![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia
Received March 17, 2003; accepted August 12, 2003
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionConclusionsReferences |
|---|
, 1999; McKeon et al., 1999). Therefore, understanding the factor(s) that initiate and control glial scarring and astrocyte proliferation may lead to therapeutic advances in the treatment of CNS injury. Because activators of protease receptors and lysophospholipid receptors are present in blood, we have examined the biochemical mechanisms that could potentially link activation of these receptors during blood-brain barrier breakdown to astrocyte proliferation.
Although long known for its role in blood coagulation, thrombin plays an important role in CNS injury (Gingrich and Traynelis, 2000; Xi et al., 2003). The thrombin precursor prothrombin, its activating enzyme Factor Xa, as well as thrombin inhibitors and thrombin receptors are all present in the CNS (Deschepper et al., 1991; Dihanich et al., 1991; Vaughan and Cunningham, 1993; Weinstein et al., 1995; Shikamoto and Morita, 1999). Direct infusion of thrombin into rat caudate nucleus causes an increase in reactive gliosis that resembles scar formation (Nishino et al., 1993). The cellular actions of thrombin can often be attributed to the presence of protease-activated receptors (PARs). Of the four known PAR family members, thrombin can activate PAR-1, PAR-3, and PAR-4. Studies on cultured astrocytes demonstrate that thrombin is mitogenic (Grabham and Cunningham, 1995) and suggest that proliferation is probably mediated via activation of PAR-1 but not PAR-3 or PAR-4 (Wang et al., 2002a,b). PAR-1 is a pleiotropic G protein-coupled receptor capable of activating members of the Gi, Gq, and G12/13 families of G proteins (Offermanns et al., 1994; Ogino et al., 1996; Mao et al., 1998). Therefore, a large variety of cellular responses including Gq/11-mediated phosphoinositide turnover and Ca2+ signaling, Gi/o-mediated activation of mitogen-activated protein kinase (MAPK) cascades and G12-mediated activation of Rho-serum response element (SRE) pathways have been attributed to PAR-1 (see Macfarlane et al., 2001, for review). Although previous studies indicate that recruitment of the MAPK, phospholipase C
, and Gi/o pathways seem to be involved in the coupling of PAR-1 to astrocyte proliferation (Wang et al., 2002a), a full understanding of the signal transduction mechanisms used by PAR-1 to mediate proliferation remains to be elucidated.
In platelets, thrombin activation results in the production of the bioactive lipids, lysophosphatidic acid (LPA), and sphingosine-1-phosphate (S1P) (Sano et al., 2002). These lipids are transported in blood bound to albumin and are thus normally excluded from the CNS. Many of the biological effects of LPA and S1P are attributable to a family of lysophospholipid (LPL) receptors formerly known as EDG receptors. Three LPL receptors designated LPA-1, -2, and -3 display specificity for LPA and five LPL receptors designated S1P-1 through S1P-5 display specificity for S1P, although high concentrations of LPA can activate the S1P-1 receptor (Lee et al., 1998; Fukushima et al., 2001; Takuwa et al., 2002). Similar to PARs, the LPL receptors are coupled to multiple intermediary G protein pathways. Whereas LPA-1 and LPA-2 couple to Gi, Gq, and G12, LPA-3 seems to couple to Gi/o and Gq/11 but not G12 (Fukushima et al., 2001; Takuwa et al., 2002). Similarly, all of the S1P receptors seem to recruit Gi/o. S1P-2 and S1P-3 additionally activate Gq/11 and G12, whereas the S1P-4 and S1P-5 receptors additionally activate G12 but not Gq/11 (Siehler and Manning, 2002). LPA and S1P have been shown to regulate a wide variety of cellular events including cytoskeletal reorganization, differentiation, chemotaxis, and secretion. However, a hallmark of LPA and S1P action in many cells is the induction of proliferation, and these lipids are thus considered major growth factors of serum.
During short-term injury, the CNS is exposed not only to thrombin but also to the products of thrombin's action on platelets, LPA, and S1P. In addition, increasing evidence supports a role for LPA and S1P as paracrine or autocrine mediators released from multiple cell types (Johnson et al., 2002; Xie et al., 2002a,b). LPA is abundant in rat brain (Das and Hajra, 1989), and physiologically active concentrations of LPA have been detected in cerebrospinal fluid in an experimental model of hemorrhagic injury (Tigyi et al., 1995). LPA and S1P have been shown to induce proliferation of astrocytes in culture (Keller et al., 1997; Ramakers and Moolenaar, 1998; Tabuchi et al., 2000; Pebay et al., 2001; Steiner et al., 2002; Yamagata et al., 2003), although some reports indicate that LPA is not a mitogen for astrocytes (Tigyi et al., 1994; Fuentes et al., 1999; Pebay et al., 1999).
In this study, we examine in parallel the capacity of these three classes of receptor (PAR-1, LPA, S1P) to induce mitogenesis in cultured astrocytes and reactive astrogliosis in vivo. We further compare in parallel their coupling to intracellular signaling pathways. Our results suggest that PAR-1, LPA, and S1P each recruit multiple parallel G protein pathways and implicate roles for a Gi/o-MAPK pathway and Rho-SRE pathway in coupling each of these agents to astrocyte proliferation.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Preparation of LPA and S1P. Lipids were prepared fresh in lipid-free 0.1% BSA on the day of the experiment. For S1P, the dry powder was suspended in chloroform and then evaporated under nitrogen. The appropriate volume of water (containing 0.1% lipid-free BSA to stabilize the lipids) was added. Lipids were resuspended by sonication and further diluted to 100x stocks in 0.1% lipid-free BSA. Addition of lipid-free BSA (0.1%) at a 1:100 dilution had no effect compared with water alone for all of the assays tested.
