1 Department of Pediatrics and 3 Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908; and 2 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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ABSTRACT |
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The distal epithelium of the developing
lung exhibits high-level expression of protein phosphatase 2A (PP2A), a
vital signaling enzyme. Here we report the discovery that in the lung,
the PP2A regulatory subunit B56 is expressed in a discrete
developmental period, with the highest protein levels at embryonic day
(e) 17, but no detectable protein in the newborn or adult. By
in situ hybridization, B56
was highly expressed in the distal
epithelium of newly forming airways and in mesenchymal cells. In
contrast, expression of B56
was quite low in the bronchial
epithelium and vascular smooth muscle. Transgenic expression of B56
using the lung-specific promoter for surfactant protein C (SP-C)
resulted in neonatal death. Examination of lungs from SP-C-B56
transgenic e18 fetuses revealed proximal airways and normal blood
vessels, but the tissue was densely populated with epithelial-type
cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway,
-catenin, was developmentally
regulated in the normal lung and was absent in lung tissue from B-56
transgenic fetuses. We propose that B56
is expressed at a particular
stage of lung development to modulate PP2A action on the
Wnt/
-catenin signaling pathway during lung airway morphogenesis.
lung development; Wnt; -catenin; PP2A
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INTRODUCTION |
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THE LUNG FORMS AS AN OUTPOUCHING from the primitive foregut endoderm into the surrounding splanchnic mesoderm. Timely airway growth and development are orchestrated by reciprocal interactions between the epithelium and surrounding mesoderm. A complex process of interactions is involved, including cytokines, growth factors, extracellular matrix, cell membrane receptors, and transcription factors (8, 9). The signaling processes linking extracellular events to the nucleus, mediating lung growth and development, are largely unknown.
The phosphorylation of signaling molecules is key to the propagation of a growth signal. Most phosphorylation of intracellular proteins occurs on Ser and Thr residues, and the enzymes protein phosphatase (PP) 1 and PP2A account for the bulk of phosphatase activity against these targets (10, 18). PP2A is clearly necessary for normal development, because mice lacking the PP2A catalytic subunit due to homologous recombination deletion suffer early fetal demise at embryonic day (e) 6 (1). PP2A activity and level of expression are developmentally regulated in the lung (19, 21). In whole fetal lung cultures, PP2A inhibition with pharmacological inhibitors blocked branching morphogenesis and induced a G2/M cell cycle arrest (15). Despite these results, the potential functions for PP2A in the developing lung remain unclear.
Most PP2A is a heterotrimer composed of a catalytic (C) and a scaffold (A, PR65) subunit, plus one of several regulatory (B) subunits (10, 18). More than a dozen unique regulatory B subunits have been identified, and evidence indicates they confer distinctive properties to the PP2A. The B56 family of regulatory subunits is unique, because various family members show specific tissue expression, and some of the B56 family members direct PP2A to the nucleus (13, 16). To date, the expression and function of the PP2A B56 regulatory subunits in the developing lung are unknown.
The Wnt signaling pathway modulates many developmental processes
(reviewed in Refs. 17, 20) and is regulated by PP2A at multiple levels (12, 14). PP2A binds axin
(3), and B56 subunits can bind adenomatous polyposis coli
protein (APC), with overexpression of B56 resulting in decreased levels
of -catenin (14).
-Catenin, a key Wnt
signaling intermediate, is required for normal mammalian development.
Mice null for
-catenin die at the gastrula stage because of defects
in epithelial formation (2).
-Catenin, through its
interaction with endothelial cadherin, may also play an important role
in the regulation of cell migration during epithelial tubulogenesis
(11), a process necessary for distal lung
formation. Although these observations indicate that Wnt
signaling to
-catenin is important in development, the expression of
-catenin in the developing lung and its regulation by PP2A are unexplored.
In the present study, we explored the expression of the PP2A regulatory
subunit B56. Using a combination of Western blotting and in situ
hybridization, we found B56
to be developmentally regulated in the
rat lung. Using a transgenic approach, we showed that B56
overexpression in the fetal lung resulted in severe alterations in
fetal lung branching morphogenesis that correlated with suppression of
-catenin levels.
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METHODS |
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Western blotting.
Affinity-purified polyclonal antibodies were raised against the
carboxy-terminal B56 peptide by coupling 405-EKLKEKLKMK-415 to
keyhole limpet hemocyanin for immunization and using the same peptide
for purification (Research Genetics, Huntsville, AL). Lungs were
homogenized in ice-cold lysis buffer (50 mM HEPES, pH 7.5, 10 mM EDTA,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 µM
leupeptin) with a Polytron (Brinkman Instruments, Westbury, NY).
