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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Increased smooth muscle mass due to hyperplasia and hypertrophy of airway smooth muscle (ASM) cells
is a common feature in asthma. Angiotensin II (Ang II), a potent vasoconstrictor and mitogen for a wide
variety of cells, has recently been implicated in bronchoconstriction in asthmatics. However, a possible
mitogenic role as well as underlying molecular mechanisms of this octapeptide in human ASM cells are
not yet known. We studied the effects of Ang II on ASM cell proliferation and growth and on the expression of three transcription factors, egr-1, c-fos, and c-jun, as well as a cytokine, transforming growth factor-
1 (TGF-1). Human ASM cells were isolated by enzymatic digestion of bronchial smooth muscle obtained from lung resection tissue. Confluent cells were growth-arrested and subsequently incubated with
Ang II (100 nM) for different time periods and processed for the measurement of cell growth and gene expression. Ang II significantly induced DNA and protein synthesis in human ASM cells at 8 h, resulting in
a net increase in the accumulation of protein over DNA (i.e., cellular hypertrophy) at 16 h of incubation.
Cell counts and MTT-reduction assay, however, showed no increase in cell number as a result of Ang II
stimulation. Ang II stimulated the expression of egr-1 and c-fos as early as 15 min, reaching maximum
levels at 45 min, whereas the expression of c-jun peaked at 2 h of Ang II exposure. Furthermore, steady-state mRNA levels of TGF-1 were upregulated by Ang II after 4 h and reached peak levels at 16 h of incubation. Secretion of biologically active TGF-1 from human ASM cells was significantly (P 
0.02) enhanced by Ang II incubation after 8 h, which remained elevated until 24 h. Our results suggest that the Ang
II-induced transient early expression of transcription factors may regulate autocrine genes like TGF-1, of
which the subsequent late upregulation could contribute to cellular hypertrophy during, for example, airway remodeling in asthma.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Airway remodeling with inflammatory cell infiltration, epithelial shedding, basement membrane thickening, and increased mass of airway smooth muscle (ASM) is an important determinant of bronchial obstruction and hyperresponsiveness in asthma (1). Studies using detailed computer models of human airways indicate that increased ASM mass is by far the most important abnormality responsible for excessive airway narrowing and compliance of the airway wall in asthma (1, 5). In analogy to vascular smooth muscle accumulation in hypertension and atherosclerosis, ASM growth in asthma is a complex phenomenon of which the underlying mechanisms are difficult to investigate in vivo. The increased amount of ASM in asthmatics is an indication of abnormal cell proliferation and growth. Both hyperplastic (i.e., increase in cell number) and hypertrophic (i.e., increase in cell size) changes contribute to the increased smooth muscle content of the airway wall (1, 2, 6, 7), but little is known regarding the molecular mechanisms and factors that regulate ASM cell proliferation and growth in asthma.
Recently, a number of growth factors and cytokines derived from inflammatory cells have been implicated in ASM cell division and growth (8). The potent circulating hormone of the renin-angiotensin system, angiotensin II (Ang II) has been implicated in bronchoconstriction in mild asthmatics (13). Plasma levels of Ang II have been shown to be elevated in patients with acute severe asthma (14). At plasma levels similar to those observed in acute asthma, Ang II was found to enhance methacholine-evoked bronchoconstriction, both in human bronchi in vitro and in mild asthmatics in vivo, thus suggesting a novel role for Ang II as a putative mediator in asthma (13). Although Ang II has been shown to stimulate proliferation and/or hypertrophy in a wide variety of cells, such as cardiac myocytes, cardiac fibroblasts, and vascular smooth muscle cells (15), it remains to be established whether this octapeptide stimulates proliferation and growth of ASM cells.
