Osteopontin Induces Airway Remodeling and Lung
Fibroblast Activation in a Murine Model of Asthma
Martin Kohan1, Raphael Breuer1, and Neville Berkman1
1Lung Cellular and Molecular Biology Laboratory, Institute of Pulmonology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
Airway remodeling is a central feature of asthma; however, the
mechanisms underlying its development have not been fully eluci-
dated. We have demonstrated that osteopontin, an inflammatory
cytokine and an extracellular matrix glycoprotein with profibrotic
properties, is up-regulated in a murine model of allergen-induced
airway remodeling. In the present study, we determined whether
osteopontin plays a functional role in airway remodeling. Osteo-
pontin (OPN)-deficient (OPN2/2) and wild-type mice were sensi-
Collagen production, peribronchial smooth muscle area, mucus-
producing cell number, and bronchoalveolar cell counts were
from OVA-treated OPN2/2and from wild-type mice were studied
using ex vivo cultures. OVA-treated OPN2/2mice exhibited reduced
lung collagen content, smooth muscle area, mucus-producing cells,
and inflammatory cell accumulation as compared with wild-type
transforming growth factor-b1 and vascular endothelial growth
from OVA-treated OPN2/2mice showed reduced proliferation,
migration, collagen deposition, and a–smooth muscle actin expres-
sion in comparison with OVA-treated wild-type lung fibroblasts.
Thus, OPN is key for the development of allergen-induced airway
remodeling in mice. In response to allergen, OPN induces the
switching of lung fibroblasts to a pro-fibrogenic myofibroblast
Keywords: airway remodeling; fibroblast; mouse model; myofibro-
Airway remodeling is a prominent pathophysiologic feature of
chronic asthma. It is characterized by changes in tissue struc-
tural components, including damage and shedding of airway
epithelium, mucus gland hypertrophy, increased myofibroblast
number, subepithelial fibrosis, increase of airway smooth mus-
cle (ASM) mass, and neovascularity (1–4). The poor response to
treatment seen in patients with refractory asthma may be
a consequence of the ongoing airway remodeling that leads to
fixed airway obstruction (1, 2). Despite advances in understand-
ing the inflammatory and immunologic features of asthma,
there is limited understanding of the cellular and molecular
mechanisms underlying the remodeling changes seen in chronic
An increased number of myofibroblasts has been reported in
airways of patients with chronic asthma (5) and in lungs of mice
chronically exposed to allergen (6). Fibroblasts/myofibroblasts
are a major source of collagens and glycoproteins, and after
activation they account for the enhanced extracellular matrix
(ECM) deposition that leads to the subepithelial fibrosis (7, 8).
Among these up-regulated ECM proteins, collagen I, fibronec-
tin, tenascin, laminins, and vitronectin have been shown to play
a direct role in the adhesion, migration, and proliferation of
lung structural cells and in the development of airway remodel-
Osteopontin (OPN), a 44-kD ECM glycoprotein that functions
both as an ECM molecule and as a cytokine, has been associated
with inflammation and fibrosis/tissue repair in lung diseases (11,
12). OPN contributes to tissue fibrosis and wound healing by
regulation of cell–ECM interactions, by modulation of trans-
forming growth factor-b1 (TGF-b1) and matrix metalloproteinase
(MMP) expression, and by enhancing angiogenesis (12–14).
OPN has recently been implicated in allergic inflammation. It
regulates dendritic cell activation in a murine model of allergen-
a role for OPN in the pathogenesis of acute airway inflammation
in early stages of asthma; however, whether OPN plays a role in
airway remodeling in chronic asthma has not been evaluated.
We have recently demonstrated that OPN is up-regulated in
a murine model of allergen-induced airway remodeling and is
expressed by eosinophils, T cells, and macrophages (17). In
addition, we observed a significant correlation between lung
OPN levels and airway collagen deposition and smooth muscle
area (17). In the present study, we show that OPN contributes to
the pathogenesis of airway remodeling and induces lung fibro-
blast activation and differentiation in a murine model of chronic
allergen-induced experimental asthma.
