European Cytokine Network

, Volume 29, Issue 1, pp 36–47 | Cite as

Interleukin-32 plays an essential role in human calcified aortic valve cells

  • Chung-Lin Tsai
  • Ying-Ming Chiu
  • Yi-Ju Lee
  • Chin-Tung Hsieh
  • Dong-Chen Shieh
  • Gregory J. Tsay
  • Da-Tian Bau
  • Yi-Ying Wu
Research Article


Interleukin-32 (IL-32) is an inflammatory cytokine produced mainly by T, natural killer, and epithelial cells. Previous studies on IL-32 have primarily investigated its proinflammatory properties. The IL-32 also has been described as an activator of the p38 mitogen-activated protein kinase (MAPK) and NF-κB, and induces several cytokines. In this study, we hypothesized that the inflammatory regulators NF-κB, MAP kinase, STAT1, and STAT3 are associated with the expression of the IL-32 protein in human calcified aortic valve cells. This study comprised aortic valve sclerotic patients and control group patients without calcified aortic valve. Increased IL-32 expression in calcified aortic valvular tissue was shown by immunohistochemical staining and western blotting. There was an increase in NF-κB p65 level, p-ERK, p-JNK, and p-p38 MAPK activation underlying IL-32 expression in the study. The level of p-STAT3 but not p-STAT1 was found to be increased in calcified aortic valve tissue. In cultured primary human aortic valve interstitial cells, inhibition of NF-κB or MAPK kinase pathways results in a decrease of IL-32 expression. Treatment of recombinant IL-32 induced the levels of TNF-α, IL-6, IL-1β, and IL-8. Our findings demonstrate that IL-32 may be an important pro-inflammatory molecule involved in calcific aortic valve disease.