It is known that growth and differentiation factors stimulate signal transduction pathways in the cell leading to
the expression of nuclear proto-oncogenes that include the
Fos, Jun, and Egr families (18). Fos and Jun proteins constitute the heterodimer AP-1 (19). The AP-1 and egr-1
transcription factors can regulate a number of target genes
during growth factor stimulation and thereby influence
cellular growth and differentiation (16, 20, 21). Ang II has
been shown to induce a rapid increase in the expression of
c-fos and c-jun (members of the leucine zipper family) and
egr-1 (a member of the zinc finger family) in vascular
smooth muscle cells as well as in cardiac fibroblasts prior
to hypertrophy and/or hyperplasia (16, 20, 22). Furthermore, an important cytokine, transforming growth factor-1 (TGF-1), has synergistically been associated with Ang
II. TGF-1 is synthesized as a biologically inactive propeptide which must be cleaved to form the active peptide, by
proteases such as plasmin (23), before it can exert its function in tissue repair after injury or act as a regulatory peptide in remodeling processes in various tissues, including
the heart and the lung (16, 24, 25). Also, TGF-1 stimulates the synthesis of extracellular matrix components such
as collagens and fibronectin in response to Ang II in vascular smooth muscle cells (17, 25, 26).
To elucidate the potential role of Ang II in ASM growth
in asthma, we investigated hyperplasia and hypertrophy in
cultured human ASM cells and examined the expression
pattern of three proto-oncogenes, namely, egr-1, c-fos, and
c-jun. Additionally, we studied the expression of the extracellular matrix regulator TGF-1 both at mRNA and protein levels in human ASM cells treated with Ang II.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
Bovine serum albumin (BSA), collagenase, elastase, apo-transferrin, ascorbate, insulin, non-enzymatic cell dissociation solution, mouse monoclonal anti-
smooth muscle
actin and anti-smooth muscle myosin, antimouse IgG fluorescein isothiocyanate (FITC) conjugate, 4',6-diamidino-2-phenylindole-2HCl, calf thymus DNA, herring sperm DNA, and trypan blue were purchased from Sigma-Aldrich BV (Zwijndrecht, The Netherlands). Hanks' balanced
salt solution (HBSS), Dulbecco's modified Eagle's medium
(DMEM), sodium pyruvate, MEM nonessential amino
acids, gentamicin, penicillin:streptomycin, Fungizone/amphotericin B, and trypsin-ethylenediamenetetraacetic acid
(EDTA) were purchased from Life Technologies BV
(Breda, The Netherlands). Fetal bovine serum (FBS) was
purchased from Bio-Whitaker BV (Verviers, Belgium). [Methyl-3H]thymidine, [methyl-3H]leucine, (-P32)-dCTP,
hybond-N, and redi-prime labeling kit were obtained from Amersham Nederland BV ('s-Hertogenbosch, The Netherlands). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-reduction assay kit was obtained
from Boehringer Mannheim GmbH (Mannheim, Germany).
The TGF-1 Emax ImmunoAssay System was procured from
Promega Corp. BNL (Leiden, The Netherlands). Anti- cytokeratin and anti--CD31 antibodies were obtained
from Dako A/S (Glostrup, Denmark). The cDNA probe
for human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from American Type Culture Collection (Rockville, MD). Tissue culture plasticware was obtained from Life Technologies BV. All other chemicals were of molecular biology and/or tissue culture grade
and were procured from local suppliers.
Cell Culture
Human airway smooth muscle cells were isolated and cultured according to the methods described previously (10, 27). Briefly, bronchial smooth muscle was dissected from the lobar or main bronchus from lung resection specimens obtained from patients undergoing surgery for lung cancer. After removal of the epithelium, pieces of macroscopically normal smooth muscle were dissected free of adherent connective and parenchymal tissue under aseptic conditions. Smooth muscle pieces were incubated in HBSS containing 10 mg/ml of BSA (fraction V), collagenase (type XI, 1 mg/ml), and elastase (type IV, 3.3 U/ml) at 37°C in a humidified ASSAB model T154 CO2 incubator (Clean Air Techniek BV, Woerden, The Netherlands). After enzymatic digestion, the tissue was centrifuged and the resultant pellet was washed in DMEM containing 10% (vol/vol) FBS supplemented with sodium pyruvate (1 mM), nonessential amino acid mixture (1:100), gentamicin (45 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (1.5 µg/ml), and subsequently seeded at 2 × 105 cells per 35-mm dish. Fresh medium was replaced every 72 h. After 10-14 d in culture, ASM cells grew to confluence and were then removed from the plastic base of each dish using non-enzymatic cell dissociation buffer and subcultured into tissue culture flasks. At confluency, cells were further passaged by means of trypsinization. Confluent cells in the fifth passage were used for experiments.
