From the
Cultured aortic smooth muscle cells (SMC) exhibit morphological
and phenotypic modulation characterized by a change from a substrate
attached monolayer culture to a multilayered nodular cell culture in
which SMC are imbedded into the extracellular matrix. Associated with
nodule formation is a change in the pattern of SMC gene expression
including increased expression of a well characterized marker of smooth
muscle cell differentiation, SM
The principal function of aortic medial smooth muscle cells
(SMC)
In culture, explanted SMC
also appear to modulate from a contractile to a synthetic phenotype
under the influence of serum factors, cytokines, and extracellular
matrix components
(11, 12, 13) . In
post-confluent and density inhibited cultures, SMC initiate phenotypic
changes such as increased expression of the smooth muscle (SM) specific
genes SM
After subcultivation in serum containing
medium, medial SMC initially form a confluent cell monolayer. Within
the monolayer multicellular foci form which subsequently develop into
morphologically and molecularly distinct nodules that contain
differentiated non-proliferating SMC embedded in a carbohydrate-rich
extracellular matrix
(22, 23, 24) . In contrast
to representative cells in monolayer culture, cells in the nodule
contain myofilaments and dense bodies. A comparison of the proteins
synthesized and secreted by nodular SMC cultures with those secreted by
monolayer SMC cultures revealed increased expression of two
glycoproteins: clusterin
(20, 21, 25) and a
heparin-binding glycoprotein of 38-kDa (gp38k)
(26, 27) .
The factors regulating differential expression of those genes, and SMC
modulation, in general, are not presently known. However, it is known
that cultured SMC respond to soluble factors including cytokines,
growth factors, and extracellular matrix
macromolecules
(28, 29, 30, 31, 32, 33, 34, 35, 36) .
It is possible that SMC modulation is mediated by proteins secreted by
SMC in response to unidentified signals
(23, 29) .
In
order to characterize the porcine SMC gp38k and begin to develop an
understanding of its role in SMC modulation, we used degenerate
oligonucleotides, based on polypeptide sequence information to isolate
a cDNA encoding full-length gp38k from a nodular SMC cDNA library. The
deduced amino acid sequence of the gp38k cDNA indicates that it encodes
a protein composed of 383 amino acid residues, has a putative signal
sequence, contains a single consensus sequence for N-linked
glycosylation, and a heparin-binding consensus sequence. The deduced
sequence reveals it to be a member of a family of related proteins
whose expression is not limited to SMC. Homologues have been sequenced
from human, bovine, murine, and Drosophila cDNAs and all of
the family members show partial sequence homology to a group of
proteins which cleave the invertebrate matrix polysaccharide chitin
(37-40).
Expression of gp38k mRNA is correlated with
multilayered SMC growth and nodule formation and the steady state level
of the mRNA is increased during conditions that facilitate nodule
formation. Addition of nodular cell culture conditioned medium or
seeding SMC onto a preformed reconstituted extracellular matrix promote
expression of gp38k and mRNA. Although the role of this protein in
phenotypic modulation of SMC remains to be elucidated, this report
provides strong support for the conclusion that gp38k is a marker of
SMC differentiation by demonstrating that its expression parallels that
of SM
Culture morphology was monitored using a Videoscope
imaging camera attached to an Olympus CK2 inverted microscope fitted
with a 2
Polyacrylamide gel
electrophoresis was performed using a 10% separating gel and a 5%
stacking gel containing 1% SDS. Gels were prepared using the system of
Laemmli
(41) and run at a constant current of 16 mA for
1.5-2 h. Molecular weight standards were myosin (200,000),
phosphorylase b(97, 400) , bovine serum albumin
(69,000), carbonic anhydrase
(30, 0) , trypsin
inhibitor
(21, 500) , and
lysozyme
(14, 300) . Gel lanes were loaded with either
equal volumes of conditioned medium or equal amounts of protein
(Bio-Rad protein assay).
To obtain internal sequences, samples were concentrated in
50 mM NH
From this analysis we obtained a single amino-terminal sequence and
5 internal sequences. Degenerate oligonucleotides were synthesized
based on conserved regions in gp38k and mammalian homologues and
porcine codon usage
(47) . Degeneracy was reduced by substituting
inosine at the third position of the triplet coding for leucine in the
sense primer. To allow directional cloning, the sense primer was
designed with a BamHI adaptor at the 5` end followed by
TTYGAYGGNYTIGAYYT-3 and the antisense primer with an EcoRI
adaptor at the 5` end followed by TCRTCRTANCCNACCCA-3`. The primers
were synthesized and trityl-specific purified by Ransom Hill Bioscience
(Ramona, CA). Amplification of the region between these two primers
included nucleotide sequences encoding three of the polypeptide
sequences that we obtained by direct amino acid sequencing of purified
gp38k.
