Research Article
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Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia

Year 2021, Volume: 5 Issue: 1, 6 - 13, 26.02.2021

Kemal Uğur Tüfekci , Halil Ateş Şermin Genç

https://doi.org/10.30621/jbachs.854244

Abstract

Objectives: Microglial cells are the central regulators of inflammatory responses in the brain and spinal cord. In addition to surveillance during resting state, they become activated due to microbial molecules and pathological insults. Endogenously expressed Activated protein C (APC) is an anticoagulant molecule with anti-inflammatory and cytoprotective roles, mediated by one of its receptors, Endothelial protein C receptor (EPCR). This study aimed to examine the basal and inducible expression of EPCR and unravel the regulatory mediators of its expression in microglia.
Methods: We studied probable effects of Lipopolysaccharide (LPS), Peptidoglycan (PGN), and Polyinosinic–polycytidylic acid [Poly(I:C)] on EPCR mRNA and protein levels in N9 mouse microglial cells by qPCR and flow cytometry. Then, Cyclosporin A (CsA) and Mithramycin A (MMA) were used to inhibit transcription factors in the promoter region of the EPCR gene, which are Nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1), and specificity protein 1 (Sp1).
Results: As a result, the Sp1 transcription factor’s chemical inhibition impaired the upregulating effects of LPS and PGN on EPCR expression.
Conclusion: Thus, our data suggest that LPS and PGN gave rise to increased EPCR levels in microglia, mainly through the Sp1 transcription factor.

Keywords

microglia endothelial protein c receptor lipopolysaccharide peptidoglycan Sp1 transcription factor

Supporting Institution

The Scientific and Technological Research Council of Turkey (TUBITAK)

Project Number

114Z577

Thanks

The authors thank Dr. Erden Eren for his technical assistance.

