(Received for publication, May 17, 1995; and in revised form, July 5, 1995)
From the
Vascular smooth muscle cells (VSMC) are the principal cellular
component of the blood vessel wall. Atherosclerosis, hypertension, and
angiogenesis are associated with abnormal VSMC growth. Angiotensin II
is hypertrophic for cultured adult rat aortic VSMC, whereas
platelet-derived growth factor and serum are hyperplastic. To identify
changes in specific proteins associated with either hyperplastic or
hypertrophic growth, high resolution two-dimensional gel
electrophoresis was performed on extracts from quiescent rat aortic
VSMC and from VSMC exposed for 24 h to growth factors (10% fetal calf
serum, platelet-derived growth factor, or angiotensin II). 12 proteins
were up-regulated and 5 down-regulated by treatment with growth
factors. Eight of the up-regulated and one of the down-regulated
proteins were identified by internal protein microsequencing from
electroblotted two-dimensional gels or by co-electrophoresis of
purified proteins in two-dimensional gels. Four of the proteins
up-regulated by growth factors were identified as mediators of protein
folding. These were heat shock proteins, HSP-60 and HSP-70, protein
disulfide isomerase, and protein disulfide isomerase isozyme Q-2.
Additional proteins were identified as elongation factor EF-1, a
component of the protein synthesis apparatus, and calreticulin, another
putative molecular chaperone. Vimentin and actin were also
up-regulated, whereas an isoform of myosin heavy chain was
down-regulated. Hyperplastic and hypertrophic growth were accompanied
by similar changes in protein expression, suggesting that both types of
growth require up-regulation of the protein synthesis and folding
machinery.
Growth and migration of VSMC ()are considered to be
key events in the pathogenesis of atherosclerosis, hypertension, and
angiogenesis(1) . VSMC display two distinct growth responses:
hyperplasia, characterized by increased DNA and protein synthesis as
well as cell division, and hypertrophy, characterized by increased cell
size and protein content without DNA synthesis or cell
division(2) . Atherogenesis is characterized by the
hyperplastic response, involving the migration of VSMC from the vessel
media to the intima and the proliferation of medial and intimal
VSMC(1) . Chronic hypertension involves predominantly
enlargement of preexisting VSMC (hypertrophy) within the media of the
blood vessel as well as proliferation(2) . In cell culture, the
nature of the growth stimulus, rather than intrinsic differences in
growth responsiveness of distinct cell subpopulations, appears to
determine whether VSMC undergo hyperplasia or hypertrophy(2) .
PDGF and serum mediate a hyperplastic response in adult rat aortic
VSMC(3) . In the same cells, Ang, arginine vasopressin, and
thrombin more typically mediate a hypertrophic
response(3, 4, 5, 6, 7, 8, 9) .
Considerable progress has been made in elucidating the molecular events associated with hypertrophic and hyperplastic VSMC growth. Many studies have focused on examining mRNAs induced by growth factor activation of VSMC. These have identified mRNAs encoding a variety of proto-oncogenes, proteins involved in the cell cycle, components of the extracellular matrix, cytokines, and growth factors(9, 10, 11, 12, 13, 14, 15) . Such studies were designed to identify growth factor-responsive proteins based upon differences in mRNA accumulation and would not identify proteins whose levels were altered predominantly by changes in translation. Studies based upon alterations in protein levels have been limited by the difficulty of displaying and identifying the large number of proteins expressed by cultured cells. We have utilized high resolution two-dimensional gel electrophoresis and protein microsequencing to examine differences in protein expression among serum-deprived cultured rat aortic VSMC and VSMC subjected to hyperplastic (PDGF, 10% serum) and hypertrophic (Ang) growth stimuli. This has led to the identification of nine proteins whose levels were either increased (eight) or decreased (one) after a 24-h exposure to growth agonists. The majority of these proteins are different from those previously identified using mRNA screening strategies and include proteins involved in the protein synthesis and folding machinery as well as components of the cytoskeleton.
