Departments of 1 Pediatrics and 2 Immunology, University of Kentucky Medical School, Lexington, Kentucky 40536
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ABSTRACT |
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After alveolar formation, >20% of
interstitial lung fibroblasts undergo apoptosis, a process that
is of critical importance for normal lung maturation. The immature lung
contains two morphologically distinct fibroblast populations,
lipid-filled interstitial fibroblasts (LIF) and non-LIF (NLIF), which
differ with respect to contractile protein content, proliferative
capacity, and expression of mRNAs for fibronectin and types I and
III collagen, but not tropoelastin. After alveolarization,
apoptosis occurs in only one fibroblast population, the LIF.
Using flow cytometry to analyze fibroblasts stained with a lipophilic,
fluorescent dye, we identified a subset, designated LIF(), that
contained fewer lipid droplets. Unlike LIF that retain lipid, LIF(+),
the LIF(
) do not undergo apoptosis after alveolarization. In
LIF(+), apoptosis was correlated with downregulation of
insulin-like growth factor I receptor (IGF-IR) mRNA and cell surface
protein expression. Treatment with anti-IGF-IR decreased total lung
fibroblast survival (P = 0.05) as did treatment with
the phosphatidylinositol 3-kinase inhibitor LY-294002 and the
ras-raf-mitogen-activated protein kinase inhibitor PD-98059 (P < 0.002), which block IGF-I/insulin receptor
survival pathways. These observations implicate downregulation of
IGF-IR expression in fibroblast apoptosis after alveolar formation.
lung development; PI 3-kinase; ras-raf-MAP kinase; flow cytometry; lipid interstitial fibroblasts
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INTRODUCTION |
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DURING DEVELOPMENT, CELLS that were once critical for the growth of an organ are often removed by apoptosis when their continued presence might adversely impact the function of the mature organ. In the immature lung, >20% of interstitial fibroblasts undergo apoptosis after the period of bulk alveolarization, resulting in a substantial reduction in interstitial volume (1, 6, 15, 25). The thinning of alveolar walls decreases the diffusion distance for inspired oxygen, thus enhancing gas exchange. In addition, the removal of a substantial fraction of interstitial fibroblasts facilitates the formation of intercellular contacts between endothelial cells of adjacent capillary layers, a process thought to promote the subsequent maturation of the lung vasculature, an integral part of which is the transformation from a double to a single capillary network in alveolar walls (25).
Apoptosis was first implicated as a contributing factor in lung development only recently. In the fetal rat lung, apoptotic mesenchymal cells were evident at days 14-18 of gestation, whereas apoptotic epithelial cells appeared somewhat later at 18 days of gestation (17, 24). The apoptotic index was observed to increase from <1% in the fetal lung to 9% at birth, followed by a decrease to 1.5% on postnatal day 2 (17). We and others have shown that after alveolar formation, the incidence of apoptotic lung fibroblasts increases sharply, reaching peak levels that exceed 20% on postnatal days 17-19 (6, 25). The onset of apoptosis was reported by Schittny and colleagues (25) to be delayed by several days when litter size exceeded 14 pups, however, suggesting the possible involvement of multiple factors in the regulatory control of this developmental apoptotic process.
After alveolar formation, apoptosis occurs in only one of two morphologically distinct rat lung fibroblast populations, the lipid-filled interstitial fibroblast (LIF). This developmental apoptotic event results in the shift from a predominantly LIF population before septation to one that consists primarily of nonlipid interstitial fibroblasts (NLIF) in the mature lung (1, 20). LIF and NLIF both express tropoelastin mRNA (1, 21), a connective tissue protein that is of critical importance for both normal fetal airway (29) and postnatal alveolar (2) development. These two subsets appear to have little else in common, however (1, 7). In addition to differences with respect to intracellular lipid content (21), LIF and NLIF differ substantially with respect to size (20), contractile protein content (20), expression of mRNAs for fibronectin (1) and types I and III collagen (3), and proliferative capacity (1), suggesting the potential for unique roles for each of these subsets during development and in response to injury.