Soluble proteins were recovered by centrifugation at 10,000 g, assayed for protein concentration, frozen, and stored at
70°C. Soluble proteins (50 µg) were resolved by 10% SDS-PAGE under denaturing conditions and transferred to a nitrocellulose membrane (Trans-Blot; Bio-Rad Laboratories, Hercules, CA). Specific immunodetection with the B56
antibody (1:500 dilution) was by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ) following the manufacturer's protocol. A duplicate membrane was immunoblotted with anti-actin (Santa Cruz Biotechnology, Santa Cruz,
CA) as a control for loading and specific expression.
In situ hybridization.
In situ hybridization was as previously described (21).
Briefly, 350-bp sense and antisense digoxigenin-cRNA probes against the
5' coding region were generated from the human B56 cDNA. Probes were
incubated with sections overnight at 37°C and washed at 42°C,
and specific hybridization was detected with anti-digoxigenin- conjugated antibody plus nitro blue tetrazolium staining.
Transgenic SP-C-B56 mice.
The full-length B56
human cDNA was subcloned in frame into a plasmid
construct containing 3.7 kb of the human surfactant protein (SP)-C
promoter and the SV40 small T intron and poly A signal (generous gift
of Jeffrey Whittset) (4). Injections of the linearized
transgene into the pronuclei of C57BL/6 mouse eggs and implantation
into pseudopregnant host mice were performed at the University of
Virginia Transgenic Mouse Facility. The genotype of embryos was
determined by Southern blotting of SacI/SalI
digests of genomic DNA extracted from mouse placenta. A 3.7-kb SP-C
promoter fragment was [32P]dCTP labeled by random priming
(Ready-to-Go Beads; Amersham Pharmacia) and hybridized at 65°C
overnight. Specific hybridization was determined by phosphorimage
analysis (Molecular Dynamics, Sunnyvale, CA).
Histology/Immunohistochemistry.
For histological analysis, lungs were fixed at room temperature in 4%
buffered paraformaldehyde for 1 h. Lungs were cryoprotected overnight in 20% sucrose and frozen in OCT medium (Miles, Elkhart, IN). Sections of 5-7 µM were cut and stained with
hematoxylin and eosin for microscopic analysis. Immunohistochemical
detection for -catenin was performed using a rabbit polyclonal
antibody (Santa Cruz) at a dilution of 1:300. Controls were slides
incubated with nonspecific mouse IgG (Vector Laboratories, Burlingame,
CA) at the same concentration as the primary antibody. Sections were blocked for 1 h at room temperature with horse serum followed by incubation for 1 h with the primary antibody. After washing the
sections for 30 min with PBS plus 0.5% Tween 20, we observed immunodetection using the avidin-biotin complex method
(Vector) with an alkaline phosphatase substrate (Vector red) as a color substrate. All slides were examined and photographed on an
Olympus-BX41 microscope coupled to an Olympus DP11 digital camera.
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RESULTS |
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Developmental expression of PP2A subunit B56.
We measured the relative amounts of B56
regulatory subunit of PP2A
in rat lung at different stages of development by Western blotting an
equivalent amount of total tissue protein with a specific carboxy-terminal anti-peptide antibody (Fig.
1). The 56-kDa protein was evident at e17
and e19, but its levels dropped sharply at e20 and in the newborn to
background levels. The B56
subunit was not detected in the adult
lung, even though the A and C subunits of PP2A were abundantly
expressed in this tissue (not shown). A duplicate membrane
immunoblotted for actin demonstrated equivalent loading and expression
of actin. We concluded that expression of the regulatory subunit B56
was regulated during fetal development, with a significant decrease in
expression at term.
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Epithelium-targeted transgenic overexpression of B56.
To test if the transient expression of B56
was critical for the
process of airway morphogenesis, we overexpressed B56
in the fetal
lung under control of the epithelium-specific promoter for SP-C. Over
200 injections were performed to produce transgenic animals, without
production of a single viable founder animal. We suspected that
transgenic animals were likely dying at or shortly after birth and
therefore were not recovered. To examine this possibility, we performed
repeat injections and killed the pseudopregnant host animals and
fetuses at day 18 of gestation to examine lung morphology.
We identified positive transgenic animals by genotyping the placenta by
Southern blot analysis. As shown in Table
1, out of 57 fetal mice examined, seven
were transgenic for SP-C-B56
. The gross appearance of the lungs in
the transgenics was much smaller in four and modestly reduced in three,
compared with nontransgenic littermates (Table 1). Lung wet weights
were lower in all SP-C-B56
transgenics, with the greatest difference
in the smallest lungs compared with nontransgenic (Table 1).
Microscopic examination of fetal lung sections demonstrated a
complete absence of distal lung differentiation that might account for
the difference in the gross size of the lungs from B56
transgenic
animals. As shown in Fig. 3,
representative lung sections from B56
transgenic animals with
grossly small lungs demonstrated a dense cellular lung, lacking peripheral airway formation (Fig. 3, C-F). Proximal
bronchiole architecture was intact, with branching into the lung
periphery, but lacked obvious budding airways. The pulmonary
vasculature was readily evident, with large muscular arteries and
capillaries present in the lung.
