(Received for publication, January 19, 1996; and in revised form, March 9, 1996)
From the
In order to identify the form of phosphorylase kinase catalytic
subunit expressed in developing lung, degenerate polymerase chain
reaction primers were designed based on conserved domains of the two
known catalytic subunits, expressed primarily in muscle and testis.
Amplification of cDNA from day 19 fetal rat lung followed by cloning
and sequence analyses indicated that only the testis isoform of
phosphorylase kinase (PhK-T) was detectable in fetal lung. In
situ hybridization analyses indicated that expression of
PhK-
T RNA in developing lung tissue was widespread and not
restricted to Type II epithelial cells; PhK-
T protein expression
was temporally and spatially correlated with expression of PhK-
T
RNA. PhK-
T RNA and protein expression was also characterized in
the PhK-deficient glycogen storage disease (gsd) rat. PhK-
T RNA
levels were similar in Type II cells isolated from wild type and
gsd/gsd fetuses; in contrast, PhK-
T protein was virtually
undetectable in gsd/gsd Type II cells and enzyme activity was very low.
These results suggest that PhK-
T plays a critical role in
mobilization of glycogen during fetal lung development and that failure
to catabolize glycogen in the gsd/gsd rat is related to an
untranslatable PhK-
T RNA or unstable protein.
The epithelium of the fetal respiratory tree is characterized by the accumulation of glycogen during the last part of gestation. Glycogen mobilization is both temporally and spatially regulated such that catabolism in the proximal airways proceeds at or close to birth, whereas catabolism in the distal airway occurs earlier coinciding with maturation of the Type II epithelial cell(1) . On day 17 of gestation, the respiratory epithelium of the fetal rat lung consists of undifferentiated columnar cells that contain small amounts of glycogen; lamellar bodies, the intracellular storage granules for surfactant, are not detectable(2) . On day 19 of gestation, the epithelium consists of cuboidal cells which contain large pools of glycogen and a few lamellar bodies. By day 21 of gestation, the epithelium approximates that observed in postnatal life consisting of thin Type I cells and cuboidal Type II cells which are devoid of glycogen and contain numerous lamellar bodies. Kikkawa and co-workers (3) noted the inverse relationship between glycogen and lamellar bodies and speculated that glycogenolysis might be associated with surfactant synthesis. A direct link between the breakdown of glycogen on day 20 of gestation and surfactant synthesis was demonstrated by Farrell and Bourbon (4) who showed that glucose, originating from glycogen, provided substrate for fatty acid and glycerol synthesis which, in turn, were incorporated into surfactant phosphatidylcholine. Both biochemical and morphological studies therefore support the hypothesis that glycogen breakdown may be an important event in prenatal surfactant phospholipid synthesis.
The initial step in glycogen catabolism is the phosphorolysis of
glycogen to glucose 1-phosphate by phosphorylase a, the
activated form of glycogen phosphorylase. Glycogen phosphorylase enzyme
activity has been shown to increase in late gestation fetal lung
concomitant with initiation of glycogen degradation(5) . In
contrast to the developmentally regulated increase in enzyme activity,
the level of RNA encoding brain glycogen phosphorylase, the predominant
isoform of glycogen phosphorylase in developing rabbit lung, does not
change during the perinatal period(6) , suggesting that enzyme
activity is regulated by allosteric activation and/or by
post-translational phosphorylation. Activation of glycogen
phosphorylase by phosphorylation is regulated by phosphorylase kinase
(PhK). ()The importance of this enzyme in glycogen
mobilization is demonstrated by the gsd/gsd rat (7) which lacks
detectable PhK activity in developing lung tissue(8) .
Phosphorylase kinase deficiency in gsd/gsd fetuses leads to an
accumulation of the inactive form glycogen phosphorylase and failure to
mobilize pulmonary glycogen, ultimately resulting in reduced
phosphatidylcholine synthesis(8) . These results suggest that
post-translational phosphorylation of glycogen phosphorylase by PhK is
a critical step in the regulation of pulmonary glycogen catabolism.
