moreover, apoptotic cell death may involve mitochondrial dysfunction and activation of caspase-3. Apoptosis induced by LPA might be mediated by LPA1. These data suggest that LPA exerts dual proliferative and proapoptotic actions mediated by specific LPA receptor subtypes.     

Lysophosphatidic acid increases phosphatidic acid formation, phospholipase D activity and degranulation by human neutrophils

I-oleoyl-sn-glycero-3-phosphate, a lysophosphatidic acid (LPA), in serum is a biologically active lipid and has multiple functions depending on the cell types. Several studies have shown that LPA stimulates phospholipase D (PLD) activity in fibroblasts and prostate cancer cells in culture. PLD plays a central role in regulating neutrophil functions. One of the functions of the lipid product, phosphatidic acid (PA), of PLD action in neutrophils is to promote degranulation. In the present study, we examined the effect of LPA on PLD activity and degranulation by human neutrophils. The results show that exogenous LPA increased PA formation, PLD activity and degranulation by human neutrophils in a time and concentration dependent manner. These findings suggest that LPA released from activated platelets during blood clotting may participate in bacterial killing and wound healing process. On the other hand, augmented LPA production might be involved in inflammation, causing damage of the host tissues.     

Serum lysophosphatidic acid is produced through diverse phospholipase pathways

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A(2) (sPLA(2)-IIA) and phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA(1), sPLA(2)-IIA, LCAT, and lysoPLD.     

Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo

The lipid mediator lysophosphatidic acid (LPA) regulates cell proliferation and enhances cell motility in vitro, both of which are important events in wound healing. To evaluate the effects of LPA in vivo, it was applied to a full-thickness wound of rat skin. LPA in micromolar concentrations, or solvent, was applied daily. Animals were killed at 1, 3, 6, and 9 days after wounding and processed for histological evaluation, including hematoxylin-eosin staining and histochemical markers for macrophage-histiocytes, proliferating cells, and capillary endothelial cells. LPA treatment accelerated wound closing and increased neoepithelial thickness. Cytological evaluation showed no evidence for a secondary inflammation-mediated injury, infection, or increased keloid formation. Whereas LPA caused only a modest dose-dependent increase in proliferating cells, a marked increase in the immigration of histiocyte-macrophage cells was observed as early as day 1. The peaks of several cytological features and immunohistological markers preceded those of the untreated side. Our data suggest that exogenously applied LPA in this model promotes healing and that macrophage-histiocytes are the primary LPA-responsive cells in vivo.     

Lysophosphatidic acid: a "bioactive" phospholipid

Lysophosphatidic acid (LPA) is a "bioactive" phospholipid able to generate growth factor-like activities in a wide variety of normal and malignant cell types. LPA is proposed to play an important role in normal physiological situations such as wound healing, vascular tone, vascular integrity, or reproduction. In parallel, LPA could also be involved in the etiology of some diseases such as atherosclerosis, cancer, or obesity. The bioactivity of LPA is mediated by the activation of specific G-protein coupled receptors (LPA1, LPA2, and LPA3) leading to the activation of a number of intracellular effectors. LPA is present in solution (bound to albumin) in various extracellular fluids (blood, ascites, aqueous humor), and is released in vitro by some cell types such as platelets, cancer cells, or adipocytes. LPA is a rather polar phospholipid, which cannot easily diffuse throughout plasma membrane, and its presence outside the cells requires soluble phospholipases (secreted phospholipase A2 and soluble lysophospholipase D/autotaxin), which synthesize LPA directly in the extracellular milieu, from precursors such as phosphatidic acid and lysophosphatidylcholine. In the future, LPA receptors, as well as the enzymes involved in LPA metabolism, will constitute promising pharmacological and transgenic targets to determine the physiopathological relevance of "bioactive" LPA in vivo.     

Differential effects of sphingosine 1-phosphate and lysophosphatidic acid on endothelial cells

This review discusses multiple effects of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) on endothelial cells and proposes that S1P and LPA are important regulators of the vascular system. Two physiologic sources of S1P and LPA are platelets and lipoproteins. S1P is an inducer of angiogenesis in vivo whereas LPA is not. S1P and LPA act through endothelial cell surface Edg receptors. S1P stimulates endothelial cell migration, but inhibits migration of most nonendothelial cells. Edg1 and Edg3 receptors, working through G(i), play an important role in regulation of S1P-stimulated endothelial cell migration. LPA effects on endothelial cells are more restricted than the effects of S1P on endothelial cells. LPA stimulates migration of certain endothelial cells on certain extracellular matrix proteins. However, LPA acts like S1P in its effects on the endothelial cell cytoskeleton, proliferation, cell-cell adhesion molecule expression, and vascular permeability. LPA receptors on endothelial cells are likely Edg2 and Edg4. Future studies should better delineate the roles of Edg receptors and downstream pathways on effects of extracellular S1P and LPA and the contributions of intracellularly generated S1P and nitric oxide (NO).     