White colonies were picked and grown
overnight at 37 °C in LB medium containing 50 µg/ml ampicillin.
Minipreps were performed to isolate plasmid DNA, and the DNA was
displayed on a 1% agarose gel next to 2 µg of pBluescript SK-
without an insert. The insert was partially sequenced using the chain
termination method
(45) with [
Each plaque lift was
transferred onto nitrocellulose (Schleicher & Schuell, Inc.) and
denatured by submerging in 1.5 M NaCl, 0.5 M NaOH for
2 min. After neutralization, the DNA was cross-linked to the filters
for 30 s using a Stratalinker UV cross-linker (Stratagene).
Filters
were prehybridized at 42 °C for 2 h in a solution containing 50%
deionized formamide, 0.5% SDS, 2
DNA sequencing utilized new primers after approximately
every 200 nucleotides. During the course of sequencing, the BLAST
e-mail server at the National Center for Biotechnology Information was
used to search for homologous sequences in various data
bases
(48) . In addition, the PC GENE package (Intelligenetics,
Mountain View, CA) and FASTA were used to search for homologies against
various data bases.
After denaturation in
0.4 M NaOH containing 0.6 M NaCl the gel was
incubated twice for 15 min in a neutralization solution (0.5 M
Tris-HCl, pH 7.8, 1.5 M NaCl). Size fractionated DNA fragments
were transferred onto nylon membrane (GeneScreen, DuPont) using
downward capillary transfer for 16 h
(49) . The membrane was then
washed in neutralization solution and the DNA cross-linked to the
membrane using a Stratalinker (Strategene, La Jolla, CA).
The
membrane was prehybridized in buffer containing 5
A 38,000-Da glycoprotein (gp38k) was previously isolated and
identified as a heparin-binding glycoprotein secreted by cultured
vascular smooth muscle cells that had undergone conversion from
monolayer to nodular cell culture
(23, 26, 27) .
To determine if gp38k was also present in the nodule layer we stained
well developed nodules with antibodies to gp38k and antibodies to the
smooth muscle specific isoform of
The protein
encoded by this cDNA has a calculated theoretical isoelectric point of
9.28, contains 6 cysteines, and a cluster of basic amino acids, RRDKRH,
located at positions 144-149, which represent the heparin binding
consensus sequence reported for a number of other heparin-binding
molecules
(58) . The cDNA sequence predicts a secreted protein
with a conventional signal sequence corresponding to the first 21 amino
acids. The mature protein is predicted to begin with the YKLVCYYTSWQYRE
sequence and this is in agreement with the results obtained from direct
amino acid sequencing of the 38-kDa protein secreted by SMC cultures.
The process of SMC phenotypic modulation in vitro is
correlated with increased expression and secretion of a heparin-binding
glycoprotein with M
Recent studies
indicate that the pattern of gene expression is modified in nodular
cell cultures. Clonal populations of human and bovine nodular cells
derived from aortic media were reported to express bone morphogenetic
protein-2a and osteocalcin
(22) . Porcine SMC nodules derived
from aortic media also show an altered pattern of gene expression that
includes an increase in clusterin and
gp38k
(21, 23, 25, 53, 54) . In
order to investigate the expression of gp38k mRNA and its correlation
with SMC differentiation we have now cloned and sequenced a full-length
cDNA that encodes the porcine gp38k and report that its expression is
correlated with that of SM
The
deduced amino acid sequence of gp38k includes a putative signal
sequence of 21 amino acid residues, a single consensus sequence for
N-linked glycosylation, and a putative heparin binding site.
Previously we demonstrated that gp38k is a heparin-binding glycoprotein
that is eluted from heparin-Sepharose by 400 mM
NaCl
(27) . Presently it is not known if the interaction with
heparin is related to a functional interaction with glycosaminoglycan
components of the extracellular matrix. gp38k contains 6 cysteine
residues, however, our previous work indicates that they are not
involved in disulfide bonding since the apparent molecular weight of
gp38k is similar in reduced and nonreduced samples examined by
SDS-polyacrylamide gel electrophoresis
(27) . It is not known if
its homology with chitinase has any functional significance since
chitinase activity has not been detected in two of the gp38k
homologues
(37, 40) .
