Skip to main content
SpringerLink
Log in

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity and inflammatory regulation. Here, DMBT1 expression was analyzed by immunohistochemistry in postmortem lung sections from patients with MAS. The lung epithelial cell line A549, stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty expression plasmid (DMBT1− cells), was exposed to meconium. NO was determined in dependence of aminoguanidine (inducible NO synthase inhibitor), steroids and lipopolysaccharide (LPS). DMBT1 is highly expressed in lungs with MAS. In the absence of meconium, DMBT1+ cells showed a higher NO production than the DMBT1− cells (p = 0.0090). Meconium led in DMBT1− and DMBT1+ cells to elevated NO levels (p < 0.0001), but with a higher NO level in DMBT1+ cells (p < 0.0001). Aminoguanidine, an iNOS inhibitor, reduced the higher NO production in DMBT1+ cells (p = 0.0476), but NO levels remained above NO production from DMBT1− cells (p = 0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p = 0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p < 0.0001). In comparison to exposure with only meconium, the combined addition of LPS and meconium to the cells increased NO levels in both DMBT1− cells (p = 0.0030) and DMBT1+ cells (p = 0.0028). In conclusion, basal and meconium-induced NO production in lung epithelial cells is positively regulated by DMBT1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Asano K, Chee CB, Gaston B, Lilly CM, Gerard C, Drazen JM, Stamler JS (1994) Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc Natl Acad Sci USA 91:10089–10093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bikker FJ, Ligtenberg AJ, End C, Renner M, Blaich S, Lyer S, Wittig R, van’t Hof W, Veerman EC, Nazmi K, de Blieck-Hogervorst JM, Kioschis P, Nieuw Amerongen AV, Poustka A, Mollenhauer J (2004) Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains. J Biol Chem 279:47699–47703

    Article  CAS  PubMed  Google Scholar 

  • Bouhafs RKL, Jarstrand C (2000) Interaction between lung surfactant and nitric oxide production by alveolar macrophages stimulated by group B streptococci. Pediatr Pulmonol 30:106–113

    Article  CAS  PubMed  Google Scholar 

  • Braidotti P, Nuciforo PG, Mollenhauer J, Poustka A, Pellegrini C, Moro A, Bulfamante G, Coggi G, Bosari S, Pietra GG (2004) DMBT1 expression is down-regulated in breast cancer. BMC Cancer 4:46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cayabyab RG, Kwong K, Jones C, Minoo P, Durand M (2007) Lung inflammation and pulmonary function in infants with meconium aspiration syndrome. Pediatr Pulmonol 42:898–905

    Article  PubMed  Google Scholar 

  • Chakravortty D, Hensel M (2003) Inducible nitric oxide synthase and control of intracellular bacterial pathogens. Mikrobes Infect 5:621–627

    Article  CAS  Google Scholar 

  • Cheng H, Bjerknes M, Chen H (1996) CRP-ductin: a gene expressed in intestinal crypts and in pancreatic and hepatic ducts. Anat Rec 244:327–343

    Article  CAS  PubMed  Google Scholar 

  • Cheung W, Darfler MM, Alvarez H, Hood BL, Conrads TP, Habbe N, Krizman DB, Mollenhauer J, Feldmann G, Maitra A (2008) Application of a global proteomic approach to archival precursor lesions: deleted in malignant brain tumors 1 and tissue transglutaminase 2 are upregulated in pancreatic cancer precursors. Pancreatology 8:608–616

    Article  PubMed  PubMed Central  Google Scholar 

  • De Beaufort AJ, Bakker AC, van Tol MJD, Poorthius BJ, Schrama AJ, Berger HM (2003) Meconium is a source of pro-inflammatory substances and can induce cytokine production in cultured A549 epithelial cells. Pediatr Res 54:491–495

    Article  PubMed  Google Scholar 

  • Dinarello CA (2010) Anti-inflammatory agents: present and future. Cell 140:935–950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • End C, Lyer S, Renner M, Stahl C, Ditzer J, Holloschi A, Kuhn HM, Flammann HT, Poustka A, Hafner M, Mollenhauer J, Kioschis P (2005) Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1. Protein Expr Purif 41:275–286