In Vivo Injection of Agonist and GFAP Immunostaining. All procedures involving the use of animals were reviewed and approved by the Emory University IACUC. C57BL/6 male mice (90 days old) from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA) were anesthetized with 8% chloral hydrate administered i.p. at 0.4 g/kg. Animals were placed on a homeothermic blanket, and rectal temperature was maintained between 36.2 and 37°C. The head was immobilized in a small animal stereotaxic device (SAS 75; Cartesian Research Inc., Sandy, OR), a scalpel was used to expose the skull, and a hole was drilled with a 0.0087-inch carbide bit at 1.000 mm rostrally, 2.150 mm laterally, and 0.500 mm ventrally to bregma. A 33-gauge needle was inserted vertically 3.450 mm into a predrilled hole and allowed to reside in place for 2 min. Injections of 0.1% lipid-free BSA in phosphate-buffered saline (PBS) at pH 7.4 containing 1 nmol of S1P, 10 nmol of LPA, or 1 pmol of thrombin were made at 0.1 µl/min for 5 min (total volume, 0.5 µl). After injection, the needle was left in place for 5 min, removed, and the skin was sutured. Animals were allowed to survive 5 days and were then injected with sodium pentobarbital (390 mg/ml) and transcardially perfused with ice-cold 4% paraformaldehyde in PBS for 10 min. The brain was removed and placed in 4% paraformaldehyde for 24 h and cryoprotected in 20% sucrose at 4°C for 24 h. The brain was embedded with Tissue Tec, and stored at -50°C. Embedded tissue was warmed to -20°C, and 10-µm sections were cut on a cryostat (CM3050; Leica, Wetzlar, Germany) and placed on gelatin-coated glass slides. Slices were incubated in 4% paraformaldehyde for 10 min and washed three times in PBS. Sections were incubated in blocking solution composed of 3% goat serum, 0.5% BSA, and 0.25% Triton X-100 in PBS for 1 h at 23°C. Sections were incubated with 1:100 rabbit anti-GFAP in blocking buffer overnight at 4°C. Sections were subsequently washed three times in PBS and incubated with streptavidin-conjugated Texas Red secondary antibody in blocking buffer for 1 h at 23°C. Sections were washed three times, coverslip-coated using Vectashield fluorescent mounting media, and stored in the dark at -20°C until fluorescent images were acquired with a Hamamatsu C4742-95 camera using Openlab software (Improvision).
Primary Culture of Astrocytes. Cultured astrocytes were prepared from P1-P3 postnatal mice. The cerebral cortex was dissected free of adherent meninges, and the tissue was minced and dissociated into single-cell suspension by trituration through a Pasteur pipette. Dissociated cells were plated into multiwell plastic plates coated with 100 µg/ml poly-D-lysine using Dulbecco's modified Eagle medium supplemented with 25 mM glucose, 10% heat inactivated horse serum, 10% heat inactivated (55°C for 30 min) fetal bovine serum, 2 mM glutamine, and a 1:100 dilution of penicillin-streptomycin. After 2 to 4 days in culture, the cells were vigorously shaken to remove all loosely adherent cells. Cultures were maintained at 37°C in a humidified 5% CO2-containing atmosphere until they reached 50% confluence. Homogenous populations of astrocytes were verified by immunofluorescence staining with GFAP (data not shown).
[3H]Thymidine Incorporation and Cell Proliferation. Primary cultures of astrocytes grown in 24-well plates were serum-starved for 18 to 24 h in the absence or presence of pharmacological inhibitors as noted. Cells were then challenged with agonist for a 24-h period. During the final 2 h of agonist challenge, [3H]thymidine was added to a final concentration of 1 µCi/ml. Cells were washed in ice-cold PBS before the addition of 20% trichloroacetic acid for 30 min at 4°C. Cells were again washed in PBS, and the acid-insoluble material was lysed in 0.1 N NaOH/1% SDS. [3H]Thymidine in lysates was measured by scintillation counting. For measurement of cell proliferation, primary cultures of astrocytes were seeded at equal density in 24-well plates and challenged with agonist for 24 h. Astrocytes were resuspended by treatment with trypsin and counted directly by visual inspection using a hemacytometer.
RT-PCR for Receptor mRNA. Total RNA was extracted from primary astrocyte cultures in 100-mm culture dishes with TRIzol reagent per the manufacturer's directions. Poly A+ mRNA was purified from total RNA using Oligotex mRNA Kits (QIAGEN, Valencia, CA) and suspended in a 60-µl volume. Reverse transcription was performed on 10-µl aliquots of astrocyte mRNA in the presence or absence of SuperScript II RNase H- reverse transcriptase per the manufacturer's directions using oligo(dT), dNTP, and RNase OUT from Invitrogen. Aliquots of cDNA samples (2 µl) were used for PCR using PCR SuperMix. Sense and antisense primers (1 µM) designed for murine PAR and LPL receptors are shown in Table 1. Samples were cycled by an initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 0.5 min, annealing at 55°C for 0.5 min, and elongation at 72°C for 1 min, and a primer extension at 72°C for 5 min. PCR fragments were separated by agarose gel electrophoresis and identified by ethidium bromide staining.
|
[3H]Inositol Phosphate Production. Primary astrocytes were serum-starved for 18 to 24 h in complete Dulbecco's modified Eagle medium containing 4 µCi/ml [3H]inositol in the absence or presence of pharmacological inhibitors as described in the text. Cells were placed in media supplemented with 5 mM LiCl, challenged with agonist, and [3H]inositol phosphates were isolated by ion exchange chromatography as described previously (Heximer et al., 1999).
