Page
1 of 27 in PresS. Am J Physiol Lung Cell Mol Physiol (September 15, 2006). doi:10.1152/ajplung.00049.2006
Articles
1
Regulation of rat alveolar type 2 cell proliferation in vitro
involves type II cAMP-dependent protein kinase
Jan T. Samuelsen1,2, Per E. Schwarze1, Henrik S. Huitfeldt3, E. Vibeke Thrane4, Marit Låg1, Magne
Refsnes1, Ellen Skarpen3 and Rune Becher1
1
Norwegian Institute of Public Health, Division of Environmental Medicine, Oslo, Norway
2
Nordic Institute of Dental Materials, Haslum, Norway
3
Laboratory for Toxicopathology, Institute of Pathology, University of Oslo, Rikshospitalet University
Hospital, Oslo, Norway
4
Poison information centre, Oslo, Norway
Running title: Type 2 cell proliferation in vitro involves PKA II.
Key words: cAMP, PKA, proliferation, type 2 cells
Corresponding author:
Jan Tore Samuelsen
Nordic Institute of Dental Materials
PO Box 70
N-1305 HASLUM
Norway
Telephone: +47 67 51 22 00
Fax: +47 67 59 15 30
[email protected]
Copyright © 2006 by the American Physiological Society.
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Abstract
To elucidate the role of cAMP and different cAMP-dependent protein kinases (PKA; A-kinase) in lung cell
proliferation, we investigated rat alveolar type 2 cell proliferation in relation to activation or inhibition of
PKA and PKA regulatory subunits (RII and RI ). Both the number of proliferating type 2 cells and the level
of different regulatory subunits varied during seven days of culture. The cells exhibited a distinct peak of
proliferation after 5 days of culture. This proliferation peak was preceded by a rise in RII protein level. In
contrast, an inverse relationship between RI and type 2 cell proliferation was noted. Activation of PKA
increased type 2 cell proliferation if given at peak RII expression. Furthermore, PKA inhibitors lowered the
rate of proliferation only when a high RII level was observed. An antibody against the anchoring region of
RII showed cell cycle dependent binding in contrast to antibodies against other regions, possibly related to
altered binding to A-kinase anchoring protein (AKAP). Following activation of PKA, relocalisation of RII
was confirmed by immunocytochemistry. In conclusion, it appears that activation of PKA II is important in
regulation of alveolar type 2 cell proliferation.
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Introduction
Extensive cell proliferation takes place during lung development. In the rat lung, the percentage of
proliferating cells decreases to a low level shortly after birth and only proliferation to replace damaged or
dying cells occurs. The population of proliferating cells in the alveolar epithelium consists mainly of type 2
cells (49). These cells are the progenitor cells of the respiratory type 1 cells and are also able to proliferate in
culture (24,27,30).
The cAMP-dependent pathway stimulates cell proliferation in some cell types, while inhibiting cell growth in
others (8,37). The cAMP dependent signaling pattern involved in the regulation of lung cell proliferation has
been elucidated only to a minor extent. Previous findings using cultured lung tissue have indicated cAMP as
a positive regulator in type 2 cell proliferation (55). Furthermore, involvement of PKA in the regulation of
type 2 cell differentiation, surfactant production and secretion, has also been indicated. The major receptor for
cAMP in eukaryotes is the cAMP-dependent protein kinase (PKA; A-kinase). PKA activity and PKA subunit
mRNA expression have been found to be developmentally regulated in fetal lung (1). It has been suggested
that this results in an optimal PKA activity at the time of type 2 cell differentiation, presumably in preparation
for gas exchange (1).
The PKA holoenzyme consists of two catalytic (C) subunits, whose activity is inhibited by a dimer of
regulatory subunits (R2). Upon binding of cAMP, the holoenzyme dissociates into a regulatory subunit dimer
and two active catalytic subunits. Active C subunits phosphorylate specific substrates that can be translocated
to the nucleus, where they activate cAMP responsive genes (18, 19, 33). Proteins involved in regulation of
proliferation, such as cdc2 (47), cyclin A (13), the RB protein and D-cyclins (54, 34) have been found to be
modulated by PKA activation. However, these proteins did not seem to be phosphorylated directly by PKA.
