FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1978391012> ?p ?o ?g. }

• W1978391012 endingPage "31183" @default.
• W1978391012 startingPage "31177" @default.
• W1978391012 abstract "We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-α (TNF-α) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-α. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFκB activation may be responsible for PAR4AP-induced TNF-α production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations. We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-α (TNF-α) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-α. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFκB activation may be responsible for PAR4AP-induced TNF-α production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations. The inflammatory response is part of the normal defensive processes that may produce both beneficial and detrimental effects in tissues. In the central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; GPCR, G-protein-coupled receptor; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; PARAP, PAR agonist peptide; TNF-α, tumor necrosis factor-α; WB, Western blot; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin and eosin; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay. microglia are the major immune effector cells and, reactive microgliosis has been implicated in the pathogenesis of a broad range of CNS disorders. These include not only infectious CNS diseases but also acute CNS injuries such as traumatic brain injury (TBI) (1Csuka E. Hans V.H. Ammann E. Trentz O. Kossmann T. Morganti-Kossmann M.C. Neuroreport. 2000; 11: 2587-2590Crossref PubMed Scopus (93) Google Scholar), spinal cord injury (2Popovich P.G. Prog. Brain Res. 2000; 128: 43-58Crossref PubMed Scopus (96) Google Scholar), stroke, and brain ischemia (3Stoll G. Jander S. Schroeter M. Prog. Neurobiol. 1998; 56: 149-171Crossref PubMed Scopus (709) Google Scholar). In addition, microgliosis is also intimately involved in several chronic neurodegenerative disorders such as Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) (4McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (303) Google Scholar, 5Benveniste E.N. J. Mol. Med. 1997; 75: 165-173Crossref PubMed Scopus (475) Google Scholar). Accompanying the focal accumulation of activated microglia are elevated inflammatory mediators in the brain, such as tumor necrosis factor-α (TNF-α) and several classes of proteases, which are believed to contribute to the delayed or extended neuronal degeneration (1Csuka E. Hans V.H. Ammann E. Trentz O. Kossmann T. Morganti-Kossmann M.C. Neuroreport. 2000; 11: 2587-2590Crossref PubMed Scopus (93) Google Scholar, 2Popovich P.G. Prog. Brain Res. 2000; 128: 43-58Crossref PubMed Scopus (96) Google Scholar, 3Stoll G. Jander S. Schroeter M. Prog. Neurobiol. 1998; 56: 149-171Crossref PubMed Scopus (709) Google Scholar, 4McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (303) Google Scholar, 5Benveniste E.N. J. Mol. Med. 1997; 75: 165-173Crossref PubMed Scopus (475) Google Scholar). Since neurons are largely irreplaceable, microglial-based inflammatory damage in the CNS has attracted considerable attention recently. Therefore, efforts to understand the fundamental mechanisms of microglial activation, and unique proinflammatory mediators, may identify novel therapeutic strategies to eliminate microglial deleterious effects. Among a myriad of microglial regulatory factors, the multi-functional serine protease, thrombin, is a recently discovered microglial activator. Prothrombin is highly concentrated in blood, circulating at micromolar levels (6Fenton J.W.d. Ann. N. Y. Acad. Sci. 1986; 485: 5-15Crossref PubMed Scopus (193) Google Scholar). Prothrombin activation and extravasation of active thrombin into CNS parenchyma have been implicated in a number of CNS disorders such as TBI, stroke, ischemia, AD, and ALS (7Akiyama H. Ikeda K. Kondo H. McGeer P.L. Neurosci. Lett. 1992; 146: 152-154Crossref PubMed Scopus (166) Google Scholar, 8Festoff B.W. Smith R.A. Handbook of Amyotrophic Lateral Sclerosis. Vol. 12. Marcel Dekker, New York1992: 661-685Google Scholar, 9Cunningham D.D. Pulliam L. Vaughan P.J. Thromb. Haemost. 1993; 70: 168-171Crossref PubMed Scopus (61) Google Scholar, 10Nishino A. Suzuki M. Ohtani H. Motohashi O. Umezawa K. Nagura H. Yoshimoto T. J. Neurotrauma. 1993; 10: 167-179Crossref PubMed Scopus (212) Google Scholar, 11Ho G.J. Smirnova I.V. Akaaboune M. Hantai D. Festoff B.W. Biomed. Pharmacother. 