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DEVELOPMENTAL DYNAMICS 211:1–10 (1998)
Developmental Expression and Localization of the
Catalytic Subunit of Protein Phosphatase 2A in Rat Lung
CHUN XUE,1 FELICE HELLER,2 ROGER A. JOHNS,1 AND ALLEN D. EVERETT2*
1Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville, Virginia
2Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, Virginia
ABSTRACT
Protein phosphatase type-2A
(PP2A) is a highly conserved serine/threonine
phosphatase known to play a key role in cell
proliferation and differentiation in vitro, but the
role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of
PP2A or the relevance of PP2A expression to
mammalian development in vivo. To examine
expression of PP2A during mammalian lung development, we studied fetal rats from day 14 of
gestation (the lung bud is formed on day 12 of
gestation) to parturition. Western analysis with a
specific PP2A catalytic subunit antibody identified a single 36 kDa protein, with protein levels
two-fold higher in the 17 and 19 day embryonic
lung as compared to the adult. With in situ hybridization and immunohistochemistry, both mRNA
and protein for PP2A were localized equally to
the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14
day embryonic lung. With maturation of the lung,
PP2A becomes highly expressed in respiratory
epithelium. The highest level of expression was
in the earliest developing airways with columnar
epithelium (the pseudoglandular stage, 15–18 days
of gestation). There was a decrease in expression
with the transformation to cuboidal epithelium
by day 20 of gestation. This was most noticeable
in the developing bronchial epithelium of the 19
and 20 day gestation lungs where only an occasional cell continues to express PP2A. Mesenchymal hybridization was most obvious in early
endothelial cells of forming vascular channels at
17–19 days of gestation. PP2A respiratory epithelial expression mimicked the centrifugal development of the respiratory tree where the highest
expression was in the peripheral columnar epithelium (15–18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation.
Endothelial hybridization decreased with muscularization of large pulmonary arteries with low
levels of expression detected in bronchial or vascular smooth muscle. In the newborn lung PP2A
expression was decreased, but detectable in alveolar epithelium and vascular endothelium. In summary; 1) PP2A mRNA and protein exhibit cell
r 1998 WILEY-LISS, INC.
specific expression during rat lung development;
2) PP2A is highly expressed in the respiratory
epithelium of the fetal rat lung and is temporally
related to the maturation of the bronchial epithelium; 3) and the PP2A subunit is highly expressed
in early vascular endothelium, but not smooth
muscle of the rat lung. Dev. Dyn. 1998;211:1–10.
r 1998 Wiley-Liss, Inc.
Key words: epithelium; lung development; fetus;
serine/threonine phosphatase; endothelium; immunohistochemistry; in
situ hybridization
INTRODUCTION
Epithelial and mesenchymal interactions are necessary for the development of many organ systems including those of the gastrointestinal, integument, urogenital, and respiratory systems. Lung development serves
as a classical model for such biologic interactions
(Minoo et al., 1994). Formed initially as an outpouching
of the primitive foregut, the lung subsequently undergoes growth and branching of the primitive respiratory
epithelium into the surrounding mesenchyme to form
the bronchial tree. A complex process of interactions
among cells, cytokines, extracellular matrix, and cell
membrane receptors is necessary for lung morphogenesis and regional specification of the respiratory system. Although there must be some mechanism to link
extracellular signals to intracellular reactions such as
gene expression (signal transduction), in the developing lung, neither the components nor their functions
are well understood. An essential mechanism in the
regulation of signal transduction involves the activities
of phosphoproteins capable of reversible phosphorylation and dephosphorylation (Cohen et al., 1989; Mumby
et al., 1993; Hemmings et al., 1994). Protein phosphatases exist in eukaryotic cells to regulate the phosphoryla-
Grant sponsor: March of Dimes Basil O’Connor Fellowship Award;
Grant sponsor: National Institutes of Health; Grant numbers: K08HL02937-02, R01-HL39706, and R01-GM49111; Grant sponsor: American Heart Associate–Virginia Affiliate.
Dr. Xue is currently at the Department of Asthma and Allergy
Research, Pharmaceuticals Division, Novartis, CH 4002 Basel, Switzerland.