Two main classes of PKA have been identified, type I (PKA I) and type II (PKA II). The two holoenzymes
differ only in their regulatory subunits, but share the same type of catalytic subunits (RI2C2 and RII2C2,
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respectively). Two isoforms of each subunit have been identified in rat tissues,
and
(14). Whereas the -
isoforms are found in most tissues examined including lung, the -isoforms seem to be tissue-specifically
expressed mainly in neural and reproductive organs (6). In addition to inhibition of the catalytic subunit, the
regulatory subunits are considered to be involved in the localization of the PKA holoenzyme. The N-terminal
region of the regulatory subunit of PKA II (RII) contains a binding site for A-kinase anchoring proteins
(AKAPs) (32). Both RI and RII can exist in soluble or anchored phases depending either on cell type or phase
or state of the cell (2). The RII subunit has been found to bind with high affinity to AKAPs, whereas the RI
subunit only binds to some AKAPs and with much lower affinity (21, 28). Different AKAPs are localized
preferentially to different membrane structures, such as sarcoplasmatic reticulum (31), centrosome and Golgi
region (40) or mitochondria (41) and thereby specify PKA II colocalisation with specific substrates (12).
Several studies have shown PKA I to be directly involved in cell proliferation and neoplastic transformation
(9, 45). PKA I has also been reported to be required for the transition from the G1 to the S phase of the cell
cycle (45), as well as mediating mitogenic signals through different growth factors (10, 11, 46). Cell lines
derived from adult mouse lung epithelial cells and displaying low levels of PKA I (RI ) protein have been
found to grow faster than cell lines with higher levels indicating an inverse relationship between growth and
PKA I (29). PKA II, which is preferentially expressed in normal tissues, seems mostly to be involved in cell
growth arrest and differentiation (9, 35, 44). However, in some cell types, an association between PKA II and
cell proliferation has been reported (4, 15). Thus, PKA I and PKA II may mediate both stimulatory and
inhibitory proliferation signals, depending on the cell type.
The aim of this study was to elucidate the role of cAMP and PKA isoforms in regulating proliferation of
primary rat alveolar type 2 cells.
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Materials and Methods
Chemicals: Ethidium bromide, dibutyryl-cAMP (dbcAMP), forskolin, protease (type 1: crude), DNAse 1
(type III), RNAse, hydrocortisone, epithelial growth factor (EGF), insulin, bovine serum albumin (BSA) were
purchased from Sigma Chemical Co (St. Louis, MO, USA). PKA-inhibitors H8 and H89 were from
Seikagaku (Falmouth, MA, USA), Williams E medium from Bio Whittaker (Walkersville, MD, USA),
Hoechst 33258 from Calbiochem-Boehringer (La Jolla, CA, USA) and fetal bovine serum (FBS) from Gibco
BRL (Paisley, Scotland). All other chemicals were purchased from commercial sources at the highest purity
available.
Cell isolation and culture: Type 2 cells were isolated from male Wistar Kyoto (WKy/NHsd) rats between 6
and 8 weeks of age. The cells were isolated by the sequential use of enzymatic digestion, centrifugal
elutriation and differential attachment, as previously described (27). In brief, development of the isolation
procedure included verification of type 2 cell presence by electron microscopy, light microscopy,
immunocytochemistry and western blotting. Current confirmation of epithelial cell origin in general and type
2 cell presence in specific, included expression of epithelial cadherin, pan-keratin as well as pro-surfactant
protein C (pro-SPC). The results indicated that cells expressing these proteins dominated throughout culture.
The epithelial lung cells were cultured in Williams E medium fortified with EGF (10 ng/ml), insulin (5
µg/ml), hydrocortisone (87 ng/ml) and 5% fetal bovine serum (FBS). Cell viability was assayed by trypan
blue exclusion. Purity in the type 2 cell fraction was approximately 90% (27).