1994; 48: 296-304Crossref PubMed Scopus (31) Google Scholar, 12Lee K.R. Kawai N. Kim S. Sagher O. Hoff J.T. J. Neurosurg. 1997; 86: 272-278Crossref PubMed Scopus (346) Google Scholar). It has long been known that thrombin is a proinflammatory agent in other tissues (13Strukova S.M. Biochemistry (Mosc). 2001; 66: 8-18Crossref PubMed Scopus (79) Google Scholar) but it also can induce infiltration of inflammatory cells, edema, and reactive gliosis in the CNS in vivo (10Nishino A. Suzuki M. Ohtani H. Motohashi O. Umezawa K. Nagura H. Yoshimoto T. J. Neurotrauma. 1993; 10: 167-179Crossref PubMed Scopus (212) Google Scholar). However, specific demonstration of direct activation of microglial cells by thrombin was only reported recently (14Moller T. Hanisch U.K. Ransom B.R. J. Neurochem. 2000; 75: 1539-1547Crossref PubMed Scopus (160) Google Scholar, 15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar, 16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). From the therapeutic point of view, systemic inhibition of the thrombin proteolytic activity is certain to induce severe anti-coagulation side effects, possibly even fatal bleeding in brain and elsewhere (17Festoff B.W. Mattson M.P. Neuroprotective Signal Transduction. Humana Press, Totowa, NJ1997: 221-241Google Scholar). Therefore, identification of specific cellular/molecular target(s) mediating thrombin effect holds the key for developing safe and effective alternative therapeutic strategies. Thrombin-induced cellular effects are primarily mediated by means of G-protein-coupled receptors (GPCRs) known as protease-activated receptors (PARs) (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar). Among four PARs (PAR1–4) identified so far, thrombin activates PAR1, PAR3, and PAR4, but not PAR2 (19Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar, 20Ishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. Tram T. Coughlin S.R. Nature. 1997; 386: 502-506Crossref PubMed Scopus (804) Google Scholar, 21Xu W.F. Andersen H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6642-6646Crossref PubMed Scopus (759) Google Scholar, 22Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Crossref PubMed Scopus (833) Google Scholar). Regarding molecular mechanisms underlying thrombin-induced microglial activation, others have suggested that PAR1 is not involved in activation of microglia, using parameters such as inducible nitric-acid synthase (15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar). Our recent study demonstrated that although the prototypic PAR1 participates in thrombin-induced microglial proliferation, it does not mediate release of thrombin-induced potentially detrimental cytokines, such as TNF-α. This has cast in doubt the perspectives of PAR1 as the therapeutic target to exploit and suggested that more specific therapeutic target(s) remained to be identified. Since PAR3 has been suggested to function primarily as a chaperone for PAR4 rather than transducing signals on its own (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, 23Sambrano G.R. Weiss E.J. Zheng Y.W. Huang W. Coughlin S.R. Nature. 2001; 413: 74-78Crossref PubMed Scopus (457) Google Scholar), we have concentrated on determining just how PAR4 might participate in thrombin-induced microglial activation in this study. The evidence provided here indicates that persistent PAR4 intracellular signaling is largely responsible for thrombin-induced TNF-α release in microglia. The mechanisms involved include prolonged phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs) and subsequent NFκB activation. Materials—Recombinant human α-thrombin was a gift from John Fenton, II, Ph.D. (Wadsworth Public Health Laboratories, Albany, NY). Synthetic PAR1AP (SFFLRN) and PAR4AP (GYPGKF) were purchased from Bachem (Torrance, CA). PAR4 antisense (N185, 5′-TACAGCAGCGGCCAGCACAT-3′-c6 amine) and sense (N186, 5′-ATGTGCTGGCCGCTGCTGTA-3′-c6 amine) oligonucleotides were synthesized by ResGen, Invitrogen Corp. (Carlsbad, CA). Goat polyclonal antibody (pAb) to PAR4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific and total p44/42 MAPKs pAbs were purchased from Cell Signaling (Beverly, MA). Griffonia simplicifolia isolectin B4 (GSI-B4)-FITC and poly-l-lysine were from Sigma. A mouse TNF-α ELISA kit and a mouse TNF-α monoclonal Ab (mAb) were bought from R&D systems (Minneapolis, MN). A mAb to γ-tubulin was a gift from Dr. Robert E. Palazzo (University of Kansas, Lawrence, KS). Fluorescent-conjugated