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Signaling Events During Induction of Plasminogen Activator Inhibitor-1 Expression by Sphingosylphosphorylcholine in Cultured Human Dermal Fibroblasts

2004; Elsevier BV; Volume: 122; Issue: 6 Linguagem: Inglês

10.1111/j.0022-202x.2004.22615.x

1523-1747

Kyung‐Chae Kye, Eunkyung Chae, Yongjun Piao, Seonyang Park, Jang-Kyu Park, Chang Deok Kim, Jeung‐Hoon Lee, Ki‐Beom Suhr,

Signaling Pathways in Disease

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In a search for effectors downstream of SPC in the wound-healing process, we found that the expression of the gene for plasminogen activator inhibitor-1 (PAI-1) was significantly affected. ELISA and western blot analyses showed that SPC markedly induced PAI-1 production in human dermal fibroblasts cultured in vitro. Inhibition by pre-treatment with pertussis toxin (PTx), but not by tyrphostin A47 (a receptor tyrosine kinase inhibitor), indicated that PTx-sensitive G proteins were involved in SPC-induced PAI-1 expression. SPC elicited a rapid and transient increase in intracellular calcium levels ([Ca2+]i), measured using laser scanning confocal microscopy, which was partly mediated through PTx-sensitive G proteins. Pre-treatment with thapsigargin, but not with EGTA, abolished SPC-induced PAI-1 expression, indicating the importance of Ca2+ release from internal stores. Phorbol-12-myristate-13-acetate (PMA) induced the expression of PAI-1, and pre-treatment with Ro 31-8220 (a PKC inhibitor) markedly suppressed SPC-induced PAI-1 expression. SPC-induced PAI-1 expression was also significantly suppressed by PD98059 (a specific MAPK kinase 1/2 inhibitor). Consistent with this result, SPC stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Together, these results suggest that SPC induces PAI-1 production through a G protein-coupled calcium increase and downstream kinase signaling events in cultured human dermal fibroblasts. Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In a search for effectors downstream of SPC in the wound-healing process, we found that the expression of the gene for plasminogen activator inhibitor-1 (PAI-1) was significantly affected. ELISA and western blot analyses showed that SPC markedly induced PAI-1 production in human dermal fibroblasts cultured in vitro. Inhibition by pre-treatment with pertussis toxin (PTx), but not by tyrphostin A47 (a receptor tyrosine kinase inhibitor), indicated that PTx-sensitive G proteins were involved in SPC-induced PAI-1 expression. SPC elicited a rapid and transient increase in intracellular calcium levels ([Ca2+]i), measured using laser scanning confocal microscopy, which was partly mediated through PTx-sensitive G proteins. Pre-treatment with thapsigargin, but not with EGTA, abolished SPC-induced PAI-1 expression, indicating the importance of Ca2+ release from internal stores. Phorbol-12-myristate-13-acetate (PMA) induced the expression of PAI-1, and pre-treatment with Ro 31-8220 (a PKC inhibitor) markedly suppressed SPC-induced PAI-1 expression. SPC-induced PAI-1 expression was also significantly suppressed by PD98059 (a specific MAPK kinase 1/2 inhibitor). Consistent with this result, SPC stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Together, these results suggest that SPC induces PAI-1 production through a G protein-coupled calcium increase and downstream kinase signaling events in cultured human dermal fibroblasts. intracellular Ca2+ mitogen-activated protein kinase plasminogen activator inhibitor-1 protein kinase C pertussis toxin sphingosine-1-phosphate sphingosylphosphorylcholine urokinase-type plasminogen activator Sphingolipid metabolites, such as sphingosine, sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), and lysophosphatidic acid (LPA), are important signaling molecules in intracellular and/or extracellular communications (Liscovitch and Cantley, 1994Liscovitch M. Cantley L.C. Lipid second messengers.Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar). SPC has been shown to accelerate cutaneous wound healing, and effect believed to be mainly due to its mitogenic potential for many target cells, including keratinocytes, fibroblasts, and endothelial cells (Sun et al., 1996Sun L. Xu L. Henry F.A. Spiegel S. Nielsen T.B. A new wound healing agent-sphingosylphosphorylcholine.J Invest Dermatol. 