Developmental regulation of claudin localization by fetal
alveolar epithelial cells
Brandy L. Daugherty,1,2Madalina Mateescu,1Anand S. Patel,1Kelly Wade,3,4Shioko Kimura,5
Linda W. Gonzales,3,4Susan Guttentag,3,4Philip L. Ballard,3,4and Michael Koval1,2
Departments of1Physiology and3Pediatrics and2Institute for Environmental Medicine, University of Pennsylvania School
of Medicine;4Division of Neonatology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; and
5Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Submitted 2 December 2003; accepted in final form 30 August 2004
Daugherty, Brandy L., Madalina Mateescu, Anand S. Patel,
Kelly Wade, Shioko Kimura, Linda W. Gonzales, Susan Gutten-
tag, Philip L. Ballard, and Michael Koval. Developmental regula-
tion of claudin localization by fetal alveolar epithelial cells. Am J
Physiol Lung Cell Mol Physiol 287: L1266–L1273, 2004. First
published September 3, 2004; doi:10.1152/ajplung.00423.2003.—
Tight junction proteins in the claudin family regulate epithelial barrier
function. We examined claudin expression by human fetal lung (HFL)
alveolar epithelial cells cultured in medium containing dexametha-
sone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which
promotes alveolar epithelial cell differentiation to a type II phenotype.
At the protein level, HFL cells expressed claudin-1, claudin-3, clau-
din-4, claudin-5, claudin-7, and claudin-18, where levels of expres-
sion varied with culture conditions. DCI-treated differentiated HFL
cells cultured on permeable supports formed tight transepithelial
barriers, with transepithelial resistance (TER) ?1,700 ohm/cm2. In
contrast, HFL cells cultured in control medium without DCI did not
form tight barriers (TER ?250 ohm/cm2). Consistent with this dif-
ference in barrier function, claudins expressed by HFL cells cultured
in DCI medium were tightly localized to the plasma membrane;
however, claudins expressed by HFL cells cultured in control medium
accumulated in an intracellular compartment and showed discontinui-
ties in claudin plasma membrane localization. In contrast to claudins,
localization of other tight junction proteins, zonula occludens (ZO)-1,
ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intra-
cellular claudins expressed by undifferentiated HFL cells were local-
ized to a compartment containing early endosome antigen-1, and
treatment of HFL cells with the endocytosis inhibitor monodansylca-
daverine increased barrier function. This suggests that during differ-
entiation to a type II cell phenotype, fetal alveolar epithelial cells use
differential claudin expression and localization to the plasma mem-
brane to help regulate tight junction permeability.
tight junction; endocytosis; lung development; epithelial barrier func-
FLUID PRODUCED BY THE FETAL LUNG creates mechanical disten-
sions that are critical for air space development (22, 23). Fetal
lung fluid balance is maintained by the coordinated regulation
of epithelial ion channel activity and barrier function (3, 5, 21,
31, 32). Epithelial barrier function is primarily mediated by
tight junction proteins known as claudins. There are roughly
two dozen different claudins that regulate paracellular ion
permeability and epithelial barrier function (8, 35–37). Each
tissue shows a distinct claudin expression pattern, which en-
ables tissue-specific differences in paracellular permeability.
Previous work has shown that rodent and human lung
epithelia express claudin-1, claudin-3, claudin-4, claudin-5,
claudin-7, claudin-8, and claudin-18 (9, 24, 28, 38). The
pattern of claudin expression is sensitive to epithelial cell
phenotype. For instance, rat type II cells express more clau-
din-3 than type I cells, whereas type I cells express more
claudin-7 than type II cells (28, 38). However, expression of
claudin-4 and claudin-5 does not appear to be tightly linked to
rat alveolar epithelial cell phenotype (38). Changes in claudin
expression by lung epithelial cells have also been linked to
changes in paracellular permeability (9, 38).
A recent screen of gene expression by human fetal lung
(HFL) alveolar epithelial cells indicates that claudin-5 is one of
the major genes upregulated during differentiation in culture
(14). In addition to claudin-5, claudin-18 expression has been
demonstrated in fetal mouse lung (24). Although there is some
information about claudins expressed in the lung, little is
known about regulation of claudin expression during human
fetal lung development. Thus we examined tight junction
protein expression by HFL cells cultured under conditions
where cells either remain undifferentiated or differentiate to a
type II cell-like phenotype (2, 14). We found that HFL cells
modulate epithelial barrier function, in part, by regulating
claudin expression and localization to the plasma membrane.
MATERIALS AND METHODS
Cell culture. Enriched populations of epithelial cells were isolated
from second-trimester (13–20 wk gestation) human fetal lung tissue
under Institutional Review Board-approved protocols. After overnight
culture as explants without hormones, explants of fetal lung tissue
were digested with a combination of trypsin, collagenase, and DNase.