Lysophosphatidic acid (LPA) stimulates mouse mammary epithelial cell growth

LPA (lysophosphatidic acid) is a bioactive phospholipid having diverse effects on various types of tissues. When NMuMG (normal murine mammary gland) cells were cultured in the presence of 0-10 μM LPA, cell numbers were increased by dose dependency for the 6-day culture periods (P<0.05). In DNA synthesis assay, 10 μM LPA induced 4.5-fold more DNA synthesis compared with control (P<0.05). In addition, the cultured cell density in the given area was increased by LPA treatment. MMP (matrix metalloproteinase) inhibitor GM6001 and EGFR [EGF (epidermal growth factor) receptor] tyrosine kinase inhibitor AG1478 [tyrphostin AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline] significantly decreased LPA-induced DNA synthesis and cell growth without cell death (P<0.05). To test the hypothesis that LPA-induced cell growth is mediated through LPA subtype receptors, LPA subtype receptor gene expressions were amplified by PCR. NMuMG cells expressed LPA1 and LPA2 receptor genes in the presence of 10% FBS (fetal bovine serum). LPA treatments increased ERK1/2 (extracellular-signal-regulated kinase) phosphorylation at 30 min and then dephosphorylated at 2 h after treatment. LPA treatment phosphorylated at tyrosine residues on a variety of Gi and PI3-dependent signal transducers in NMuMG cells. These results suggest that LPA subtype receptors play a role as the active transactivator of EGFR-associated kinases as well as direct growth regulator in mammary tissues.     

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.     

Two G protein-coupled receptors activate Na+/H+ exchanger isoform 1 in Chinese hamster lung fibroblasts through an ERK-dependent pathway

The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific alpha(1)-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 microM phenylephrine and 10 microM LPA. alpha(1)-Adrenergic stimulation also led to a rise in steady-state pH(i) of 0.16+/-0.02 pH units, and incubation with LPA induced a 0.43+/-0.06 pH unit increase in pH(i). Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the alpha(1)-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.     

Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.

Fibronectin (FN) matrix assembly is a cell-dependent process mediated by cell surface-binding sites for the 70-kDa amino-terminal region of FN. We have shown recently that lysophosphatidic acid (LPA) is a stimulator of FN matrix assembly. Disruption of microtubules has been shown to mimic some of the intracellular effects of LPA including the formation of actin stress fibers and myosin light chain phosphorylation. We compared the effects of microtubule disruption and LPA on FN binding and actin cytoskeleton organization. The disruption of microtubules by nocodazole or vinblastine increased FN binding to adherent cells. The modulation of binding sites was rapid, dynamic, and reversible. Enhanced binding was due to increases in both the number and affinity of binding sites. These effects are similar to the effects of LPA on FN binding. Binding induced by nocodazole was inhibited by the microtubule-stabilizing agent Taxol but not by pretreatment with a concentration of phospholipase B that totally abolished the stimulatory effect of LPA. Fluorescence microscopy revealed a close correlation among actin stress fiber formation, cell contraction, and FN binding. Blockage of the small GTP binding protein Rho or actin-myosin interactions inhibited the effects of both nocodazole and LPA on FN binding. These observations demonstrate that Rho-dependent actin stress fiber formation and cell contraction induce increased FN binding and represent a rapid labile way that cells can modulate FN matrix assembly.     

Sphingosine 1-phosphate receptors

Sphingosine-1-phosphate (SPP) is a bioactive lipid produced from the metabolism of sphingomyelin. It is an important constituent of serum and regulates cell growth, survival, migration, differentiation and gene expression. Its mode of action has been enigmatic; however, recent findings have shown that a family of G-protein-coupled receptors (GPCR) of the endothelial differentiation gene (EDG) family serve as plasma membrane-localized receptors for SPP. Furthermore, the EDG receptors appear to be SPP receptor subtypes with distinct signaling characteristics. In vascular endothelial cells, SPP acts on EDG-1 and EDG-3 subtypes of receptors to induce cell survival and morphogenesis. Such pathways appear to be critical for SPP-induced angiogenic response in vivo. In addition, the EDG-1 gene is essential for vascular maturation in development. Moreover, developmental studies in Zebrafish have indicated that SPP signaling via the EDG-5 like receptor Miles Apart (Mil) is essential for heart development. These data strongly suggest that a physiological role of SPP is in the formation of the cardiovascular system. Despite these recent findings, much needs to be clarified with respect to the physiological role of SPP synthesis and action. This review will focus on the recent findings on SPP receptors and the effects on the cardiovascular system.     

A "traffic control" role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) beta3 levels, which are undetectable in plasma and high in serum, and by TGFbeta receptor (TbetaR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFbeta3 from serum converts serum to a plasmalike reagent. The addition of TGFbeta3 to plasma converts it to a serumlike reagent. Down-regulation of TbetaRII in dermal cells or up-regulation of TbetaRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma-->serum-->plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.