Muscle PhK is a hexadecameric protein
()
with a molecular mass of 1.3
10
Da (for review, see (9) ). Calmodulin, the
subunit, is responsible for the
Ca
-dependent activity of this enzyme; the
and
subunits regulate the activity of PhK in response to
phosphorylation by cAMP-dependent protein kinase A. Glycogen
phosphorylase is activated following phosphorylation by the catalytic
subunit,
, a 387-amino acid protein. cDNAs encoding muscle
PhK
have been cloned and characterized by a number of
laboratories(10, 11, 12) . RNAs of 2.4 and
1.6 kilobases encoding muscle PhK
were detected in mouse skeletal
muscle, brain, and cardiac tissues but not in liver, consistent with
tissue-specific expression of the muscle enzyme(10) . The
tissue-specific expression of PhK may be related to the presence of
distinct PhK isozymes. Consistent with this hypothesis, human and rat
testis PhK
(PhK-
T) cDNAs have been isolated and shown to be
67% identical to rabbit skeletal muscle PhK
at the nucleic acid
level(13, 14) . The PhK-
T cDNA detected an
abundant 2.0-kilobase RNA in testis which was expressed at much lower
levels in most other tissues including lung. PhK-
T RNA was not
detected prior to 2 months of age, indicating both temporal as well as
a tissue-specific regulation of expression. The purpose of this study
was to identify the PhK
isozyme expressed in the fetal Type II
epithelial cell and characterize its temporo-spatial pattern of
expression during the perinatal period.
Figure 1:
Isolation, cloning, and sequence of
PhK in fetal rat lung. A, degenerate primer pairs LS or
NS were used in polymerase chain reactions to amplify sequences of 434
and 335 bp, respectively, from cDNA templates encoding rat muscle (PKM) and rat testis (PKT) phosphorylase kinase
catalytic subunits. Primer pair LS was subsequently used to amplify
PhK
in cDNA pools which were synthesized by reverse transcription
of three separate preparations of poly(A)
RNA isolated
from day 19 fetal rat lung. PhK
fragments of 509 and 434 bp were
identified, cloned, and sequenced. B, nucleotide sequence
analyses of cDNA clones encoding the 434-bp fragment (open
box) indicated that this fragment corresponded exactly to nt
685-1119 of the open reading frame of PhK-
T (solid box).
cDNA clones encoding the 509-bp fragment were identical to the 434-bp
fragment with the exception of a 75-bp insert at position 736 of the
PhK-
T sequence. C, PCR primers flanking the 75-bp insert
(forward primer, nt 706-725; reverse primer, nt 780-760)
were used to amplify DNA from the 434-bp cDNA clone, the 509 cDNA
clone, day 19 fetal rat lung cDNA, and adult rat genomic DNA. Cloning
and sequence analyses of cDNA clones encoding the 150-bp band verified
the presence of the 75-bp insert in genomic DNA. Size markers appear at
the right of the figure.
Spatial expression of PhK-T RNA was assessed by in situ hybridization. Sense and antisense probes were generated as
described above, using
S-labeled UTP, and hybridized with
cryosections of lung tissues as we have previously
described(19) .
For Western blot analyses, lung
tissues or Type II epithelial cell pellets were homogenized in 10
mM Tris (pH 7.5), 0.25 M sucrose, 1 mM EDTA,
5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride,
and 10 µg/ml each of pepstatin A, aprotinin, antipain, leupeptin,
and chymostatin followed by centrifugation at 140 g for 10 min (4 °C); a cytosolic fraction was prepared by
centrifugation of the supernatant at 100,000
g for 30
min (4 °C). Equal amounts of cytosolic protein, estimated by the
method of Lowry(21) , were dissolved in electrophoresis sample
buffer, subjected to SDS-PAGE followed by electrophoretic transfer to
nitrocellulose membrane, and PhK identified by Western
blotting(22) . The specificity of the immunoreaction was
verified by prior incubation of the antiserum with 100 µg of
recombinant PhK
or by immunoblotting with preimmune serum.