secondary antibodies and dyes were bought from Molecular Probes (Eugene, OR). Alkaline phosphatase-conjugated secondary Abs were purchased from Sigma. Pre-cast sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels were from Novex (San Diego, CA). The bicinchoninic acid (BCA) method kit was from Pierce. Polyvinylidene difluoride membranes were from Millipore (Bedford, MA). Cell culture supplies, Iscoves modified Dulbecco's medium (IMDM) and Dulbecco's modified Eagle's medium (DMEM) media, fetal bovine sera, and other routinely used reagents were from Sigma, GIBCOL, and Fisher. Cell Culture—The mouse microglial cell line, N9, was a gift from Drs. Bottani and Ricciardi-Castagnoli (Università Degli Studi Di Milano-Bicocca, Milan, Italy). Primary microglial cultures from the brains of neonatal C57BL6 mice were prepared and characterized as we previously described (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). One night before treatment, we replaced serum-containing media in primary and N9 microglial cells with serum-free DMEM and IMDM (Gly/Ser-free) media, respectively. All treatments were also performed under serum-free conditions in the corresponding culture medium. RT-PCR—Cultured cells were washed and then lysed in Trizol and total RNAs were extracted according to the manufacturer's recommendations. RNA concentrations were based on absorbance at 230, 260, and 280 nm. Reverse transcription-PCR was performed with 0.5 μg of total RNA, downstream primer sequence 5′-CGTACCTTCTCCCTGAACTC and upstream primer 5′-CCCCAGCATCTACGATGATG in a 50-μl volume with 5 units of rTth polymerase (Applied Biosystems, Foster City, CA), 0.3 mm dNTPs, 2.5 mm MnOAc, 0.45 μm primer incubated at 60 °C for 30 min, 94 °C for 3 min, followed by 40 cycles of 94 °C for 1 min and 60 °C for 1 min. cDNA products were visualized after agarose gel electrophoresis and staining with ethidium bromide under UV light, and images were captured and analyzed with the aid of a CCD camera and NIH Image software. Intracortical Infusion—ALZET® Mini-osmotic pumps (Model 1003D, Alza Corp., Palo Alto, CA) were prepared according to the manufacturer's instructions. Briefly, 1 day before surgery, all minipumps were primed in sterile 0.9% saline for at least 6 h, and then weighed, filled with treatment reagents, and weighed again. The concentrations of reagents filled in each minipump were calculated according to the weight difference before and after filling the pump with saline (100 μl/pump), human α-thrombin (11 units/100 μl/pump), PAR1AP (100 μm/100 μl/pump), and PAR4AP (100 μm/100 μl/pump). All procedures were performed under sterile conditions. Twelve male Sprague-Dawley rats (3–4 months old; 250–300 gm) were grouped as: 1) saline, 2) thrombin, 3) PAR1AP, and 4) PAR4AP with 3 rats per group. For surgery, animals were anesthetized with 2% xylazine plus 10% ketamine and immobilized in a rat stereotaxic apparatus. After incising the skin and removing the skull connective tissues, a small hole was drilled followed by placing a 30-gauge cannula into the cortical region using stereotaxic coordinates of bregma: –4.80 mm, right lateral: 3.00 mm and ventral: 1.60 mm according to Paxinos' Rat Brain Stereotaxic Coordinates (36Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed. Academic Press, Sydney1986Google Scholar). The pre-filled ALZET osmotic minipump connected to the cannula was placed under the dorsal skin. Ten days following surgery, animals were re-anesthetized and perfused with saline followed by 4% paraformaldehyde before removing the brains for immunohistochemistry. Immunohistochemistry—Rat brains were routinely paraffin embedded, formalin fixed and sectioned with a thickness of 20 μm. After routine de-paraffinization, rehydration and blocking, sections were stained with mAb to TNF-α (1:250) followed by goat anti-mouse-Cy3 conjugates (1:500) and GSI-B4-FITC (1:500) staining. The slides were then mounted with Fluoromount and observed under a Nikon Bio-Radiance confocal microscope. Hematoxylin and eosin (HE) staining was performed as previously described (24Suo Z. Fang C. Crawford F. Mullan M. Brain Res. 1997; 762: 144-152Crossref PubMed Scopus (84) Google Scholar). Western Blot (WB) Analysis—WB for PAR4 (1:500 dilution of the PAR4 pAb), γ-tubulin (1:1,000), total and phospho-p44/42 MAPK, and semi-quantitative analysis of protein band density were performed routinely as previously described (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). Ca2 + Imaging—The relative [Ca2+]i changes in response to treatments of thrombin, PAR1AP and PAR4AP in primary microglial cultures were performed with Fluo-4 under a Nikon Bio-Radiance confocal microscope as previously described (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts from treated N9 microglial cells were prepared essentially as previously described (25Haglund R.E. Rothblum L.I. Mol. Cell Biochem. 