Although developmental apoptosis has been investigated extensively in many other organ systems, the cell death pathways activated in lung fibroblasts after septation have not yet been identified. To address this issue, the possible involvement of the insulin-like growth factor I receptor (IGF-IR) in the marked increase in the incidence of fibroblast apoptosis that occurs after alveolarization was considered in view of the well-documented role of this receptor as a survival factor in both fibroblasts (18) and adipocytes (22), which are similar in many respects to the LIF. Consistent with the hypothesis that decreased signaling via the IGF-IR contributes to lung fibroblast apoptosis, IGF-IR mRNA expression in whole lung homogenate decreases substantially after alveolar formation, coinciding with the onset of apoptosis in rat lung fibroblasts (30).
Both the phosphatidylinositol (PI) 3-kinase and the ras/raf/mitogen-activated protein (MAP) kinase (MAPK) pathways have been implicated in IGF-IR-mediated survival signaling (23). The IGF-IR survival pathway is initiated by ligand binding, receptor autophosphorylation, and activation of tyrosine kinase activity. Activation of the PI 3-kinase pathway involves the tyrosine phosphorylation of insulin receptor substrates (IRS-1, IRS-2), which then interact with SH2 domains of the p85 subunit of PI 3-kinase, which in turn activates Akt/protein kinase B (PKB). Activated Akt/PKB can phosphorylate Bad, which suppresses apoptosis. IGF-IR and the insulin receptor (IR) can activate the MAPK pathway either by direct interaction with Shc proteins that bind growth factor receptor-bound protein 2 (Grb2) or indirectly through IRS-1 and IRS-2, which contain binding sites for Grb2 (31). The binding of Shc and Grb2 to the activated receptor leads to the activation of the ras-MAPK signaling pathway.
In some systems, IGF-IR activation of PI 3-kinase and its downstream effector PKB/Akt is necessary and sufficient to mediate survival (18), whereas the MAPK pathway influences survival in other systems (23). Downstream of the MAPK and PI 3-kinase pathways, Bad is thought to be a target for at least three antiapoptotic protein kinases: MAPK, protein kinase A (PKA), and PKB. Each of these protein kinases appears to phosphorylate Bad at a different site, however (10). Although a proapoptotic member of the bcl-2 family, once phosphorylated, Bad can no longer bind and inactivate bcl-2 (11, 18).
In the present study we compared IGF-IR expression in LIF, which
undergo apoptosis after alveolar formation, with expression in
NLIF, which do not, to determine whether there was a correlation between IGF-IR expression and survival. We observed that IGF-IR message
and cell surface protein expression were both downregulated in LIF
after alveolar formation on postnatal days 16-18,
strongly suggesting a role for the IGF-IR in lung fibroblast survival. In further support of this concept, lung fibroblast apoptosis could be induced in vitro by treatment with IR-3, an antibody that
binds the ligand binding site on the IGF-IR. Using specific inhibitors
of the PI 3-kinase and the ras-raf-MAPK pathways, we observed a
significant increase in cell death in response to treatment with each
of these inhibitors, suggesting the involvement of both the PI 3-kinase
and MAPK pathways in postnatal lung fibroblast survival.
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METHODS |
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Fibroblast isolation.