Determining cell number: Cell culture dishes were fixed for 5 min in methanol and stained with 2% Giemsa’s
solution in neutral destilled water. The stained cells were counted using a Nikon Optiphot light microscope.
Antibodies: Antisera to rat RI and rat RII were raised against synthetic peptides corresponding to AA 1-18
and 5-21, respectively, as previously described (39). A second antibody to RII raised against AA 367-394
was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to catalytic subunit (BD-
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Bioscience, Erembodgem, Belgium) was a generous gift from Bjørn Skalhegg (Faculty of Medicine,
University of Oslo). As secondary antibodies, HRP-conjugated anti-rabbit IgG (Sigma Chemical Co, St.
Louis, USA) and Fluorrescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (DAKO A/S, Glostrup,
Denmark) were used.
Activation and inhibition of PKA in type 2 cells: To activate PKA, dbcAMP (200 µM) or forskolin (3 µM)
was added to the medium at specific time points. To inhibit PKA activity, 50 µM and 3 µM, respectively, of
the PKA inhibitors H-8 or H89 were used.
SDS-PAGE and Western analysis: Cultured type 2 cells were scraped off the dish. Total protein (15 µg) of
each sample from the cells were prepared and electrophoresed in 10 % sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE) and blotted onto nitrocellulose filters, according to standard procedures
(26). Even loading and transfer of proteins to nitrocellulose membrane was confirmed by Ponceau-S staining
of the blots. Proteins were analysed according to the Western blotting technique (48). The filters were
blocked for 30 min with 5% nonfat dry milk in TBS-Tween (0,1% Tween 20), and then incubated with
antisera diluted in TBS-Tween containing 1% nonfat dry milk. Working dilutions of 1:10000 (anti-RI ),
1:5000 (anti-RII 5-21), 1:15000 (anti-RII 367-394) and 1:2000 (anti-C) were found to be appropriate for
immunoblotting techniques. Immunoreactive protein was detected using the chemiluminescence system
(ECL, Amersham Laboratories, Buckinghamshire, UK) after incubation with a HRP-conjugated secondary
antibody.
Flow cytometric cell cycle analysis: Freshly isolated cells and cells cultured for up to 7 days were stored in
citrate buffer (29) at -70°C. Cellular DNA was stained with Hoechst 33258 (1.2 µg/ml), essentially as
previously described (50). Samples were measured using a flow cytometer (Argus 100, Skatron, Tranby,
Norway), and the DNA histograms were analysed using the Multiplus Program (Phoenix Flow Systems, San
Diego, CA, USA).
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Dual parameter DNA/PKA flow cytometry. To determine the level of regulatory subunits during different
phases of the cell cycle, cultured cells were trypsinised off the culture dish and processed according to a
previously described method (36) with small modifications. Isolated cells were fixed in 4°C ethanol for 5
min. After centrifugation, the samples were washed twice in phosphate buffered saline (PBS) containing 1%
BSA and incubated overnight at room temperature with RNase (50 µg/ml in PBS) and primary antibody to
RI
or two different RII
antibodies (diluted 1:1000 and 1:500, respectively). Samples were washed and
incubated for 2h with secondary antibody (FITC-conjugated goat anti rabbit, DAKO, Glostrup, Denmark).
Finally, the cells were stained with ethidium bromide (2 µg/ml). As control, cells were treated identically, but
with preimmune rabbit serum at the same concentration as the antibody.
Immunocytochemistry: To investigate subcellular localisation of RII and possible translocation during cell
cycle, unstimulated as well as dibutyryl treated type 2 cells were incubated with Mito-Tracker according to
the protocol provided by the manufacturer. The cells were subsequently washed in PBS and fixed in methanol
for 3 min before incubation for 20 h with rabbit anti-RII
(working dilution 1:200). After washing and
incubation for 3 h with an anti-rabbit fluorescein-isothiocyanate (FITC)-conjugated antibody, the preparations
were mounted and visualized using a Nikon Eclipse E400 microscope and a SPOT diagnostic instruments
digital camera. As controls, the anti-RII antibody was omitted.