1987; 73: 11-20Crossref PubMed Scopus (27) Google Scholar). Briefly, cells were washed in cold PBS and harvested by brief centrifugation. Cells were then swollen in hypotonic buffer (10 mm HEPES pH 7.9, 1.5 mm MgCl2, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 10 mm KCl) supplied with complete protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitors (200 pm cypermethrin, 25 μm dephostatin, 50 nm okadaic acid, 50 pm NIPP-1, 25 μm p-bromoter-ramisole oxalate, 5 μm camtharidin, 5 nm microcystin), and disrupted by homogenization with a glass Dounce homogenizer. After centrifugation at 4 °C at 15,000 × g for 10 min, nuclear pellets were collected and resuspended in half-pellet volume of low salt buffer (20 mm HEPES, 25% glycerol, 1.5 mm MgCl2, 0.02 m KCl, 0.2 mm EDTA, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol). With stirring, an additional half pellet volume of high salt buffer (1.2 m KCl in low salt buffer) was added and followed by 30 min incubation with gentle agitation at 4 °C. After centrifugation, nuclear protein extracts were dialyzed against 50-fold excess volume of dialysis buffer (20 mm HEPES, 20% glycerol, 100 mm KCl, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol). Protein concentrations were then determined by the BCA assay. For EMSA, nuclear proteins (5 μg) were incubated for 10 min at 25 °C with binding buffer (Promega) and phophatase inhibitor mixture, followed by addition of 2 μl of 175 nm CY5 fluorescent-labeled oligonucleotide (26Ruscher K. Reuter M. Kupper D. Trendelenburg G. Dirnagl U. Meisel A. J. Biotechnol. 2000; 78: 163-170Crossref PubMed Scopus (66) Google Scholar) containing specific binding sequences for NFκB: (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and incubation at room temperature for 30 min. Reactions were stopped by adding 1 μl of 10-fold TBE and the protein-DNA complexes were separated on a 6% DNA Retardation Gel (Invitrogen) and scanned by a Storm 860 Phospho-fluorImager. Measurements of TNF-α Production—TNF-α production into microglial culture media was measured and standardized as previously described (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). Statistical Analysis—All qualitative analysis was repeated at least three times. Calcium imaging data were averages taken from three separate experiments. TNF-α levels were measured in duplicate for each sample, and each treatment was quadruplicated. Quantitative data are expressed as mean ± S.E. and analyzed by ANOVA using StatView 6.0 (Abacus Systems, Mountain View, CA). Post-hoc comparisons of means were made using Scheffe's or Tukey's method where appropriate. Mouse Microglia Express PAR4 —To determine whether or not PAR4 is involved in thrombin-induced microglial activation, we first determined whether PAR4 was expressed in microglia. To accomplish this we used both primary microglial cultures derived from C57BL6 mouse cortices with a purity of 96–98%, as we previously reported (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar), as well as a clonal mouse microglial cell line, N9. Both primary and clonal microglia were cultured in normal growth media, and the cell lysates were analyzed by WB with PAR4-specific pAb. The results shown in Fig. 1 indicated that both primary and clonal mouse microglial cells constitutively express equivalent levels of PAR4 protein. In addition, we have also found PAR4 mRNA expression in the clonal N9 microglial cells (100% purity without contamination of other cell types, Fig. 1). Moreover, others have recently reported in abstract form that N9 and other clonal and primary microglial cells expressed PAR4 mRNA (27Moller T. Baslcaitis S. Xie Y. Weinstein J. Ransom B.R. Hanisch U.K. Soc. for Neurosci. Abst. 2002; 28: 101.103Google Scholar). Therefore, these studies clearly show that murine microglial cells express PAR4 at both mRNA and protein levels. PAR4AP Mimics Thrombin-induced Microglial TNF-α Production in Vitro—Having established that murine microglia express PAR4, we then assessed the effects of PAR4AP on microglial TNF-α production. For