SMC nodulation, in
vitro, is facilitated by the presence of a collagenous
extracellular matrix
(26) . Medial SMC, in vivo, are
embedded in a three-dimensional extracellular matrix and we postulated
that nodules mimic the media by containing SMC embedded in an
extracellular matrix which is elaborated by the cell cultures. During
the formation of the multilayered regions that give rise to nodules,
gp38k synthesis is initiated. The time required for nodules to form in
standard tissue culture conditions is greater than 9 days. However,
that time requirement is reduced to 24-36 h when SMC are seeded
onto a reconstituted extracellular matrix, composed of collagen gel or
Matrigel
(21, 55, 56) . Other laboratories have
reported that SMC cultured on Matrigel exhibit an altered pattern of
gene expression suggestive of SMC differentiation
(35) . In this
report, we demonstrate that gp38k mRNA expression is induced by each of
the reconstituted matrices and gp38k expression correlates well with
nodule formation and SM
Recently genes
homologous to gp38k have been cloned from three mammalian species and
Drosophila(36, 37, 38, 39) and
the mRNA identified in tissues other than SMC. The human homologue, HC
gp39k has been identified in articular chondrocytes and synovial
cells
(37) . A bovine oviduct-specific glycoprotein, with
sequence homology to gp38k, appears to correlate with changes during
ovulation
(39) . An oviduct specific homologue in baboon is
larger than the porcine gp38k and more heavily
glycosylated
(57) . The occurrence of gp38k homologues in several
species and in different tissues suggests that it may be a member of a
larger protein family. To address that question we used Southern
transfer analysis done under high stringency conditions to estimate the
number of genes for gp38k in porcine SMC genomic DNA. Restriction
digestion with HindIII and hybridization with a 600-bp cDNA
revealed a single band suggesting that porcine gp38k is present as a
single gene.
The occurrence of gp38k in SMC undergoing morphological
reorganization and the gp38k homologues in other tissues undergoing
remodeling suggests the possibility that gp38k has a regulatory role in
that process in vascular as well as other tissues. For example, in
experimental animal models of restinosis medial SMC develop a
morphology and pattern of cytoskeletal protein expression, including
change in the predominant actin iosform, suggesting a de-differentiated
phenotype. At later times after injury the SMC show partial
re-differentiation including increased amounts of vimentin, desmin,
tropomyosin, and
The
gp38k sequence is available as GenBank U19900.
We are grateful to Dr. Greg Lnenicka (Department of
Biology, University at Albany) for advice on video imaging and
providing access to the Video Imaging Facility at the University at
Albany.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-actin, and a 38-kDa glycoprotein
(gp38k). gp38k has sequence homology with proteins reported to be
correlated with tissue remodeling. To characterize the gp38k mRNA we
designed degenerate oligonucleotides based on partial polypeptide
sequencing to select a cDNA encoding the full-length gp38k. Southern
analysis indicates that porcine gp38k is present as a single copy gene.
Northern analysis indicates that the increase in gp38k is correlated
with an increase in the steady state level of gp38k mRNA; and is
present in cultures that have initiated the formation of multilayered
foci and nodules. The correlation between SMC differentiation and gp38k
expression is further established by using culture conditions that
facilitate SMC differentiation. Cultures seeded onto reconstituted
extracellular matrix show rapid formation of nodules and increased
expression of gp38k mRNA. Comparison of the gp38k and cDNA sequences
with nucleotide and protein sequences available through GenBank and
SwissProt data banks revealed that molecules homologous to gp38k were
present in human, mouse, bovine, and Drosophila tissues,
suggesting that the gp38k may be a member of a gene family. Although a
function for gp38k has not been identified, this report represents the
first report of its correlation with a specific process important in
phenotypic and morphological modulation of vascular SMC.
(
)
is to maintain the vessel wall tension
and contractility
(1, 2) . Medial SMC are embedded in an
interstitial extracellular matrix and in response to endothelial injury
migrate into the intima, proliferate, and synthesize components of the
extracellular
matrix
(3, 4, 5, 6, 7) . The
phenotypic modulation of medial SMC from a quiescent differentiated
phenotype to a migrating synthetic phenotype is considered to be a key
event in the development of atherosclerotic plaques in response to
injury
(8, 9, 10) .
-actin and SM-myosin heavy
chains
(14, 15, 16, 17, 18, 19) .