Immunocytochemical Characterization of ASM Cells
Monoclonal antibodies against -smooth muscle actin and
smooth muscle cell specific myosin (SM-1 and SM-2) were
used as markers to characterize human ASM cells in culture (28). Cells were allowed to attach to multiwell slides
for 24 h in FBS-containing medium and were subsequently
growth-arrested by incubating for 60-72 h in FBS-free
DMEM supplemented with apo-transferrin (5 µg/ml),
ascorbate (100 µM), and insulin (1 µM) prior to fixation and staining. Following two washes in ice-cold phosphate-buffered saline (PBS; 140 mM NaCl, 2.6 mM KCl, 1.4 mM
KH2PO4, 8.1 mM Na2HPO4 · 2 H2O, pH 7.4), the cells were
fixed in ice-cold methanol and permeabilized in PBS containing 0.1% Tween-20. Nonspecific binding was blocked
by incubating the cells in 1% BSA in PBS, and the cells
were then washed and subsequently incubated with anti- smooth muscle actin or anti-smooth muscle myosin antibodies. After incubation, the cells were washed twice in PBS
and further incubated with affinity purified FITC-conjugated antimouse antibody. Unbound antibody was washed
away using distilled water and the sections were dehydrated
and mounted in glycerol. Specimens were visualized under
a fluorescence microscope (Carl Zeiss BV, Weesp, The Netherlands) and photographed. Human ASM cells stained
positive for anti- smooth muscle actin and anti-smooth
muscle myosin. Additionally, the cultures were stained for
endothelial and epithelial cell contamination using anti-CD31 and anti-cytokeratin antibodies, respectively. The
staining showed that human ASM cell cultures were essentially free (> 95%) of other contaminating cell types. Under the light microscope, the human ASM cells appeared
elongated and spindle-shaped with central oval nuclei containing prominent nucleoli. Confluent human ASM cells in
culture showed a specific pattern, aligned in parallel so
that the broad nuclear region of one cell lies adjacent to
the thin cytoplasmic area of another, giving rise to a typical "hill and valley" appearance as described earlier by
Twort and van Breeman (27).
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from the ASM cells by the
guanidinium thiocynate-phenol-chloroform method described previously (16). The RNA concentration was measured by spectrophotometry. For Northern hybridization,
samples of total RNA were denatured at 65°C and size-fractionated on a 1% agarose gel containing 2.2 M formaldehyde. Ethidium bromide-stained gels were photographed
and RNA was transferred to hybond-N membrane by alkaline downward capillary transfer (29). The filters were
air-dried and ultraviolet crosslinked in a gene linker (Bio-Rad Labs BV, Veenendaal, The Netherlands). Blots were hybridized at 42°C in a buffer containing 50% deionized
formamide, 1.0 M sodium chloride, 1% sodium dodecyl
sulfate (SDS), 0.2% polyvinyl pyrrolidone, 0.2% ficoll,
0.2% BSA, 50 mM Tris-HCl (pH 7.5), 0.1% sodium pyrophosphate, 2% dextran sulphate, and denatured herring sperm DNA (2 mg/ml). The cDNA probes used for hybridization were: mouse c-fos (2.1-kb fragment), mouse c-jun
(2.6-kb fragment), mouse egr-1 (300-bp fragment of zinc finger region), and human TGF-1 (1,050-bp fragment). These
inserts were labeled with the redi-prime labeling system to
a specific activity of approximately 109 cpm/µg DNA. Filters were washed under stringent conditions, wrapped in
household plastic film, and exposed to Kodak X-OMAT
AR films (Eastman Kodak, Rochester, NY) at 
80°C for 1-
3 d. A GAPDH cDNA probe was used to rehybridize membranes for reference purposes. Hybridization signals were
quantified by scanning laser densitometry using the Ultroscan XL enhanced laser densitometer (LKB, Bromma, Sweden). Densitometric values for each gene were normalized
with respective GAPDH mRNA values and expressed as
relative optical density (OD) in Ang II-stimulated cells
versus control. An optimal concentration of Ang II (100 nM) for the early response of human ASM cells was determined by incubating growth-arrested cells for 1 h in 10
5,
106, 107, and 108 M [Sar1]-Ang II, and subsequently examining the expression pattern of the proto-oncogene egr-1.