Immunocytochemistry was performed on paraffin-embedded lung tissues
exactly as described by Vorbroker et al.(23) . Tissue
sections were incubated with either affinity-purified PhK antisera
or antiserum directed against the SP-C proprotein(23) . The
specificity of the immunoreaction was verified as described above.
Figure 2:
Expression of PhK RNA in lung tissue.
P-Labeled antisense RNA probes (pkt and pkm at right of figure) were generated from the
3`-untranslated region of the rat testis PhK
(PKT) cDNA
(see Fig. 1B) or the rat muscle PhK
(PKM)
cDNA. Labeled probes were hybridized with 10 µg of total RNA
isolated from adult rat testis (T), muscle (M), lung (AL), or day 19 fetal lung (FL). Following an
overnight incubation at 42 °C, unhybridized RNA was digested with
ribonuclease and the protected fragments were analyzed by
SDS-PAGE/autoradiography. A 268-base fragment is protected by PKT in
all four tissues, although expression in lung and muscle is very low
relative to testis; in contrast, PKM protects RNA only in muscle
tissue.
Figure 8:
Expression of PhK-T RNA and protein
in Type II epithelial cells isolated from gsd/gsd rats. A,
Type II epithelial cells were isolated from adult rat lungs and from
wild type or gsd/gsd fetal lungs on days 20 and 21 of gestation. Total
RNA was isolated from each group of Type II cells and RNase protection
assays performed with PhK-
T antisense probes, as described in the
legends to Fig. 2and Fig. 3. PhK-
T RNA was detected
at similar levels in Type II cells isolated from wild type and gsd/gsd
fetuses. B, Type II cells were isolated from gsd/gsd fetuses (lanes 1-3), or from wild type fetuses on day 20 of
gestation (lanes 4 and 5) and from adult rat lungs (lane 6). RNA was isolated from each Type II cell preparation
and analyzed by reverse transcriptase-PCR, using the LS primer pair, as
described in the legend to Fig. 1. Alternatively spliced forms
of PhK-
T RNA were detected in wild type and gsd/gsd fetal Type II
cells as well as in adult Type II cells. C, 125 µg of
protein from lung homogenates (lanes 1-3) and Type II
cell lysates (lanes 4-6) were analyzed by Western
blotting as described in the legend to Fig. 6. PhK-
T
protein, M
approximately 41,000, was detected in
wild type tissues (lanes 1, 4, and 5) but not in Type
II cells or lung tissues from gsd/gsd fetuses (lanes 2, 3, and 6).
Figure 3:
Quantitation of PhK-T RNA expression
during lung development.
P-Labeled PKT antisense RNA
probes (see Fig. 2) were hybridized with 10 µg of total RNA
isolated from fetal (days 17, 19, and 21 of gestation), postnatal (days
1, 3, and 5), and adult rat lung as well as adult rat testis. Following
an overnight incubation at 42 °C, unhybridized RNA was digested
with ribonuclease and the protected fragments, recovered by ethanol
precipitation, were analyzed by SDS-PAGE. PhK
RNA bands were
quantitated by PhosphorImager analysis of the dried gel and normalized
to an internal control (L32 RNA). Results presented represent the mean
± the S.D. of four separate
experiments.
Figure 6:
Generation and characterization of
PhK-T antisera. A, bacterial lysates expressing
recombinant rat PhK-
T protein were loaded onto a Ni-NTA column as
described under ``Materials and Methods.'' The column was
washed with 20 mM Tris (pH 7.9), 6 M urea, 500 mM NaCl and eluted with 50 mM EDTA in 20 mM Tris
(pH 7.9), 6 M urea, 150 mM NaCl. Fractions of protein
not bound to the column (lane 2) and protein eluted from the
column (lane 3) were analyzed by SDS-PAGE and silver staining.