In the appropriate environment, cultured SMC can express proteins that
appear to be related to cell differentiation, including
clusterin
(20, 21) and bone morphogenetic
protein
(22) .
-actin
(18, 19) and demonstrates for the
first time that gp38k expression can be regulated by components of the
extracellular matrix.
Materials
Medium 199 was obtained from Life
Technologies, Inc. (Grand Island, NY); fetal bovine sera from HyClone
Laboratories, Inc. (Logan, UT); [P]dCTP(3000
Ci/mmol) from Amersham, Inc.; Klenow fragment of DNA polymerase from U.
S. Biochemical Corp. (Cleveland, OH); GeneScreen plus from Biotech
System (Boston, MA); electrophoresis reagents from Bio-Rad; and TRI
reagent from Molecular Research Center, Inc. (Cincinnati, OH).
Cell Culture
Porcine aortic smooth muscle cell
cultures were initiated using explants of the luminal face of the
thoracic aorta and cultivated in medium 199 supplemented with 10% fetal
bovine serum (HyClone, Logan, UT) and 3% CO, 97% air as
described previously
(21, 23) . Cells were routinely
subcultivated by treatment of confluent monolayer cultures with EDTA
and trypsin in isotonic saline. At each passage the cell number was
determined using a Coulter counter (Coulter Electronics, Hialeah, FL)
and the cultures replated at 2
10
cells/cm
in fresh serum containing medium. Nodular cell cultures were
generated by replacing the medium of confluent cultures with fresh
medium 199 supplemented with 5% fetal bovine serum. Under these
conditions, nodules begin to form after 5-9 days and well
nodulated cultures (>70 nodules/cm
of surface) are
present within 2-3 weeks.
Substrate Preparation
Freshly harvested cells were
mixed with equal volumes of soybean trypsin inhibitor, centrifuged for
5 min at 1000 rpm, and immediately suspended in M199 containing 10%
serum. These cells were seeded at a density of 7
10
cells/cm
on various substrata. Reconstituted
basement membrane gel, Matrigel (Collaborative Biomedical Products,
Bedford, MA), was used after 1:1 dilution, on ice, in cold serum-free
medium M199. Collagen gel-coated cluster dishes were also prepared on
ice using a collagen I solution consisting of 8 parts rat tail collagen
I (Collaborative Biomedical Products), 1 part 0.01 M NaOH, 1
part 10
phosphate-buffered saline and the pH brought to 7.4
with 0.1 M NaOH. Each well of a 6-well cluster dish (Falcon,
Becton Dickinson, Bedford, MA) was coated with 400 µl of either gel
solution and allowed to gel for 30 min at 37 °C before addition of
SMC
(21) .
objective and the image processed with Metamorph
imaging software (Universal Imaging Corp., West Chester, PA). The
numbers of nodules are calculated by the software directly from the
recorded image.
Preparation of Conditioned Media
Serum-free
conditioned media from monolayer and nodular smooth muscle cell
cultures were prepared by washing cultures 3 with Hank's
balanced salt solution before incubation for 24 h in serum-free medium
199. The medium was collected into 1 mM phenylmethylsulfonyl
fluoride and 1% aprotinin (Trasylol; Meloy Labs, NY), centrifuged for
15 min at 37,000
g, and either used immediately or
stored frozen
(23, 26) .
Gel Electrophoresis
For polyacrylamide gel
electrophoresis the conditioned medium was precipitated with 15%
trichloroacetic acid at 4 °C for 1 h. Greater than 95% of the total
protein was precipitated by this procedure (as estimated from Bio-Rad
Protein assays) and all of the immunodetectable gp38k was precipitated
(trichloroacetic acid-soluble fractions were concentrated in Amicon
ultrafiltrations units and examined by immunoassay). Electrophoresis
grade reagents were obtained from Bio-Rad.
Antibody Preparations and
Immunoblotting
Polyclonal antibodies to rat clusterin (Sertoli
cell clusterin) were generously provided by Drs. Michael Griswold and
Steven Sylvester, Washington State University
(42) . Polyclonal
antibodies to gp38k were prepared in this laboratory as described
previously
(27) . Antibodies to gp38k were affinity purified by
adsorption to purified gp38k fixed to nitrocellulose
membrane
(43, 44) . Proteins were separated on minigels
and transferred from the gel onto nitrocellulose paper (Schleicher
& Schuell) in 192 mM glycine, 20% methanol, 25 mM
Tris-HCl, pH 8.3, using a Genie electroblotter (Idea Scientific).