Fibroblasts were isolated from the lungs of fetal and postnatal
Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) on
postnatal days 3-30 according to a procedure previously described in detail (5). Cells were cultured in either
complete medium or serum-free (S-F) medium as indicated. Complete
medium contained 1:1 (vol/vol) Dulbecco's modified Eagle's medium
(DMEM)-Ham's F-12, 10% fetal bovine serum, penicillin (100 U/ml),
streptomycin (10 mg/ml), and glutamine (292 µg/ml). S-F medium
contained 1:1 (vol/vol) DMEM-Ham's F-12 medium, 1 mg/ml fatty
acid-free bovine serum albumin (Sigma, St. Louis, MO), 10 µg/ml
iron-saturated transferrin (Sigma), 2 × 108 M
selenium (Sigma), penicillin (100 U/ml), streptomycin (10 mg/ml), and
glutamine (292 µg/ml). Unless otherwise indicated, enzymes and tissue
culture reagents were purchased from GIBCO BRL (Life Technologies,
Grand Island, NY).
Quantitation and sorting of LIF by flow cytometry.
Adherent fibroblasts were detached from flasks using 0.05%
trypsin-0.02% EDTA and then either fixed in paraformaldehyde (0.5% final concentration) or resuspended in PBS. Both fixed and unfixed cells were maintained at 4°C during centrifugation at 1,200 rpm for
10 min, filtration through nylon mesh (70 µm), and treatment with
Nile red (9-diethylamino-5H-benzo[]phenoxazine-5-one),
a fluorescent, lipophilic dye (Molecular Probes, Eugene, OR) to stain
intracellular lipid droplets, as previously described (1). The Nile red stock solution (1.0 mg/ml DMSO) was diluted in saline and
then added immediately to the fibroblast suspension to achieve a final
concentration of 0.1 µg Nile red/ml.
Analysis of cell surface IGF-IR expression by flow cytometry.
Lung cell homogenate was plated for 1 h in DMEM:Ham's F-12
containing 10% autologous rat serum and then detached from flasks by
treatment with nonenzymatic Cell Dissociation Solution (Sigma) on a
rocking platform for 10 min at 37°C. The cells were then fixed
briefly at 4°C with an equal volume of 0.2% paraformaldehyde to
preserve cell integrity during subsequent centrifugation steps. The
fixed cells were rinsed in RPMI containing 1.0% BSA, resuspended in
anti-IGF-IR (clone IR-3) (8.0 µg/ml in RPMI-1.0% BSA), incubated on ice for 2.5 h, rinsed in RPMI-1.0% BSA, and stained with an R-phycoerythrin-conjugated F(ab') fragment of goat anti-mouse IgG (100 µg/ml) (Dako, Carpinteria, CA) for 45 min on ice, rinsed with
RPMI-1.0% BSA, and stored in 1.0% paraformaldehyde at 4°C until
they were assayed by flow cytometry. The monoclonal antibody
IR-3,
which binds reversibly to the ligand binding site on the IGF-IR
(18), was obtained from Oncogene Science (Uniondale, NY).
An unstained sample and a sample stained only with the secondary antibody were analyzed in each experiment. Emission spectra for Nile
red overlap with the emission spectra for phycoerythrin; thus it was
necessary to stain an aliquot of each cell suspension with Nile red
alone to identify the appropriate regions for each fibroblast subset.
Cell surface-bound fluorescence was analyzed on a Becton Dickinson FACS
Calibur flow cytometer using a 15-mW argon-ion laser operated at 488 nm. Phycoerythrin fluorescence was collected through a 585-nm band pass
filter. Data were analyzed using CyCLOPS Summit software obtained from Cytomation.
Extraction of total RNA and RT-PCR. Total RNA extracted from freshly isolated fibroblasts sorted under sterile conditions was purified and reverse transcribed as described previously (5). The number of rats used in each assay varied with postnatal age. In each of the three experiments conducted on 4-day rat pups, fibroblasts were obtained from 6-8 rats, and at 17 days each of four experiments was conducted on fibroblasts obtained from 10-12 rats. The PCR products were resolved by electrophoresis on 12.5% polyacrylamide gels and stained with SYBR-Gold (Molecular Probes). Fluorescent PCR products were quantitated on a Storm 840 using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The identities of amplified cDNAs were confirmed on an automatic sequencer (Perkin-Elmer) located in the Macromolecular Structure Analysis Facility at the University of Kentucky.