*Correspondence to: Dr. Allen D. Everett, M.D., University of
Virginia Health Sciences Center, MR4 Building, Box 14, Charlottesville, VA 22908.
Received 4 June 1997; Accepted 11 September 1997
2
XUE ET AL.
tion state of phosphoproteins, and are largely divided
into protein tyrosine and serine/threonine phosphatases as determined by their amino acid substrates. Of
the serine/threonine protein phosphatases, protein phosphatase type-2A (PP2A) is highly conserved in all
eukaryotic cells and accounts for a large portion of total
cellular phosphatase activity (Cohen et al., 1989; Hemmings et al., 1994). PP2A is essential for a variety of
cellular functions including signal transduction, cell
cycle regulation, cell transformation, and cell fate determination (Hemmings et al., 1994).
PP2A is a trimeric holoenzyme, composed of a core
dimer plus a third subunit. The holoenzyme consists of
a 36 kDa catalytic (C) subunit (Stone et al., 1987;
Khew-Goodall et al., 1991), a 63 kDa structural (A)
subunit (Hemmings et al., 1990), and a third variable,
regulatory (B) subunit (Hemmings et al., 1994; Tehrani
et al., 1996; McCright et al., 1996). The B subunit is
variable, ranging from 54 to 130 kDa and serves to
confer distinct properties on the enzyme for substrate
specificity (Hemmings et al., 1994; Kamibayaashi et al.,
1992; Cegielska et al., 1994). The catalytic subunit of
PP2A is highly conserved (Hemmings et al., 1994), the
human structure having 70% homology to yeast Saccharomyces cerevisiae (Arndt et al., 1989) and 95% identity
with Drosophila (Mayer-Jaekel et al., 1992, 1993).
There is no evidence that the free catalytic subunit
exists in cells (Mumby et al., 1993).
Although the in vitro biochemical aspects of PP2A are
well known, the role of PP2A in mammalian development is largely unexplored. For PP2A to play a role in
lung development it would be expected that there be
developmental differences in PP2A subunit expression
and activity. As shown by Warburton and Cohen (1988),
PP2A activity is developmentally upregulated in the rat
lung. To date no information exists on the developmental, cell specific expression of PP2A in any organ,
including the lung. To begin to explore a potential role
for PP2A in lung development, the present study determined the cellular expression pattern of the 36 kDa
catalytic subunit of PP2A (PP2A) in development of the
rat fetal lung. As the catalytic subunit does not exist
alone in nature but always as an active enzyme coupled
at least to the A structural subunit (Mumby et al., 1993)
we used PP2A as a marker for expression of the PP2A
holoenzyme in the developing lung using Western
analysis, in situ hybridization, and immunohistochemistry.
RESULTS
Western Analysis of PP2A
Western analysis of whole lung homogenates for
PP2A protein was performed as shown by the representative blot in Figure 1A. Using a specific polyclonal
peptide antibody against the carboxy terminal portion
of PP2A a single 36 kDa protein was detected that
co-migrated with the PP2A control protein (not shown)
as previously reported for PP2A (Stone et al., 1987;
Khew-Goodall et al., 1991; Martin et al., 1994). As
A
Fig. 1. Representative Western analysis of PP2A protein levels in the
developing lung. A: Total lung protein (50 µg) from 14 day (E14, n 5 29),
17 day (E17, n 5 27), 19 day (E19, n 5 18), and 20 day (E20, n 5 17)
gestation, newborn (NB, n 5 9) and adult (AD, n 5 2) rats was analyzed
by SDS-PAGE with Western blotting for PP2A protein levels. Protein
standard markers are shown on left. B: Densitometric analysis of PP2A
protein levels is shown.
shown (Fig. 1A,B), PP2A protein is abundant in the
developing lung with protein levels two-fold higher in
the 17 and 19 day embryonic lung as compared to the
adult. Therefore PP2A protein demonstrates significant
developmental regulation in the lung with protein
levels highest at the time of maximal growth of the
lung.
In Situ Hybridization
and Immunohistochemistry
14 day gestation lung. To map the cell specific
mRNA and protein expression of PP2A in the developing lung, in situ hybridization and immunohistochemical methods were utilized. PP2A mRNA was readily
detected in the developing lung at 14 day of gestation
(Fig. 2, the lung bud is formed during day 12 of
gestation). PP2A mRNA (Fig. 2A,B) and protein (Fig.