References

  • 1) Fukudome K, Esmon CT. Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR. J Biol Chem. 1995;270(10):5571-5577.
  • 2) Esmon CT. The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness. Crit Care. 2001;5(2):S7-12.
  • 3) Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost. 2006;32 Suppl 1:49-60.
  • 4) Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas. 2004;47(4):305-314.
  • 5) Gleeson EM, O'Donnell JS, Preston RJ. The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling. Cell Mol Life Sci. 2012;69(5):717-726.
  • 6) Iwasaki H, Arai F, Kubota Y, Dahl M, Suda T. Endothelial protein C receptor-expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver. Blood. 2010;116(4):544-553.
  • 7) Park SY, Lee HE, Li H, Shipitsin M, Gelman R, Polyak K. Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer. Clin Cancer Res. 2010;16(3):876-887.
  • 8) Murakami K, Okajima K, Uchiba M, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol. 1997;272(2 Pt 1):L197-202.
  • 9) Yuksel M, Okajima K, Uchiba M, Horiuchi S, Okabe H. Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factor-kappa B and activator protein-1 in human monocytes. Thromb Haemost. 2002;88(2):267-273.
  • 10) Iwaki T, Cruz DT, Martin JA, Castellino FJ. A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse. Blood. 2005;105(6):2364-2371.
  • 11) Li W, Zheng X, Gu J, et al. Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin. J Thromb Haemost. 2005;3(7):1351-1359.
  • 12) Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor. Biochem J. 2003;373(Pt 1):65-70.
  • 13) Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005;105(8):3178-3184.
  • 14) Bae JS, Yang L, Manithody C, Rezaie AR. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. Blood. 2007;110(12):3909-3916.
  • 15) Nayak RC, Sen P, Ghosh S, et al. Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications. Blood. 2009;114(9):1974-1986.
  • 16) Nayak RC, Keshava S, Esmon CT, Pendurthi UR, Rao LV. Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa. PLoS One. 2013;8(3):e59304.
  • 17) Griffin JH, Zlokovic BV, Mosnier LO. Protein C anticoagulant and cytoprotective pathways. Int J Hematol. 2012;95(4):333-345.
  • 18) Thiyagarajan M, Fernandez JA, Lane SM, Griffin JH, Zlokovic BV. Activated protein C promotes neovascularization and neurogenesis in postischemic brain via protease-activated receptor 1. J Neurosci. 2008;28(48):12788-12797.
  • 19) Zlokovic BV, Zhang C, Liu D, Fernandez J, Griffin JH, Chopp M. Functional recovery after embolic stroke in rodents by activated protein C. Ann Neurol. 2005;58(3):474-477.
  • 20) Guo H, Liu D, Gelbard H, et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004;41(4):563-572.
  • 21) Zhong Z, Ilieva H, Hallagan L, et al. Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells. J Clin Invest. 2009;119(11):3437-3449.
  • 22) Deane R, LaRue B, Sagare AP, Castellino FJ, Zhong Z, Zlokovic BV. Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier. J Cereb Blood Flow Metab. 2009;29(1):25-33.
  • 23) Righi M, Mori L, De Libero G, et al. Monokine production by microglial cell clones. Eur J Immunol. 1989;19(8):1443-1448.
  • 24) Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-408.
  • 25) Gu JM, Fukudome K, Esmon CT. Characterization and regulation of the 5'-flanking region of the murine endothelial protein C receptor gene. J Biol Chem. 2000;275(17):12481-12488.
  • 26) Lee MY, Garvey SM, Ripley ML, Wamhoff BR. Genome-wide microarray analyses identify the protein C receptor as a novel calcineurin/nuclear factor of activated T cells-dependent gene in vascular smooth muscle cell phenotypic modulation. Arterioscler Thromb Vasc Biol. 2011;31(11):2665-2675.
  • 27) Balcaitis S, Xie Y, Weinstein JR, et al. Expression of proteinase-activated receptors in mouse microglial cells. Neuroreport. 2003;14(18):2373-2377.
  • 28) Bushell TJ, Plevin R, Cobb S, Irving AJ. Characterization of proteinase-activated receptor 2 signalling and expression in rat hippocampal neurons and astrocytes. Neuropharmacology. 2006;50(6):714-725.
  • 29) Ubl JJ, Reiser G. A novel proteolytic mechanism for termination of the CA2+ signalling evoked by proteinase-activated receptor-1 (PAR-1) in rat astrocytes. Adv Exp Med Biol. 2000;477:323-329.
  • 30) Esmon CT. The endothelial protein C receptor. Curr Opin Hematol. 2006;13(5):382-385.
  • 31) Galligan L, Livingstone W, Volkov Y, et al. Characterization of protein C receptor expression in monocytes. Br J Haematol. 2001;115(2):408-414.
  • 32) Nakamura M, Gabazza EC, Imoto I, et al. Anti-inflammatory effect of activated protein C in gastric epithelial cells. J Thromb Haemost. 2005;3(12):2721-2729.
  • 33) Sturn DH, Kaneider NC, Feistritzer C, Djanani A, Fukudome K, Wiedermann CJ. Expression and function of the endothelial protein C receptor in human neutrophils. Blood. 2003;102(4):1499-1505.
  • 34) Song D, Ye X, Xu H, Liu SF. Activation of endothelial intrinsic NF-{kappa}B pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice. Blood. 2009;114(12):2521-2529.
  • 35) Kerschen EJ, Fernandez JA, Cooley BC, et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med. 2007;204(10):2439-2448.
  • 36) Zheng X, Li W, Song Y, et al. Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia. J Thromb Haemost. 2007;5(7):1394-1400.
  • 37) von Drygalski A, Furlan-Freguia C, Ruf W, Griffin JH, Mosnier LO. Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol. 2013;33(4):769-776.
  • 38) Seol JW, Lee YJ, Jackson CJ, Sambrook PN, Park SY. Activated protein C inhibits bisphosphonate-induced endothelial cell death via the endothelial protein C receptor and nuclear factor-kappaB pathways. Int J Mol Med. 2011;27(6):835-840.
  • 39) Hayashi T, Nakamura H, Okada A, et al. Organization and chromosomal localization of the human endothelial protein C receptor gene. Gene. 1999;238(2):367-373.
  • 40) Lagrange J, Li Z, Fassot C, et al. Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice. FASEB J. 2014;28(5):2062-2072.