Calcium Mobilization in Intact Astrocytes. Cultured astrocytes were incubated with 5 µM Fura2 acetoxymethyl ester for 30 min at room temperature. Cells were then placed on a microscope stage for imaging of intracellular calcium concentration. External solution contained 150 mM NaCl, 10 mM HEPES, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5 mM glucose, pH 7.3, and 325 mOsm. Intensity images of 510 nm wavelength were taken at 340- and 380-nm excitation wavelengths, and the two resulting images were taken from individual cells for ratio calculations. Axon Imaging Workbench version 2.2.1 (Axon Instruments, Union City, CA) was used for acquisition of intensity images and conversion to ratios.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis. Primary cultures of astrocytes were grown in six-well format and serum-starved in the absence or presence of inhibitors as indicated before the addition of the agonists as described. Intracellular signaling reactions were stopped by the addition of 300 µlofhot (80°C) sample buffer. Aliquots (50 µl) of lysates were boiled in SDS sample buffer, subjected to SDS-polyacrylamide gel (12%) electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked at room temperature for 1 h in Tris-buffered saline containing 5% nonfat milk. Antibody to total ERK 1/2 (1:300), phosphorylation-specific ERK 1/2 antibody (1:1000), phosphorylation-specific SAPK/JNK antibody (1:250), or phosphorylation-specific p38 MAPK antibody (1:250) was diluted in Tris-buffered saline supplemented with 0.1% Tween (TBS-T) and incubated with the membranes overnight at 4°C. Membranes were washed with TBS-T and incubated for 1 h in horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 in TBS-T). Membranes were again washed and immunoreactive proteins detected by enhanced chemiluminescence. Total ERK 1/2 immunoreactivity was equal under each experimental condition within each experiment. For experiments with GFAP antibody, cells were scraped into lysis buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Samples were assayed for protein, and equal aliquots (20 µg) of lysate were boiled in SDS sample buffer before SDS-polyacrylamide gel electrophoresis and transfer to Immobilon-P membranes. Immunoblotting was determined as described above with 1:1000 rabbit anti-GFAP primary antibody.
Transcription Factor Luciferase Reporters. Infectious retroviral supernatants were prepared from Phoenix producer cells transfected with retroviral luciferase reporter plasmids containing binding elements for NFAT and SRE as described previously (Abbott et al., 2000). Green fluorescent protein-containing retroviral plasmids were run in parallel and served as controls. Primary astrocytes in six-well format were infected with three rounds of retrovirus exposure resulting in 30 to 70% infection efficiency as assessed by green fluorescent protein. Two to 3 days after infection, astrocytes were passaged (1 to 4 by surface area) onto poly-D-lysine-coated 24-well plates. Two to 7 days after passage, cells were serum-starved overnight before application of agonist for the time indicated in the text. Reactions were stopped, and luciferase activity was monitored on a luminometer using a luciferase assay system (Promega).
Dose Response Analysis. Dose response curves were analyzed using Prism software (GraphPad, San Diego, CA) and data fit by nonlinear regression to a variable slope sigmoidal dose-response curve.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
) with thrombin (1 pmol/0.5 µl) markedly reduced the gliotic response in mice (n = 3, data not shown); BMS200261 alone had no effect on GFAP immunoreactivity (n = 2). Cresyl violet staining in parallel slices was homogenous, thereby confirming that these changes in astrocyte reactivity were not the result of nonspecific injury at the site of injection (data not shown).
|
Thrombin, TFLLR, LPA, and S1P Stimulate Astrocyte Proliferation in Vitro. A key aspect of astrocyte reactivity is a proliferative response. Therefore, the capacity of thrombin, LPA, and S1P to induce a mitogenic response was evaluated in astrocytes in culture. Previous studies have determined that PAR-1, but not PAR-3 or PAR-4, is primarily responsible for proliferative responses to thrombin in astrocytes; activation of the thrombin-insensitive trypsin receptor PAR-2 initiates a moderate proliferative response (Wang etal., 2002a). Therefore, we also compared the effects of the selective PAR-1-activating peptide TFLLR, which mimics the new N terminus revealed by thrombin cleavage at Arg41 (Hollenberg et al., 1997). Primary cultures of astrocytes were challenged with either 50 pM thrombin, 30 µM TFLLR, 30 µM LPA, or 10 µM S1P, and proliferation was monitored by the incorporation of [3H]thymidine and direct counting of cells. Thrombin, TFLLR, and LPA induced a 5- to 6-fold increase in [3H]thymidine incorporation, whereas S1P induced only a 3-fold increase (Fig. 2). The increase caused by S1P was measurably smaller than that caused by TFLLR and LPA (statistically significant, p < 0.05, Neuman-Keuls Test). The number of astrocytes also increased measurably over 24 h in response to the same concentration of each agonist [thrombin, 2.0 ± 0.4-fold (S.E.M.) compared with basal; TFLLR, 2.0± 0.4-fold; LPA 1.8 ± 0.3-foldl; and S1P, 1.6 ± 0.4-fold]. No changes in normalized GFAP expression (immunoreactive GFAP/total protein) were observed by immunoblot analysis in cultured astrocytes treated with these agents (data not shown). Because thrombin and TFLLR have similar effects on astrocyte proliferation and evidence suggests that thrombin actions are mediated by PAR-1 (Wang et al., 2002a), subsequent signaling studies focused on the comparative effects of TFLLR, LPA, and S1P on astrocyte signaling events.
|
Multiple PAR, LPA, and S1P Receptor Subtype mRNAs Are Expressed in Astrocytes. Although TFLLR requires PAR-1 to induce a proliferative response and is a specific PAR-1 agonist, the effects of LPA and S1P on proliferation could be mediated by receptor-dependent and/or receptor-independent effects of these agents (Van Brocklyn et al., 1998; Spiegel and Milstien, 2000; Hooks et al., 2001). Therefore, the presence of mRNA for these receptors was analyzed by RT-PCR. mRNA for all four known PAR subtypes and all three known LPA subtypes was detected in the astrocytes (Fig. 3, A and B). Of the five known S1P receptors, mRNA for S1P1, S1P3, S1P4, and S1P5 was detected (Fig. 3C). Although the mRNA for S1P2 was readily detected in NIH3T3 fibroblasts (data not shown), this receptor mRNA was not detected in cortical astrocytes (Fig. 3C).