To measure DNA and protein synthesis, VSMC
were plated in 12-well dishes. At 25% confluence, cultures were
incubated in nongrowth medium for 48 h and then treated for 24 h with
either PDGF or Ang in the presence of
[
H]thymidine or [
H]leucine
(1 µCi/well). Triplicate experiments were performed using 3 wells
per treatment per experiment. Incorporation of the radiolabeled
material was determined by liquid scintillation spectrometry of the
trichloroacetic acid-precipitable material as previously
described(5) .
Spots of interest were
excised from the nitrocellulose membrane and subjected to in situ proteolytic cleavage for 3 h at 37 °C using 0.5 µg of
trypsin (sequencing grade from Boehringer Mannheim) in 50 µl of 100
mM NHHCO
, supplemented with 0.3% Tween
80 (Calbiochem). The resulting peptide mixture was reduced and S-alkylated with 0.1%
-mercaptoethanol (Bio-Rad) and 0.3%
4-vinylpyridine (Aldrich), respectively, and fractionated by
narrow-bore reversed phase high performance liquid chromatography
(HPLC) using a 2.1-mm Vydac C4 (214TP54) column (The Separations Group,
Hesperia, CA). HPLC system configuration and solvents were as described (28) ; a flow rate of 100 µl/min was used. Fractions were
collected by hand and stored at -70 °C before sequence
analysis. Trypsin blanks were analyzed as controls.
Selected peak
fractions were analyzed by automated Edman degradation using an Applied
Biosystems model 477A sequencer. Stepwise liberated
phenylthiohydantoin-amino acids were identified using an
``on-line'' 120A HPLC system (Applied Biosystems) equipped
with a phenylthiohydantoin C18 (2.1 220 mm; 5-micron particle
size) column (Applied Biosystems). The standard AB method was optimized
for sub-pmole phenylthiohydantoin-amino acid analysis as
described(25, 29) . After storage, column fractions
were supplemented with neat trifluoroacetic acid (10% final
concentration) before loading onto the sequencing disc. Peptide
sequences were compared to data base entries (PIR, SwissProt, GenBank).
Figure 1:
DNA and protein synthesis in
rat aortic VSMC. Quiescent VSMC (incubated in nongrowth medium for 48
h) were incubated in fresh nongrowth medium or treated with 1
µM Ang or 5 half-maximal units/ml of PDGF for 24 h in the
presence of either [H]thymidine (A) or
[
H]leucine (B). Incorporation of the
radiolabel into DNA or protein was determined by liquid scintillation
spectrometry of trichloroacetic acid precipitates as
described(5) . Data are expressed as percentage increase
relative to quiescent VSMC and represents the average (±S.E.) of
three experiments, each performed on triplicate wells. Experiments
shown were performed at passage 5. Essentially identical results have
been found using cells from passages 3-13 (data not
shown).
Figure 2: Two-dimensional electrophoretic protein map of serum-stimulated rat aortic smooth muscle cells. Large format gels were used, and proteins were visualized by silver staining (see ``Materials and Methods''). Proteins that were either up- or down-regulated by treatment with serum, Ang, or PDGF are marked with the spotnumber (coordinates and additional information are listed in Table 1). Additional marker proteins (``landmarks'' on the map) were located by co-electrophoresis of purified proteins (labeled with name). Three of the annotated proteins (and actin) were identified by co-electrophoresis and five by internal microsequencing.
Figure 3: Detailed display of two-dimensional protein patterns of rat aortic smooth muscle cells grown in defined medium or stimulated with Ang. Magnified, matching regions of computerized gel images (full size in Fig. 2) are shown. Four of the down-regulated and one of the up-regulated proteins appear in this section of the gels (listed in Table 1). Spots 111 and 109 represent HSP-60 and a fragment of myosin heavy chain, respectively; the others are unidentified.