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B56 overexpression regulates
-catenin levels in the fetal
lung.
Overexpression of PP2A B56 subunits in cells in vitro results in
decreased levels of
-catenin (14). We performed
immunohistochemical mapping of
-catenin developmental expression in
the e18 and adult lung. As shown in Fig.
4A,
-catenin was present at
high levels in the e18 fetal lung. All but an occasional (Fig.
4A) alveolar epithelial cell were positive for
-catenin.
Vascular endothelial cells also stained positive for
-catenin. By
contrast, the adult lung
-catenin expression was limited to a small
number of alveolar epithelial cells (Fig. 4B). At both ages,
-catenin also was present in the adventitial layer of pulmonary
blood vessels and the basement membrane of bronchi (not shown). We
concluded that
-catenin is highly expressed in the fetal lung
relative to the adult. Lung sections from SP-C-B56
transgenic and
nontransgenic littermates were stained for
-catenin by
immunohistochemistry. As shown in the representative section from the
nontransgenic littermate in Fig.
5A,
-catenin was broadly
expressed in the e18 airway epithelium of the lung, with both a
cytoplasmic and nuclear distribution. However, in the SP-C-B56
transgenic lung with severe airway defects, no
-catenin could be
detected (Fig. 5B).
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DISCUSSION |
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The balance of phosphorylation/dephosphorylation by protein
phosphatases and kinases is key to the regulation of cell
proliferation, differentiation, and growth. PP2A is an abundant
serine/threonine protein phosphatase present in cells as a collection
of distinct holoenzymes composed of the same catalytic and structural
subunits plus multiple regulatory B subunits. The B56 family of PP2A
regulatory subunits includes five widely expressed paralogous genes
(,
,
,
, and
) (6, 7, 16).
We found that B56 expression is developmentally regulated in the rat
lung, with expression highest in the fetal lung in contrast to barely
detectable protein expression in the perinatal and adult lung. B56
was predominately expressed by the developing airway epithelium in the
fetal lung. Because the catalytic subunit of PP2A is expressed in a
similar developmental pattern in the lung (21), one can
imagine that PP2A with a B56
subunit has a specific role in
regulation of fetal lung airway development.
We demonstrate that B56 overexpression under control of the
epithelium-specific SP-C promoter resulted in small lungs with a lack
of peripheral air spaces. The importance of PP2A in normal growth and
development has been shown by homologous recombination knockout of the
catalytic subunit of PP2A, resulting a lack of fetal development past
e5-6 (1). In the lung in particular, PP2A plays a
positive role in fetal lung development because PP2A inhibitors
severely impaired fetal lung growth in culture (15). The
fidelity and specificity of PP2A for substrates are determined by the
various regulatory subunits such as B56
. Thus appropriate expression
of B56
in the developing lung is essential for normal branching
morphogenesis to occur. The alteration in normal airway formation
caused by B56
overexpression was likely not secondary to cell loss
or apoptosis. The densely cellular lung seen in the B56
mice
contrasts with the massive bleb-like spaces within the lung seen upon
expression of diphtheria toxin under control of the SP-C promoter
(4).
The Wingless/Wnt family of secreted glycoproteins regulates important
body axis determination and patterning events (17, 20).
Wnt signaling results in an accumulation of -catenin in the nucleus
and its binding to the Lef-1/Tcf family of factors to activate
transcription of target genes. At least two Wnt proteins, Wnt-5a and
Wnt-11, are expressed in the developing airway epithelium (13), suggesting that Wnt signaling may be important for
lung development. We found that
-catenin was highly expressed in the e18 lung compared with the adult lung. SP-C-B56
transgenic
overexpression resulted in suppression of
-catenin levels during
this period of development. Similarly, B56 overexpression in cultured
cells resulted in phosphorylation-induced proteosomal degradation of
-catenin, likely through increased activation of glycogen synthase kinase-3 (14). Importantly, B56 expression in
cultured cell lines also decreased downstream transcriptional
activation of at least the Lef-1/Tcf1 target gene siamois
(5). Together these observations indicate that B56
subunits target PP2A and thereby promote degradation of
-catenin.
PP2A can also play a positive role in regulating Wnt signaling
(12). With the use of microinjection assays in
Xenopus laevis, the catalytic subunit of PP2A (PP2A-C) was
found to potentiate Wnt signaling, whereas a B56 subunit inhibited signaling. These observations suggest that different B regulatory subunits of PP2A can redirect the phosphatase to provide both positive
and negative input into the Wnt signaling cascade.