PCR primers. To amplify the IGF-IR 364-bp product, the 5'-forward and 3'-reverse complement primer sequences used were 5-ATGAGTACAACTACCGCTGC-3' and 5'TACATGCTCTGGGTGCTGTT-3, respectively. The primer pair used to amplify cyclophilin has been published previously (5).
Survival assay.
Freshly isolated fibroblasts were allowed to adhere for 1 h and
then were thoroughly rinsed to remove nonadherent cells. To assess the
influence of IGF-IR expression on fibroblast survival, cells were
cultured for 24 and 48 h either in complete or S-F medium in the
presence of monoclonal antibodies (2.0 µg/ml) directed against the
IGF-IR (clone IR-3) or against the
-subunit of the IR (clone
CT-3) (Oncogene Science) (18). To assess the influence of
specific kinase pathways on lung fibroblast survival, cells were
treated with specific inhibitors of PI 3-kinase (20 µM LY-294002, Cayman Chemical, Ann Arbor, MI) and MAPK kinase (MEK; 20 µM PD-98059, CalBiochem, San Diego, CA) for 24 h in complete or S-F
media. Viability was assessed after staining cells with 1.0 µg/ml propidium iodide and 1.0 µg/ml calcein AM (Molecular Probes)
to identify dead and live cells, respectively. Cells were viewed on a
Nikon Diaphot 300 microscope equipped with filter sets for fluorescence.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Chromatin fragmentation was assessed using a fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection Kit, Boehringer Mannheim, Indianapolis, IN). In brief, adherent treated and control cells were fixed in freshly prepared 4% paraformaldehyde (pH 7.4) for 1 h at room temperature, rinsed in PBS, permeabilized for 2 min on ice, and rinsed twice with PBS. Next, the TUNEL reaction mix was applied to each slide, covered with Parafilm, incubated for 1 h in the dark at 37°C, and rinsed 3× with PBS. To assess chromatin condensation, slides were counterstained for 10 min with 1.0 µg/ml Hoechst 33258 (Molecular Probes), rinsed with PBS, mounted in glycerol-PBS (1:2), and viewed under a Nikon Diaphot 300 microscope equipped with filter sets for fluorescence [Chroma fluorescein isothiocyanate filter cube for TUNEL; Nikon 4'-6-diamidino-2-phenylindole filter cube for Hoechst].
Statistical analysis of data.
Values are presented as means ± SD. The significance of decreases
in the relative percentage of LIF and in cell surface expression of
IGF-IR with postnatal age was evaluated by Student's
t-test. To assess the effects on fibroblast survival of
treatment with specific kinase inhibitors in the presence and absence
of serum, we used a two-factor ANOVA with Fisher's least-significant
difference correction for multiple comparisons (Systat version 9.0, SPSS, Chicago, IL). Values were considered to be significantly
different when P 0.05.
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RESULTS |
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Intracellular lipid content of a subpopulation of LIF decreases during postnatal lung development. In the late fetal and early postnatal rat lung, the LIF is the predominant fibroblast subset, accounting for 80% of lung fibroblasts on postnatal day 4 (1). At the end of the period of bulk alveolar formation, LIF undergo apoptosis (1), resulting in a fibroblast population in the mature lung that consists primarily of NLIF. In addition to the decrease in the relative percentage of LIF during postnatal development, there is also a substantial loss of lipid droplets from many of the LIF that remain in the lung (1, 9, 20). One objective of this investigation was to further characterize the population of LIF that loses intracellular lipid droplets as a function of postnatal age.