Fibroblasts were removed by preferential adherence to 60-mm plastic
culture dishes (14). The resultant nonadherent cells are ?83% epi-
thelial as assessed by cytokeratin staining (14). Nonadherent cells
were subsequently plated onto 35-mm plastic culture dishes, glass
coverslips, or 12-well Transwell permeable supports (Corning Costar,
Corning, NY) in Waymouth medium containing 10% fetal calf serum.
After overnight culture (day 1), the cells were washed and cultured for
an additional 3–5 days in 1 ml of serum-free Waymouth control
medium or Waymouth medium containing 10 nM dexametha-
sone plus 0.1 mM 8-bromo-cAMP and 0.1 mM IBMX (DCI). These
concentrations maximally induce surfactant components in human
lung explant cultures, as previously assessed by mRNA expression
analysis (14). Induction of the type II cell phenotype by DCI was
routinely assessed using Nile red to label lamellar bodies (Fig. 1).
Address for reprint requests and other correspondence: M. Koval, Univ. of
Pennsylvania School of Medicine, Dept. of Physiology, B-400 Richards
Bldg./6085, 3700 Hamilton Walk, Philadelphia, PA 19104 (E-mail: mkoval
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Am J Physiol Lung Cell Mol Physiol 287: L1266–L1273, 2004.
First published September 3, 2004; doi:10.1152/ajplung.00423.2003.
6. Buse P, Woo PL, Alexander DB, Reza A, and Firestone GL. Glucocor-
ticoid-induced functional polarity of growth factor responsiveness regu-
lates tight junction dynamics in transformed mammary epithelial tumor
cells. J Biol Chem 270: 28223–28227, 1995.
7. Chen Y, Lu Q, Schneeberger EE, and Goodenough DA. Restoration of
tight junction structure and barrier function by down-regulation of the
mitogen-activated protein kinase pathway in ras-transformed Madin-
Darby canine kidney cells. Mol Biol Cell 11: 849–862, 2000.
8. Colegio OR, Van Itallie CM, McCrea HJ, Rahner C, and Anderson
JM. Claudins create charge-selective channels in the paracellular pathway
between epithelial cells. Am J Physiol Cell Physiol 283: C142–C147,
9. Coyne CB, Gambling TM, Boucher RC, Carson JL, and Johnson LG.
Role of claudin interactions in airway tight junctional permeability. Am J
Physiol Lung Cell Mol Physiol 285: L1166–L1178, 2003.
10. Coyne CB, Ribeiro CM, Boucher RC, and Johnson LG. Acute mech-
anism of medium chain fatty acid-induced enhancement of airway epithe-
lial permeability. J Pharmacol Exp Ther 305: 440–450, 2003.
11. Das Sarma J, Meyer RA, Wang F, Abraham V, Lo CW, and Koval M.
Heteromeric connexin interactions prior to the trans Golgi network. J Cell
Sci 114: 4013–4024, 2001.
12. Farshori P and Kachar B. Redistribution and phosphorylation of occlu-
din during opening and resealing of tight junctions in cultured epithelial
cells. J Membr Biol 170: 147–156, 1999.
13. Gonzales LW, Angampalli S, Guttentag SH, Beers MF, Feinstein SI,
Matlapudi A, and Ballard PL. Maintenance of differentiated function of
the surfactant system in human fetal lung type II epithelial cells cultured
on plastic. Pediatr Pathol Mol Med 20: 387–412, 2001.
14. Gonzales LW, Guttentag SH, Wade KC, Postle AD, and Ballard PL.
Differentiation of human pulmonary type II cells in vitro by glucocorticoid
plus cAMP. Am J Physiol Lung Cell Mol Physiol 283: L940–L951, 2002.
15. Hansen SH, Sandvig K, and van Deurs B. Clathrin and HA2 adaptors:
effects of potassium depletion, hypertonic medium, and cytosol acidifica-
tion. J Cell Biol 121: 61–72, 1993.
16. Ishizaki T, Chiba H, Kojima T, Fujibe M, Soma T, Miyajima H,
Nagasawa K, Wada I, and Sawada N. Cyclic AMP induces phosphor-
ylation of claudin-5 immunoprecipitates and expression of claudin-5 gene
in blood-brain-barrier endothelial cells via protein kinase A-dependent and
-independent pathways. Exp Cell Res 290: 275–288, 2003.
17. Ivanov AI, Nusrat A, and Parkos CA. Endocytosis of epithelial apical
junctional proteins by a clathrin-mediated pathway into a unique storage
compartment. Mol Biol Cell 15: 176–188, 2004.