[3H]Thymidine and [3H]Leucine Incorporation Assays
Effects of Ang II on DNA and protein biosynthesis were
assessed by incorporation of [methyl-3H]thymidine and
[methyl-3H]leucine, respectively. An optimum concentration of Ang II (100 nM) was determined by incubating
growth-arrested ASM cells for 24 h in 106, 107, 108, and
109 M [Sar1]-Ang II, and subsequently examining [3H]thymidine and [3H]leucine incorporation. Confluent cells were
washed in PBS, detached by trypsinization, and transferred into 24-well plates at a seeding density of 2 × 104
cells/well. After 5 d in culture the cells were growth-arrested as described above. Cells were incubated with [methyl-
3H]thymidine or [methyl-3H]leucine (1 µCi/well) in either
fresh FBS-free DMEM (control), FBS-containing DMEM
(+ serum), or DMEM containing 100 nM [Sar1]-Ang II for
4, 8, 16, 24, or 48 h. After stimulation, the cells were
washed in PBS, fixed with ice-cold methanol, and exposed to ice-cold trichloroacetic acid (5% wt/vol). The acid-insoluble fraction was lysed in 0.3 M NaOH and the incorporated radioactivity was determined in a Packard 1500 Tri-carb liquid scintillation analyzer (Packard-Becker BV, Delft,
The Netherlands) (9).
Assessment of Cellular Hypertrophy and Hyperplasia
Protein/DNA ratio. ASM cell hypertrophy in relation to Ang II was assessed by calculating the ratio of total protein to DNA content. Cells were plated at a seeding density of 2 × 104 cells/well in 24-well plates. After 5 d the cells were growth-arrested as described above, and then incubated in either fresh FBS-free DMEM (control), FBS-containing DMEM (+ serum), or DMEM containing 100 nM [Sar1]-Ang II for 4, 8, 16, 24, or 48 h. After stimulation, the cells were washed in ice-cold PBS and lysed in 0.3 M NaOH. The total DNA content was determined fluorimetrically using the method described by Kapuscinski and Skoczylas (30). The total protein content was estimated colorimetrically using the method described by Bradford (31). Serial concentrations of calf thymus DNA and BSA were used for the calibration curves.
MTT assay.
In order to assess cellular hyperplasia in
response to Ang II, MTT assays were performed. Cells in
the fifth-sixth passage were transferred to 24-well plates at
a seeding density of 2 × 104 cells/well. After 24 h, the cells
were growth-arrested as described above and subsequently
incubated in either fresh FBS-free DMEM or DMEM
containing 100 nM [Sar1]-Ang II for 4, 8, 16, 24, or 48 h.
Proliferation was assessed using the MTT dye technique,
which relies on the specific metabolic reduction of the tetrazolium salt MTT by living cells as described earlier (32).
Following incubations, the cells were washed twice in PBS,
200 µl of MTT in DMEM (final concentration 0.5 µg/ml) was added to each well, and the cells were incubated for
5 h at 37°C. After incubation, the blue formazan product
was solubilized by the addition of 200 µl of solubilization
solution (10% SDS in 0.01 M HCl) to each well and incubating for a further 16 h at 37°C. An aliquot of 200 µl from
each duplicate well was then transferred to a 96-well microtiter plate, and the OD was determined by automated
dual wavelength spectrophotometry (Bio-Rad Labs) at a
test wavelength of 595 nm and a reference of 690 nm. The change in optical density (
OD), which correlates directly
with change in cell numbers, was plotted against time as
described previously by Hirst and colleagues (10).
Hemocytometry. Cells were seeded into 24-well plates and treated as described above. Following stimulation, the medium was removed and the cell monolayers were resuspended in 100 µl trypsin-EDTA at room temperature for 10 min. An equal volume of trypan blue was added to the cell suspension before counting in a Bürker hemocytometer (Marienfeld GmbH, Marienfeld, Germany). At least 120 cells were counted from each well to minimize counting error.