Figure 4: Quantitative analysis of the nine identified polypeptides regulated by serum, PDGF, and Ang. Passage 2-5 rat aortic VSMC were made quiescent for 48 h and then treated for 24 h with 1) fresh ``nongrowth'' medium or fresh medium containing 2) 1 µM Ang, 3) PDGF (5 half maximal units/ml), or 4) 10% CS. Each bar represents the average (±S.D.) of 3-4 different two-dimensional gels. The y axis is presented in integrated optical density units and provides a measure of each protein's relative abundance.
This study reports the use of high resolution two-dimensional gel electrophoresis to identify proteins regulated by growth factors in cultured rat aortic VSMC. Among the proteins identified were two classes of proteins known to assist in the protein folding process, molecular chaperones and protein disulfide isomerases. These proteins have not been previously shown to be regulated by growth factors in cultured VSMC and have not been identified in VSMC utilizing RNA-based screening strategies.
Post-translational or co-translational folding
is a necessary aspect of manufacturing new proteins (reviewed in (31) ). Molecular chaperones interact with a wide array of
polypeptides from the onset of translation until attainment of the
final folded state(32, 33, 34) . The two
families of heat shock proteins most intimately associated with general
protein folding are HSP-60 and HSP-70(35) . HSP-70 has been
shown to be inducible by serum mitogens in several cell
types(36, 37) . HSP-70 mRNA has also been found to be
induced in intact aorta after treatment with sympathomimetic or
hypertensive agents and in cultured aortic VSMC by hypertrophic
stimuli(38, 39, 40, 41, 42) .
Yeast strains lacking a class of cytoplasmic HSP-70 show defects in
protein synthesis(33, 34) . The growth defect can be
overcome by overproduction of a protein related to the translation
elongation factor, EF-1. The rate of synthesis of EF-1
increases 6-fold following mitogenic stimulation (43) . The
present study would not have detected changes in EF-1
levels
because this protein has an apparent isoelectric point of 10.0 (44) , well outside the pH range (pH 4.0-7.5) of the
two-dimensional gels. However, another component of the elongation
factor complex, EF-1
, was found to be up-regulated by Ang, PDGF,
and CS.
Protein disulfide isomerases are capable of catalyzing a wide range of protein disulfide oxidoreduction reactions(45, 46) . Protein disulfide isomerases bind newly synthesized proteins in the process of folding in the endoplasmic reticulum and are also found in stable association with misfolded molecules in this compartment(45, 47) . The enzyme has also been shown to facilitate the oxidative refolding of lysozyme in vitro, provided it is present in amounts comparable to that found in the endoplasmic reticulum(48) . At least one protein disulfide isomerase isoform has been found to be expressed at high levels in normal cells engaged in the synthesis of large amounts of secreted proteins(49) .
Calreticulin acts as a major
Ca-binding protein in the lumen of the endoplasmic
reticulum(50) . Calreticulin shares significant homology to
calnexin, an 88-kDa endoplasmic reticulum-associated protein determined
to be a novel molecular chaperone participating in the assembly of
murine class I histocompatibility molecules(47, 51) .
Calreticulin has been found in the nucleus and recently has been shown
to modulate gene expression by binding to the glucocorticoid
receptor(52, 53) , further suggesting it serves as a
nuclear chaperone. We have recently found that antisense
oligonucleotides to calreticulin mRNA inhibited PDGF-mediated growth of
cultured rat aortic VSMC by
80%, suggesting that calreticulin may
play a critical role in the growth response of these
cells(69) . Several similarities between the 5`-flanking
regions of calreticulin, protein disulfide isomerase, GRP78, and GRP94
suggest that they may be similarly regulated(54) . GRP78 is
approximately 60% homologous to HSP-70. No growth-induced changes in
GRP78 were observed in this study.
A variety of cytoskeleton
proteins have been previously identified as being growth
factor-responsive in VSMC using RNA-based
strategies(10, 55, 56, 57, 58) .