18. Koval M, Harley JE, Hick E, and Steinberg TH. Connexin46 is retained
as monomers in a trans-Golgi compartment of osteoblastic cells. J Cell
Biol 137: 847–857, 1997.
19. Lawrence DW, Comerford KM, and Colgan SP. Role of VASP in
reestablishment of epithelial tight junction assembly after Ca2?switch.
Am J Physiol Cell Physiol 282: C1235–C1245, 2002.
20. Matsuda M, Kubo A, Furuse M, and Tsukita S. A peculiar internal-
ization of claudins, tight junction-specific adhesion molecules, during the
intercellular movement of epithelial cells. J Cell Sci 117: 1247–1257,
21. McCray PB Jr, Bettencourt JD, and Bastacky J. Secretion of lung fluid
by the developing fetal rat alveolar epithelium in organ culture. Am J
Respir Cell Mol Biol 6: 609–616, 1992.
22. McCray PB Jr and Welsh MJ. Developing fetal alveolar epithelial cells
secrete fluid in primary culture. Am J Physiol Lung Cell Mol Physiol 260:
23. Moessinger AC, Harding R, Adamson TM, Singh M, and Kiu GT.
Role of lung fluid volume in growth and maturation of the fetal sheep lung.
J Clin Invest 86: 1270–1277, 1990.
24. Niimi T, Nagashima K, Ward JM, Minoo P, Zimonjic DB, Popescu
NC, and Kimura S. Claudin-18, a novel downstream target gene for the
T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and
stomach-specific isoforms through alternative splicing. Mol Cell Biol 21:
25. Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CL III,
and Sontag E. Protein phosphatase 2A associates with and regulates
atypical PKC and the epithelial tight junction complex. J Cell Biol 158:
26. Olver RE, Walters DV, and M Wilson S. Developmental regulation of
lung liquid transport. Annu Rev Physiol 66: 77–101, 2004.
27. Romero IA, Radewicz K, Jubin E, Michel CC, Greenwood J, Couraud
PO, and Adamson P. Changes in cytoskeletal and tight junctional
proteins correlate with decreased permeability induced by dexamethasone
in cultured rat brain endothelial cells. Neurosci Lett 344: 112–116, 2003.
28. Sandoval AJ, Zhou B, Liebler JM, Kim KJ, Ann DK, Crandall ED,
and Borok Z. Claudin expression and localization in alveolar epithelial
cells (Abstract). Am J Respir Crit Care Med 165: A74, 2002.
29. Sasaki H, Matsui C, Furuse K, Mimori-Kiyosue Y, Furuse M, and
Tsukita S. Dynamic behavior of paired claudin strands within apposing
plasma membranes. Proc Natl Acad Sci USA 100: 3971–3976, 2003.
30. Schlegel R, Dickson RB, Willingham MC, and Pastan IH. Amantadine
and dansylcadaverine inhibit vesicular stomatitis virus uptake and recep-
tor-mediated endocytosis of alpha 2-macroglobulin. Proc Natl Acad Sci
USA 79: 2291–2295, 1982.
31. Schneeberger EE and Lynch RD. Structure, function, and regulation of
cellular tight junctions. Am J Physiol Lung Cell Mol Physiol 262: L647–
32. Strang LB. Fetal lung liquid: secretion and reabsorption. Physiol Rev 71:
33. Stuart RO and Nigam SK. Regulated assembly of tight junctions by
protein kinase C. Proc Natl Acad Sci USA 92: 6072–6076, 1995.
34. Tsukamoto T and Nigam SK. Role of tyrosine phosphorylation in the
reassembly of occludin and other tight junction proteins. Am J Physiol
Renal Physiol 276: F737–F750, 1999.
35. Tsukita S, Furuse M, and Itoh M. Multifunctional strands in tight
junctions. Nat Rev Mol Cell Biol 2: 285–293, 2001.
36. Turksen K and Troy TC. Barriers built on claudins. J Cell Sci 117:
37. Van Itallie C, Rahner C, and Anderson JM. Regulated expression of
claudin-4 decreases paracellular conductance through a selective decrease
in sodium permeability. J Clin Invest 107: 1319–1327, 2001.
38. Wang F, Daugherty B, Keise LL, Wei Z, Foley JP, Savani RC, and
Koval M. Heterogeneity of claudin expression by alveolar epithelial cells.
Am J Respir Cell Mol Biol 29: 62–70, 2003.
39. Wong V and Gumbiner BM. A synthetic peptide corresponding to the
extracellular domain of occludin perturbs the tight junction permeability
barrier. J Cell Biol 136: 399–409, 1997.
DEVELOPMENTAL REGULATION OF CLAUDINS
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