Using flow cytometry to quantitate the intracellular lipid content in freshly isolated rat lung fibroblasts stained with the lipophilic fluorescent dye Nile red, we identified, at the beginning of the second postnatal week, a separate population consisting of fibroblasts that stained less intensely with Nile red than did LIF (Fig. 1A). Light microscopic analysis of fibroblasts sorted by flow cytometry on the basis of Nile red fluorescence intensity and then stained with oil red O, indicated that cells in this subpopulation were similar in appearance to LIF but contained substantially fewer lipid droplets than LIF. Accordingly, these cells were designated LIF(
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Temporal correlation of developmental apoptosis with a
decrease in steady-state expression of IGF-IR mRNA in LIF vs. NLIF.
To explore the possibility that downregulation of IGF-IR mRNA
expression might be a factor in the developmental apoptosis seen in LIF, we compared IGF-IR mRNA expression in NLIF vs. LIF obtained both before (postnatal day 4) and during
developmental apoptosis (day 17). Freshly isolated
fibroblasts were stained with Nile red and sorted by flow cytometry,
based on their intracellular lipid content and on FCS and SSC
characteristics. Cells in the region of overlap between the NLIF,
LIF(+), and LIF() peaks were excluded to ensure the collection of
highly enriched populations of each subset. IGF-IR mRNA expression
levels in the sorted subsets were then assessed by RT-PCR. As shown in
Fig. 3, IGF-IR mRNA expression in NLIF
was essentially the same on postnatal days 4 and
17. In LIF, however, IGF-IR mRNA expression was markedly lower on day 17, during apoptosis, than on day
4, before the onset of apoptosis associated with the
postseptation period. Expressed as a ratio of LIF to NLIF, mean mRNA
values for IGF-IR normalized to cyclophilin were 2.37 ± 0.84 at 4 days (n = 3) vs. 0.46 ± 0.26 at 17 days
(n = 4), indicating a significant decrease in the
relative expression of IGF-IR mRNA that was temporally correlated with the onset of apoptosis after alveolar formation
(P = 0.007).
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Cell surface IFG-IR expression decreases in
LIF(+) after alveolarization.
We then assessed cell surface IGF-IR expression on lung
fibroblasts obtained during the initial phase of alveolar formation (postnatal days 3, 7, and 8) and after
alveolar formation (days 16-18) to determine whether
there was a correlation between the onset of apoptosis and
downregulation of cell surface receptor expression. Freshly isolated
adherent fibroblasts were removed from tissue culture flasks using a
nonenzymatic cell dissociation solution and then stained with IR-3.
Cell surface IGF-IR protein expression was evaluated by flow cytometry,
and expression levels in NLIF, LIF(+), and LIF(
) were compared before
and after the onset of apoptosis. Because the broad emission
spectra of Nile red precluded dual labeling with phycoerythrin, the
regions containing NLIF, LIF(+), and LIF(
) were identified in an
aliquot of cells stained with Nile red alone by flow cytometric
analysis of fluorescence plotted on a log scale, and SSC plotted on
linear scale. In lung fibroblasts obtained from 3- to 8-day rats,
IGF-IR expression was greater in LIF than in NLIF (Fig.
4). Surface IGF-IR expression remained
essentially unchanged in NLIF from days 3 through
18 and in LIF(
) obtained from rats at 8-18 days of
age. In contrast, there was a significant decrease in the mean value
for cell surface IGF-IR expression for LIF(+) from 20.5 ± 4.1 on
days 3-8 to 1.2 ± 2.1 on days
16-18 (P = 0.005).
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IGF-IR is a survival factor for rat lung fibroblasts.
To determine the extent to which rat lung fibroblasts are dependent on
survival signaling via the IGF-IR, cells obtained from the lungs of 7- to 9-day rats were treated with IR-3, a specific blocking antibody
for this receptor. Fibroblast survival was assessed by quantitating the
percentage of calcein AM-positive (live) vs. propidium iodide-positive
(dead) cells in response to treatment. After treatment with
IR-3 for
24 h in the absence of serum, the mean value for the survival rate
in three separate experiments was 52 ± 17.8%, which was
significantly lower than that in untreated S-F control cultures,
80 ± 3.6% (P = 0.05). When cells were treated with both
IR-3 and the blocking antibody to the IR in combination, the additive effect was negligible. In a separate experiment, we
documented the presence of apoptotic cells seen in response to
treatment of 8-day rat lung fibroblasts with
IR-3 and with both
IR-3 and anti-IR antibodies in combination (Fig.