These include phospholamban, smooth muscle isoforms of -actin and
-actin, CHIP28 (a channel protein),
-calponin, SM22
(a
member of the calponin family), tropoelastin, vinculin, and smooth
muscle isoforms of myosin heavy chain. The approach taken in the
current study was similar to a recently published two-dimensional gel
study examining the hypertrophic effects of Ang and arginine
vasopressin on rat aortic VSMC(59) . In that report, a
2-3-fold increase in actin content, a 2.5-7-fold increase
in vimentin content, and a 3-6-fold increase in tropomyosin
content were observed upon stimulation of quiescent VSMC with
hypertrophic agents for 96 h. We found similar increases in total actin
and vimentin content in response to 24 h of either hyperplastic or
hypertrophic stimuli. In contrast, total VSMC-specific tropomyosin and
myosin light chain content were altered little by 24 h of stimulation
(data not shown).
Both PDGF and Ang were used as agonists to
stimulate protein synthesis. As demonstrated in Fig. 1, Ang acts
as a hypertrophic agent for adult rat aortic VSMC, whereas PDGF is
hyperplastic(6, 7) . Ang and PDGF share a variety of
intracellular signaling pathways, including the activation of
phospholipase C, the induction of the mitogen-activated protein kinase
system, and the activation of the Na-H
antiporter (reviewed in (60) ). In addition, both
agonists induce similar sets of early response genes, including
c-fos and c-jun (reviewed in (13) ). Previous
attempts by this laboratory to identify differences in gene expression
in response to Ang and PDGF using differential screening (11) have been unsuccessful. Despite the relatively large
number of proteins available for evaluation in the current study, no
differences were seen in the 24-h expression of specific proteins in
response to Ang or PDGF. This underscores the similarities in signaling
between the two agonists and further suggests that there are a limited
number of molecular events that distinguish the hypertrophic and
hyperplastic responses of VSMC. In this regard, it should be noted that
Dzau and co-workers (61) have provided evidence that the
secretion of transforming growth factor-
in response to Ang may be
the critical event in converting a hyperplastic response to a
hypertrophic one.
The present study describes a model system for analyzing approximately 1,000 proteins expressed in VSMC. Protein spots on two-dimensional gels must contain at least 15-20 pmol (combined from a maximum of 5 gels) of material to be suitable for identification by microsequencing. State-of-the-art approaches are required to allow for amino acid identification at the sub-pmol level(25, 28, 29) . We estimate that based on their abundance on two-dimensional gels, about 10% of these proteins can be identified by microsequencing. Indeed, eight proteins regulated by growth agonists in VSMC were present at levels below that necessary for even the most sensitive sequencing approaches currently available. These proteins could potentially be identified with specific antibodies or by comparison with the migration patterns of other known proteins on two-dimensional gels. Increasing the amount of total protein analyzed on two-dimensional gels should increase the percentage of proteins suitable for identification by sequencing. However, this usually results in very distorted gel patterns with minor spots being obscured by the more abundant proteins. Thus, pre-gel enrichment of the proteins of interest would be necessary.
In summary, high resolution two-dimensional gel electrophoresis and microsequencing has provided information on a group of proteins involved in protein synthesis and folding that had not previously been appreciated as growth factor-regulated molecules in VSMC. This underscores the power of this approach to identify classes of proteins whose regulation is not based on changes in steady state mRNA levels easily identifiable by mRNA- and cDNA-based screening strategies. Of note, this study did not identify the protein products of many of the early response genes, such as the proto-oncogenes or cell cycle-related genes found by others using analyses of mRNA. This is likely due in many cases to the relatively low concentration of these proteins in whole cell extracts and in some to the transient nature of the increase in protein levels. The two-dimensional gel approach should complement those involving mRNA and cDNA in providing a comprehensive analysis of molecular events associated with VSMC growth.