Immunologic Research

, Volume 66, Issue 4, pp 445–461 | Cite as

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

  • Alexandru Tatomir
  • Cosmin A. Tegla
  • Alvaro Martin
  • Dallas Boodhoo
  • Vinh Nguyen
  • Adam J. Sugarman
  • Armugam Mekala
  • Freidrich Anselmo
  • Anamaria Talpos-Caia
  • Cornelia Cudrici
  • Tudor C. Badea
  • Violeta Rus
  • Horea RusEmail author
Original Article


Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.


RGC-32 Astrocyte Multiple sclerosis Experimental autoimmune encephalomyelitis Extracellular matrix 



alpha smooth muscle actin


collagen type I alpha 1


collagen type IV alpha 1


collagen type V alpha 1


experimental autoimmune encephalomyelitis


extracellular matrix


epithelial to mesenchymal transition




glial fibrillary acidic protein




myelin oligodendrocyte glycoprotein


multiple sclerosis


normal-appearing gray matter


normal-appearing white matter


perivascular space


response gene to complement 32


rho-associated coiled-coil-containing protein kinase





We thank Dr. Deborah McClellan for editing this manuscript.


This work was supported in part by Veterans Administration Merit Award I01BX001458 (to H.R.).

Compliance with ethical standards

Conflict of interest

H.R. has received a grant from TEVA Neuroscience (CNS-2014-174). All other authors declare that they have no conflict of interest.

Supplementary material

12026_2018_9011_MOESM1_ESM.doc (45 kb)
Supplemental Table 1 (DOC 45 kb)
12026_2018_9011_Fig14_ESM.png (70 kb)
Supplemental Fig. 1

Quantification of ECM deposits in MS and control brains Collagen I–V deposits in MS and normal brains were quantified by two independent investigators. Statistically significant higher staining intensity was found in MS brains when compared with normal brains. Results are expressed as mean ± SEM. **p < 0.01; ***p < 0.001; ****p < 0.0001 (PNG 70 kb)

12026_2018_9011_MOESM2_ESM.tif (134 kb)
High Resolution Image (TIF 133 kb)