1996; 1: 232-237Google Scholar; Wakita et al., 1998Wakita H. Matsushita K. Nishimura K. Tokura Y. Furukawa F. Takigawa M. Sphingosylphosphorylcholine stimulates proliferation and upregulates cell surface-associated plasminogen activator activity in cultured human keratinocytes.J Invest Dermatol. 1998; 110: 253-258Crossref PubMed Scopus (57) Google Scholar). In addition, several lines of evidence suggest that SPC has other important functions in the wound-healing process; examples include the stimulation of endothelial cell migration and morphogenesis, the enhancement of fibroblast contraction, and the induction of extracellular matrix (ECM) component (Boguslawski et al., 2000Boguslawski G. Lyons D. Harvey K.A. Kovala A.T. English D. Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis.Biochem Biophys Res Commun. 2000; 272: 603-609Crossref PubMed Scopus (69) Google Scholar; Suhr et al., 2000Suhr K.B. Tsuboi R. Ogawa H. Sphingosylphosphorylcholine stimulates contraction of fibroblast-embedded collagen gel.Br J Dermatol. 2000; 143: 66-71Crossref PubMed Scopus (16) Google Scholar,Suhr et al., 2003Suhr K.B. Tsuboi R. Seo E.Y. Piao Y.J. Lee J.H. Park J.K. Ogawa H. Sphingosylphosphorylcholine stimulates cellular fibronectin expression through upregulation of IL-6 in cultured human dermal fibroblasts.Arch Dermatol Res. 2003; 294: 433-437PubMed Google Scholar). Despite accumulating evidence for various functional roles, the precise mechanism underlying the stimulation of wound healing by SPC remains to be elucidated. In an effort to find downstream effectors of SPC, we have performed intensive in vivo and in vitro screening tests. We found that the expression of plasminogen activator inhibitor-1 (PAI-1) was greatly influenced by SPC treatment. PAI-1, a member of the serine protease inhibitor (serpin) superfamily, is known to have a pivotal role in the fibrinolytic system. During fibrinolysis, tissue-type plasminogen activator (tPA) converts the proenzyme plasminogen into active plasmin, a broad-spectrum proteolytic enzyme that degrades the fibrin clot. By binding to its active site, PAI-1 neutralizes tPA activity and thereby regulates the production of plasmin (Huber, 2001Huber K. Plasminogen activator inhibitor type-1 (part one): Basic mechanisms, regulation, and role for thromboembolic disease.J Thromb Thrombolys. 2001; 11: 183-193Crossref PubMed Scopus (63) Google Scholar). In addition to this critical role in the fibrinolytic system, there is evidence supporting the involvement of PAI-1 in several other biological phenomena, including wound healing (Romer et al., 1991Romer J. Lund L.R. Eriksen J. et al.Differential expression of urokinase-type plasminogen activator and its type-1 inhibitor during healing of mouse skin wounds.J Invest Dermatol. 1991; 97: 803-811Abstract Full Text PDF PubMed Google Scholar; Jensen and Lavker, 1996Jensen P.J. Lavker R.M. Modulation of the plasminogen activator cascade during enhanced epidermal proliferation in vivo.Cell Growth Differ. 1996; 7: 1793-1804PubMed Google Scholar; Irigoyen et al., 1999Irigoyen J.P. Munoz-Canoves P. Montero L. Koziczak M. Nagamine Y. The plasminogen activator system: Biology and regulation.Cell Mol Life Sci. 1999; 56: 104-132Crossref PubMed Scopus (341) Google Scholar; Providence et al., 2000Providence K.M. Kutz S.M. Staiano-Coico L. Higgins P.J. PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair.J Cell Physiol. 2000; 1: 269-280Crossref Scopus (46) Google Scholar). Interestingly, it has been reported that PAI-1 level is significantly elevated under conditions associated with tissue fibrosis and excessive fibrin accumulation, such as sclerosis and keloid formation, supporting its role as an important regulator of proteolytic environment in tissue remodeling (Tuan et al., 1996Tuan T.L. Zhu J.Y. Sun B. Nichter L.S. Nimni M.E. Laug W.E. Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.J Invest Dermatol. 1996; 106: 1007-1011Crossref PubMed Scopus (80) Google Scholar; Higgins et al., 1999Higgins P.J. Slack J.K. Diegelmann R.F. Staiano-Coico L. Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibrotic phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar; Tuan et al., 2003Tuan T.L. Wu H. Huang E.Y. et al.Increased plasminogen activator inhibitor-1 in keloid fibroblasts may account for their elevated collagen accumulation in fibrin gel cultures.Am J Pathol. 