5). Chromatin fragmentation was assessed
using a fluorescent DNA end-labeling TUNEL technique, and chromatin
condensation was detected using a Hoechst counterstain.
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Inhibitors of the PI 3-kinase and MAPK pathways decrease fibroblast
survival.
Both IGF-IR and the IR can signal via either the PI 3-kinase pathway or
the MAPK pathway to promote fibroblast survival. To assess the
contributions of each of these downstream pathways in 7- to 9-day rat
lung fibroblasts, we assessed survival in cells treated with 20 µM
LY-294002, a specific inhibitor of the PI 3-kinase pathway and with 20 µM PD-98059, a specific inhibitor of MEK in the MAPK pathway (Fig.
6). Treatment with LY-294002 and
PD-98059, alone and in combination, resulted in a significant increase
in cell death, both in the presence of serum (P = 0.002) and in the absence of serum (P < 0.0001). The
presence of serum conferred protection against cell death induced by
treatment with PI 3-kinase and MAPK inhibitors, but only in the case of
treatment with PD-98059 did the degree of protection achieve
statistical significance (P = 0.007). Treatment with
both inhibitors in combination resulted in a small increase in cell
death relative to the effect seen after treatment with either agent
alone. This increase was not significant, however. Lung fibroblasts in
which apoptosis was induced by treatment with inhibitors of PI
3-kinase and MAPK are shown in Fig. 7.
Chromatin fragmentation was assessed using a fluorescent DNA
end-labeling TUNEL technique. Chromatin condensation was detected in
the same cells counterstained with Hoechst.
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DISCUSSION |
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Within several days after the completion of the period of bulk alveolarization in the postnatal rat lung, >20% of interstitial fibroblasts undergo apoptosis (1, 6, 17, 25). This substantial decline in fibroblast number serves to decrease alveolar wall thickness, which, in turn, enhances gas exchange and may also facilitate the maturation of the microvasculature from a double to a single capillary layer (25). Our initial observations that the relative percentage of LIF decreased during the first month of life suggested that apoptosis occurred primarily, if not exclusively, in LIF (1). The occurrence of apoptosis in LIF, but not NLIF, was verified using the TUNEL assay to identify DNA strand breaks in fibroblasts shown by flow cytometric analysis of intracellular lipid content and relative size and structural complexity to be LIF (1).
In the present study, we extended these observations by further
characterizing the LIF removed by apoptosis. Flow cytometric analysis of the intracellular lipid content of lung fibroblasts obtained during the second postnatal week demonstrated the presence of
a population of LIF, designated LIF(), that had lost a substantial fraction of their lipid droplets. LIF(
) could be distinguished from
NLIF and from LIF that retained lipid droplets, LIF(+), by flow
cytometric quantitation of intracellular lipid content and by the
analysis of FCS and SSC characteristics, indicators of relative cell
size and structural complexity, respectively. On day 9,
LIF(
) constituted ~20% of total lung fibroblasts, a relative percentage that did not change substantially throughout the remainder of the first month of life. In contrast, the relative percentage of
LIF(+) decreased from 45% of total fibroblasts on days
9-12 to 6% at 1 mo. Taken together with flow cytometric
evidence of changes consistent with apoptosis only in LIF(+),
these data indicate that the apoptosis previously shown to
occur in LIF (1) occurs primarily in those cells that
retain lipid droplets, LIF(+).