interleukin 32 MAPK NF-κB STAT3 calcific aortic valve disease 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Mohler ER 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation 2001; 103: 1522–8.CrossRefPubMedGoogle Scholar
  2. 2.
    New SE, Aikawa E. Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification. Circ Res 2011; 108: 1381–91.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Netea MG, Azam T, Ferwerda G, et al. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-1β and IL-6 production through a caspase 1-dependent mechanism. Proc Natl Acad Sci U S A 2005; 102: 16309–14.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nold MF, Nold-Petry CA, Pott GB, et al. Endogenous IL-32 controls cytokine and HIV-1 production. J Immunol 2008; 181: 557–65.CrossRefPubMedGoogle Scholar
  5. 5.
    Kim SH, Han SY, Azam T, Yoon DY, Dinarello CA. Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 2005; 22: 131–42.PubMedGoogle Scholar
  6. 6.
    Cagnard NLF, Essabbani A, Devauchelle V, et al. Interleukin-32, CCL2, PF4F1 and GFD10 are the only cytokine/chemokine genes differentially expressed by in vitro cultured rheumatoid and osteoarthritis fibroblast-like synoviocytes. Eur Cytokine Netw 2005; 16: 289–92.PubMedGoogle Scholar
  7. 7.
    Joosten LA, Netea MG, Kim SH, et al. IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc Natl Acad Sci U S A 2006; 103: 3298–303.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kobayashi H, Lin PC. Molecular characterization of IL-32 in human endothelial cells. Cytokine 2009; 46: 351–8.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hu Y, Cheng L, Hochleitner BW, Xu Q. Activation of mitogenactivated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 1997; 17: 2808–16.CrossRefPubMedGoogle Scholar
  10. 10.
    Lai K, Wang H, Lee WS, Jain MK, Lee ME, Haber E. Mitogenactivated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation. J Clin Invest 1996; 98: 1560–7.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Rahaman SO, Lennon DJ, Febbraio M, Podrez EA, Hazen SL, Silverstein RL. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell Metab 2006; 4: 211–21.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhang S, Ren J, Khan MF, Cheng AM, Abendschein D, Muslin AJ. Grb2 is required for the development of neointima in response to vascular injury. Arterioscler Thromb Vasc Biol 2003; 23: 1788–93.CrossRefPubMedGoogle Scholar
  13. 13.
    Webber JL, Tooze SA. Coordinated regulation of autophagy by p38alpha MAPK through mAtg9 and p38IP. EMBO J 2010; 29: 27–40.CrossRefPubMedGoogle Scholar
  14. 14.
    Orlowski RZ, Baldwin ASJ. NF-kappaB as a therapeutic target in cancer. Trends Mol Med 2002; 8: 385–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Hernández-Presa MA, Ortego M, Tu˜nón J, et al. Simvastatin reduces NF-kappaB activity in peripheral mononuclear and in plaque cells of rabbit atheroma more markedly than lipid lowering diet. Cardiovasc Res 2003; 57: 168–77.CrossRefPubMedGoogle Scholar
  16. 16.
    Bromberg JF. Activation of STAT proteins and growth control. Bioessays 2001; 23: 161–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Darnell JEJ. STATs and gene regulation. Science 1997; 277: 1630–5.CrossRefPubMedGoogle Scholar
  18. 18.
    Ihle JN. STATs: signal transducers and activators of transcription. Cell 1996; 84: 331–4.CrossRefPubMedGoogle Scholar
  19. 19.
    Imada K, Leonard WJ. The Jak-STAT pathway. Mol Immunol 2000; 37: 1–11.CrossRefPubMedGoogle Scholar
  20. 20.
    Takeda K, Akira S. STAT family of transcription factors in cytokine-mediated biological responses. Cytokine Growth Factor Rev 2000; 11: 199–207.CrossRefPubMedGoogle Scholar
  21. 21.
    Williams JG. STAT signalling in cell proliferation and in development. Curr Opin Genet Dev 2000; 10: 503–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Ng DC, Court NW, dos Remedios CG, Bogoyevitch MA. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts. Cardiovasc Res 2003; 57: 333–46.CrossRefPubMedGoogle Scholar
  23. 23.
    Podewski EK. Alterations in Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in patients with endstage dilated cardiomyopathy. Circulation 2003; 107: 798–802.CrossRefPubMedGoogle Scholar
  24. 24.
    Chmielewski S, Olejnik A, Sikorski K, et al. STAT1-dependent signal integration between IFNgamma and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis. PLoS One 2014; 9: e113318.CrossRefGoogle Scholar
  25. 25.
    Lim W-S, Timmins JM, Seimon TA, et al. STAT1 is critical for apoptosis in macrophages subjected to endoplasmic reticulum stress in vitro and in advanced atherosclerotic lesions in vivo. Circulation 2008; 117: 940–51.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zachary I, Georgia G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res 2001; 49: 568–81.CrossRefPubMedGoogle Scholar
  27. 27.
    Wang M, Zhang W, Crisostomo P, et al. Endothelial STAT3 plays a critical role in generalized myocardial proinflammatory and proapoptotic signaling. AmJ Physiol Heart Circ Physiol 2007; 293: H2101-8.CrossRefGoogle Scholar
  28. 28.
    Wang SG, Xu Y, Chen JD, Yang CH, Chen XH. Astragaloside IV stimulates angiogenesis and increases nitric oxide accumulation via JAK2/STAT3 and ERK1/2 pathway. Molecules 2013; 18: 12809–19.CrossRefPubMedGoogle Scholar
  29. 29.
    Waxman AB, Mahboubi K, Knickelbein RG, et al. Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death. Am J Respir Cell Mol Biol 2003; 29: 513–22.CrossRefPubMedGoogle Scholar
  30. 30.
    Wang Y-M, Lu T-L, Hsu P-N, et al. Ribosome inactivating protein Bchain induces osteoclast differentiation from monocyte/macrophage lineage precursor cells. Bone 2011; 48: 1336–45.CrossRefPubMedGoogle Scholar
  31. 31.
    Meng X, Ao L, Song Y, et al. Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol 2008; 294: C29–35.CrossRefPubMedGoogle Scholar
  32. 32.
    Messier RHJ, Bass BL, Aly HM, et al. Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblast. J Surg Res 1994; 57: 1–21.CrossRefPubMedGoogle Scholar
  33. 33.
    Wang YM, Lu TL, Hsu PN, et al. Ribosome inactivating protein Bchain induces osteoclast differentiation from monocyte/macrophage lineage precursor cells. Bone 2011; 48: 1336–45.CrossRefPubMedGoogle Scholar
  34. 34.
    Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, Ungvari Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells. Role of nuclear factor-β activation by tumor necrosis factor-α, H2O2, and high intravascular pressure. Circulation 2005; 111: 2364–72.PubMedGoogle Scholar
  35. 35.
    Szelag M, Piaszyk-Borychowska A, Plens-Galaska M, Wesoly J, Bluyssen HA. Targeted inhibition of STATs and IRFs as a potential treatment strategy in cardiovascular disease. Oncotarget 2016; 7: 48788–812.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Kao JT, Feng CL, Yu CJ, et al. IL-6, through p-STAT3 rather than p-STAT1, activates hepatocarcinogenesis and affects survival of hepatocellular carcinoma patients: a cohort study. BMC Gastroenterol 2015; 15: 50.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Zeng Q, Song R, Fullerton DA, et al. Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice. Proc Natl Acad Sci U S A 2017; 114: 1631–6.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Sádaba JR, Martínez-Martínez E, Arrieta V, et al. Role for galectin-3 in calcific aortic valve stenosis. J Am Heart Assoc 2016; 5: e004360.CrossRefGoogle Scholar
  39. 39.
    Babu AN, Meng X, Zou N, et al. Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis. Ann Thorac Surg 2008; 86: 71–6.CrossRefPubMedGoogle Scholar
  40. 40.
    Pawade TA, Newby DE, Dweck MR. Calcification in aortic stenosis: the skeleton key. J Am Coll Cardiol 2015; 66: 561–77.CrossRefPubMedGoogle Scholar
  41. 41.
    Yu Z, Seya K, Daitoku K, Motomura S, Fukuda I, Furukawa KI. Tumor necrosis factor-α accelerates the calcification of human aortic valve interstitial cells obtained from patients with calcific aortic valve stenosis via the BMP2-Dlx5 pathway. J Pharmacol Exp Ther 2011; 337: 16–23.CrossRefPubMedGoogle Scholar
  42. 42.
    Kaden JJ, Kilic R, Sarikoc A, et al. Tumor necrosis factor alpha promotes an osteoblast-like phenotype in human aortic valve myofibroblasts: a potential regulatory mechanism of valvular calcification. Int J Mol Med 2005; 16: 869–72.PubMedGoogle Scholar
  43. 43.
    El Husseini D, Boulanger MC, Mahmut A, et al. P2Y2 receptor represses IL-6 expression by valve interstitial cells through Akt: implication for calcific aortic valve disease. J Mol Cell Cardiol 2014; 72: 146–56.CrossRefPubMedGoogle Scholar
  44. 44.
    Kaden JJ, Dempfle CE, Grobholz R, et al. Interleukin-1 beta promotes matrix metalloproteinase expression and cell proliferation in calcific aortic valve stenosis. Atherosclerosis 2003; 170: 205–11.CrossRefPubMedGoogle Scholar
  45. 45.
    Nadlonek N, Lee JH, Reece TB, et al. Interleukin-1 beta induces an inflammatory phenotype in human aortic valve interstitial cells through nuclear factor kappa beta. Ann Thorac Surg 2013; 96: 155–62.CrossRefPubMedGoogle Scholar