4A) were widely expressed in the epithelium and mesenchyme of the developing lung bud.
17 day gestation lung. By 17 days of gestation (Fig.
2C–G) the lung airway has undergone extensive branch-
PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG
Fig. 2. PP2A mRNA localization in the 14 and 17 day gestation lung. In
situ hybridization for PP2A in 14 day gestation rat lung is shown in A and
B and 17 day lung in C–G. The anti-sense cRNA probe was used in A, C,
E–G, and the control sense probe in B and D. A,B: The respiratory
epithelium in the 14 day lung bud is identified by arrows. C–G: Hybridization to small developing airways (small arrows), larger bronchioles (large
3
arrows), endothelial cells (arrow heads), and the pulmonary artery (long
arrow) is shown. F is a magnification of E and demonstrates hybridization
to endothelial cells of developing vascular channels (arrow heads). G
demonstrates high expression of PP2A in the respiratory epithelium of
immature forming airways (arrows) and decreased expression in epithelium lining bronchioles (B).
4
XUE ET AL.
ing. At this stage, PP2A mRNA hybridization was
increased relative to the 14 day lung (Fig. 2A) and
continues to map predominately to the now glandular
airway epithelium (Fig. 2C–G). A phenotypic gradient
of PP2A mRNA expression was demonstrated between
the more mature central bronchiolar and peripheral
(least mature) respiratory epithelial cells. As shown in
Figure 2E–G PP2A is highly expressed in the respiratory epithelium of the least mature peripheral airway
epithelium. Whereas, expression is markedly decreased in the more mature bronchiolar (‘‘B’’ in panel G
and large arrow panel E, Fig. 2) epithelial cells. Mesenchymal expression of PP2A in the 17 day lung localized
predominately to endothelial cells of forming vascular
channels (Fig. 2F) identified by the presence of red
blood cells. The specificity of the in situ hybridization is
shown in the control section incubated with the digoxigenin labeled mRNA probe (Fig. 2D). As shown, there is
an absence of nonspecific hybridization in the fetal lung
demonstrating the specificity of the cRNA probe. PP2A
mRNA expressing cells in the 17 day gestation lung
were also seen to contain PP2A protein (Fig. 4B).
Immunocytochemistry with a PP2A specific antibody
(Fig. 4B) demonstrated that respiratory epithelial cells
as well as the endothelial cells of forming vascular
channels were all immunopositive for PP2A (Fig. 4B).
19 day gestation lung. At 19 days of gestation, as
the airway branches extensively, PP2A mRNA continues to be expressed at a very high level in the airway
epithelium (Fig. 3A,C–E). However, the pattern of
expression is higher within the epithelium of small
canalicular structures in the peripheral and less mature portions of the lung (small arrows in Fig. 3D,E)
rather than in the large, central more mature airways
(large arrows in Fig. 3D,E). The quantity of PP2A
mRNA hybridization clearly decreased in the transition
from a more cuboidal to flattened epithelial cell type in
the more mature airways (Fig. 3A,C–E). In particular
the bronchiolar epithelial cells (‘‘B’’ in Fig. 3A,C,D)
showed very little PP2A mRNA hybridization relative
to the more glandular respiratory epithelial cells. There
did appear to be a gradient of expression in the bronchiolar epithelium with higher mRNA expression in the
peripheral than in the central bronchiolar epithelium
(Fig. 3C). Expression in the mainstem bronchus epithelium was higher in cells nearest the lumenal surface of
the bronchus (Fig. 3D). Intense hybridization to endothelial cells lining forming vascular channels (Fig. 3E
arrow heads) was seen. However endothelial and smooth
muscle cells of mature muscular pulmonary arteries
(Fig. 3A arrows and D ‘‘PA’’) expressed PP2A at background levels. The specificity of the in situ hybridization is shown in Figure 3B using the control sense
probe. As shown incubation with the control sense
probe demonstrated a complete lack of hybridization to
the airway epithelium and lung parenchyma.