Year 2021, Volume: 5 Issue: 1, 6 - 13, 26.02.2021

Kemal Uğur Tüfekci , Halil Ateş Şermin Genç

https://doi.org/10.30621/jbachs.854244

Abstract

Project Number

114Z577

References

  • 1) Fukudome K, Esmon CT. Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR. J Biol Chem. 1995;270(10):5571-5577.
  • 2) Esmon CT. The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness. Crit Care. 2001;5(2):S7-12.
  • 3) Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost. 2006;32 Suppl 1:49-60.
  • 4) Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas. 2004;47(4):305-314.
  • 5) Gleeson EM, O'Donnell JS, Preston RJ. The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling. Cell Mol Life Sci. 2012;69(5):717-726.
  • 6) Iwasaki H, Arai F, Kubota Y, Dahl M, Suda T. Endothelial protein C receptor-expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver. Blood. 2010;116(4):544-553.
  • 7) Park SY, Lee HE, Li H, Shipitsin M, Gelman R, Polyak K. Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer. Clin Cancer Res. 2010;16(3):876-887.
  • 8) Murakami K, Okajima K, Uchiba M, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol. 1997;272(2 Pt 1):L197-202.
  • 9) Yuksel M, Okajima K, Uchiba M, Horiuchi S, Okabe H. Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factor-kappa B and activator protein-1 in human monocytes. Thromb Haemost. 2002;88(2):267-273.
  • 10) Iwaki T, Cruz DT, Martin JA, Castellino FJ. A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse. Blood. 2005;105(6):2364-2371.
  • 11) Li W, Zheng X, Gu J, et al. Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin. J Thromb Haemost. 2005;3(7):1351-1359.
  • 12) Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor. Biochem J. 2003;373(Pt 1):65-70.
  • 13) Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005;105(8):3178-3184.
  • 14) Bae JS, Yang L, Manithody C, Rezaie AR. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. Blood. 2007;110(12):3909-3916.
  • 15) Nayak RC, Sen P, Ghosh S, et al. Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications. Blood. 2009;114(9):1974-1986.
  • 16) Nayak RC, Keshava S, Esmon CT, Pendurthi UR, Rao LV. Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa. PLoS One. 2013;8(3):e59304.
  • 17) Griffin JH, Zlokovic BV, Mosnier LO. Protein C anticoagulant and cytoprotective pathways. Int J Hematol. 2012;95(4):333-345.
  • 18) Thiyagarajan M, Fernandez JA, Lane SM, Griffin JH, Zlokovic BV. Activated protein C promotes neovascularization and neurogenesis in postischemic brain via protease-activated receptor 1. J Neurosci. 2008;28(48):12788-12797.
  • 19) Zlokovic BV, Zhang C, Liu D, Fernandez J, Griffin JH, Chopp M. Functional recovery after embolic stroke in rodents by activated protein C. Ann Neurol. 2005;58(3):474-477.
  • 20) Guo H, Liu D, Gelbard H, et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004;41(4):563-572.
  • 21) Zhong Z, Ilieva H, Hallagan L, et al. Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells. J Clin Invest. 2009;119(11):3437-3449.
  • 22) Deane R, LaRue B, Sagare AP, Castellino FJ, Zhong Z, Zlokovic BV. Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier. J Cereb Blood Flow Metab. 2009;29(1):25-33.
  • 23) Righi M, Mori L, De Libero G, et al. Monokine production by microglial cell clones. Eur J Immunol. 1989;19(8):1443-1448.
  • 24) Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-408.
  • 25) Gu JM, Fukudome K, Esmon CT. Characterization and regulation of the 5'-flanking region of the murine endothelial protein C receptor gene. J Biol Chem. 2000;275(17):12481-12488.
  • 26) Lee MY, Garvey SM, Ripley ML, Wamhoff BR. Genome-wide microarray analyses identify the protein C receptor as a novel calcineurin/nuclear factor of activated T cells-dependent gene in vascular smooth muscle cell phenotypic modulation. Arterioscler Thromb Vasc Biol. 2011;31(11):2665-2675.
  • 27) Balcaitis S, Xie Y, Weinstein JR, et al. Expression of proteinase-activated receptors in mouse microglial cells. Neuroreport. 2003;14(18):2373-2377.
  • 28) Bushell TJ, Plevin R, Cobb S, Irving AJ. Characterization of proteinase-activated receptor 2 signalling and expression in rat hippocampal neurons and astrocytes. Neuropharmacology. 2006;50(6):714-725.
  • 29) Ubl JJ, Reiser G. A novel proteolytic mechanism for termination of the CA2+ signalling evoked by proteinase-activated receptor-1 (PAR-1) in rat astrocytes. Adv Exp Med Biol. 2000;477:323-329.
  • 30) Esmon CT. The endothelial protein C receptor. Curr Opin Hematol. 2006;13(5):382-385.
  • 31) Galligan L, Livingstone W, Volkov Y, et al. Characterization of protein C receptor expression in monocytes. Br J Haematol. 2001;115(2):408-414.
  • 32) Nakamura M, Gabazza EC, Imoto I, et al. Anti-inflammatory effect of activated protein C in gastric epithelial cells. J Thromb Haemost. 2005;3(12):2721-2729.
  • 33) Sturn DH, Kaneider NC, Feistritzer C, Djanani A, Fukudome K, Wiedermann CJ. Expression and function of the endothelial protein C receptor in human neutrophils. Blood. 2003;102(4):1499-1505.
  • 34) Song D, Ye X, Xu H, Liu SF. Activation of endothelial intrinsic NF-{kappa}B pathway impairs protein C anticoagulation mechanism and promotes coagulation in endotoxemic mice. Blood. 2009;114(12):2521-2529.
  • 35) Kerschen EJ, Fernandez JA, Cooley BC, et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med. 2007;204(10):2439-2448.
  • 36) Zheng X, Li W, Song Y, et al. Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia. J Thromb Haemost. 2007;5(7):1394-1400.
  • 37) von Drygalski A, Furlan-Freguia C, Ruf W, Griffin JH, Mosnier LO. Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol. 2013;33(4):769-776.
  • 38) Seol JW, Lee YJ, Jackson CJ, Sambrook PN, Park SY. Activated protein C inhibits bisphosphonate-induced endothelial cell death via the endothelial protein C receptor and nuclear factor-kappaB pathways. Int J Mol Med. 2011;27(6):835-840.
  • 39) Hayashi T, Nakamura H, Okada A, et al. Organization and chromosomal localization of the human endothelial protein C receptor gene. Gene. 1999;238(2):367-373.
  • 40) Lagrange J, Li Z, Fassot C, et al. Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice. FASEB J. 2014;28(5):2062-2072.
There are 40 citations in total.