|
Cellular Signaling Responses to TFLLR, LPA, and S1P in Astrocytes. The presence of functional LPA and S1P receptors was determined by monitoring the inositol phosphate production that occurs in response to receptor-mediated activation of the Gq/11-phospholipase C (PLC) pathway. Application of either LPA or S1P induced a time-dependent accumulation of inositol phosphates (Fig. 4A) demonstrating the presence of Gq/11-coupled LPA and S1P receptors. Similarly, TFLLR induced activation of the Gq/11-PLC pathway. Although S1P had the lowest relative activity in [3H]thymidine assays, S1P displayed the highest intrinsic activity for inositol phosphate production (Figs. 2 and 4B). TFLLR, LPA, and S1P also stimulated a marked rise in intracellular Ca2+ levels in individual astrocytes (Fig. 4C). Stimulated increases in intracellular Ca2+ were similar in both magnitude (5-fold over resting basal) and duration for each agonist. Intracellular Ca2+ levels peaked within 30 s and returned to baseline levels after 4 min for TFLLR, LPA (Fig. 4C); after S1P treatment, Ca2+ levels also peaked within 30 s and declined but remained elevated after 4 min (Fig. 4C).
|
To further probe the interrelationship between inositol phosphate production and [3H]thymidine incorporation in astrocytes, dose response relationships were determined. For TFLLR, the potency to induce the downstream proliferative response is approximately 10-fold greater than potency for activation of the upstream PLC response. This leftward shift in the dose-response curve is predicted by signal amplification. In contrast, LPA and S1P are less potent in activating [3H]thymidine incorporation compared with activation of PLC (Fig. 5).
|
To further assess the signaling pathways activated by TFLLR and the LPLs, immunoblot analysis was used to determine the ability of these agents to induce phosphorylation of MAPK. Phospho-specific antibodies to ERK 1/2, stress activated protein kinase/c-jun N-terminal kinase (SAPK/JNK), and p38 MAPK were used to determine changes in the phosphorylation state of these MAPK family members. All of the agonists tested induced phosphorylation of ERK1/2 and SAPK/JNK, but no changes in p38 phosphorylation were detected. Although the onset of phosphorylation induced by each of these agents was rapid (2 to 5 min), kinetic differences in the sustained activation of the MAPK pathways were noted. LPA induced a prolonged phosphorylation of both MAPK pathways, whereas the effects of S1P were more transient and returned to baseline values at 1 to 2 h. TFLLR displayed differential effects on the MAPKs such that phosphorylation of ERK1/2 was prolonged and that of SAPK/JNK was transient (Fig. 6).
|
MAPKs and Rho Kinase couple TFLLR, LPA and S1P Responses to Astrocyte Proliferation. To dissect the pathway(s) responsible for the coupling of TFLLR, LPA, or S1P activation of PAR-1 and LPL receptors to astrocyte proliferation, cultured astrocytes were treated with pharmacological inhibitors of MEK/ERK, Gi/o, PLC, inositol phosphate receptor, protein kinase C (PKC), or Rho pathways before agonist challenge. Treatment of cells with 10 µM of the MEK inhibitor U0126 blocked the coupling of each agonist to both ERK1/2 and SAPK/JNK phosphorylation (Fig. 7A). Similarly, pretreatment of cells for 24 h with 0.1 µg/ml PTX to disrupt receptor coupling to Gi partially inhibited the coupling of TFLLR, LPA, and S1P to these MAPK pathways (Fig. 7B). These data suggest that Gi/o proteins contribute to TFLLR- and LPL-mediated activation of MAPK pathways. However, studies using the Rho kinase inhibitor, Y27632, demonstrate that activation of this Rho effector is not required for coupling TFLLR, LPA, or S1P to the phosphorylation of either ERK1/2 or SAPK/JNK (Fig. 7C). In addition, PTX partially inhibits the coupling of TFLLR, LPA, and S1P to accumulation of inositol phosphates, indicating that both Gq/11 and Gi/o mechanisms for phosphoinositide turnover exist in astrocytes. In contrast, Y27632 does not affect the coupling of the agents to phosphoinositide turnover (data not shown).
|
Although these inhibitors had differential effects on agonist-induced phosphoinositide turnover as well as phosphorylation of MAPK pathways, each disrupted agonist-induced activation of proliferative responses. The MEK inhibitor U0126 completely inhibited TFLLR-, LPA-, or S1P-induced increases in [3H]thymidine incorporation, indicating that the activation of MAPK pathways is necessary for these agonists to activate proliferation in astrocytes. TFLLR-, LPA-, and S1P-induced activation of proliferation was sensitive to both PTX and Y27632. PAR-1 mediated proliferation was inhibited 70% to 80% in the presence of either PTX or Y27632, whereas LPA and S1P responses were inhibited by 40% to 50% in the presence of these inhibitors (Fig. 8).
|
To further probe the role of Gq-PLC pathways in mediating TFLLR or LPL effects on proliferation, cells were treated with various pharmacological inhibitors of this pathway. Treatment of astrocytes with 10 µM of the PLC inhibitor U73112
[GenBank]
, the minimal concentration we found necessary to block inositol phosphate production, or with 100 µM of the inositol 1,4,5-trisphosphate receptor inhibitor 2-amino ethoxydiphenyl borate, the reported concentration needed to block Ca2+ mobilization, was cytotoxic to cultured astrocytes after 6 h and prohibited further proliferative studies with these agents. Treatment of astrocytes with 1 µM concentrations of the PKC inhibitor bisindolylmaleimide, a concentration that completely blocks the activity of many PKC isoforms (Toullec et al., 1991), was not cytotoxic and did not alter the capacity of TFLLR, LPA, or S1P to stimulate astrocyte proliferation (data not shown).