    Article  CAS  PubMed  Google Scholar 

  • End C, Bikker F, Renner M, Bergmann G, Lyer S, Blaich S, Hudler M, Helmke B, Gassler N, Autschbach F, Ligtenberg AJ, Benner A, Holmskov U, Schirmacher P, Nieuw Amerongen AV, Rosenstiel P, Sina C, Franke A, Hafner M, Kioschis P, Schreiber S, Poustka A, Mollenhauer J (2009) DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands. Eur J Immunol 39:833–842

    Article  CAS  PubMed  Google Scholar 

  • Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL (1998) Characterization of the A549 cell lines as a type II pulmonary epithelial cell model for drug metabolism. Exp Cell Res 243:359–366

    Article  CAS  PubMed  Google Scholar 

  • Hoehn T, Huebner J, Paboura E, Krause M, Leititis JU (1998) Effect of therapeutic concentrations of nitric oxide on bacterial growth in vitro. Crit Care Med 26:1857–1862

    Article  CAS  PubMed  Google Scholar 

  • Hong H, Jang BC (2014) Prednisone inhibits the IL-1β-induced expression of COX-2 in HEI-OC1 murine auditory cells through the inhibition of ERK-1/2, JNK-1 and AP-1 activity. Int J Mol Med 34:1640–1646

    CAS  PubMed  Google Scholar 

  • Jeng M-J, Soong W-J, Lee Y-S, Tsao P-C, Yang C-F, Chiu S-Y, Tang R-B (2010) Meconium exposure dependent cell death and apoptosis in human alveolar epithelial cells. Pediatr Pulmonol 45:816–823

    Article  PubMed  Google Scholar 

  • Kamosinska B, Radomski A, Man SFP, Radomski MW, Duszyk M (2000) Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells. J Pharm Exp Ther 295:500–505

    CAS  Google Scholar 

  • Kao YJ, Piedra PA, Larsen GL, Colasurdo GN (2001) Induction and regulation of nitric oxide synthase in airway epithelial cells by respiratory syncytial virus. Am J Respir Crit Care Med 163:532–539

    Article  CAS  PubMed  Google Scholar 

  • Khan AM, Lally KP, Elidemir O, Colasurdo GN (2002a) Meconium enhances the release of nitric oxide in human airway epithelial cells. Biol Neonate 81:99–104

    Article  CAS  PubMed  Google Scholar 

  • Khan AM, Lally KP, Larsen GL, Colasurdo GN (2002b) Enhanced release of thromboxane A2 after exposure of human airway epithelial cells to meconium. Pediatr Pulmonol 33:111–116

    Article  PubMed  Google Scholar 

  • Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ (1994) Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343:133–135

    Article  CAS  PubMed  Google Scholar 

  • Kusaka E, Sugiyama M, Senoo N, Yamamoto A, Sugimoto Y (2013) Genomic and non-genomic effects of glucocorticoids on allergic rhinitis model in mice. Int Immunopharmacol 16:279–287

    Article  CAS  PubMed  Google Scholar 

  • Kytölä J, Kääpä P, Uotila P (2003) Meconium aspiration stimulates cyclooxygenase-2 and nitric oxide synthase-2 expression in rat lungs. Pediatr Res 53:731–736

    Article  PubMed  Google Scholar 

  • Ligtenberg AJM, Veerman ECI, Nieuw Amerongen AV, Mollenhauer J (2007) Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer. Biol Chem 388:1275–1289

    Article  CAS  PubMed  Google Scholar 

  • MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QS, Sokol K, Hutchinson N, Chen H, Mudget JS (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641–650

    Article  CAS  PubMed  Google Scholar 

  • Misko TP, Moore WM, Kasten TP, Nickols GA, Corbett JA, Tilton RG, McDaniel ML, Williamson JR, Curie MG (1993) Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol 233:119–125