Immunocytochemically PP2A continued to map to
endothelial cells of forming vascular channels (Fig. 4C
arrow heads) and airway epithelium (Fig. 4C,D, small
arrows). The PP2A protein was expressed in both
cytoplasmic and nuclear compartments of endothelial
(Fig. 4C, arrowheads) and respiratory epithelial cells
(Fig. 4C,D, small arrows). The nuclear localization of
PP2A protein in respiratory epithelial cells is shown in
Figure 4D (small arrows) and decreases in cells of
larger airways (Fig. 4D, large arrow). Likewise endothelial cells of more mature blood vessels (Fig. 4D, long
arrow) expressed less PP2A protein.
Newborn lung. After birth, alveolar cells continued
to express PP2A mRNA (Fig. 5A, arrows) whereas most
of the bronchial epithelial cells in the aerated lung were
negative for PP2A mRNA (Fig. 5C, arrows). Endothelial
cells lining capillaries and small peripheral arteries
(Fig. 5A,E, arrowheads) continued to express PP2A
mRNA at high levels however expression in large
muscular arteries was less obvious (Fig. 5F, arrowheads). Hybridization to the sense control probe is
shown (Fig. 5B,D) and demonstrates the specificity of
the results. PP2A protein expression was decreased as
compared to the 19 day gestation lung but continued to
be detectable in alveolar epithelial and endothelial cells
(Fig. 4E). No immunostaining was detected in a serial
section using nonspecific rabbit IgG as a control
(Fig. 4F).
DISCUSSION
Considering the conserved nature of PP2A and the
abundant number of regulatory units available Mumby
and Walter (1993) have proposed that the catalytic
subunit would not show cell and tissue specific regulation. However, the present study shows regulated expression at high levels of PP2A mRNA and protein in
the developing bronchiolar epithelium and endothelial
cells. In addition western analysis demonstrated a two
fold decrease in PP2A protein from 19 day gestation to
the adult. Thus paralleling the maturation of the lung
airway. Similarly, endothelial cells of the pulmonary
blood vessels demonstrate increased expression of PP2A
protein and mRNA in the fetus as compared to the
newborn. Therefore, PP2A demonstrates cell specific
regulation in expression during development, an unexpected result based on existing knowledge.
Developmental regulation of PP2A mRNA has been
demonstrated previously by Northern analysis in whole
Drosophila (Mayer-Jaekel et al., 1993) and Xenopus
(Van Hoof et al., 1995) embryos although cell and tissue
specific expression of PP2A was not demonstrated. This
cell specific and maturational associated localization of
PP2A to developing lung epithelium suggests a role for
PP2A in the epithelial-mesenchymal interactions necessary for normal lung development. Embryologically the
lung is of endodermal origin forming as an outpouching
of the primitive gut into the surrounding mesenchyme.
Although a great number of growth factors and their
respective receptors have been identified that affect
lung development (Minoo et al., 1994) the complex
cascade of down stream events mediating their effect
are largely unknown. The complex process of lung
PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG
Fig. 3. PP2A mRNA localization in the 19 day gestation lung. In situ
hybridization for PP2A in 19 day gestation rat lung is shown with the
antisense probe in A, C–E, and the sense control probe in B. Hybridization to bronchiolar epithelium (B), larger more mature airways (large
arrows), small forming airways (small arrows), pulmonary arteries (long
arrows and PA), and endothelium of small forming vascular channels
5
(arrow heads) is shown in A–E. D demonstrates the minimal hybridization
to the vascular smooth muscle and endothelium of the pulmonary artery
(PA). E demonstrates the high level of expression of PP2A in the small
forming airways (small arrows) with decreased expression in larger
airways (large arrows) and endothelium of forming vascular channels
(arrow head).
Fig. 4. Mapping PP2A protein expression in the 14, 17, 19 day
gestation and newborn lung. PP2A protein was detected by immunocytochemistry in the 14 day (A), 17 day (B), 19 day (C and D) gestation, and
newborn (E) rat lung. F is a slide of newborn lung incubated with
nonspecific IgG as a control. Arrows in A indicate staining of the
respiratory epithelium in the 15 day lung bud. Staining of the respiratory
epithelium of small forming airways (short arrows), endothelium of
forming vascular channels (arrow heads), larger more mature airways
(large arrows) and arteries (long arrows) is shown in B–F. D demonstrates
PP2A protein localized to the nucleus of epithelial cells lining developing
airways (short arrows). In D, expression of PP2A protein is decreased in
the endothelial of larger blood vessels (long arrow) and the epithelium of
larger airways (large arrows). Staining of alveolar cells (arrows) and
capillary endothelium (arrow head) in the newborn lung is shown in E.
PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG
7
Fig. 5. PP2A mRNA expression in the newborn lung. In situ hybridization with a anti-sense probe (A,C,E, and F) and control sense probe (B
and D) is shown. In A hybridization to alveolar cells (arrows) and capillary
endothelial cells (arrowhead) was detected. A serial section hybridized
with the sense control probe (B) demonstrated no background hybridiza-
tion. Hybridization to bronchial epithelium was very low as shown by the
arrows in the antisense probe in C and control sense probe in D. Hybridization
was still detectable in endothelium of small muscular arteries in the periphery of
the lung (E, arrowheads) whereas little hybridization was detected in the
endothelium (F, arrowhead) or smooth muscle of larger arteries (F).
morphogenesis requires the coordinate signal transduction of information from the plasma membrane to the
cell interior. Reversible phosphorylation and dephos-
phorylation of proteins is an essential mechanism
regulating the activities of proteins involved in cell
signaling (Mumby et al., 1993). PP2A is a significant
8
XUE ET AL.
down stream signaling molecule playing a role in cell
fate determination (Uemura et al., 1993) and serves as
a negative regulator of progression through the cell
cycle (Clarke et al., 1993) and growth and proliferation
(Sontag et al., 1993). A plausible explanation is that
increased PP2A expression is related to PP2A regulation of gene transcription (Alberts et al., 1993). Increased PP2A at the time of branching morphogenesis
and the decline in PP2A at its completion suggests that
PP2A is regulating expression of genes possibly necessary for the differentiation of the respiratory epithelial
cells. This theory is supported by the identification of
nuclear and cytoplasmic localization of PP2A protein in
endothelium and respiratory epithelial cells in this
study and the recent identification of specific B regulatory subunits that target PP2A to the nucleus (Tehrani
et al., 1996; McCright et al., 1996). Furthermore, PP2A
has been shown to regulate gene transcription by
dephosphorylating the cyclic AMP-regulatory element
binding protein (CREB; Wadzinski et al., 1993) and
activation of c-Jun a component of the AP-1 transcription factor (Alberts et al., 1993).
PP2A is highly expressed in the endothelium of
developing vascular channels of the lung whereas expression in endothelium of large, muscular pulmonary
arteries is barely detectable. The vasculature of the
lung develops as a combination of angiogenesis and
vasculogenesis (Coffin et al., 1988; Noden, 1989; Risau,
1995). Blood vessels begin to form de novo in the
mesenchyme of the developing lung (vasculogenesis) by
the poorly understood process whereby angioblasts
proliferate and become endothelial lined vascular channels. Lung vasculature development is completed by
ingrowth of large pulmonary arteries (angiogenesis) as
a remnant of the sixth aortic arch with connection to
the peripheral vascular tree (Noden, 1989). Although
the role of PP2A in endothelial cell development is
unknown, the high expression of PP2A in the endothelium of early vascular channels suggests a role for PP2A
in the regulation of pulmonary vasculogenesis and not
angiogenesis. This is especially intriguing as angiogenesis in the lung involves a epithelial-mesenchymal
interaction between vascular endothelial growth factor
(VEGF) secreting endoderm and mesodermal cells containing its receptor (VEGF-2R) similar to the model of
lung epithelial development (Risau, 1995). Future studies are required to determine this potential role of PP2A
in pulmonary vasculogenesis.
The role of serine/threonine protein phosphatases in
regulating organ morphogenesis has been clearly demonstrated in the rat kidney where okadaic acid an
inhibitor of PP2A and PP1, was shown in vitro to inhibit
the normal tubular branching morphogenesis of the
kidney in a dose dependent manner (Svennilson et al.,
1995). Importantly, the kidney shares a similar developmental origin and centrifugal morphologic pattern of
development as the lung.