Pre-stained molecular weight marker proteins appear in lane 1.
B, preimmune serum (lane 1) and immune serum (lanes
1-3) generated against purified, recombinant PhK-
T
protein were used in Western blot analyses of purified rabbit muscle
PhK (lane 3), and lung homogenates of day 21 fetal lung (lane 1), and adult lung (lane 2). Prior incubation
of immune serum with recombinant PhK-
T protein blocked detection
of the major immunoreactive band, M
of
approximately 41,000 (not shown). 125 µg of protein was analyzed
for each lung homogenate; 100 ng of muscle PhK was analyzed (lane
3).
Figure 4:
Expression of PhK-T RNA in developing
lung.
S-Labeled sense and antisense probes were generated
from the 3`-untranslated region of the rat testis PK
cDNA. In
situ hybridization with antisense probes indicated that PK
RNA was expressed predominantly in the epithelium of developing airways
on day 17 of gestation (panel A). By day 19 of gestation,
virtually all cells in the developing lung expressed PhK
RNA (panel B). Widespread expression was maintained in lung
tissues from day 21 of gestation (panel C) and postnatal day
21 rats (panel D). In situ hybridization with sense
probes resulted in relatively little signal in day 17 gestation lung (panel E); however, significant signal was observed in the
epithelium of some airways in day 21 gestation lung (panel F).
Sense signal was not detected with irrelevant sense probes and was also
detected with a second sense probe generated from the 3` end of the
coding sequence of the rat PK
cDNA (not
shown).
Hybridization with control sense probes resulted in significant
signal in the bronchiolar epithelium and blood vessel walls of fetal
(not shown) and postnatal lung (Fig. 4F). The
specificity of PhK-T antisense RNA expression was confirmed by: 1) in situ hybridization with two separate sense probes from the
3`-untranslated or 3`-coding region of PhK-
T; 2) by in situ hybridization with irrelevant sense probes (cdk-4 and cdk-1); and
3) by RNase protection assays with labeled PhK-
T sense probes (Fig. 5). Antisense PhK-
T RNA was consistently detected in
both fetal and adult lung but not in adult testis or skeletal muscle (Fig. 5). These results suggest that PhK-
T antisense RNA is
expressed in lung in a spatially restricted manner.
Figure 5:
Expression of antisense PhK-T RNA in
fetal and adult lung.
P-Labeled sense PK-
T
RNA probes were generated from the 3`-untranslated region of the rat
PK
cDNA and hybridized with 10 µg of total RNA isolated from
fetal (day 17 and 21 of gestation) rat lung, adult rat lung (L), adult rat testis (T), and adult rat muscle (M). Following overnight incubation at 42 °C, unhybridized
RNA was digested with RNase and the protected fragments were analyzed
by SDS-PAGE/autoradiography.
Figure 7:
Expression of PhK-T protein in adult
lung. Adult lungs were perfusion-fixed and embedded in paraffin. Tissue
sections were incubated with either affinity purified PK
antiserum (left panel) or antiserum directed against the SP-C proprotein (right panel). SP-C expression is restricted to alveolar Type
II cells, whereas expression of PK
is detected in both Type II
cells as well as bronchiolar epithelial
cells.
The present study focused to the identification and
characterization of PhK expression in lung and, in particular, the
Type II epithelial cell during the perinatal period. PCR conditions
were designed to identify both known and closely related novel isoforms
of PhK
. The collective results of PCR, RNase protection, and in situ hybridization studies indicate that the testis isoform
was the only detectable form of PhK
in fetal lung tissue.