2003; 162: 1579-1589Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The function of PAI-1 in wound repair is thought to be tightly regulated in a spatiotemporal manner. Temporal changes in the expression and/or the localization of PAI-1 are particularly likely to affect the stability of cell-to-ECM adhesive complexes; increased synthesis or availability of PAI-1 may inhibit cell detachment from substrates, contributing to the stabilization of cell-to-matrix contact sites (Ciambrone and McKeown-Longo, 1990Ciambrone G.J. McKeown-Longo P.J. Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells.J Cell Biol. 1990; 111: 2183-2195Crossref PubMed Scopus (77) Google Scholar; Germer et al., 1998Germer M. Kanse S.M. Kirkegaard T. Kjoller L. Felding-Habermann B. Goodman S. Preissner K.T. Kinetic analysis of integrin-dependent cell adhesion on vitronectin—the inhibitory potential of plasminogen activator inhibitor-1 and RGD peptides.Eur J Biochem. 1998; 253: 669-674Crossref PubMed Scopus (40) Google Scholar). The involvement of PAI-1 in SPC-stimulated wound healing raises the possibility that PAI-1 plays such a role during this process. Although independently, both SPC and PAI-1 have been clearly implicated in wound healing; the relationship between these two molecules has not been characterized. At present, limited data are available to support the hypothesis that plasminogen activator systems are involved in an SPC-stimulated wound-healing process. For instance, SPC upregulates the cell-surface plasminogen activity, and at the same time increases the cell surface expression of both urokinase-type plasminogen activator (uPA) and its receptor in keratinocytes (Wakita et al., 1998Wakita H. Matsushita K. Nishimura K. Tokura Y. Furukawa F. Takigawa M. Sphingosylphosphorylcholine stimulates proliferation and upregulates cell surface-associated plasminogen activator activity in cultured human keratinocytes.J Invest Dermatol. 1998; 110: 253-258Crossref PubMed Scopus (57) Google Scholar). In this study, we demonstrate for the first time that SPC induces the expression of PAI-1 in cultured human dermal fibroblast, and that a G protein-coupled signaling cascade is coordinately involved in this process. The healing effects of several sphingolipid metabolites and related derivatives (S1P, SPC, LPA, N-acetylphytosphingosine (NAPS) and tetraacetylphytosphingosine (TAPS)) were tested using a well-established rabbit ear wound model. Of these, SPC demonstrated the most powerful promotion of wound healing. Control wounds healed slowly, showing incomplete closure of wounded areas at 4 d after injury, whereas almost all SPC-treated areas were closed Fig 1A and B. These results were consistent with those of previous reports (Sun et al., 1996Sun L. Xu L. Henry F.A. Spiegel S. Nielsen T.B. A new wound healing agent-sphingosylphosphorylcholine.J Invest Dermatol. 1996; 1: 232-237Google Scholar; Wakita et al., 1998Wakita H. Matsushita K. Nishimura K. Tokura Y. Furukawa F. Takigawa M. Sphingosylphosphorylcholine stimulates proliferation and upregulates cell surface-associated plasminogen activator activity in cultured human keratinocytes.J Invest Dermatol. 1998; 110: 253-258Crossref PubMed Scopus (57) Google Scholar). Immunohistochemical examination showed that treatment with SPC significantly increased granulation tissue, and induced PAI-1 production in the dermis Fig 1C. To investigate the effect on PAI-1 production, SPC was added to the fibroblast cells in serum-free cultures. As PAI-1 is a secretary protein, we first measured PAI-1 in conditioned media using an ELISA technique. As shown in Fig 2A, SPC significantly induced the release of PAI-1 in cultured human dermal fibroblast cells in a time-dependent manner. In addition, SPC also induced the release of uPA, the regulation of which is closely related to that of PAI-1 production (Irigoyen et al., 1999Irigoyen J.P. Munoz-Canoves P. Montero L. Koziczak M. Nagamine Y. The plasminogen activator system: Biology and regulation.Cell Mol Life Sci. 1999; 56: 104-132Crossref PubMed Scopus (341) Google Scholar). The levels of PAI-1 were, however, several-fold higher than those of uPA. Consistent with this observation, western blot analysis showed that SPC treatment