TFLLR, LPA, and S1P Activate Two Transcriptional Pathways. Transcription factors serve as downstream effectors of many signal transduction pathways. Activation of the PLC/Ca2+ pathway and subsequent activation of calcineurin can initiate transcription through NFAT (Macian et al., 2001). Although each of the agonists studied couples to inositol phosphate production and Ca2+ mobilization (Fig. 4), only LPA and S1P can couple to the prolonged activation of NFAT pathways as measured by an NFAT-luciferase reporter, whereas TFLLR does not appreciably couple to activation of NFAT (Fig. 9A). These studies indicate that activation of NFAT pathways is not required for GPCR-mediated proliferative responses in general. However, each of the agonists tested can couple to activation of the SRE transcription factor, a downstream effector of Rho pathways (Fromm et al., 1997; Mao et al., 1998). Time courses for agonist coupling to SRE demonstrate that activation of SRE occurs within 2 h of application of each agonist. TFLLR and S1P coupling to SRE is maximal by 2 to 4 h and returns toward baseline, whereas LPA coupling to SRE is sustained out to 12 h (Fig. 9B).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
; Ramakers and Moolenaar, 1998; Tabuchi et al., 2000; Pebay et al., 2001; Steiner et al., 2002; Wang et al., 2002a; Yamagata et al., 2003), in this study we have sought to link in vivo effects of PAR-1, LPA, and S1P on reactive astrogliosis with shared signaling pathways for these receptors that mediate proliferative responses in vitro.
PAR-1, LPA, and S1P Receptors Share Intracellular Signaling Pathways. Although multiple PAR mRNAs are present in cultured astrocytes (Fig. 3A), previous studies have determined that PAR-1 plays a major role in activating proliferative responses in astrocytes, whereas PAR-3 and PAR-4 are not mitogenic; activation of the thrombin-insensitive trypsin receptor PAR-2 initiates a moderate proliferative response (Wang et al., 2002a). Therefore, the present studies have focused on PAR-1 signaling events using a selective peptide activator (TFLLR) of PAR-1 that mimics the new N terminus revealed by thrombin cleavage at Arg41. Dose-response curves for TFLLR reveal that the concentrations that induce relatively modest changes in upstream signaling events (phosphoinositide turnover) can induce robust downstream signaling in vitro (proliferation). This result is predicted by amplification of signaling pathways that is often evident on downstream effects. In contrast, higher doses of LPA and S1P were required to induce proliferation in vitro compared with doses required for phosphoinositide turnover (Fig. 5). These results seem to uncouple LPL-induced proliferative responses from phosphoinositide turnover. In support of this idea, treatment of astrocytes with a selective inhibitor of PKC also had no effect on agonist-mediated cell proliferation in culture. These data support previous results demonstrating that Gi/o proteins are important for astrocyte proliferation induced by S1P but activation of PKC is not (Pebay et al., 2001). Because phosphoinositide turnover is mediated through activation of Gq/11, these results suggest that the LPA- and S1P-mediated effects on proliferation are mediated by Gi/o and/or G12 pathways or are receptor-independent (Van Brocklyn et al., 1998; Spiegel and Milstien, 2000; Hooks et al., 2001). However, several alternate explanations of these data exist. For example, the dose-response relationship for phosphoinositide turnover induced by LPA or S1P occurs over a span of greater than 2 log and is better predicted by a variable slope fit versus a one-site fit (for LPA, R2 = 0.98 versus 0.96; for S1P, R2 = 0.94 versus 0.91 for variable slope versus one-site, respectively) with Hill slopes of less than 1. The identification of multiple mRNAs for different receptor subtypes is consistent with the idea that multiple LPA or S1P receptor subtypes mediate phosphoinositide turnover; perhaps only the low-affinity subset of those receptors is involved in proliferative responses. In addition, LPA and S1P may not be stable over the prolonged incubation times required for proliferative responses (24 h). Although we verified that LPA and S1P are biologically active while stored in aqueous 0.1% BSA after 24 h, the stability of these lipids in the presence of astrocytes is unknown. Therefore, the doses added to initiate these experiments may be much higher than the effective doses required for proliferation.
Similar to TFLLR, LPA and S1P both couple to the activation of the ERK1/2 and SAPK/JNK members of the MAPK family, and inhibition of these pathways with the MEK inhibitor U0126 completely inhibits proliferative responses to these agonists. These findings indicate an important role for ERK1/2 activation in the proliferation of astrocytes. Consistent with this idea, TFLLR and LPA each induced a sustained activation of ERK1/2 and a robust stimulation of [3H]thymidine uptake, whereas S1P induced a transient activation of ERK1/2 (peak within 5 min) and a considerably more modest uptake of [3H]thymidine. Together, these findings suggest that sustained activation of MAPK pathways may underlie proliferation of astrocytes. The coupling of TFLLR, LPA, and S1P to MAPK and proliferation can be further dissected by pharmacological inhibitors of the intermediary signaling pathways. PTX and Y27632 were used to define roles for Gi/o and Rho pathways, respectively, in these processes. Rho pathways are stimulated by G13-mediated activation of p115RhoGEF (Hart et al., 1998; Kozasa et al., 1998), although recent studies demonstrate that Rho pathways are also activated by Gq/11 independent of G12/13 (Sagi et al., 2001; Vogt et al., 2003). Although our studies did not directly measure activation of Rho in astrocytes, partial blockade of thymidine uptake by Y27632 indicates Rho involvement in astrocyte proliferation. Whereas both PTX and Y27632 disrupt proliferation, the coupling of these agents to MAPKs is sensitive only to PTX but not Y27632. Taken together, these studies suggest a similar mechanism of action for TFLLR, LPA, and S1P in coupling to MAPK that involves a Gi/o-dependent pathway. Although Y27632 attenuated proliferative responses, this agent had no effect on the capacity of TFLLR, LPA, or S1P to initiate MAPK cascades. Therefore, the involvement of Rho pathways in mediating proliferative responses seems to be independent of effects on the MAPK pathway (Fig. 10).