The probes and antibodies used in the present study
cannot differentiate between the a and b isoform of the
catalytic subunit of PP2A therefore the results of this
study reflect total catalytic subunit expression (a and
b). The sequences of these two isoforms are 81%
homologous in the coding region and are coded for by
two separate genes. Although the a and b isoform has a
similar expression pattern in adult tissues (KhewGoodall, 1988), the a isoform was always expressed at a
level five to 12 times higher than the b. The physiologic
significance of these two isoforms still remains to be
determined.
In summary, we have demonstrated PP2A developmental regulation in the lung with specific mapping of
the mRNA and protein to the developing respiratory
epithelium and endothelium. We speculate that PP2A
is an important mediator in the complex process of
epithelial development in the lung and possibly other
organs such as the gut and kidney where epithelialmesenchymal interactions are important in normal
organogenesis.
EXPERIMENTAL PROCEDURES
Tissue Preparations
Sixteen time-dated pregnant Sprague-Dawley rats,
ranging in gestational age from 13 to 20 days (term is
22 days), and 23 newborn rats (Hilltop Laboratory
Animals, Inc., Scottsdale, PA) were sacrificed and their
lungs rapidly removed. Segments of the fetal chest and
neonatal lung were cut as cross or longitudinal pieces
approximately 1.5 mm thick. The specimens were then
immersed into fixative solution containing 4% w/v
paraformaldehyde in phosphate buffered saline (PBS).
After 90 min of fixation, the specimens were dehydrated in an increasing gradient of sucrose in PBS, then
quickly frozen by immersion in liquid nitrogen and
embedded in a 1:1 solution containing OCT compound
(Miles Inc., Elkhart, IN) and 20% sucrose in PBS. Two
to four µm sections were cut (at least 12 sections or six
slides for each lung) and thaw-mounted onto precleaned Superfrost Plus slides (Fisher Scientific Inc.,
Springfield, NJ).
In Situ Hybridization
Sections as above were washed in PBT (PBS, 0.3%
Triton X-100), digested in Proteinase K (1 µg/µl) for 10
min at 37°C, washed three times in PBT, re-fixed in 4%
paraformaldehyde for 2 min, washed again in PBT, and
acetylated for 10 min in 0.25% acetic anhydride, 100
mM triethanolamine HCl, and 0.09% NaCl (Sigma
Chemical Co., St. Louis, MO). Sections were then
dehydrated in an ethanol series (70%, 80%, 90%, 95%,
100%, and 100%), delipidated in chloroform for 15 min,
washed again in 100% and 95% ethanol, and air-dried.
Digoxigenin-labeled sense and antisense RNA probes
were synthesized with T3 and T7 RNA polymerase
(RNA Labeling Kit, Boehringer-Mannheim, Indianapolis, IN) from a template consisting of an 840 bp EcoR
I/Sst I restriction fragment of the rat PP2A a isoform of
the catalytic subunit (Posas et al., 1989; bp 70–910)
coding region subcloned into Bluescript plasmid vector
PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG
(Stratagene, La Jolla, CA). This coding region probe has
81% homology with PP2A b isoform of the catalytic
subunit and will detect both PP2Aa and b isoforms.
Subsequently the probes were hydrolyzed for 35 min at
60°C in hydrolysis buffer (80 mM NaHCO3, 120 mM
Na2CO3) to give probes between 150–250 bp in length.
Sections were pre-hybridized in hybridization buffer
(HB; 50% formamide, 4 3 SSC, 1 3 Denhardts solution, 500 µg/ml herring sperm DNA, 250 µg/ml tRNA,
10% dextran sulfate) for 30 min at 37°C. Probes were
denatured at 65°C and added to the HB at a final
concentration of 1 µg/ml. Hybridization took place
overnight at 37°C in a humid chamber. Slides were
washed twice in 2 3 SSC, then treated with RNase A
(20 µg/µl) at 37°C for 30 min, and washed again in 2 3
SSC 1 0.3% Triton X-100 three times. High stringency
washes were done twice in 0.23 SSC at 42°C for 15 min
each, followed by 13 SSC. Slides were rinsed in PBS,
incubated for 1 hr in PBS 1 10% heat inactivated sheep
serum, and then incubated for 1 hr in anti-digoxigenin
antibody diluted 1:2,000 in the PBS/serum solution.