resulted in high-level induction of intracellular PAI-1 protein, reaching a maximum at 12 h and declining slightly by 24 h (Fig 2B, upper panel). SPC-induced PAI-1 expression was also confirmed by reverse fibrin autography, which revealed that PAI-1 activity also increased concomitantly (Fig 2B, lower panel). As shown in Fig 2C and D, SPC significantly increased release, cellular content and enzyme activity of PAI-1 in a concentration-dependent manner. Northern blot analysis revealed a time-dependent increase in PAI-1 mRNA level in response to SPC Fig 3. These results clearly demonstrated that SPC induces the PAI-1 expression, at both the transcriptional and translational levels, in cultured human dermal fibroblasts.Figure 3Northern blot analysis of PAI-1 expression in cultured human dermal fibroblasts. Cells were treated with 5 μM SPC at the indicated time points, and total RNA was isolated. Aliquots of 10 μg of total RNA were loaded in each lane, and the ethidium bromide-stained gel was photographed as a loading control.View Large Image Figure ViewerDownload (PPT) In other systems, SPC is known to affect downstream molecular events through G protein-coupled signaling pathway (Seufferlein and Rozengurt, 1995Seufferlein T. Rozengurt E. Sphingosylphosphorylcholine activation of mitogen-activated protein kinase in Swiss 3T3 cells requires protein kinase C and a pertussis toxin-sensitive G protein.J Biol Chem. 1995; 270: 24334-24342Crossref PubMed Scopus (75) Google Scholar; Zhu et al., 2001Zhu K. Baudhuin L.M. Hong G. et al.Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4.J Biol Chem. 2001; 276: 41325-41335Crossref PubMed Scopus (208) Google Scholar; Xu, 2002Xu Y. Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction.Biochim Biophys Acta. 2002; 1: 81-88Crossref Scopus (197) Google Scholar). These findings led us to investigate whether the G protein-coupled signaling events were also involved in SPC-induced PAI-1 expression in cultured human dermal fibroblasts. After pre-incubation with pertussis toxin (PTx), an inhibitor for Gi/o subfamily, a marked inhibition of SPC-induced PAI-1 expression was observed Fig 4A. On the other hand, pre-treatment of tyrphostin A47, a receptor tyrosine kinase inhibitor, had no effect on PAI-1 induction by SPC Fig 4B. As SPC is known to affect the intracellular calcium level ([Ca2+]i) via a G protein-dependent pathway (Okajima and Kondo, 1995Okajima F. Kondo Y. Pertussis toxin inhibits phospholipase C activation and Ca2+ mobilization by sphingosylphosphorylcholine and galactosylsphingosine in HL60 leukemia cells.J Biol Chem. 1995; 270: 26332-26340Crossref PubMed Scopus (75) Google Scholar; Chin and Chueh, 1998Chin T.Y. Chueh S.H. Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway.Am J Physiol. 1998; 275: 1255-C1263PubMed Google Scholar), we measured [Ca2+]i by laser scanning confocal microscopy. Treatment with SPC led to a robust [Ca2+]i increase, peaking at 7 s then decreasing to 50% of the maximum level at 15 s Fig 5A. As anticipated, this response was significantly diminished by pre-incubation with PTx Fig 5B, indicating that Ca2+ mobilization by SPC was partly mediated through PTx-sensitive G proteins. The rapid and transient nature of the Ca2+ increase suggested release from intracellular Ca2+ stores, rather than influx from extracellular sources. To test this assumption, cells were pre-treated with thapsigargin (an intracellular Ca2+ chelator) or EGTA (to chelate extracellular Ca2+). As shown in Fig 6A, pre-treatment with thapsigargin, but not with EGTA, completely inhibited SPC-induced PAI-1 expression. The calcium ionophore A23187 also induced PAI-1, and SPC interacted synergistically to enhance PAI-1 induction over a longer period than was seen with A23187 alone Fig 6B.Figure 5Measurements of intracellular Ca2+ level. Cells were loaded with 4 μM Fluo-3, AM for 40 min, and then subjected to confocal laser scanning microscopy. SPC (5 μM) was added alone (A) or following pre-treatment with 100 ng per mL pertussis toxin (PTx) (B). Results are presented as relative fluorescence intensity (RFI).View Large Image Figure ViewerDownload (PPT)Figure 6Effects of Ca2+ on SPC-induced PAI-1 expression. (A) Fibroblasts were pre-treated with the intracellular Ca2+ chelator thapsigargin (Thap) at 30 nM or with the extracellular chelator EGTA (1 mM) for 30 min. SPC (5 μM) was then added and incubation was continued for 12 h. (B) The calcium ionophore A23187 (1 μM) was added to cultures, and cellular extracts were prepared at the indicated time points (upper panel). In cotreatment experiments, SPC (5 μM) was added 12 h before the treatment of A23187 (1 μM) (lower panel). Western blot analyses were performed with 100 μg of cellular protein per lane.View Large Image Figure ViewerDownload (PPT) It has been well established that an increase in [Ca2+]i led to the activation of protein kinase C (PKC) in many systems (Desai et al., 1993Desai N.N. Carlson R.O. Mattie M.E. et al.Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.J Cell Biol. 1993; 121: 1385-1395Crossref PubMed Scopus (115) Google Scholar; Bitar and Yamada, 1995Bitar K.N. Yamada H. Modulation of smooth muscle contraction by sphingosylphosphorylcholine.Am J Physiol. 1995; 269: 370-G377PubMed Google Scholar; Seufferlein and Rozengurt, 1995Seufferlein T. Rozengurt E. Sphingosylphosphorylcholine activation of mitogen-activated protein kinase in Swiss 3T3 cells requires protein kinase C and a pertussis toxin-sensitive G protein.J Biol Chem. 1995; 270: 24334-24342Crossref PubMed Scopus (75) Google Scholar). Cells were treated with phorbol-12-myristate-13-acetate (PMA) for 30 min (PKC activation) or for 4 h (PKC depletion) prior to SPC treatment. PKC activation alone induced the PAI-1 production, which was not further enhanced by SPC addition Fig 7A. Pre-treatment with PMA for a longer period had no effect on PAI-I expression. Pre-treatment with Ro 31-8220, a PKC inhibitor, however, resulted in marked suppression of SPC-induced PAI-1 expression, indicating the involvement of PKC activation in the process Fig 7B. Cells were pre-treated with PD98059, a specific MAPK kinase 1/2 inhibitor. As shown in Fig 8A, PD98059 significantly suppressed PAI-1 induction by SPC. SPC was also shown to induce the rapid phosphorylation of p42/44 extracellular signal-regulated kinase (ERK) Fig 8B. These results demonstrated that an MAPK signaling pathway is involved in SPC-induced PAI-1 expression. In this study, we demonstrated that SPC, which stimulates wound healing, induces PAI-1 production in human dermal fibroblasts. Although SPC is a well-known potent mitogen for a number of target cells, the effect of SPC is unlikely to be explained simply by its impact on cell proliferation. Wound healing is a multistep process, in which distinct yet interrelated phases overlap. These include the inflammatory phase, the proliferative phase, and the regeneration phase (Chan et al., 2001Chan J.C. Duszczyszyn D.A. Castellino F.J. Ploplis V.A. Accelerated skin wound healing in plasminogen activator inhibitor-1-deficient mice.Am J Pathol. 2001; 159: 1681-1688Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). There is accumulating evidence that components of the fibrinolytic system are indispensable to one or more of these phases. For instance, plasminogen-deficient mice display delayed wound healing after skin injury, which is related to impaired keratinocyte migration (Bugge et al., 1996Bugge T.H. Kombrinck K.W. Flick M.J. Daugherty C.C. Danton M.J. Degen J.L. Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.Cell. 1996; 87: 709-719Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar; Romer et al., 1996Romer J. Bugge T.H. Pyke C. Lund L.R. Flick M.J. Degen J.L. Dano K. Impaired wound healing in mice with a disrupted plasminogen gene.Nat Med. 1996; 2: 287-292Crossref PubMed Scopus (479) Google Scholar). It has also been reported that downregulation of PAI-1 synthesis, in HaCaT cells transfected with an inducible antisense vector, markedly impairs both the rate and extent of wound closure (Li et al., 2000Li F. Goncalves J. Faughnan K. et al.Targeted inhibition of wound-induced PAI-1 expression alters migration and differentiation in human epidermal keratinocytes.Exp Cell Res. 2000; 258: 245-253Crossref PubMed Scopus (31) Google Scholar). Additional study has revealed that plasmin induces the degradation of collagen, thereby regulating fibroblast-mediated tissue remodeling during wound healing (Pins et al., 2000Pins G.D. Collins-Pavao M.E. Van De Water L. Yarmush M.L. Morgan J.R. Plasmin triggers rapid contraction and degradation of fibroblast-populated collagen lattices.J Invest Dermatol. 