Statistics
The data were analyzed using either one-way ANOVA with pairwise multiple comparison procedures
according to the Holm-Sidak method, Kruskal-Wallis One way ANOVA on Ranks or Mann-Whitney Rank
Sum test to evaluate the difference between groups. P values less than 0.05 were considered significant.
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Results
Lung cell proliferation
The proliferative behaviour of type 2 cells in primary culture was monitored over a period of 7 days. Figure 1
a shows the percentage of type 2 cells in S- and G2/M- phase of the cell cycle as measured by flow cytometry.
The percentage of type 2 cells in these phases increased significantly from day 3 of culture and reached a
proliferation peak at day 5 with more than 25% of the cells in S- and G2-/M- phase. The cell number
increased approximately 3-fold from day 1 to day 7 (Fig 1b). The largest increase was from day 5 to day 7.
To study the possible involvement of PKA in type 2 cell proliferation, PKA activity was stimulated with
dibutyryl cAMP (db-cAMP) or inhibited with the PKA inhibitor H8 at different time points (Fig. 2a). The
inhibitor and the stimulator had no detectable effect on the percentage cells in S- and G2/M-phase early (day
1) or late (day 7) in the culture. In contrast, dbcAMP exerted a statistically significant stimulatory effect on
the number of S- and G2/M-phase cells just prior to and at the proliferation peak at days 3 and 5. During the
same period H8 had a statistically significant inhibitory effect (Fig. 2a).
Figure 2b shows a comparison of the relative effects of different stimulators and inhibitors of PKA on day 3
of the culture. The PKA inhibitor H8 significantly lowered the number of proliferating cells by about 3-4%
(20% reduction compared to control). Also the PKA inhibitor H89 appeared to lower the number of
proliferating cells although we did not find this decrease statistically significant. Cells incubated with
dbcAMP, 8-bromo-cAMP or forskolin showed a significant increase of 3 to 4% in S/ G2/M –phase cells (a
relative increase of 18 to 25% compared to control). Dibutyryl-cAMP seemed to exert a slightly stronger
effect than forskolin or 8-bromo-cAMP.
Furthermore, the number of cells in culture increased significantly 48 h after PKA activation, whereas PKAinhibition significantly reduced that number relative to untreated control (Fig 2 c). Cells simultaneously
exposed to PKA stimulators and inhibitors exhibited the same reduction in proliferating cells as with the
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inhibitors alone (data not shown). After prolonged incubation (48 h), the percentage of S/ G2/M cells was
similar in control cells and cells with stimulated PKA (data not shown).
PKA-regulatory subunit protein levels
To investigate if the differential effects of PKA activity modulators resulted from changes in PKA regulatory
subunit levels, we measured their levels in type 2 cells during 7 days of culture by immunoblotting.
Immunoblotting of PKA-RI detected a protein at MW 49 kD (Fig 3a). Freshly isolated type 2 cells exhibited
a relatively high level of RI that declined after the first 24 h in culture. The significantly lowest levels were
observed at days 3 to 5. Subsequently, the amount of RI , increased approximately to the same as found in
24 h cultured type 2 cells (Fig. 3a). Immunoblotting of RII detected a protein at MW 52 kD (Fig 3b). The
RII level in type 2 cells increased from very low levels at day 0 to significantly higher levels between days
1 and 5 of culture and with an apparent peak at day 3. Thereafter, the level of RII declined to levels similar
to the level at day 1 (fig. 3b). Thus, the time curve for stimulation with cAMP analogues and appearance of
increased RII levels correlated with the proliferation curve, whereas the time curve for RI levels showed an
inverse correlation.