  1. 1.
    Badea TC, Niculescu FI, Soane L, Shin ML, Rus H. Molecular cloning and characterization of RGC-32, a novel gene induced by complement activation in oligodendrocytes. J Biol Chem. 1998;273:26977–81.CrossRefPubMedGoogle Scholar
  2. 2.
    Badea T, Niculescu F, Soane L, Fosbrink M, Sorana H, Rus V, et al. RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into S-phase. J Biol Chem. 2002;277:502–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Vlaicu SI, Tatomir A, Boodhoo D, Ito T, Fosbrink M, Cudrici C, et al. RGC-32 is expressed in the human atherosclerotic arterial wall: role in C5b-9-induced cell proliferation and migration. Exp Mol Pathol. 2016;101:221–30.CrossRefPubMedGoogle Scholar
  4. 4.
    Fosbrink M, Cudrici C, Tegla CA, Soloviova K, Ito T, Vlaicu S, et al. Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells. Exp Mol Pathol. 2009;86:87–94.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rus H, Cudrici C, Niculescu F, Shin ML. Complement activation in autoimmune demyelination: dual role in neuroinflammation and neuroprotection. J Neuroimmunol. 2006;180:9–16.CrossRefPubMedGoogle Scholar
  6. 6.
    Tegla CA, Cudrici CD, Azimzadeh P, Singh AK, Trippe R 3rd, Khan A, et al. Dual role of response gene to complement-32 in multiple sclerosis. Exp Mol Pathol. 2013;94:17–28.CrossRefPubMedGoogle Scholar
  7. 7.
    Rus V, Nguyen V, Tatomir A, Lees JR, Mekala AP, Boodhoo D, et al. RGC-32 promotes Th17 cell differentiation and enhances experimental autoimmune encephalomyelitis. J Immunol. 2017;198:3869–77.CrossRefPubMedGoogle Scholar
  8. 8.
    Vlaicu SI, Cudrici C, Ito T, Fosbrink M, Tegla CA, Rus V, et al. Role of response gene to complement 32 in diseases. Arch Immunol Ther Exp. 2008;56:115–22.CrossRefGoogle Scholar
  9. 9.
    Huang W-Y, Li Z-G, Rus H, Wang X, Jose PA, Chen S-Y. RGC-32 mediates transforming growth factor-β-induced epithelial-mesenchymal transition in human renal proximal tubular cells. J Biol Chem. 2009;284:9426–32.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Guo X, Jose PA, Chen S-Y. Response gene to complement 32 interacts with Smad3 to promote epithelial–mesenchymal transition of human renal tubular cells. Am J Physiol Cell Physiol. 2011;300:C1415–C21.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sofroniew MV. Astrogliosis. Cold Spring Harb Perspect Biol. 2015;7:a020420.CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Voskuhl RR, Peterson RS, Song B, Ao Y, Morales LB, Tiwari-Woodruff S, et al. Reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the CNS. J Neurosci. 2009;29:11511–22.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Anderson MA, Ao Y, Sofroniew MV. Heterogeneity of reactive astrocytes. Neurosci Lett. 2014;565:23–9.CrossRefPubMedGoogle Scholar
  14. 14.
    Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, Giffard RG, et al. Genomic analysis of reactive astrogliosis. J Neurosci. 2012;32:6391–410.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cudrici C, Ito T, Zafranskaia E, Weerth S, Rus V, Chen H, et al. Complement C5 regulates the expression of insulin-like growth factor binding proteins in chronic experimental allergic encephalomyelitis. J Neuroimmunol. 2008;203:94–103.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tegla CA, Azimzadeh P, Andrian-Albescu M, Martin A, Cudrici CD, Trippe R 3rd, et al. SIRT1 is decreased during relapses in patients with multiple sclerosis. Exp Mol Pathol. 2014;96:139–48.CrossRefPubMedGoogle Scholar
  17. 17.
    Hoffman WH, Cudrici CD, Zafranskaia E, Rus H. Complement activation in diabetic ketoacidosis brains. Exp Mol Pathol. 2006;80:283–8.CrossRefPubMedGoogle Scholar
  18. 18.
    Rus HG, Kim LM, Niculescu FI, Shin ML. Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus. J Immunol. 1992;148:928–33.PubMedGoogle Scholar
  19. 19.
    Tegla CA, Cudrici CD, Nguyen V, Danoff J, Kruszewski AM, Boodhoo D, et al. RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp Mol Pathol. 2015;98:328–37.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Rus HG, Niculescu F, Shin ML. Sublytic complement attack induces cell cycle in oligodendrocytes. J Immunol. 1996;156:4892–900.PubMedGoogle Scholar
  21. 21.
    Mohan H, Krumbholz M, Sharma R, Eisele S, Junker A, Sixt M, et al. Extracellular matrix in multiple sclerosis lesions: fibrillar collagens, biglycan and decorin are upregulated and associated with infiltrating immune cells. Brain Pathol. 2010;20:966–75.PubMedGoogle Scholar
  22. 22.