Details

Primary Language English
Subjects Health Care Administration
Journal Section Research Article
Authors

Kemal Uğur Tüfekci

Halil Ateş This is me 0000-0002-0112-5505

Şermin Genç 0000-0001-6126-7460

Project Number 114Z577
Publication Date February 26, 2021
Submission Date January 6, 2021
Published in Issue Year 2021 Volume: 5 Issue: 1

Cite

APA Tüfekci, K. U., Ateş, H., & Genç, Ş. (2021). Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia. Journal of Basic and Clinical Health Sciences, 5(1), 6-13. https://doi.org/10.30621/jbachs.854244
AMA Tüfekci KU, Ateş H, Genç Ş. Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia. JBACHS. February 2021;5(1):6-13. doi:10.30621/jbachs.854244
Chicago Tüfekci, Kemal Uğur, Halil Ateş, and Şermin Genç. “Endothelial Protein C Receptor Expression Is Regulated by Sp1 Transcription Factor in Murine Microglia”. Journal of Basic and Clinical Health Sciences 5, no. 1 (February 2021): 6-13. https://doi.org/10.30621/jbachs.854244.
EndNote Tüfekci KU, Ateş H, Genç Ş (February 1, 2021) Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia. Journal of Basic and Clinical Health Sciences 5 1 6–13.
IEEE K. U. Tüfekci, H. Ateş, and Ş. Genç, “Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia”, JBACHS, vol. 5, no. 1, pp. 6–13, 2021, doi: 10.30621/jbachs.854244.
ISNAD Tüfekci, Kemal Uğur et al. “Endothelial Protein C Receptor Expression Is Regulated by Sp1 Transcription Factor in Murine Microglia”. Journal of Basic and Clinical Health Sciences 5/1 (February 2021), 6-13. https://doi.org/10.30621/jbachs.854244.
JAMA Tüfekci KU, Ateş H, Genç Ş. Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia. JBACHS. 2021;5:6–13.
MLA Tüfekci, Kemal Uğur et al. “Endothelial Protein C Receptor Expression Is Regulated by Sp1 Transcription Factor in Murine Microglia”. Journal of Basic and Clinical Health Sciences, vol. 5, no. 1, 2021, pp. 6-13, doi:10.30621/jbachs.854244.
Vancouver Tüfekci KU, Ateş H, Genç Ş. Endothelial Protein C Receptor Expression is Regulated by Sp1 Transcription Factor in Murine Microglia. JBACHS. 2021;5(1):6-13.
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