|
PAR-1, LPA, and S1P Receptors Regulate Transcription in Astrocytes. Signaling pathways can mediate global cellular changes via activation of transcription factors that regulate gene expression. Two such transcription factors that can be activated by Gi/o, Gq/11, or G12 signaling pathways are NFAT and SRE. NFAT is activated by the phosphatase calcineurin, which is in turn sensitive to changes in intracellular Ca2+. Therefore, activation of PLC pathways, as well as other Ca2+ regulatory pathways, can induce activation of NFAT, which can bind DNA in cooperation with nuclear partners such as AP-1, a downstream component of MAPK cascades (Macian et al., 2001). NFAT has been shown to be involved in the regulation of a number of cellular events including proliferative responses in myocytes and endothelial cells (Horsley et al., 2001; Horsley and Pavlath, 2002; Johnson et al., 2003). Similar to NFAT, transcription through SRE is regulated by the cooperative binding of multiple transcription factors. SRE is bound by a ternary complex consisting of the serum response factor and cofactors such as Elk-1, a downstream component of MAPK cascades (Yordy and Muise-Helmericks, 2000). GPCRs have previously been shown to couple to activation of SRE through Rho-dependent mechanisms. Guanine-nucleotide exchange factors for Rho can be activated by both G12- and Gq/11-dependent pathways (Fromm et al., 1997; Chikumi et al., 2002; Booden et al., 2002). However, in NIH 3T3 fibroblasts, thrombin and LPA activate SRE in the absence of Gq/11 (Mao et al., 1998). Together, these studies identify NFAT as a downstream effector of Gq/11-PLC and MAPK pathways, whereas SRE is activated by Rho and MAPK pathways.
In our studies, NFAT activation was most robust in the presence of S1P followed by LPA. Although these results are internally consistent with the enhanced activity of S1P to stimulate PLC pathways (compare Figs. 4 and 9), TFLLR coupling to NFAT was not detectable despite TFLLR coupling to PLC. These findings identify a point of divergence in PAR-1 and LPL receptor signaling and raise the possibility that PAR-1 recruits a distinct pathway that serves to limit NFAT activation. All three of the agents activated transcription through SRE. In general, agonists that coupled more robustly to SRE (LPA and TFLLR) also had higher intrinsic activity for proliferative responses. Together, these data favor a role for SRE pathways as important components of mitogenic responses in astrocytes.
|
Conclusions |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: CNS, central nervous system; PAR, protease activated receptor; MAPK, mitogen activated protein kinase; LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; LPL, lysophospholipid; BSA, bovine serum albumin; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenyltio)butadiene; Y27632, R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide; U73122 [GenBank] , 1-(6-{[17-3-methoxyestra-1,3.5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione; PTX, pertussis toxin; ERK, extracellular signal-regulated kinase; SAPK/JNK, stress activated protein kinase/c-jun N-terminal kinase; PCR, polymerase chain reaction; GFAP, glial fibrillary acidic protein; TBS-T, Tris-buffered saline-Tween 20; NFAT, nuclear factor of activated T cells; SRE, serum response element; PLC, phospholipase C; PKC, protein kinase C; MEK, mitogen-activated protein kinase kinase; GPCR, G protein-coupled receptor; RT, reverse transcriptase.
Address correspondence to: Dr. John R. Hepler, Emory University School of Medicine, Department of Pharmacology, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322-3090. E-mail: jhepler{at}pharm.emory.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Bernatowicz MS, Klimas CE, Hartl KS, Peluso M, Allegretto NS, and Seiler SM (1996) Development of potent thrombin receptor antagonist peptides. J Med Chem 39: 4879-4887.[CrossRef][Medline]
Booden MA, Siderovski DP, and Der CJ (2002) Leukemia-associated Rho guanine nucleotide exchange factor promotes G
q-coupled activation of RhoA. Mol Cell Biology 22: 4053-4061.
Chikumi H, Vazquez-Prado J, Servitja J-M, Miyazaki H, and Gutkind JS (2002) Potent activation of RhoA by Gq and Gq-coupled receptors. J Biol Chem 277: 27130-27134.
Das AK and Hajra AK (1989) Quantification, characterization and fatty acid composition of lysophosphatidic acid in different rat tissues. Lipids 24: 329-333.[CrossRef][Medline]
Davies SJ, Field PM, and Raisman G (1996) Regeneration of cut adult axons fails even in the presence of continuous aligned glial pathways. Exp Neurol 142: 203-216.[CrossRef][Medline]
Davies SJ, Goucher DR, Doller C, and Silver J (1999) Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J Neurosci 15: 5810-5822.
Deschepper CF, Bigornia V, Berens ME, and Lapointe MC (1991) Production of thrombin and antithrombin III by brain and astroglial cell cultures. Brain Res Mol Brain Res 11: 355-358.[Medline]
Dihanich M, Kaser M, Reinhard E, Cunningham DD, and Monard D (1991) Prothrombin mRNA is expressed by cells of the nervous system. Neuron 6: 575-581.[CrossRef][Medline]
Fuentes E, Nadal A, and McNaughton PA (1999) Lysophospholipids trigger calcium signals but not DNA synthesis in cortical astrocytes. Glia 28: 272-276.[Medline]
Fromm C, Coso OA, Montaner S, Xu N, and Gutkind JS (1997) The small GTP-binding protein Rho links G protein-coupled receptors and G12 to the serum response element and to cellular transformation. Proc Natl Acad Sci USA 94: 10098-10103.
Fukushima N, Ishii I, Contos JJA, Weiner JA, and Chun J (2001) Lysophospholipid receptors. Ann Rev Pharmacol Toxicol 41: 507-534.[CrossRef][Medline]
Gingrich MB and Traynelis SF (2000) Serine proteases and brain damage—is there a link? Trends Neurosci 23: 399-407.[CrossRef][Medline]
Grabham P and Cunningham DD (1995) Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation. J Neurochem 64: 583-591.[Medline]
Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, and Bollag G (1998) Direct stimulation of the guanine nucleotide exchange activity of p115RhoGEF by G13. Science (Wash DC), 280: 2112-2114.
Heximer SP, Srinivasa SP, Bernstein LS, Bernard JL, Linder ME, Hepler JR, and Blumer KJ (1999) G protein selectivity is a determinant of RGS2 function. J Biol Chem 274: 34253-34259.
Hollenberg MD, Saifeddine M, Al-Ani G, and Kawabata A (1997) Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity and receptor selectivity of receptor-activating peptides. Can J Physiol Pharmacol 77: 832-841.