PKA subunit levels after PKA activation: PKA may regulate the levels of its subunits as a “feedback”
inhibition. Immunoblotting was used to monitor the PKA subunit level after activation with either db-cAMP
or forskolin. After PKA activation, the level of C was significantly reduced (Fig 4 a) whereas RI
was
significantly upregulated after treatment with dbcAMP (Fig 4 b). However, also forskolin appeared to cause
an increase in RI subunit level, although we did not find this statistically significant. The level of RII was
not affected (not shown).
Cell cycle-dependent level of the regulatory subunits: Dual parameter flow cytometry was used to measure
the binding of antibodies to RI or RII during the cell cycle. Analysis with RI
antibody and ethidium
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bromide (EB) showed that the antibody binding was proportional to the DNA content of the cells (Fig. 5a).
There was twice as much antibody binding in G2/M-phase cells as in G1-phase cells, indicating that the
increase in the RI
level followed average protein doubling. The antibody raised against the N-terminal
region of RII (aa 5-21), which also is the AKAP binding region of the RII protein, was bound to the cells
in a cell cycle specific manner. Antibody binding was relatively low in G0/G1 phase, but increased severalfold
in the G1- to S-phase transition. Thereafter, the level increased proportionally to the DNA-content through Sand G2/M-phase (Fig 5b). The antibody raised against aa 367-394 of RII was also used to see if RII levels
varied abnormally or if the changes was due to differences in antigen availability during a certain period of
the cell cycle. This antibody did not show any cell cycle specific binding (Fig 5c) suggesting the latter
possibility.
Immunocytochemistry:
Our observation that the antibody against the anchoring region of RII showed cell cycle dependent binding
could imply altered binding to A-kinase anchoring protein (AKAP) and a change in RII localisation during
cell cycle. Such a possibility was confirmed immunohistochemically where mitochondrial location of RII
was observed in unstimulated cells (Fig 6 a) whereas stimulation by dibutyryl cAMP for 1 h caused a more
diffuse cytoplasmatic localisation of RII (Fig 6 b).
Discussion
Cyclic AMP has the dual ability to act both as a negative and a positive regulator of cell proliferation. By
using stimulators of cAMP, it has been shown that cyclic AMP acts as a positive regulator in proliferation of
lung tissues (55). These latter results further indicated that cAMP induced proliferation mainly occurred in
alveolar regions, where the type 2 cells are the main proliferating cell population. Most likely, the effect of
cAMP was mediated through activation of PKA since no other cAMP receptors have been reported in
epithelial cells.
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Many studies exploring the role of PKA isozymes have related PKA I to proliferation and PKA II to growth
arrest and differentiation (8). However, we found an inverse pattern between the wave of type 2 cell cell
proliferation in vitro and changes in RI protein levels. This is in agreement with the results of Lange-Carter
and coworkers (29) reporting an inverse correlation with the RI subunit and proliferation of cell lines
derived from mouse lung type 2 cells. Also, in other cell types a similar correlation between increased levels
of RI
and decreased proliferation rate has been reported (38). Some studies have also suggested a
correlation between RII
and proliferation (4,15). Interestingly, we find that the wave of type 2 cell
proliferation in vitro and the RII protein levels of these cells mainly display a corresponding pattern.
In their study of RI expression in tumour cell lines of lung epithelial origin, Lange-Carter and co-workers
did not observe any changes in RII protein levels (29). We find that peak RII protein levels preceded the
peak of proliferating type 2 cells. This may indicate an involvement of the kinase early in the cell cycle of
type 2 cells. The data suggest that PKA may be involved in the G1-phase or early S-phase of the type 2 cell
cycle, consistent with what has been reported in thyroid cells and liver cells (5,15). This would also be in
agreement with previous findings indicating a role for cAMP and probably PKA in regulating
phosphorylation status of Rb protein and D-type cyclins (54). These proteins appear to have central roles
during the G1-phase and the entrance into the S-phase of the cell cycle. In addition, cyclin A exerts its effect
during the G1-S phase of the cell cycle and PKA is shown to have a role in the regulation of cyclin A2 level
in hepatocytes (13). The involvement of PKA II early in the cell cycle is supported by our results showing
that the level of RII antibody binding in G2/M-phase is severalfold higher than in the G1-phase and not two
times as expected for the average of proteins. Activation of PKA II is a result of conformational changes in
the N-terminal region of RII dimer after cAMP binding. This region is also known to bind to PKA anchoring
proteins (AKAPs), which have been suggested to dissociate from RII upon cAMP binding. Only the antibody
raised against this region of RII showed cell cycle specific binding. This further suggests that the increase in
antibody binding in the G1/S is due to activation of PKA II in this period.