Furthermore, PhK-
T RNA was readily detected in fetal Type II
epithelial cells and was present at comparable levels in wild type and
gsd/gsd fetuses; however, in contrast to wild type animals, PhK-
T
protein and enzyme activity were significantly decreased in gsd/gsd
fetal Type II cells. Given these results, it is likely that the lack of
PhK-
T protein is directly responsible for impaired glycogen
mobilization in gsd/gsd Type II cells during the perinatal period.
It is unclear if the small amount of enzyme activity detected in
fetal gsd/gsd Type II cells is due to residual testis PhK isozyme, to
contamination of the Type II cell preparation with some other cell
types, or to the presence of a minor, novel PhK isozyme. Support for a
novel PhK isozyme comes from the unexpected finding that PhK activity
is significantly increased in adult Type II cells and is only slightly
decreased in adult gsd/gsd Type II cells. PhK in adult Type II
cells is immunologically distinct and is sufficiently divergent in
nucleic acid sequence from the muscle and testis isoforms such that it
is not detected in fetal Type II cells by PCR with degenerate primers.
The identity and function of this putative novel PhK isozyme remains
unknown but is particularly intriguing given the paucity of glycogen in
adult Type II epithelial cells.
Apart from modulation of and
regulatory subunit expression, PhK-
T enzyme activity in
developing lung may be regulated at several levels. Differential
splicing of the PhK-
T gene gives rise to a least one alternatively
spliced RNA transcript which was detected in fetal and adult Type II
cells from both wild type and gsd/gsd animals. The alternatively
spliced PhK-
T transcript encodes a 75-bp in-frame insertion which
corresponds positionally to the E intron of the rat muscle PhK
gene(27) . Insertion of 25 amino acids into the catalytic
domain of PhK-
T could conceivably reduce enzyme activity or alter
substrate specificity. Differential splicing resulting in deletion,
replacement, or insertion of specific sequences has previously been
reported for muscle
and
and liver
subunits (28, 29) and may play a role in regulating PhK enzyme
activity.
Spatial distribution of PhK-T may be regulated in
part by antisense RNA expression. Antisense transcripts were detected
by RNase protection and were localized by in situ hybridization to portions of the bronchiolar epithelium and the
blood vessel wall. There was no detectable change in antisense
expression in fetal and adult lung; antisense expression was not
detected in testis or muscle, consistent with a tissue-specific
response. However, it was not clear from these studies if PhK-
T
protein and/or enzyme activity was reduced at the site of antisense
expression.
The PhK-T RNA transcript contains a number of
elements consistent with translational control of expression. First
there is an AUG located at position -82, upstream from the major
open reading frame, which is followed by an in-frame STOP codon at
position 27-29 within the coding sequence of PhK-
T. The
sequence context of the upstream AUG is similar to the authentic start
codon and may therefore be recognized by the ribosome resulting in
inhibition of translation (for review, see (30) ). Second, an
RNA folding algorithim (31) predicts stable secondary
structures within the 5` leader sequence with the potential to inhibit
ribosomal scanning(32) . It is possible that alternate sites of
PhK-
T transcription initiation (14) or differential
splicing of the primary transcript may lead to loss of inhibitory
elements in the leader region resulting in more efficient translation.
In this regard, translational control may account for similar levels of
PhK-
T protein in lung and testis despite the fact that PhK-
T
RNA levels are approximately 100-fold higher in testis.
The testis
isoform of PhK is the major source of PhK activity in the fetal
Type II epithelial cells. The importance of PhK-
T in glycogen
mobilization in the prenatal lung is underscored by parallel changes in
PhK-
T protein, enzyme activity, and glycogen catabolism in gsd/gsd
fetal Type II cells. The temporo-spatial regulation of PhK-
T
expression in fetal lung is likely to be very complex and may involve
differential splicing, antisense expression, and translational controls
in addition to interaction with various regulatory subunits. Despite a
clear role for PhK-
T in fetal lung, the function of this enzyme
and the identity of other PhK
isozyme(s) in postnatal lung remains
unknown.