Hooks SB, Santos WL, Im DS, Heise CE, Macdonald TL, and Lynch KR (2001) Lysophosphatidic acid-induced mitogenesis is regulated by lipid phosphate phosphatase and is Edg-receptor independent. J Biol Chem 276: 4611-4621.
Horsley V, Friday BB, Matteson S, Kegley KM, Gephart J, and Pavlath GK (2001) Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway. J Cell Biol 153: 329-338.
Horsley V and Pavlath GK (2002) NFAT: ubiquitous regulator of cell differentiation and adaption. J Cell Biol 156: 771-774.
Johnson EN, Lee YM, Sander TL, Rabkin E, Schoen FJ, Kaushal S, and Bischoff J (2003) NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells. J Biol Chem 278: 1686-1692.
Johnson KR, Becker KP, Facchinetti MM, Hannun YA and Obeid LM (2002) PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA). J Biol Chem 277: 35257-35262.
Keller JN, Steiner MR, Holtsberg FW, Mattson MP, and Steiner SM (1997) Lysophosphatidic acid induced proliferation-related signals in astrocytes. J Neurochem 69: 1073-1084.[Medline]
Kozasa T, Jian X, Hart MJ, Sternweis PC, Hart MJ, Singer WD, Gilman AG, Bollag G, and Sternweis PC (1998) p115RhoGEF, a GTPase activating protein for G12 and G13. Science (Wash DC), 280: 2109-2111.
Lee MJ, Thangada S, Liu CH, Thompson BD, and Hla T (1998) Lysophosphatidic acid stimulates the G-protein-coupled receptor EDG-1 as a low affinity agonist. J Biol Chem 273: 22105-22112.
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R (2001) Proteinase-activated receptors. Pharmacol Rev 53: 245-282.
Macian F, Lopez-Rodriguez C, and Rao A (2001) Partners in transcription: NFAT and AP-1. Oncogene 20: 2476-2489.[CrossRef][Medline]
Mao J, Yuan H, Xie W, Simon MI, and Wu D (1998) Specific involvement of G proteins in regulation of serum response factor-mediated gene transcription by different receptors. J Biol Chem 273: 27118-27123.
McKeon RJ, Jurynec MJ, and Buck CR (1999) The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar. J Neurosci 19: 10778-10788.
Nishino A, Suzuki M, Ohtani H, Motohashi O, Umezawa K, Nagura H, and Yoshimoto T (1993) Thrombin may contribute to the pathophysiology of central nervous system injury. J Neurotrauma 10: 167-179.[Medline]
Offermanns S, Laugwitz KL, Spicher K, and Schultz G (1994) G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 91: 504-508.
Ogino Y, Tanaka K, and Shimizu N (1996) Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells. Eur J Pharmacol 316: 105-109.[CrossRef][Medline]
Pebay A, Torrens Y, Toutant M, Cordier J, Glowinski J, and Tence M (1999) Pleiotropic effects of lysophosphatidic acid on striatal astrocytes. Glia 28: 25-33.[Medline]
Pebay A, Toutant M, Premont J, Calvo C-F, Venance L, Cordier J, Glowinski J, and Tence M (2001) Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades. Eur J Neurosci 13: 2067-2076.[CrossRef]
Ramakers GJA and Moolenaar WH (1998) Regulation of astrocyte morphology by RhoA and lysophosphatidic acid. Exp Cell Res 245: 252-262.[CrossRef][Medline]
Sagi SA, Seaholtz TM, Kobiashaili M, Wilson BA, Toksoz D, and Brown JH (2001) Physical and functional interactions of Gq with Rho and its exchange factors. J Biol Chem 276: 15445-15452.
Sano T, Baker D, Virag T, Wada A, Yatomi Y, Kobayashi T, Igarashi Y, and Tigyi G (2002) Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in the blood. J Biol Chem 277: 21197-21206.
Siehler S and Manning DR (2002) Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors. Biochim Biophys Acta 1582: 94-99.[Medline]
Shikamoto Y and Morita T (1999) Expression of factor Xa in both the rat brain and cells of the central nervous system. FEBS Lett 463: 387-389.[CrossRef][Medline]
Spiegel S and Milstien S (2000) Sphingosine-1-phosphate: signaling inside and out. FEBS Lett 476: 55-57.[CrossRef][Medline]
Steiner MR, Urso JR, Klein J, and Steiner SM (2002) Multiple astrocyte responses to lysophosphatidic acids. Biochim Biophys Acta 1582: 154-160.[Medline]
Tabuchi S, Kume K, Aihara M, and Shimizu T (2000) Expression of lysophosphatidic acid receptor in rat astrocytes: mitogenic effect and expression of neurotrophic genes. Neurochemical Res 25: 573-582.[CrossRef][Medline]
Takuwa Y, Takuwa N, and Sugimoto N (2002) The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities. J Biochem 131: 767-771.
Tigyi G, Dyer DL, and Miledi R (1994) Lysophosphatidic acid possesses dual action in cell proliferation. Proc Natl Acad Sci USA 91: 1908-1912.
Tigyi G, Hong L, Yakubu M, Parfenova H, Shibata M, and Leffler CW (1995) Lysophosphatidic acid alters cerebrovascular reactivity in piglets. Am J Physiol 268: H2048-H2055.
Toullec D, Pianetti P, Coste H, Bellevrgue P, Grand-Perrett T, Ajakane M, Baudet V, Boissin P, Loriolle F, Duhame L, et al. (1991) The bisindolyleimide 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266: 15771-15781.
Van Brocklyn JR, Lee MJ, Menzeleev R, Olivera A, Edsall L, Cuvillier O, Thomas DM, Coopman PJ, Thangada S, Liu CH, et al. (1998) Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 142: 229-240.
Vaughan PJ and Cunningham DD (1993) Regulation of protease nexin-1 synthesis and secretion in cultured brain cells by injury-related factors. J Biol Chem 268: 3720-3727.
Vogt S, Grosse R, Schultz G, and Offermanns S (2003) Receptor dependent RhoA activation in G12/13 deficient cells: Genetic evidence for an involvement of Gq/11. J Biol Chem 278: 28743-28749.