Detection of TGF-1 Protein in Conditioned Media
Confluent cells in the fifth passage were growth-arrested
as described above, and then incubated in either fresh
FBS-free DMEM (control), FBS-containing DMEM (+ serum), or DMEM containing 100 nM [Sar1]-Ang II for 4, 8, 16, or 24 h. Media were removed and stored at 20°C until
assay. Biologically active TGF-1 protein was quantified by enzyme-linked immunosorbent assay (ELISA) using a
TGF-1 Emax ImmunoAssay System (Promega Corp. BNL,
Leiden, The Netherlands) following the suppliers' instructions.
Statistical Analysis
Data are given as means ± SEM. Statistical analysis was
performed by using the Student's t test. Significance was
accepted at P 0.05.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Expression of Transcription Factors in Relation to Ang II
Representative Northern blots showing the expression pattern of three proto-oncogenes, following Ang II stimulation of human ASM cells, are shown in Figures 1A-1C. Ang II induced the expression of mRNAs encoding egr-1 (panel A) and c-fos (panel B) as early as 15 min, while c-jun expression was increased after 45 min of Ang II stimulation (panel C ). Figure 1D shows the quantitative analysis, by scanning laser densitometry, of the Northern blots for each proto-oncogene. Densitometric analysis revealed that the induction of c-fos and egr-1 was transient and reached a maximum at 45 min followed by an abrupt decline; whereas the expression of c-jun was also transient but reached a maximum at 120 min and gradually returned to basal levels after 4 h. The relative OD values for the levels of mRNA encoding egr-1, c-fos, and c-jun increased from 0 to their maximum values of 1.97, 2.66, and 0.67, respectively, after Ang II stimulation of human ASM cells.
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Effects of Ang II on ASM Proliferation and Growth
The initial aim of this study was to determine whether Ang II induces an increase in DNA and protein synthesis in ASM cells made quiescent in defined serum-free medium. Biosynthesis of DNA and protein from their respective labeled precursors (thymidine and leucine) was calculated as specific activity and represented as dpm/ng DNA (Figure 2A) or dpm/µg protein (Figure 2B). Data show that the biosynthesis of both DNA and protein was minimal until 8 h of Ang II incubation. However, a significant increase was observed in both DNA and protein biosynthesis at 16 h of Ang II stimulation and these levels remained elevated until 48 h as compared with controls.
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In order to assess whether the DNA biosynthesis in human ASM cells, treated with Ang II, was accompanied
with an increase in cell number, we performed hemocytometry and MTT assays. Data on hemocytometry showed
that Ang II incubation did not significantly increase the
cell number with time as compared with respective controls. However, serum already induced a significant (P 0.05) increase in cell numbers at 16 h as compared with
controls, and at 48 h cell numbers were considerably
higher (P 0.001) as compared with respective control
and Ang II-treated cells (Figure 3A). MTT-reduction assay data showed no significant change in OD values at 4, 8, 16, 24, or 48 h of Ang II incubation as compared with respective controls, indicating that Ang II did not cause
ASM cell proliferation (Figure 3B).
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In a separate set of experiments, the total DNA and
protein contents of the ASM cells were determined in relation to Ang II stimulation with time. Figure 4 shows the
effect of Ang II on the protein/DNA ratio in human ASM
cells. In order to assess Ang II-induced hypertrophy in human ASM cells, we calculated the protein-to-DNA ratio at
different time points. After 16 h of Ang II incubation, the
protein/DNA ratio of the ASM cells increased significantly (P 0.001) compared with controls, indicating that
the cells were synthesizing more protein than DNA, suggesting cellular hypertrophy.
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Effects of Ang II on TGF-1 Expression
To evaluate the effects of Ang II on TGF-1 expression,
we analyzed total cellular RNA from serum-fed ASM cells
and cells stimulated with 100 nM Ang II for various time
intervals and compared the expression pattern with respective controls. Using a human-specific cDNA probe encoding TGF-1, we detected an mRNA species of 2.4 kb in
human ASM cells (Figure 5A). TGF-1 expression was
drastically increased (P 0.001) in serum-fed cells as compared with the control cells. Scanning laser densitometric
analysis of TGF-1 expression showed that Ang II significantly induced (P 0.001) the steady-state mRNA levels
which became apparent after 8 h and reached a maximum
level at 16 h (P 0.001) as compared with controls (Figure
5B). To evaluate whether the increase in mRNA levels for TGF-1 were accompanied with an increase in secreted
protein, biologically active TGF-1 was measured in the
conditioned medium after stimulation of the ASM cells
with Ang II for various time intervals. Indeed, a significant
increase in TGF-1 protein was observed after 8 h, which
remained elevated at 24 h (Table 1).