MATERIALS AND METHODS
OPN2/2(B6.Cg-Spp1tm1Blh/J) and C57Bl/6 mice were purchased
from The Jackson Laboratory (Bar Harbor, ME) and from Harlan
Laboratories Ltd (Jerusalem, Israel). Mice were housed under specific
pathogen–free conditions. The Hebrew University-Hadassah Medical
School Animal Ethics Committee approved all experimental animal
protocols. Ten mice per group were used.
Allergen Challenge Protocol and Lung Pathology
Mice were sensitized with intraperitoneal ovalbumin (OVA) (Sigma-
Aldrich, St. Louis, MO) (10 mg OVA/1 mg Al (OH)3[Sigma] in 0.5 ml
0.9% saline), or saline on Days 1 and 10, and then challenged with
inhaled saline or OVA (2% weight/volume diluted in 0.9% saline: 4 ml/
inhalation) three times a week, starting on Day 15, for 5 weeks as
described (17). Twenty-four hours after the last challenge, mice were
anesthetized with 150 ml pentobarbitone sodium (CTS Chemical Ind.,
Tel Aviv, Israel) and bronchoalveolar lavage (BAL) was performed as
described (17). Mice were then killed by abdominal aortic transection
and exsanguination. The right lung was removed and frozen in liquid
nitrogen for collagen content (upper lobe) and enzyme-linked immu-
nosorbent assay (ELISA) determination (lower lobe). Theleftlungwas
fixed by intrabronchial infusion with 4% formaldehyde (Biolab, Jerusa-
lem, Israel). Sections of the left lung were stained with hematoxylin and
eosin (H&E), Masson Trichrome, and Periodic Acid–Schiff (PAS).
(Received in original form June 8, 2008 and in final form December 17, 2008)
This study was supported by a grant from the Chief Scientist Office of the
Ministry of Health, Israel (Grant N8 5878–1) and the Israel Lung Association, Tel
Correspondence and requests for reprints should be addressed to Neville Berkman,
MBBCh, FRCP, Institute of Pulmonology, Hadassah-Hebrew University Medical
Center, POB 12000, Jerusalem, Israel 91120. E-mail: Neville@hadassah.org.il
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2008-0307OC on January 16, 2009
Internet address: www.atsjournals.org
Vol 41. pp 290–296, 2009
effect on human lung fibroblasts. J Allergy Clin Immunol 2006;117:103–
20. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and
inexpensive method for analysis of cell migration in vitro. Nat
21. Sugiura H, Liu X, Duan F, Kawasaki S, Togo S, Kamio K, Wang XQ,
Mao L, Ahn Y, Ertl RF, et al. Cultured lung fibroblasts from
ovalbumin-challenged ‘‘asthmatic’’ mice differ functionally from
normal. Am J Respir Cell Mol Biol 2007;37:424–430.
22. Cohn L, Elias JA, Chupp GL. Asthma: mechanisms of disease persis-
tence and progression. Annu Rev Immunol 2004;22:789–815.
23. Ophascharoensuk V, Giachelli CM, Gordon K, Hughes J, Pichler R,
Brown P, Liaw L, Schmidt R, Shankland SJ, Alpers CE, et al.
Obstructive uropathy in the mouse: role of osteopontin in interstitial
fibrosis and apoptosis. Kidney Int 1999;56:571–580.
24. Trueblood NA, Xie Z, Communal C, Sam F, Ngoy S, Liaw L, Jenkins
AW, Wang J, Sawyer DB, Bing OH, et al. Exaggerated left
ventricular dilation and reduced collagen deposition after myocar-
dial infarction in mice lacking osteopontin. Circ Res 2001;88:
25. Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM, Hogan BL.
Altered wound healing in mice lacking a functional osteopontin gene
(spp1). J Clin Invest 1998;101:1468–1478.
26. Kumagai K, Ohno I, Okada S, Ohkawara Y, Suzuki K, Shinya T, Nagase
H, Iwata K, Shirato K. Inhibition of matrix metalloproteinases
prevents allergen-induced airway inflammation in a murine model
of asthma. J Immunol 1999;162:4212–4219.
27. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth
and activation of promatrix metalloproteinase-2 through nuclear
factor-kappa B-mediated induction of membrane type 1 matrix
metalloproteinase in murine melanoma cells. J Biol Chem 2001;276:
28. Kelly MM, Leigh R, Gilpin SE, Cheng E, Martin GE, Radford K, Cox
G. Gauldie. Cell-specific gene expression in patients with usual
interstitial pneumonia. Am J Respir Crit Care Med 2006;174:557–565.