Wang H, Ubl JJ, Stricker R and Reiser G (2002a) Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways. Am J Physiol 283: C1351-C1364.
Wang H, Ubl JJ, and Reiser G (2002b) Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia 37: 53-63.[CrossRef][Medline]
Weinstein JR, Gold SJ, Cunningham DD, and Gall CM (1995) Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 15: 2906-2919.[Abstract]
Xi G, Reiser G, and Keep RF (2003) The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective? J Neurochem 84: 3-9.[CrossRef][Medline]
Xie Y, Gibbs TC, Mukhin YV, and Meier KE (2002a) Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells. J Biol Chem 277: 32516-32526.
Xie Y, Gibbs TC, and Meier KE (2002b) Lysophosphatidic acid as an autocrine and paracrine mediator. Biochim Biophys Acta 1582: 270-281.[Medline]
Yamagata K, Tagami M, Torii Y, Takenaga F, Tsumagari S, Itoh S, Yamori Y, and Nara Y (2003) Sphingosine 1-phosphate induces the production of glial cell line-derived neurotrophic factor and cellular proliferation in astrocytes. Glia 41: 199-206.[Medline]
Yordy JS and Muise-Helmericks RC (2000) Signal transduction and the Ets family of transcription factors. Oncogene 19: 6503-6513.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  T. J. Wilson, K. C. Nannuru, and R. K. Singh Cathepsin G Recruits Osteoclast Precursors via Proteolytic Activation of Protease-Activated Receptor-1 Cancer Res., April 1, 2009; 69(7): 3188 - 3195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Gonzalez-Cabrera, E. Jo, M. G. Sanna, S. Brown, N. Leaf, D. Marsolais, M.-T. Schaeffer, J. Chapman, M. Cameron, M. Guerrero, et al. Full Pharmacological Efficacy of a Novel S1P1 Agonist That Does Not Require S1P-Like Headgroup Interactions Mol. Pharmacol., November 1, 2008; 74(5): 1308 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-P. Wu, K. Mizugishi, M. Bektas, R. Sandhoff, and R. L. Proia Sphingosine kinase 1/S1P receptor signaling axis controls glial proliferation in mice with Sandhoff disease Hum. Mol. Genet., August 1, 2008; 17(15): 2257 - 2264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Mannaioni, A. G. Orr, C. E. Hamill, H. Yuan, K. H. Pedone, K. L. McCoy, R. B. Palmini, C. E. Junge, C. J. Lee, M. Yepes, et al. Plasmin Potentiates Synaptic N-Methyl-D-aspartate Receptor Function in Hippocampal Neurons through Activation of Protease-activated Receptor-1 J. Biol. Chem., July 18, 2008; 283(29): 20600 - 20611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. d. S. e Spohr, J. W. Choi, S. E. Gardell, D. R. Herr, S. K. Rehen, F. C. A. Gomes, and J. Chun Lysophosphatidic Acid Receptor-dependent Secondary Effects via Astrocytes Promote Neuronal Differentiation J. Biol. Chem., March 21, 2008; 283(12): 7470 - 7479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen, E. Guerriero, K. Lathrop, and N. SundarRaj Rho/ROCK Signaling in Regulation of Corneal Epithelial Cell Cycle Progression Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 175 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. B. Hoelzinger, T. Demuth, and M. E. Berens Autocrine Factors That Sustain Glioma Invasion and Paracrine Biology in the Brain Microenvironment J Natl Cancer Inst, November 7, 2007; 99(21): 1583 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Kassel, N. A. Schulte, S. M. Parker, A. D. Lanik, and M. L. Toews Lysophosphatidic Acid Decreases Epidermal Growth Factor Receptor Binding in Airway Epithelial Cells J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 109 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Citro, S. Malik, E. A. Oestreich, J. Radeff-Huang, G. G. Kelley, A. V. Smrcka, and J. H. Brown Phospholipase C{varepsilon} is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation PNAS, September 25, 2007; 104(39): 15543 - 15548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Hamill, W. M. Caudle, J. R. Richardson, H. Yuan, K. D. Pennell, J. G. Greene, G. W. Miller, and S. F. Traynelis Exacerbation of Dopaminergic Terminal Damage in a Mouse Model of Parkinson's Disease by the G-Protein-Coupled Receptor Protease-Activated Receptor 1 Mol. Pharmacol., September 1, 2007; 72(3): 653 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Lee, G. Mannaioni, H. Yuan, D. H. Woo, M. B. Gingrich, and S. F. Traynelis Astrocytic control of synaptic NMDA receptors J. Physiol., June 15, 2007; 581(3): 1057 - 1081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, A. Bialkowska, R. Rusovici, S. Chanchevalap, H. Shim, J. P. Katz, V. W. Yang, and C. C. Yun Lysophosphatidic Acid Facilitates Proliferation of Colon Cancer Cells via Induction of Kruppel-like Factor 5 J. Biol. Chem., May 25, 2007; 282(21): 15541 - 15549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Croft and M. F. Olson The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1, and p27Kip1 Levels by Distinct Mechanisms Mol. Cell. Biol., June 15, 2006; 26(12): 4612 - 4627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Nicole, A. Goldshmidt, C. E. Hamill, S. D. Sorensen, A. Sastre, P. Lyuboslavsky, J. R. Hepler, R. J. McKeon, and S. F. Traynelis Activation of Protease-Activated Receptor-1 Triggers Astrogliosis after Brain Injury J. Neurosci., April 27, 2005; 25(17): 4319 - 4329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Itagaki, K. B. Kannan, and C. J. Hauser Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils J. Leukoc. Biol., February 1, 2005; 77(2): 181 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Martin, R. Goggel, A.-R. Ressmeyer, and S. Uhlig Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L250 - L257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Gruber, B. Kandler, C. Jindra, G. Watzak, and G. Watzek Dental Pulp Fibroblasts Contain Target Cells for Lysophosphatidic Acid Journal of Dental Research, June 1, 2004; 83(6): 491 - 495. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, onset of drug applications. Results are representative of two experiments.