Copyright information

© John Libbey Eurotext 2018

Authors and Affiliations

  • Chung-Lin Tsai
    • 1
    • 2
  • Ying-Ming Chiu
    • 3
    • 4
  • Yi-Ju Lee
    • 5
  • Chin-Tung Hsieh
    • 6
  • Dong-Chen Shieh
    • 4
  • Gregory J. Tsay
    • 7
    • 8
  • Da-Tian Bau
    • 9
  • Yi-Ying Wu
    • 10
  1. 1.Division of Cardiovascular SurgeryTaichung Veterans General HospitalTaichungTaiwan
  2. 2.Graduate Institute of Clinical Medical ScienceChina Medical UniversityTaichungTaiwan
  3. 3.Division of Allergy, Immunology & RheumatologyChanghua Christian HospitalChanghuaTaiwan
  4. 4.Department of Nursing, College of Medicine & NursingHungkuang UniversityTaichungTaiwan
  5. 5.Institute of Biochemistry, Microbiology and ImmunologyChung Shan Medical UniversityTaichungTaiwan
  6. 6.Department of PediatricsLotung Poh-Ai HospitalI-LanTaiwan
  7. 7.Division of Immunology and Rheumatology, Department of Internal MedicineChina Medical University HospitalTaichungTaiwan
  8. 8.School of Medicine, College of MedicineChina Medical UniversityTaichungTaiwan
  9. 9.Graduate Institute of Biomedical SciencesChina Medical UniversityTaichungTaiwan
  10. 10.Department of Medical Laboratory Science and BiotechnologyChina Medical UniversityTaichungTaiwan

Personalised recommendations