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The subcellular localization of signaling molecules is suggested to be an important mechanism for regulating
specificity of signal transduction pathways. Most associations between AKAPs and R subunits as well as
other signaling proteins are likely to be conditional. In this context, phosporylation of RII and its association
with the centrosome has been shown to change with the phase of the cell cycle (25). In thyroid cells, cAMPdependent regulation of cell cycle genes has been linked to intracellular PKA II translocation (15). We find
that cAMP stimulation of PKA II resulted in transient proliferation and relocation of RII from mitochondria
to the cytoplasm in alveolar type 2 cells. A kinase anchoring proteins (AKAPs) direct the subcellular
localization of PKA by binding to the regulatory (R) subunits. Two AKAPs (DAKAP1 and 2) localized to
mitochondria of several tissues including lungs and having affinity for both RI and RII have been identified
(41). Although the physiological relevance of PKA and AKAPs within mitochondria is not fully understood,
it has been recognized that PKA plays a critical role in mitochondrial physiology. In this context, it has been
suggested that mitochondrial AKAP create a junction at which cAMP and tyrosine kinase signal transduction
converge and interact (16, 51). Other proposed physiological responses by mitochondria-anchored PKA
include involvement in the apoptotic process through phosphorylation and inactivation of the proapoptotic
protein bad (20). Interestingly, we found that the antibody against the anchoring region of RII showed cell
cycle dependent binding. This could indicate altered binding to AKAP and a change in RII localisation
during cell cycle. We confirmed this immunohistochemically as mitochondrial location of RII was observed
in unstimulated cells whereas stimulation by dibutyryl cAMP caused diffuse cytoplasmatic localisation of
RII .
Although the initial events associated with type 2 cell proliferation are likely to involve complex signaling
pathways in addition to cAMP and PKA, our results support that PKA II is an important regulator in the
process. In this regard, the observed increase in RII
level just prior to proliferation may indicate a
prerequisite for PKA II before proliferation may be initiated. For subsequent entry into the cell cycle, we
suggest that the already increased level of PKA II requires activation. This is supported by our results
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showing that stimulation of PKA lead to an increased percentage of proliferating cells as well as an increased
number of cells. However, this effect could only be achieved when PKA II was already the dominating
isoform. Similar, inhibition of PKA reduces the percentage of proliferating cells, as well as lowers the
increase in cell number, but only when the PKA II isoform dominates. Morerover, stimulation of PKA at high
PKA II levels, appears to induce increased RI levels whereas the RII levels remained stable. Upregulation
of cytoplasmatic RI may function as a negative feedback regulating mechanism on proliferative signals by
PKA II. This upregulation together with a cytoplasmatic translocation of RII similar to what we observe,
and which most likely localize the PKA holoenzyme to other substrate proteins, could then have an inhibitory
influence on proliferation.
Cyclic AMP regulates PKA activity through altering the binding affinity between C and R. However, PKA
may also regulate its own activity by altering the R:C ratio (23, 42). In normal adult tissues, the ratio between
R and C is 1:1 (22). We find reduced level of C and increased level of R after PKA stimulation, thus
increasing the R/C ratio. This implies that higher cAMP threshold levels would be needed for PKA
activation. Such a possibility is suggested by our observation that prolonged PKA activation results in
proliferative activity similar to unstimulated levels.