this purpose, primary microglial cultures were treated with increasing doses of either human α-thrombin, PAR1AP, or PAR4AP for 24 h as indicated (Fig. 2). TNF-α production in the culture media measured by ELISA showed that both thrombin (p < 0.001, dose versus thrombin) and PAR4AP (p < 0.01, dose versus PAR4AP), but not PAR1AP, dose-dependently induced TNF-α release from the microglial cultures. A peptide containing the same amino acids as PAR4AP, but in a scrambled sequence (KPGFYG), did not induce significant increase of TNF-α production, even at 100 μm, in a parallel experiment (data not shown). These results indicated that PAR4AP, but not PAR1AP, was able to mimic the thrombin-induced microglial TNF-α production in vitro. Thrombin and PAR4AP Activate Microglia in Vivo—To confirm this in vitro finding and extend it in vivo, we infused either saline, α-thrombin (11 units/100 μl), PAR1AP (100 μm/100 μl) or PAR4AP (100 μm/100 μl) separately into rat cerebral cortices with the aid of a pre-set osmotic pump (at 100 μl/72 h) to control the infusion rate. Ten days after the initial stereotactic surgery, brain sections were obtained for histological staining with a microglial marker, GSI-B4, along with a mAb to TNF-α (Fig. 3). We found that thrombin infusion induced significant microgliosis compared with saline infusion (restricted to the needle track). A significant proportion of the reactive microglial cells stained positively for TNF-α. In contrast, although PAR1AP induced a mild microglial proliferation, we were unable to detect TNF-α-positive cells with this peptide. On the other hand, we were surprised to find that PAR4AP induced a massive TNF-α-positive microgliosis, even greater than that observed with α-thrombin. HE staining indicated that PAR1AP induced a mild increase of dark blue-stained small nuclei (assumed microglial cells) without significant lesion, while both thrombin and PAR4AP induced significant increase of microglial cells along with apparent cortical lesions. These results indicate that both thrombin and PAR4AP, but not PAR1AP, were able to induce TNF-α-positive microgliosis in vivo. As such, they generally agree with our in vitro findings. Therefore, these novel findings indicate that PAR4AP can mimic thrombin-induced microglial TNF-α production both in vitro and in vivo. Down-regulation of PAR4 Reduces Thrombin- and PAR4AP-induced TNF-α—Although PAR4AP has been shown to specifically activate PAR4 in other cell types (28Faruqi T.R. Weiss E.J. Shapiro M.J. Huang W. Coughlin S.R. J. Biol. Chem. 2000; 275: 19728-19734Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), to further confirm that PAR4AP-induced TNF-α production in microglial cells was indeed mediated via activation of PAR4, we attempted to down-regulate PAR4 and measured TNF-α production. For this purpose, we synthesized an antisense oligonucleotide (N185) that spanned the initial coding sequence of the murine PAR4 gene as well as a sense oligonucleotide (N186) with a complementary sequence to N185 (“Experimental Procedures”). Following 24-hour treatment of N9 microglial cells, WB of PAR4 revealed a dose-dependent (p < 0.01) reduction of PAR4 expression induced by the antisense N185. In contrast, the sense N186, at a concentration similar to the highest (1 μm) used for N185, failed to show significant effect (Fig. 4). When we increased the concentration of the antisense N185 further to 5 μm, no additional effect on PAR4 down-regulation (∼20% PAR4 remained after 1 μm N185 treatment, data not shown) was found. In addition, after stripping, re-probing with anti-γ-tubulin, a centrosome marker, showed equivalent amount of γ-tubulin expression among different lanes. The results confirmed that the loss of PAR4 expression is not related to cell death or an anti-proliferative response to the oligonucleotides. In parallel with the PAR4 WB analysis, the same PAR4 oligonucleotide-treated microglial cells were further treated with a single dose of either thrombin (100 nm) or PAR4AP (100 μm) for an additional 24 h. TNF-α production in the culture media was quantified by ELISA (Fig. 4). We found that the PAR4 antisense N185 induced a dose-dependent decrease of TNF-α secretion induced by thrombin (p < 0.001, N185 dose versa thrombin) and PAR4AP (p < 0.001, N185 dose versa PAR4AP). In contrast, the PAR4 sense N186 showed no effects on TNF-α secretion induced either by thrombin or PAR4AP. These results indicate that activation of PAR4 specifically mediated