Finally, slides were washed five times in PBS and two
times in Genius buffer 3 (100 mM Tris-HCl, 100 mM
NaCl, 50 mM MgCl2; pH 9.5), and incubated in the color
substrate solution (NBT/X-phosphate plus 0.3% Triton
X-100) in the dark for 24 hr. Color solution was removed
by two washes in PBS, and the slides were mounted in
aqueous medium (Geltol, Lipshaw, Detroit, MI) and
photographed using an Olympus Vanox AHBS3 bright
field microscope (Olympus, Lake Success, NY).
Immunohistochemistry
The immunohistochemical method employed has been
previously described (Xue et al., 1996), with slight
modification. After preincubating with 1% goat serum
(Vector Laboratories, Burlingame, CA) for 1 hr, tissue
cryostat sections were washed (2 3 10 min in PBS) and
incubated at room temperature with a peptide rabbit
polyclonal antibody recognizing the C-terminal portion
of PP2Aa and b (Martin et al., 1994; 1:100 dilution).
The control slides were incubated with PBS alone or
nonspecific rabbit IgG. After removal of unbound primary antibodies by washing with PBS, the sections
were incubated for 1 hr with a biotinylated anti-rabbit
antibody (1:250 dilution; Vector Laboratories), followed
by incubation in avidin-biotin-horseradish peroxidase
complex (1:50 dilution for 45 min; Vector Laboratories).
The peroxidase activity was visualized by a color reaction using diaminobenzidine (DAB; 0.5 mg/ml; Sigma)
as the substrate (brown). Finally, the slides were
mounted and then examined under an Olympus Vanox
AHBS3 bright field microscope.
Western Blot
Western blotting was performed by a method described previously with minor modifications (Xue et al.,
1996). Fetal lungs at day 14 (19 fetuses), 17 (27 fetuses),
19 (18 fetuses), and 20 (17 fetuses) of gestation, newborn (nine rats) and adult (90 days of age, six rats)
9
lungs were dissected, pooled and stored in 280°C.
Lungs were homogenized in ice-cold 50 mM Tris-HCl
buffer (pH:7.4) containing 0.1 mM EDTA, 0.1 mM
EGTA, 0.1% 2-mercaptoethanol, 1 mM PMSF, 2 µM
leupeptin, and 1 µM pepstatin A (Sigma). The homogenate was centrifuged at 1,000g for 10 min at 4°C and
the pellet was discarded. Equal quantities of lung
homogenate protein (50 µg each) and purified PP2A
protein (Martin et al., 1994; 0.1 µg, provided by Dr.
David Brautigan, University of Virginia, Charlottesville, VA) were loaded and separated on a 10% SDSPAGE gel, followed by blotting the proteins onto nitrocellulose (Bio-rad, Hercules, CA). Blots were
subsequently stained with Ponceau Red to confirm
equal loading and transfer. The blots were blocked with
buffer: 50 mM Tris-HCI (pH 7.4), 0.15 M NaCl, 2% BSA,
and 0.1% Tween-20, for 1 hr at room temperature. Then
the blots were incubated with the anti-PP2A Cterminal polyclonal antibody (Martin et al., 1994; 1:
1,000 dilution) for 1 hr at room temperature. The blots
were washed six times with PBS (5 min each) and then
incubated for 1 hr with anti-rabbit IgG antibodies
conjugated with horse radish peroxidase (Bio-Rad) at
room temperature. The blots were washed six times
with PBS (5 min each), followed by detection of immunoreactive proteins by enhanced chemiluminescence
(ECL System, Amersham). Relative protein differences
were determined using a densitometer (Personal Densitometer, Molecular Dynamics, Sunnyvale, CA) and
analysis software (ImageQuant, Molecular Dynamics).
ACKNOWLEDGMENTS
The authors are grateful to Dr. David Brautigan for
the gift of the PP2A antibody and Dr. Keith Norman for
his critical review of the manuscript. The authors also
wish to thank Mrs. Nan Zhou and Miss Tamara Stoops
for technical assistance. This study was supported by
March of Dimes Basil O’Connor Fellowship Award (to
A.E.), NIH grants K08-HL02937-02 (to A.E.), RO1HL39706 (to R.A.J.), RO1-GM49111 (to R.A.J.), and by
a research award from the American Heart AssociationVirginia Affiliate (to C.X.).
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