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Introduction
Materials & Methods
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This is the first study showing that Ang II, in the absence of serum, induces hypertrophy in human airway smooth muscle cells in vitro. In an attempt to discern whether Ang II could induce cell hypertrophy, cell hyperplasia, or both, we used several biochemical markers. On the basis of [3H]leucine incorporation, protein over DNA accumulation in the cell (33), MTT-reduction assays, and cell number determinations, our results clearly indicate a hypertrophic response to Ang II in human ASM cells. Our data also show that Ang II induced an increase in [3H]thymidine incorporation into DNA without significantly altering the cell number in human ASM cells. Furthermore, the magnitude of increase in [3H]thymidine incorporation was much lower (55%) than that seen with FBS, suggesting that Ang II may act as a weak mitogen for human ASM cells.
Studying vascular smooth muscle cells in vitro, several groups have demonstrated cell proliferation (34) whereas others have shown only cellular hypertrophy after Ang II stimulation (15, 17, 35). Ang II-induced hypertrophy in vascular smooth muscle cells has been shown to be associated with an increase in expression and secretion of the autocrine growth factor platelet-derived growth factor-AA (PDGF-AA) (15, 36). PDGF is a potent mitogen for vascular smooth muscle cells and it is quite possible that in analogy to these cells, human ASM cells express and secrete PDGF in response to Ang II, resulting in an increase in DNA synthesis. Moreover, Ang II induces the synthesis and release of other autocrine-paracrine mitogenic factors such as endothelin-1 and basic fibroblastic growth factor (8, 15). Therefore, in this study, Ang II-induced DNA synthesis may be attributed to the autocrine induction of such growth factors, although it does not appear that the cells are capable of completing the cell cycle (i.e., no increase in cell number). In this regard, Jahan and associates (37) demonstrated that Ang II did not induce the transition of aortic smooth muscle cells in the G0 phase to the G1 phase; rather, it acted as a progression factor stimulating cells remaining in the G1 phase to synthesize protein and DNA. Additionally, they found that PDGF stimulated the entry of cells in the G0 phase into the G1 phase without a further progression into the S and M phases, whereas Ang II incubation stimulated the progression of these PDGF-pretreated G1 cells to the S and M phases. Perhaps, in our study, DNA repair could also be partly responsible for the increase in [3H]thymidine incorporation into DNA with time in the Ang II-treated cells.
In this study, we chose 60 h of serum-free culture conditions in order to synchronize and growth-arrest human ASM cells. Fluorescent-activated cell sorter analysis verified that more than 85% of cells were in the G0/G1 phase of the cell cycle after 60 h of serum deprivation, allowing us to examine the direct effects of Ang II on cell division and growth. Others have reported earlier that 48 h of serum deprivation was successful in growth-arresting ASM cells in culture (10, 38). The data presented here demonstrate that Ang II is a weak mitogen and a potent hypertrophic stimulus for human ASM cells in vitro. However, the cellular mechanisms involved in Ang II-stimulated mitogenesis in ASM cells are undefined and may be distinct from vascular smooth muscle cells.
Previous studies have established that Ang II can induce a rapid increase of the growth-associated nuclear proto-oncogenes c-myc, c-fos, c-jun, and egr-1 in a variety of cell types (20, 22, 39). Similarly, in human ASM cells, nuclear events associated with Ang II activation appear to be the induction of immediate early genes (c-fos, c-jun, and egr-1). The observed increase in DNA biosynthesis could be attributed to the transient expression of the transcription factors c-fos, c-jun, and egr-1, which have previously been reported to be involved in cell growth and differentiation (18, 20, 21, 39). Additionally, the Fos and Jun proteins form a heterodimeric complex known as the AP-1 transcription factor, which activates gene transcription by binding to an AP-1 site in target genes that may subsequently contribute to cell proliferation and growth (19, 21, 40). Previous studies have demonstrated that Ang II stimulates Ang II type-1 (AT1) receptor-mediated hypertrophy in cardiac myocytes and hyperplasia in cardiac fibroblasts (18, 41). In vascular smooth muscle cells, proliferative effects of Ang II are attributed to the AT1 receptor which is coupled to G-proteins and classic intracellular second messenger systems (42). In contrast, the function and the signal transduction pathways for the AT2 receptor, which exhibits only 32% homology to the AT1 receptor are not fully understood. However, AT2 receptor-mediated growth inhibition in endothelial cells treated with Ang II has been reported (43). We might speculate that the Ang II-induced upregulation of immediate early genes in human ASM cells may be mediated via AT1 receptors.