effects of thrombin and PAR4AP on microglial TNF-α induction. PAR4 Activation Induces Prolonged [Ca2 + ] i Increase and p44/42 MAPK Activation—Since both PAR1 and PAR4 appear to be expressed and fully functional in microglia, we went further to investigate why these two thrombin receptors might mediate distinct functions, particularly regarding TNF-α induction. Previous data from fibroblasts indicated that soon after activation, PAR1 was immediately desensitized while PAR4 was shut off much more slowly than PAR1 (29Shapiro M.J. Weiss E.J. Faruqi T.R. Coughlin S.R. J. Biol. Chem. 2000; 275: 25216-25221Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). We have recently found that, once activated, microglial PAR1 is rapidly desensitized by G-protein-coupled receptor kinase-5 and -2, while a relatively nonspecific (unrelated to agonist stimulation), sluggish but constitutive PAR4 desensitization may take place in microglial cells. 2Z. Suo, M. Wu, B. A. Citron, G. T. Wong, and B. W. Festoff, manuscript in preparation. To determine if the differential desensitization of these receptors might be responsible for their distinct functions, we compared PAR1 and PAR4 downstream signaling in parallel. Increase of [Ca2+]i is one such downstream signal following PAR activation. When microglial cells were treated with 100 μm PAR1AP, a sharp increase of [Ca2+]i was followed by an immediate and complete decrease of [Ca2+]i to baseline (Fig. 5). Compared with PAR1AP, 100 μm PAR4AP induced a similar steep, although smaller, increase of [Ca2+]i followed by a significantly slower decline curve to ∼300 nm. It then remained at 200 nm above baseline until the end of the record (at least 4 min). The similar upwardly rising curves induced by PAR1AP and PAR4AP indicated that the ability for the two peptides to activate their corresponding receptors was not significantly different. However, the distinct downward curves implied that these two receptors were shut off differently. Changes of [Ca2+]i induced by 100 nm thrombin, which are sufficient to activate both PAR1 and PAR4, showed a curve that appeared to reflect a combination of effects of both PAR1 and PAR4 activation: a sharp upwardly rising curve and a downward curve, comprised of a rapid declining phase followed by a sustained phase. Together, these data suggested that PAR4 activation induced a prolonged [Ca2+]i increase as opposed to PAR1 activation, supporting the concept that PAR4 desensitization is considerably slower than PAR1. In addition to [Ca2+]i increase, we have previously shown that PAR1 activation in microglial cells induced a transient p44/42 MAPK activation with the peak occurring at 30 min (16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). To further define differences between PAR1 and PAR4 signaling, we analyzed levels of phosphospecific and total p44/42 MAPKs in microglial cells at different time points (Fig. 6). We found that thrombin (100 nm) and PAR1AP (100 μm), as well as PAR4AP (100 μm), induced significant elevation of phosphospecific p44/42 MAPKs at 30 min. However, PAR1AP-induced p44/42 MAPK phosphorylation returned to control level while thrombin- and PAR4AP-induced p44/42 MAPK phosphorylation remained elevated at least 6–24 h after treatments. These results suggested that as compared with PAR1 signaling, PAR4 activation not only induced a prolonged [Ca2+]i increase but also a prolongation of p44/42 MAPK activation, which further supports the observation of “sluggish” PAR4 desensi" @default.
• W1978391012 created "2016-06-24" @default.
• W1978391012 creator A5008786196 @default.
• W1978391012 creator A5031090962 @default.
• W1978391012 creator A5032159481 @default.
• W1978391012 creator A5062350439 @default.
• W1978391012 creator A5090297236 @default.
• W1978391012 date "2003-08-01" @default.
• W1978391012 modified "2023-10-11" @default.
• W1978391012 title "Persistent Protease-activated Receptor 4 Signaling Mediates Thrombin-induced Microglial Activation" @default.
• W1978391012 cites W1266806140 @default.
• W1978391012 cites W1623022149 @default.
• W1978391012 cites W1811402656 @default.
• W1978391012 cites W1842710166 @default.
• W1978391012 cites W1964499190 @default.
• W1978391012 cites W1994551148 @default.
• W1978391012 cites W1995712454 @default.
• W1978391012 cites W1997086569 @default.
• W1978391012 cites W1999190313 @default.
• W1978391012 cites W2006331624 @default.
• W1978391012 cites W2031850340 @default.
• W1978391012 cites W2033852349 @default.