    Article  CAS  PubMed  Google Scholar 

  • Mollenhauer J, Wiemann S, Scheurlen W, Korn B, Hayashi Y, Wilgenbus KK, von Deimling A, Poustka A (1997) DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours. Nat Genet 17:32–39

    Article  CAS  PubMed  Google Scholar 

  • Mollenhauer J, Helmke B, Müller H, Kollender G, Lyer S, Diedrichs L, Holmskov U, Ligtenberg T, Herbertz S, Krebs I, Wiemann S, Madsen J, Bikker F, Schmitt L, Otto HF, Poustka A (2002a) Sequential changes of the DMBT1 expression and location in normal lung tissue and lung carcinomas. Genes Chromosomes Cancer 35:164–169

    Article  CAS  PubMed  Google Scholar 

  • Mollenhauer J, Müller H, Kollender G, Lyer S, Diedrichs L, Helmke B, Holmskov U, Ligtenberg T, Herbertz S, Krebs I, Madsen J, Bikker F, Schmitt L, Wiemann S, Scheurlen W, Otto HF, von Deimling A, Poustka A (2002b) The SRCR/SID region of DMBT1 defines a complex multi-allele system representing the major basis for its variability in cancer. Genes Chromosomes Cancer 35:242–255

    Article  CAS  PubMed  Google Scholar 

  • Mollenhauer J, Deichmann M, Helmke B, Müller H, Kollender G, Holmskov U, Ligtenberg T, Krebs I, Wiemann S, Bantel-Schaal U, Madsen J, Bikker F, Klauck SM, Otto HF, Moldenhauer G, Poustka A (2003) Frequent downregulation of DMBT1 and galectin-3 in epithelial skin cancer. Int J Cancer 105:149–157

    Article  CAS  PubMed  Google Scholar 

  • Mollenhauer J, Helmke B, Medina D, Bergmann G, Gassler N, Müller H, Lyer S, Diedrichs L, Renner M, Wittig R, Blaich S, Hamann U, Madsen J, Holmskov U, Bikker F, Ligtenberg A, Carlén A, Olsson J, Otto HF, O’Malley B, Poustka A (2004) Carcinogen inducibility in vivo and down-regulation of DMBTI during breast carcinogenesis. Genes Chromosomes Cancer 39:185–194

    Article  CAS  PubMed  Google Scholar 

  • Müller H, End C, Renner M, Helmke BM, Gassler N, Weiss C, Hartl D, Griese M, Hafner M, Poustka A, Mollenhauer A, Poeschl J (2007) Deleted in malignant brain tumors 1 (DMBT1) is present in hyaline membranes and modulates surface tension of surfactant. Respir Res 8:69

    Article  PubMed  PubMed Central  Google Scholar 

  • Müller H, End C, Weiss C, Renner M, Bhandiwad A, Helmke BM, Gassler N, Hafner M, Poustka A, Mollenhauer A, Poeschl J (2008) Respiratory Deleted in Malignant Brain Tumours 1 (DMBT1) levels increase during lung maturation and infection. Clin Exp Immunol 151:123–129

    Article  PubMed  PubMed Central  Google Scholar 

  • Müller H, Renner M, Helmke BM, End C, Weiss C, Poeschl J, Mollenhauer J (2009) Deleted in malignant brain tumors 1 is up-regulated in bacterial endocarditis and binds to components of vegetations. J Thorac Cardiovasc Surg 138:725–732

    Article  PubMed  Google Scholar 

  • Müller H, Hu J, Popp R, Schmidt MHH, Müller-Decker K, Mollenhauer J, Fisslthaler B, Eble JA, Fleming I (2012) Deleted in malignant brain tumors 1 is present in the vascular extracellular matrix and promotes angiogenesis. Arterioscler Thromb Vasc Biol 32:442–448

    Article  PubMed  Google Scholar 

  • Müller H, Nagel C, Weiss C, Mollenhauer J, Poeschl J (2015) Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells. BMC Pulm Med 15:32