In the present study we have demonstrated that Ang II
induces TGF-1 mRNA expression and the secretion of
biologically active TGF-1 protein in human ASM cells.
Our findings are in agreement with previous reports where
Ang II has been shown to stimulate TGF-1 both at
mRNA and protein levels in vascular smooth muscle cells
(17, 26, 34). One explanation for the induction of TGF-1
seems to be the positive regulation by the AP-1 transcription factor. The TGF-1 promoter region contains an AP-1
site which can modulate TGF-1 gene expression, suggesting that the Ang II-induced expression of c-fos and c-jun
may participate in the induction of TGF-1 gene expression (17, 44). Several groups have shown that the incubation of vascular smooth muscle cells with TGF-1 induces
cellular hypertrophy and inhibits mitogen-stimulated proliferation (34, 45, 46). Apart from the mechanisms involving induction of growth factors such as TGF-1 and
PDGF, Ang II-induced hypertrophy in human ASM cells
may be attributed partly to changes in the contractile phenotype of these cells in response to Ang II stimulation. Koibuchi and colleagues have shown that vascular smooth
muscle cell growth is mediated by the autocrine production of active TGF-1 (26). This may also be the case in
ASM cells. The autocrine production of biologically active
TGF-1 is a major determinant of whether vascular smooth
muscle cells grow by hypertrophy or hyperplasia (17). It
remains to be determined whether the direct incubation of
human ASM cells with biologically active TGF-1 can stimulate cellular hypertrophy.
In conclusion, we have established that Ang II induces
DNA biosynthesis and protein biosynthesis, with the net
effect being the accumulation of protein over DNA indicating hypertrophy in human ASM cells. Caution must be
exercised in extrapolating from in vitro studies to in vivo
observations, but our data suggest a potentially important
role for Ang II in ASM growth in airway remodeling in
asthma. This possibility is supported by the findings that
plasma levels of Ang II are elevated in patients with acute severe asthma (13, 14). Our results also demonstrate that the ASM cells are activated by Ang II and that egr-1, c-fos,
and c-jun appear to be the primary response genes. The
transient expression of these transcription factors is evidence in favor of induction of the nuclear transcriptional
machinery that can subsequently be involved in cellular
hypertrophy, since egr-1, c-fos, and c-jun upregulation generally precede the induction of growth-associated genes in
a wide variety of cells. Furthermore, the late and sustained induction of TGF-1 in response to Ang II could contribute, in an autocrine manner, to the process of hypertrophy
in human ASM cells. Hence, Ang II activates a proliferative pathway (increase in DNA synthesis), as well as a possible antiproliferative and hypertrophic pathway (TGF-1
upregulation), such that the net effect is hypertrophy in
human ASM cells.
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Footnotes |
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Address correspondence to: Hari S. Sharma, Ph.D., Institute of Pharmacology, Erasmus University, Faculty of Medicine and Health Sciences, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: SHARMA{at}FARMA.FGG.EUR.NL
(Received in original form February 10, 1997 and in revised form November 3, 1997).
Acknowledgments:
This study was supported by The Netherlands Asthma
Foundation, grant no. NAF 95.46. The authors are grateful to Drs. Rolf Müller
(c-fos), Rodigro Bravo (c-jun), Vikas Sukhatme (egr-1), and Rik Derynck
(TGF-1) for generously providing the cDNA probes. They also thank Ms.
Marion de Haas for her technical assistance.
Abbreviations
Ang II, angiotensin II;
ASM, airway smooth muscle;
BSA, bovine serum albumin;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
OD, optical density;
PBS, phosphate-buffered saline;
PDGF, platelet-
derived growth factor;
TGF-1, transforming growth factor-1.
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References |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Lambert, R. K.,
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