2000; 114: 647-653Crossref PubMed Scopus (54) Google Scholar). These results clearly indicate that the function of the fibrinolytic system is fundamental to proper wound healing. Our results strengthen the notion that PAI-1 is one important downstream effector in SPC stimulation of the wound-healing process. The sphingolipid metabolite S1P has been shown to act as an intracellular second messenger (Olivera and Spiegel, 1993Olivera A. Spiegel S. Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens.Nature. 1993; 365: 557-560Crossref PubMed Scopus (814) Google Scholar; Mattie et al., 1994Mattie M. Brooker G. Spiegel S. Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway.J Biol Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar; Spiegel, 1999Spiegel S. Sphingosine 1-phosphate: A prototype of a new class of second messengers.J Leukoc Biol. 1999; 65: 341-344Crossref PubMed Scopus (160) Google Scholar). Many investigations, however, have revealed effects of S1P that are sensitive to PTx, suggesting the presence of cognate G protein-coupled receptors (Goodemote et al., 1995Goodemote K.A. Mattie M.E. Berger A. Spiegel S. Involvement of a pertussis toxin-sensitive G protein in the mitogenic signaling pathways of sphingosine 1-phosphate.J Biol Chem. 1995; 270: 10272-10277Crossref PubMed Scopus (160) Google Scholar; Wu et al., 1995Wu J. Spiegel S. Sturgill T.W. Sphingosine 1-phosphate rapidly activates the mitogen-activated protein kinase pathway by a G protein-dependent mechanism.J Biol Chem. 1995; 2: 11484-11488Crossref Scopus (182) Google Scholar; Zhu et al., 2001Zhu K. Baudhuin L.M. Hong G. et al.Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4.J Biol Chem. 2001; 276: 41325-41335Crossref PubMed Scopus (208) Google Scholar). Indeed, S1P was demonstrated to activate the heterotrimeric G protein-coupled orphan receptor Edg-1, which was originally cloned as an endothelial differentiation gene (Lee et al., 1998Lee M.J. Van Brocklyn J.R. Thangada S. et al.Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.Science. 1998; 279: 1552-1555Crossref PubMed Scopus (886) Google Scholar). Several groups have identified other members of a closely related family of G protein-coupled orphan receptors that are functional receptors for sphingolipid metabolites (Masana et al., 1995Masana M.I. Brown R.C. Pu H. Gurney M.E. Dubocovich M.L. Cloning and characterization of a new member of the G-protein coupled receptor EDG.Receptor Channel. 1995; 3: 255-262PubMed Google Scholar; Yamaguchi et al., 1996Yamaguchi F. Tokuda M. Hatase O. Brenner S. Molecular cloning of the novel human G protein-coupled receptor (GPCR) gene mapped on chromosome 9.Biochem Biophys Res Commun. 1996; 227: 608-614Crossref PubMed Scopus (106) Google Scholar; Ancellin and Hla, 1999Ancellin N. Hla T. Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDG-1, EDG-3, and EDG-5.J Biol Chem. 1999; 274: 18997-19002Crossref PubMed Scopus (228) Google Scholar; Gonda et al., 1999Gonda K. Okamoto H. Takuwa N. et al.The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways.Biochem J. 1999; 337: 67-75Crossref PubMed Scopus (180) Google Scholar). Thus, it is now believed that sphingolipid metabolites exert their actions from the extracellular environment via G protein-coupled receptors. Recently, the specific high-affinity receptors for SPC, OGR1, and GPR4, have been identified (Xu et al., 2000Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.Nat Cell Biol. 2000; 2: 261-267Crossref PubMed Scopus (176) Google Scholar; Zhu et al., 2001Zhu K. Baudhuin L.M. Hong G. et al.Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4.J Biol Chem. 2001; 276: 41325-41335Crossref PubMed Scopus (208) Google Scholar). OGR1 and GPR4 are highly homologous, and have similar high affinities for SPC. It has been suggested, however, that they act through different G protein pathways. SPC induced the ERK activation in a PTx-insensitive manner in OGR1-transfected cells, whereas the activation of ERK was sensitive to PTx in GPR4-transfected cells. Our results suggest that GPR4 may medicate SPC-induced PAI-1 expression in cultured dermal fibroblasts. The presence and involvement of this type of receptor, however, remain to be demonstrated. Evidences in other systems have indicated that SPC induces increases in [Ca2+]i, thereby affecting cell proliferation (Seufferlein and Rozengurt, 1995Seufferlein T. Rozengurt E. Sphingosylphosphorylcholine activation of mitogen-activated protein kinase in Swiss 3T3 cells requires protein kinase C and a pertussis toxin-sensitive G protein.J Biol Chem. 1995; 270: 24334-24342Crossref PubMed Scopus (75) Google Scholar; Chin and Chueh, 1998Chin T.Y. Chueh S.H. Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway.Am J Physiol. 1998; 275: 1255-C1263PubMed Google Scholar). We hypothesized that an increase in [Ca2+]i was also involved in SPC-induced PAI-1 expression. As expected, our confocal laser scanning microscopy data clearly showed that SPC induced a rapid and transient increase in [Ca2+]i. The involvement of [Ca2+]i was further supported by experiments showing both PAI-1 induction by calcium ionophore and complete suppression of PAI-1 induction following intracellular Ca2+ depletion by thapsigargin. Interestingly, pre-treatment with PTx could not completely suppress either the increase in [Ca2+]i or the induction of PAI-1 expression. These results raise the possibility that another pathway, together with PTx-sensitive G protein, may be involved in SPC effect. One candidate is Rho signaling pathway. In a preliminary study, PTx and C3-exoenzyme (a specific inhibitor of Rho) each independently caused partial inhibition of the induction of PAI-1 by SPC. Marked synergistic enhancement of suppression was seen when they were used together (data not shown). We are currently investigating the possible involvement of the Rho signaling pathway in SPC-induced PAI-1 expression. Increased intracellular Ca2+ levels can activate numerous downstream signaling targets, including PKC and MAPK pathway. For example, SPC-induced MAPK activation was shown to be critically dependent on increases in [Ca2+]i in porcine aortic smooth muscle cells, and PKC activation was also shown to be involved in this process (Chin and Chueh, 1998Chin T.Y. Chueh S.H. Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway.Am J Physiol. 1998; 275: 1255-C1263PubMed Google Scholar). In this study, we demonstrated that the activations of PKC and MAPK pathway were involved in SPC-induced PAI-1 expression. Together with previous reports, our results raise the possibility that the activation of PKC and MAPK pathway may be functional downstream signaling events following SPC-induced increases in [Ca2+]i. The putative relationship between these signaling cascades, however, must be investigated further. In summary, we have demonstrated that SPC promotes both wound healing and PAI-1 production, and that a G protein-coupled Ca2+ increase and additional downstream signaling events are involved in this process. SPC was purchased from Matreya (Pleasant, California). PAI-1 and uPA ELISA kits, monoclonal PAI-1 antibody, and purified PAI-1 were obtained from American Diagnostica (Greenwich, Connecticut). Pertussis toxin, tyrphostin A47, thapsigargin, EGTA, A23187, PMA, Ro 31-8220, and PD 98059 were obtained from Calbiochem (La Jolla, California). FBS, penicillin/streptomycin solution, trypsin/EDTA, and DMEM were from Gibco BRL (Rockville, Maryland), and plasminogen and thrombin were from Sigma (St Louis, Missouri). Anti-phospho-p42/44 ERK antibody was purchased from New England BioLabs (Beverly, Massachusetts). Fluo-3, AM was supplied by Molecular Probes (Eugene, Oregon). Five Netherlands rabbits, between 10 and 12 wk of age, were anesthetized with phenobarbital. All tests were approved by IRB (Institute of Medical Research, College of Medicine, Chungnam National University). Full-thickness wounds, approximately 6 mm in diameter that did not cross the muscle fascia were made in the inner surface of the ear using a biopsy punch. The wounds were not sutured, but were dressed with a transparent plastic film. Experimental groups received daily injections of 5 μM SPC solution into the wounded areas, which were then re-covered with plastic film. Rabbits were euthanized after 4 d of treatment, and the wounded areas were dissected out and embedded in paraffin. Sections were stained with hematoxylin and eosin to examine cell migration and remodeling. Immunohistochemical staining was performed as follows. After brief washing in PBS, sections were blocked in PBS containing 0.5% skim milk, and incubated for 2 h at