Overall, PKA isotypes most likely play a partial role only for regulation of lung cell growth and
differentiation (7, 52, 53). In line with this, we find that inhibitors and stimulators of PKA modulate type 2
cell proliferation only to a certain extent and only at specific time points. Other signaling pathways suggested
to be of relevance in regulating type 2 cell proliferation in vitro is the extracellular signal-regulated kinase
(ERK) cascade (43). The ERK pathway could be controlled in both PKA- dependent and PKA-independent
ways (3, 17) although the relevance of this in lung cell proliferation has not been elucidated.
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In conclusion, our results indicate that cAMP and the activation of PKA II participate in the stimulation of rat
alveolar type 2 cell proliferation in vitro, whereas RI
may have other functions in this cell type. The
activation of PKA II seems to be specific to the G1 to S transition of the cell cycle.
Acknowledgments
The financial support by The Research Council of Norway is gratefully acknowledged. Furthermore, the
expert technical assistence of Edel M. Lilleaas is greatly appreciated. We are also thankful to Dr. Richard
Wiger for useful discussion and comments on the manuscript.
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Figure legends
Figure 1. Proliferation of alveolar type 2 cells in primary culture. a: Proportion of cells in S-and G2/Mphase of the cell cycle during culture. A peak level of close to 30% cells is seen at day 5 of culture. b: Cell
density (cells/mm2) increases in during the seven days of culture. The highest increase is seen after day 5.
All values are the means + SD of three or more separate experiments. Asterisks depict values significantly
higher compared to day 0 (fig. a) or day 1 (fig. b) (p < 0.05).
Figure 2. Effect of PKA activators and inhibitors on type 2 cell proliferation. a: Proportion of cells in Sand G2/M-phase different from control after 24 h treatment. b: Proportion of cells in S- and G2/M-phase
different from control after 24h treatment with more PKA activators and inhibitors at day 3 of culture. c: Cell
density after treatment with different PKA activators and inhibitors. The activators or inhibitors were added at
day 2, and cells counted at day 4 of culture. All values are the means + SD of three or more separate
experiments. Asterisks depict values significantly different from control (p < 0.05).
Figure 3. Western analysis of the regulatory subunits of PKA in primary culture of alveolar type 2
cells. a: RI protein level in freshly isolated type 2 cells and cells cultured up to 7 days. Relatively high
protein level is seen in freshly isolated cells. At days 3-5 the level is barely detectable, but the level increases
again at day 7. b: RII protein level increases from barely detactable in freshly isolated cells to a peak level at
days 3-5. The western blot results each show a representative experiment. All values in the bar chart are the
means + SD of three or more separate experiments. Asterisks depict values significantly different from day 0
(p < 0.05).
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Figure 4. Level of PKA subunit levels after activation with dbcAMP or forskolin. a: The level of
catalytic subunit is clearly down regulated after 24h PKA activation with dbcAMP or forskolin. b: The level
of RI regulatory subunit is upregulated after 24 h activation of PKA. The increase is higher in cells activated
with dbcAMP than in cells activated with forskolin. The western blot results each show a representative
experiment. All values in the bar chart are the means + SD of three or more separate experiments. Asterisks
depict values significantly different from day 0 (p < 0.05).
Figure 5. Level of regulatory subunits of PKA during cell cycle. a: Dual parameter flow cytometry show
linear relationship between DNA content and RI protein level in type 2 cells. b: The level of RII protein
measured with antibody raised to N-terminal region of the protein show very low binding in G0/G1 cells. The
level increases several fold to early S-phase and thereafter increases proportional with the DNA level. C: The
level of RII
protein measured with antibody raised to AA 367-394 is proportional to the DNA content
during the cell cycle.
Figure 6: Localization of regulatory subunit RII . Staining of mitochondria with Mito-Tracker (red) and
the RII regulatory subunit (green). Upper panel shows similar staining pattern for mitochondria (a) and RII
(b) indicating their colocalisation in unstimulated cells. Following stimulation by dibutyryl, no changes in
staining pattern of mitochondria occured (c) whereas a more diffuse cytoplasmatic localisation of RII
observed (d).
was
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