    Article  PubMed  PubMed Central  Google Scholar 

  • Nathan C, Xie QW (1994) Regulation of biosynthesis of nitric oxide. J Biol Chem 269:13725–13728

    CAS  PubMed  Google Scholar 

  • Pechkovsky DV, Zissel G, Goldmann T, Einhaus M, Taube C, Magnussen H, Schlaak M, Müller-Quernheim J (2001) Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultures AEC II. Am J Physiol Lung Cell Mol Physiol 282:L684–L692

    Article  Google Scholar 

  • Rairigh RL, Parker TA, Ivy DD, Kinsella JP, Fan I-D, Abman SH (2001) Role of inducible nitric oxide synthase in the pulmonary vascular response to birth-related stimuli in the ovine fetus. Circ Res 88:721–726

    Article  CAS  PubMed  Google Scholar 

  • Renner M, Bergmann G, Krebs I, End C, Lyer S, Hilberg F, Helmke B, Gassler N, Autschbach F, Bikker F, Strobel-Freidekind O, Gronert-Sum S, Benner A, Blaich S, Wittig R, Hudler M, Ligtenberg AJ, Madsen J, Holmskov U, Annese V, Latiano A, Schirmacher P, Amerongen AV, D’Amato M, Kioschis P, Hafner M, Poustka A, Mollenhauer J (2007) DMBT1 confers mucosal protection in vivo and a deletion variant is associated with Crohn’s disease. Gastroenterology 133:1499–1509

    Article  CAS  PubMed  Google Scholar 

  • Robbins RA, Barnes PJ, Springall DR, Warren JB, Kwon OJ, Buttery LD, Wilson AJ, Geller DA, Polak JM (1994) Expression of inducible nitric oxide in human lung epithelial cells. Biochem Biophys Res Commun 203:209–218

    Article  CAS  PubMed  Google Scholar 

  • Rosenstiel P, Sina C, End C, Renner M, Lyer S, Till A, Hellmig S, Nikolaus S, Fölsch UR, Helmke B, Autschbach F, Schirmacher P, Kioschis P, Hafner M, Poustka A, Mollenhauer J, Schreiber S (2007) Regulation of DMBT1 via NOD2 and TRL4 in intestinal epithelial cells modulates bacterial recognition and invasion. J Immunol 178:8203–8211

    Article  CAS  PubMed  Google Scholar 

  • Zagariya A, Bhat R, Uhal B, Shankararao N, Freidine M, Vidyasagar D (2000) Cell death and lung cell histology in meconium aspirated newborn rabbit lung. Eur J Pediatr 159:819–826

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Lundbeckfonden Center of Excellence NanoCAN Grant, by the DAWN-2020 Project financed by Rektorspuljen SDU2020 program, and the MIO Project of the OUH Frontlinjepuljen.

Author information

Authors and Affiliations

  1. Department of Pediatrics I, Neonatology, University Hospital Essen, University Duisburg-Essen, Hufelandstr. 55, 45147, Essen, Germany

    Hanna Müller & Ursula Felderhoff-Müser

  2. Division of Neonatology, Department of Pediatrics, University of Heidelberg, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany

    Hanna Müller

  3. Institute of Medical Statistics and Biomathematics, Medical Faculty Mannheim, University of Heidelberg, Ludolf-Krehl-Straße 13-17D, 68167, Mannheim, Germany

    Christel Weiss

  4. Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany

    Marcus Renner

  5. Lundbeckfonden Center of Excellence NanoCAN and Molecular Oncology, Institute for Molecular Medicine, University of Southern Denmark, JB Winsloews Vej 25, 5000, Odense C, Denmark

    Jan Mollenhauer

Authors
  1. Hanna Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christel Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marcus Renner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ursula Felderhoff-Müser
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jan Mollenhauer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hanna Müller.

Ethics declarations

Conflict of interest

The authors declare that no conflict of interest exists.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Müller, H., Weiss, C., Renner, M. et al. DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro. Histochem Cell Biol 147, 389–397 (2017). https://doi.org/10.1007/s00418-016-1493-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-016-1493-9

Keywords

Search

Navigation