A similar decrease in intracellular lipid content of lung fibroblasts beyond postnatal day 8 was also noted by Kaplan and colleagues (4, 14). Lipid droplet content, determined at the electron microscopy (EM) level, was constant from postnatal days 4-8 (4), followed by an 88% decrease from day 8 to the adult lung (14). Although these authors concluded that there was only a modest decrease in the relative percentage of LIF in the mature lung, this apparent discrepancy with our results could be due to the fact that relatively few cells (200/lung) were evaluated at the EM level, whereas we were able to quantitate intracellular lipid in at least 10,000 cells per experiment, a number not feasible with light or EM techniques.
Although the regulatory control of intracellular lipid content in LIF
is poorly understood, lipid accumulation in a similar cell, the
adipocyte, has been well characterized. Both cell types acquire neutral
lipid droplets rich in triglycerides during development. These droplets
appear late in gestation and then decrease in number during the
perinatal period. Chen and colleagues (9) have shown in
rat LIF that expression of the peroxisome proliferator-activated receptor-gamma (PPAR-), an orphan nuclear receptor implicated in
lipid homeostasis and adipogenesis in the adipocyte, peaks shortly
before birth. This increase in PPAR-
precedes the increased expression at birth of genes containing response elements for PPAR-
that control lipid hydrolysis and transport lipoprotein lipase and
adipocyte lipid binding protein (12, 16). A role for insulin/IGF signaling in the activation of adipogenesis has also
been suggested by the observation that differentiation of 3T3-L1
preadipocytes into adipocytes requires either insulin or IGF-I
(26). However, there is, as yet, no evidence to support a
causal relationship between a decrease in ligand binding and the loss
of lipid from adipocytes.
On the basis of our results and those of other investigators
(14), it appears likely that LIF() are derived from LIF,
since both contain lipid droplets, in contrast to NLIF. In addition, both LIF(+) and LIF(
) are larger and more structurally complex than
NLIF. Our results suggest that loss of intracellular lipid in LIF(
)
at the beginning of the second postnatal week confers protection
against the marked increase in fibroblast apoptosis that occurs
after the period of bulk alveolar formation. This observation would
seem to imply that interference with the loss of intracellular lipid
may render a larger fraction of lung fibroblasts susceptible to
apoptosis, an event that could result in excessive thinning of
alveolar walls and thus compromise mature lung function. Although
beyond the scope of the current investigation, it will be of interest
to determine in future studies the mechanisms responsible for the loss
of intracellular lipid in rat lung fibroblasts. Furthermore, comparison
of adipogenic gene expression in LIF(+) vs. LIF(
) could clarify the
roles played by these genes during what appears to be a
dedifferentiation process.
Whereas the NLIF is the predominant fibroblast population in the mature rodent lung, the LIF is predominant during the late saccular and early alveolar stages of lung development, suggesting a critical role for the LIF during the late fetal and early postnatal stages. One such developmental role proposed by McGowan and Torday (21) is that of providing a source of lipid that could be used in the synthesis of surfactant phospholipid by the alveolar type II epithelial cell. Also of potential relevance to postnatal lung development is the fact that expression of mRNA for fibronectin, a major constituent of the pulmonary extracellular matrix, is ninefold higher in LIF than in NLIF during the first week of life (1). The ability of this cell-adhesive protein to influence migration, proliferation, and differentiation of specific lung cell types suggests that fibronectin production by LIF is of critical importance during the early stages of lung development.
There is, perhaps, a greater degree of uncertainty regarding the
specific roles of LIF and NLIF in the mature lung, however. It has been
assumed, although never rigorously tested, that the LIF is a precursor
of the myofibroblast, a cell identified by the presence of -smooth
muscle actin (
-SMA) and vimentin and the absence of desmin
(21). Myofibroblasts play a critical role in repair
because of their ability to proliferate and secrete abundant
extracellular matrix in response to injury. In sorted 16-day
fibroblasts, we observed that NLIF cultures were essentially devoid of
-SMA, whereas the majority of LIF(
) were
-SMA positive (M. C. Bruce, unpublished results).