29. Takahashi F, Takahashi K, Okazaki T, Maeda K, Ienaga H, Maeda M,
Kon S, Uede T, Fukuchi Y. Role of osteopontin in the pathogenesis
of bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol
30. Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, Becerril C,
Yousem S, Herrera I, Ruiz V, Selman M, et al. Up-regulation and
profibrotic role of osteopontin in human idiopathic pulmonary
fibrosis. PLoS Med 2005;2:e251.
31. Collins AR, Schnee J, Wang W, Kim S, Fishbein MC, Bruemmer D, Law
RE, Nicholas S, Ross RS, Hsueh WA. Osteopontin modulates
angiotensin II-induced fibrosis in the intact murine heart. J Am Coll
32. Richter A, Puddicombe SM, Lordan JL, Bucchieri F, Wilson SJ,
Djukanovic R, Dent G, Holgate ST, Davies DE. The contribution
of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic
unit in asthma. Am J Respir Cell Mol Biol 2001;25:385–391.
33. Lenga Y, Koh A, Perera AS, McCulloch CA, Sodek J, Zohar R.
Osteopontin expression is required for myofibroblast differentiation.
Circ Res 2008;102:319–327.
34. Kariyawasam HH, Robinson DS. The role of eosinophils in airway tissue
remodelling in asthma. Curr Opin Immunol 2007;19:681–686.
35. Hansel NN, Cheadle C, Diette GB, Wright J, Thompson KM, Barnes
KC, Georas SN. Analysis of CD41 T-cell gene expression in allergic
subjects using two different microarray platforms. Allergy 2008;63:
36. Konno S, Eckman JA, Plunkett B, Li X, Berman JS, Schroeder J, Huang
SK. Interleukin-10 and Th2 cytokines differentially regulate osteo-
pontin expression in human monocytes and dendritic cells. J In-
terferon Cytokine Res 2006;26:562–567.
37. Lloyd CM, Robinson DS. Allergen-induced airway remodelling. Eur
Respir J 2007;29:1020–1032.
38. Yue TL, McKenna PJ, Ohlstein EH, Farach-Carson MC, Butler WT,
Johanson K, McDevitt P, Feuerstein GZ, Stadel JM. Osteopontin-
stimulated vascular smooth muscle cell migration is mediated by beta
3 integrin. Exp Cell Res 1994;214:459–464.
39. Gadeau AP, Campan M, Millet D, Candresse T, Desgranges C.
Osteopontin overexpression is associated with arterial smooth muscle
cell proliferation in vitro. Arterioscler Thromb 1993;13:120–125.
40. Bosse ´ Y, Thompson C, Audette K, Stankova J, Rola-Pleszczynski M.
Interleukin-4 and interleukin-13 enhance human bronchial smooth
muscle cell proliferation. Int Arch Allergy Immunol 2008;146:138–148.
41. Shinagawa K, Kojima M. Mouse model of airway remodeling: strain
differences. Am J Respir Crit Care Med 2003;168:959–967.
42. Tanabe T, Fujimoto K, Yasuo M, Tsushima K, Yoshida K, Ise H,
Yamaya M. Modulation of mucus production by interleukin-13
receptor alpha2 in the human airway epithelium. Clin Exp Allergy
43. Leali D, Dell’Era P, Stabile H, Sennino B, Chambers AF, Naldini A,
Sozzani S, Nico B, Ribatti D, Presta M. Osteopontin (Eta-1) and
fibroblast growth factor-2 cross-talk in angiogenesis. J Immunol 2003;
44. Leali D, Moroni E, Bussolino F, Presta M. Osteopontin overexpression
inhibits in vitro re-endothelialization via integrin engagement. J Biol
45. Puxeddu I, Ribatti D, Bader R, Berkman N, Levi-Schaffer F. Osteo-
pontin is expressed and functional in human peripheral blood
eosinophils. J Allergy Clin Immunol 2006;117:S2. (abstract).
46. Denhardt DT, Noda M. Osteopontin expression and function: role in
bone remodeling. J Cell Biochem Suppl 1998;30–31:92–102.
296 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 41 2009