    Moreels M, Vandenabeele F, Dumont D, Robben J, Lambrichts I. Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1). Neuropathol Appl Neurobiol. 2008;34:532–46.CrossRefPubMedGoogle Scholar
  23. 23.
    Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425:577–84.CrossRefPubMedGoogle Scholar
  24. 24.
    Jinnin M, Ihn H, Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression. Mol Pharmacol. 2006;69:597–607.CrossRefPubMedGoogle Scholar
  25. 25.
    Chen S, Crawford M, Day RM, Briones VR, Leader JE, Jose PA, et al. RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation. J Biol Chem. 2006;281:1765–70.CrossRefPubMedGoogle Scholar
  26. 26.
    Kuivaniemi H, Tromp G, Prockop DJ. Mutations in fibrillar collagens (types I, II, III, and XI), fibril-associated collagen (type IX), and network-forming collagen (type X) cause a spectrum of diseases of bone, cartilage, and blood vessels. Hum Mutat. 1997;9:300–15.CrossRefPubMedGoogle Scholar
  27. 27.
    Marjamaa J, Tulamo R, Abo-Ramadan U, Hakovirta H, Frosen J, Rahkonen O, et al. Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: high-resolution magnetic resonance imaging, at 4.7 T, of the aorta and cerebral arteries. Magn Reson Med. 2006;55:592–7.CrossRefPubMedGoogle Scholar
  28. 28.
    van Horssen J, Dijkstra CD, de Vries HE. The extracellular matrix in multiple sclerosis pathology. J Neurochem. 2007;103:1293–301.CrossRefPubMedGoogle Scholar
  29. 29.
    Hara M, Kobayakawa K, Ohkawa Y, Kumamaru H, Yokota K, Saito T, et al. Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin–N-cadherin pathway after spinal cord injury. Nat Med. 2017;23:818–28.CrossRefPubMedGoogle Scholar
  30. 30.
    Liesi P, Kauppila T. Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar. Exp Neurol. 2002;173:31–45.CrossRefPubMedGoogle Scholar
  31. 31.
    Zhu D, Tapadia MD, Palispis W, Luu M, Wang W, Gupta R. Attenuation of robust glial scar formation facilitates functional recovery in animal models of chronic nerve compression injury. J Bone Joint Surg Am. 2017;99:e132.CrossRefPubMedGoogle Scholar
  32. 32.
    Xia M, Zhu Y. Fibronectin enhances spinal cord astrocyte proliferation by elevating P2Y1 receptor expression. J Neurosci Res. 2014;92:1078–90.CrossRefPubMedGoogle Scholar
  33. 33.
    van Horssen J, Vos CM, Admiraal L, van Haastert ES, Montagne L, van der Valk P, et al. Matrix metalloproteinase-19 is highly expressed in active multiple sclerosis lesions. Neuropathol Appl Neurobiol. 2006;32:585–93.CrossRefPubMedGoogle Scholar
  34. 34.
    Massagué J, Wotton D. Transcriptional control by the TGF-β/Smad signaling system. EMBO J. 2000;19:1745–54.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Zhao P, Gao D, Wang Q, Song B, Shao Q, Sun J, et al. Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4. Cell Mol Immunol. 2015;12:692–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Cregg JM, DePaul MA, Filous AR, Lang BT, Tran A, Silver J. Functional regeneration beyond the glial scar. Exp Neurol. 2014;253:197–207.CrossRefPubMedGoogle Scholar
  37. 37.
    Harlow DE, Macklin WB. Inhibitors of myelination: ECM changes, CSPGs and PTPs. Exp Neurol. 2014;251:39–46.CrossRefPubMedGoogle Scholar
  38. 38.
    Kawano H, Kimura-Kuroda J, Komuta Y, Yoshioka N, Li HP, Kawamura K, et al. Role of the lesion scar in the response to damage and repair of the central nervous system. Cell Tissue Res. 2012;349:169–80.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Alexandru Tatomir
    • 1
  • Cosmin A. Tegla
    • 1
    • 2
  • Alvaro Martin
    • 1
  • Dallas Boodhoo
    • 1
  • Vinh Nguyen
    • 3
  • Adam J. Sugarman
    • 1
  • Armugam Mekala
    • 1
  • Freidrich Anselmo
    • 1
  • Anamaria Talpos-Caia
    • 1
    • 4
  • Cornelia Cudrici
    • 5
  • Tudor C. Badea
    • 6
  • Violeta Rus
    • 2
    • 3
  • Horea Rus
    • 1
    • 2
    • 7
    Email author
  1. 1.Department of NeurologyUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Research ServiceVeterans Administration Maryland Health Care SystemBaltimoreUSA
  3. 3.Department of Medicine, Division of Rheumatology and Clinical ImmunologyUniversity of Maryland School of MedicineBaltimoreUSA
  4. 4.Department of Rheumatology“Iuliu Hatieganu” University of Medicine and PharmacyCluj-NapocaRomania
  5. 5.National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of HealthBethesdaUSA
  6. 6.Retinal Circuit Development and Genetics Unit, N-NRLNational Eye InstituteBethesdaUSA
  7. 7.Veterans Administration Multiple Sclerosis Center of Excellence–EastBaltimoreUSA

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