-SMA-positive filaments were absent
in the majority of LIF(+), suggesting that the LIF(
) may be the
predominant source of myofibroblast precursors. In the absence of a
specific marker for each subset, however, the rapid loss of lipid
droplets in vitro and the differential rates of proliferation of these
subsets precluded the definitive identification of the fibroblast
subset(s) that contain
-SMA-positive cells.
In addition to the unique contributions of each fibroblast subset, several lines of evidence suggest that lung fibroblast behavior may also be a function of the interactions between subsets. Sorted populations of NLIF tend to adhere more readily and to proliferate more rapidly when plated on fibronectin-coated surfaces than when plated on plastic (M. C. Bruce, unpublished observations). We have also observed that after sorting by flow cytometry, NLIF proliferate more rapidly when cocultured with LIF than when cultured alone (1). Although the increased adherence and proliferation seen in NLIF cocultured with LIF may well be the result of a complex series of interactions, the increased fibronectin production by LIF relative to that by NLIF appears likely to be a contributing factor.
The identification of LIF(+) as the fibroblast subtype that undergoes apoptosis in the postnatal lung enabled us to compare levels of expression of IGF-IR among fibroblast subsets to assess the role of this receptor as a survival factor. The IGF-IR was considered a logical survival factor candidate both because of its documented role in this capacity in fibroblasts and adipocytes and because the decreased expression of this receptor in the developing rat lung coincides with the onset of apoptosis after alveolar formation (6, 30). In the present study, a significant decrease in lung fibroblast expression of IGF-IR, at both the cell surface protein and mRNA levels, was seen only in LIF(+), implying that downregulation of this growth factor receptor is responsible, at least in part, for the developmental apoptosis that occurs in this lung fibroblast subset.
The role of the IGF-IR as a survival factor in rat lung fibroblasts was
further established in a series of experiments in which the ligand
binding region was blocked using the IR-3 antibody, a technique used
successfully by others to induce apoptosis in a variety of cell
types. Treatment with this blocking antibody substantially decreased
survival of 7- to 9-day fibroblasts. The decreased survival observed in
cells treated with blocking antibodies in S-F medium, which lacks
IGF-I, suggests that lung fibroblasts are capable of producing this
growth factor in vitro, consistent with earlier reports of IGF-I
production by primary cultures of rat lung fibroblasts
(27).
The PI 3-kinase and MAPK pathways have each been implicated in IGF-IR and IR survival signaling, although the degree to which these pathways are activated by these receptors varies considerably with cell type (18). In the present studies, blocking IGF-IR and IR survival pathways by treatment with inhibitors of the PI 3-kinase and the MAPK pathways also resulted in a significant decrease in fibroblast survival. Survival was further decreased in cells treated with both inhibitors in combination, consistent with the possibility that signaling via each of these pathways can promote fibroblast survival.
Although the concept of lung fibroblast heterogeneity has been accepted
for more than two decades, the findings presented herein suggest a
greater degree of complexity than was previously appreciated.
Significant progress has been made in recent years in the
identification of specific roles for lung fibroblast subsets during
development. However, in light of our observations that there are, in
fact, two distinct subpopulations of lipid-containing fibroblasts,
LIF(+) and LIF(), further comparisons of the developmental changes in
gene and protein expression in these subsets appear warranted. The
observed differences with respect to intracellular lipid content,
IGF-IR expression, and the incidence of apoptosis in these two
LIF subtypes suggest that these cells are likely to have distinct roles
during alveolar formation and injury repair in the mature lung.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-31172 (to M. C. Bruce).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. C. Bruce, Dept. of Pediatrics, Div. of Neonatology, Univ. of Kentucky Medical School, Lexington, KY 40536 (E-mail: mbruce{at}uky.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.00050.2001
Received 7 February 2001; accepted in final form 9 May 2001.
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