room temperature with a 1:200 dilution (5 μg per mL) of monoclonal anti-PAI-1 antibody. After further washing in PBS, sections were incubated sequentially with biotinylated rabbit anti-mouse IgG and with peroxidase-conjugated avidin. Normal human skin samples were obtained from circumcisions, in accordance with the ethical committee approval process of Chungnam National University Hospital. Specimens were briefly sterilized in 70% ethanol, minced, and then incubated in DMEM supplemented with 10% FBS and antibiotics. Dermal fibroblasts normally outgrew from the explants after 5–7 d. At confluence, cells were routinely passaged using a 1:4 split ratio. Cells were used between passages 4 and 16. For treatment with SPC, approximately 1×106 cells were seeded on 100 mm culture dishes and grown to confluence. Cells were starved of serum for 24 h, then treated with SPC in serum-free medium. Levels of PAI-1 and uPA in conditioned media were quantified using ELISA kits according to the manufacturer's recommended protocols. Measurements were repeated at least three times, with independent cell batches obtained from three different donors. Cell extracts were prepared in lysis buffer containing 62.5 mM Tris, pH 6.8, 50 mM DTT, 2% SDS, 10% glycerol, and proteinase inhibitors. Total protein was measured using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, California). Samples were run on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with anti-PAI-1 antibody (1:200 dilution in 2.5% skim milk in PBS-T) for either 2 h at room temperature or overnight at 4°C with gentle agitation. Blots were then incubated with peroxidase-conjugated anti-mouse IgG (Amersham, Buckinghamshire, UK) at a dilution of 1:2000 in 2.5% skim milk in PBS-T, and were developed by enhanced chemiluminescence (Amersham). The PAI-1 activity in cell lysates was assayed according to a slight modification of the method reported previously (Nordt et al., 1995Nordt T.K. Sawa H. Fujii S. Sobel B.E. Induction of plasminogen activator inhibitor type-1 (PAI-1) by proinsulin and insulin in vivo.Circulation. 1995; 91: 764-770Crossref PubMed Google Scholar). Samples of cell extracts (100 μg) were run on 10% SDS-polyacrylamide gels, under non-reducing conditions. Following electrophoresis, SDS was removed by washing with 2.5% Triton X-100 for 90 min at room temperature. Gels were placed on an agarose indicator containing thrombin (0.06 U per mL), purified plasminogen (5 μg per mL), fibrinogen (2 mg per mL), and tPA (0.05 U per mL). Gels were incubated overnight at 4°C to allow penetration of proteins into the fibrin-agarose, and then incubated for 3–4 h at 37°C to induce enzyme activity. PAI-1 activity was visualized by the appearance of an opaque band. Total RNA samples (10 μg) were electrophoresed on 1% agarose gels containing formaldehyde and transferred onto Hybond-N+ membranes (Amersham). Blots were prehybridized for 30 min at 62°C, then hybridized overnight at 62°C in the same solution containing 32P-labeled probe. Following hybridization, blots were washed, and then analyzed using a Fuji BAS 2500 phosphoimager (Fuji Film, Tokyo, Japan). Cells were grown on coverslips for 24 h, under serum-free conditions, then incubated with 4 μM Fluo-3, AM for 40 min. After three washes with serum-free medium, coverslips were mounted in perfusion chambers of our own design and subjected to confocal laser scanning microscopy (Zeiss LSM 410; Carl Zeiss, MicroImaging, Thornwood, New York). Scanning was performed at 1 s intervals with 488 nm excitation argon laser and 515 nm long-pass emission filter. SPC was added to the cells using an automatic pump system. All images were processed to analyze the changes in Ca2+ concentration at the cellular level. Results are expressed as relative fluorescence intensity. Data for PAI-1 and uPA ELISAs were statistically evaluated using Student's t test. Statistical significance was set at p<0.01. We thank Dr Guo Dong Zheng for providing assistance in rabbit ear wound-healing experiments. Dr Zee-won Lee, Dr Seong Woo Kim, and Professor Kwon-Soo Ha (Biomolecule Research Group, Korea Basic Science Institute, Daejeon, Korea) are greatly appreciated for providing confocal imaging and expert technical assistance. This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Korea (HMP-00-PT-21100-0004).