In summary, the developmentally expressed PP2A regulatory subunit
B56 dramatically alters lung branching morphogenesis. When B56
was expressed as a tissue-specific transgene, it suppressed
-catenin
levels. These data indicate an important role for PP2A in the
regulation of lung growth and development, possibly through regulation
of the Wnt signaling pathway.
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ACKNOWLEDGEMENTS |
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A. D. Everett was partially supported by the University of Virginia Children's Medical Center and the Virginia Thoracic Society. D. L. Brautigan was supported by United States Public Health Service Grant CA-77584, and C. Kamibayashi by American Heart Association, Texas affiliate Grant 95R-091.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. D. Everett, Univ. of Virginia Health System, Pediatric Cardiology, MR4 Bldg., PO Box 801356, Charlottesville, VA 22908-1356 (E-mail: ade5r{at}virginia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00262.2001
Received 16 July 2001; accepted in final form 15 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Gotz, J,
Probst A,
Ehler E,
Hemmings B,
and
Kues W.
Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Calpha.
Proc Natl Acad Sci USA
95:
12370-12375,
1998
2.
Haegel, H,
Larue L,
Ohsugi M,
Fedorov L,
Herrenknecht K,
and
Kemler R.
Lack of beta-catenin affects mouse development at gastrulation.
Development
121:
3529-3537,
1995
3.
Hsu, W,
Zeng L,
and
Costantini F.
Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain.
J Biol Chem
274:
3439-3445,
1999
4.
Korfhagen, TR,
Glasser SW,
Wert SE,
Bruno MD,
Daugherty CC,
NcNeish JD,
Stock JL,
Potter SS,
and
Whitsett JA.
Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice.
Proc Natl Acad Sci USA
87:
6122-6126,
1990[Abstract].
5.
Lemaire, P,
Garrett N,
and
Gurdon JB.
Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.
Cell
81:
85-94,
1995[ISI][Medline].
6.
McCright, B,
Rivers AM,
Audlin S,
and
Virshup DM.
The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm.
J Biol Chem
271:
22081-22089,
1996
7.
McCright, B,
and
Virshup DM.
Identification of a new family of protein phosphatase 2A regulatory subunits.
J Biol Chem
270:
26123-26128,
1995
8.
Mendelson, CR.
Role of transcription factors in fetal lung development and surfactant protein gene expression.
Annu Rev Physiol
62:
875-915,
2000[ISI][Medline].
9.
Minoo, P,
and
King RJ.
Epithelial-mesenchymal interactions in lung development.
Annu Rev Physiol
56:
13-45,
1994[ISI][Medline].
10.
Mumby, MC,
and
Walter G.
Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth.
Physiol Rev
73:
673-699,
1993
11.
Pollack, AL,
Barth AI,
Altschuler Y,
Nelson WJ,
and
Mostov KE.
Dynamics of beta-catenin interactions with APC protein regulate epithelial tubulogenesis.
J Cell Biol
137:
1651-1662,
1997
12.
Ratcliffe, MJ,
Itoh K,
and
Sokol SY.
A positive role for the PP2A catalytic subunit in Wnt signal transduction.
J Biol Chem
275:
35680-35683,
2000
13.
Sakiyama, J,
Yokouchi Y,
and
Kuroiwa A.
Coordinated expression of Hoxb genes and signaling molecules during development of the chick respiratory tract.
Dev Biol
227:
12-27,
2000[ISI][Medline].
14.
Seeling, JM,
Miller JR,
Gil R,
Moon RT,
White R,
and
Virshup DM.
Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.
Science
283:
2089-2091,
1999
15.
Taylor, BK,
Stoops TD,
and
Everett AD.
Protein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung cultures.
Am J Physiol Lung Cell Mol Physiol
278:
L1062-L1070,
2000
16.
Tehrani, MA,
Mumby MC,
and
Kamibayashi C.
Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle.
J Biol Chem
271:
5164-5170,
1996
17.
Uusitalo, M,
Heikkila M,
and
Vainio S.
Molecular genetic studies of Wnt signaling in the mouse.
Exp Cell Res
253:
336-348,
1999[ISI][Medline].
18.
Virshup, DM.
Protein phosphatase 2A: a panoply of enzymes.
Curr Opin Cell Biol
12:
180-185,
2000[ISI][Medline].
19.
Warburton, D,
and
Cohen P.
Ontogeny of protein phosphatases 1 and 2A in developing rat lung.
Pediatr Res
24:
25-27,
1988[Abstract].
20.
Wodarz, A,
and
Nusse R.
Mechanisms of Wnt signaling in development.
Annu Rev Cell Dev Biol
14:
59-88,
1998[ISI][Medline].
21.
Xue, C,
Heller F,
Johns RA,
and
Everett AD.
Developmental expression and localization of the catalytic subunit of protein phosphatase 2A in rat lung.
Dev Dyn
211:
1-10,
1998[ISI][Medline].