• W1978391012 cites W2037583192 @default.
• W1978391012 cites W2039222951 @default.
• W1978391012 cites W2043526402 @default.
• W1978391012 cites W2050940330 @default.
• W1978391012 cites W2071793324 @default.
• W1978391012 cites W2072101129 @default.
• W1978391012 cites W2072624957 @default.
• W1978391012 cites W2072913586 @default.
• W1978391012 cites W2074707530 @default.
• W1978391012 cites W2075914468 @default.
• W1978391012 cites W2081753893 @default.
• W1978391012 cites W2082431283 @default.
• W1978391012 cites W2101958789 @default.
• W1978391012 cites W2116644918 @default.
• W1978391012 cites W2122342801 @default.
• W1978391012 cites W2123596783 @default.
• W1978391012 cites W2126787472 @default.
• W1978391012 cites W2401781149 @default.
• W1978391012 cites W4230951225 @default.
• W1978391012 cites W4300004351 @default.
• W1978391012 doi "https://doi.org/10.1074/jbc.m302137200" @default.
• W1978391012 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12775717" @default.
• W1978391012 hasPublicationYear "2003" @default.
• W1978391012 type Work @default.
• W1978391012 sameAs 1978391012 @default.
• W1978391012 citedByCount "106" @default.
• W1978391012 countsByYear W19783910122012 @default.
• W1978391012 countsByYear W19783910122013 @default.
• W1978391012 countsByYear W19783910122014 @default.
• W1978391012 countsByYear W19783910122015 @default.
• W1978391012 countsByYear W19783910122016 @default.
• W1978391012 countsByYear W19783910122017 @default.
• W1978391012 countsByYear W19783910122018 @default.
• W1978391012 countsByYear W19783910122019 @default.
• W1978391012 countsByYear W19783910122020 @default.
• W1978391012 countsByYear W19783910122021 @default.
• W1978391012 countsByYear W19783910122022 @default.
• W1978391012 countsByYear W19783910122023 @default.
• W1978391012 crossrefType "journal-article" @default.
• W1978391012 hasAuthorship W1978391012A5008786196 @default.
• W1978391012 hasAuthorship W1978391012A5031090962 @default.
• W1978391012 hasAuthorship W1978391012A5032159481 @default.
• W1978391012 hasAuthorship W1978391012A5062350439 @default.
• W1978391012 hasAuthorship W1978391012A5090297236 @default.
• W1978391012 hasBestOaLocation W19783910121 @default.
• W1978391012 hasConcept C125268873 @default.
• W1978391012 hasConcept C149577978 @default.
• W1978391012 hasConcept C170493617 @default.
• W1978391012 hasConcept C181199279 @default.
• W1978391012 hasConcept C185592680 @default.
• W1978391012 hasConcept C203014093 @default.
• W1978391012 hasConcept C2776714187 @default.
• W1978391012 hasConcept C2777292125 @default.
• W1978391012 hasConcept C55493867 @default.
• W1978391012 hasConcept C62478195 @default.
• W1978391012 hasConcept C75184817 @default.
• W1978391012 hasConcept C86803240 @default.
• W1978391012 hasConcept C89560881 @default.
• W1978391012 hasConcept C95444343 @default.
• W1978391012 hasConceptScore W1978391012C125268873 @default.
• W1978391012 hasConceptScore W1978391012C149577978 @default.
• W1978391012 hasConceptScore W1978391012C170493617 @default.
• W1978391012 hasConceptScore W1978391012C181199279 @default.
• W1978391012 hasConceptScore W1978391012C185592680 @default.
• W1978391012 hasConceptScore W1978391012C203014093 @default.
• W1978391012 hasConceptScore W1978391012C2776714187 @default.
• W1978391012 hasConceptScore W1978391012C2777292125 @default.
• W1978391012 hasConceptScore W1978391012C55493867 @default.
• W1978391012 hasConceptScore W1978391012C62478195 @default.
• W1978391012 hasConceptScore W1978391012C75184817 @default.
• W1978391012 hasConceptScore W1978391012C86803240 @default.
• W1978391012 hasConceptScore W1978391012C89560881 @default.
• W1978391012 hasConceptScore W1978391012C95444343 @default.
• W1978391012 hasIssue "33" @default.
• W1978391012 hasLocation W19783910121 @default.
• W1978391012 hasOpenAccess W1978391012 @default.

• next