Following transfer at 25 V for 1 h the blot was incubated in a blocking
agent consisting of 5% nonfat dried milk (Carnation, Los Angeles, CA).
After incubation with primary antibody (1:2000 dilution of anti-SGP2 or
anti-38K protein in rabbit sera) for 2 h at room temperature on a
rocker, the membrane was washed and incubated with horseradish
peroxidase-conjugated donkey anti-rabbit whole antibody (1:10,000
dilution; Amersham) for 60 min at room temperature. After washing,
immobilized antigens were detected using an enhanced chemiluminescence
assay (ECL: Amersham). Following immunotransfer, each gel was stained
with Coomassie Blue to evaluate the efficiency of transfer.
Purification and Amino-terminal Sequencing of
gp38k
gp38k was purified from nodular SMC conditioned medium by
heparin affinity chromatography (Pharmacia, Inc., Pitscataway, NJ),
followed by high performance liquid chromatography. Samples were
purified using conditions to minimize amino-terminal blocking and
further purified via 5-20% linear gradients of
polyacrylamide
(44) . The gel bands were visualized by brief
staining with Ponceau S. Amino-terminal sequencing was performed on
protein eluted from gel band slices by soaking overnight at 37 °C
in 0.5 ml of 100 mM Tris, 0.1% SDS, pH 8.0, or by
electroeluting in 0.1% SDS, 0.05 M
NHHCO
. This material was loaded onto the
hydrophobic portion of a biphasic Hewlett-Packard sequencer column and
washed with 0.2% trifluoroacetic acid, and 10% acetonitrile to remove
the Tris and glycine. Sequencing was performed using automated Edman
degradation chemistry on a Hewlett-Packard G1000A Protein Sequencing
system.
HCO
, using an ISCO
electroelution protein concentrator. Each sample was diluted with 5
volumes of 50 mM NH
HCO
and
subsequently precipitated with 8 volumes of acetone at -20
°C. The pellet was then washed 3 times with 95% ethanol at
-20 °C. The pellet was resuspended in 30 µl of Lys-C
digestion buffer composed of 25 mM Tris-HCl, pH 8.5, 0.01%
SDS, 1 mM EDTA and allowed to dissolve overnight at 4 °C.
This preparation was digested for 24 h with 5 µg of Lys-C
(Boehringer Mannheim, Indianapolis, IN) and fractionated on a C
reverse phase column (Brownlee RP300) prior to sequencing.
Selection of Clone Expressing gp38k cDNA
(pBS38K)
To determine the cDNA sequence of gp38k, a strategy
employing reverse transcription-PCR and cDNA library screening was
used.
Reverse Transcription
RNA isolated from cultured
porcine aorta nodular smooth muscle cells was reverse transcribed as
follows: 2-4 µg of RNA was brought up to a final volume of
16.5 µl and incubated at 65 °C for 5-7 min. After
heating, the RNA was quenched on ice for 2 min and centrifuged briefly.
A reverse transcription reaction mixture (final volume of 13.5 µl)
containing 250 mM Tris-Cl, pH 8.3, 50 mM
MgCl, 300 mM KCl, 50 mM dithiothreitol,
50 mg/ml oligo(dT) (Pharmacia, Upsala, Sweden), 0.5 mM dNTP
mixture (U. S. Biochemical Corp.), 1 mg/ml bovine serum albumin, 667
units/ml RNasin (Promega, Madison, WI), and 900 units/ml AMV Reverse
Transcriptase (Molecular Genetic Resources, Tampa, FL) was added to the
tube containing RNA and the reaction was incubated at 42 °C for 1
h. After incubation, 20 µl of double distilled H
O was
added to the resulting cDNA before using in subsequent PCR reactions.
Polymerase Chain Reaction
Polymerase chain
reaction was performed in 50-µl samples containing 500 mM
KCl, 100 mM Tris-Cl, pH 8.3, 25 mM MgCl,
200 µM dNTP mixture, 4 mM each of the sense and
antisense degenerate primers, 80 units/ml Taq DNA polymerase
(Perkin Elmer), and 10 µl of cDNA reverse transcribed under the
conditions listed above. Each 50-µl reaction was overlaid with 1
drop of DNase-free, RNase-free mineral oil (Sigma), denatured for 5 min
at 94 °C, and cycled 30 times using the following parameters; 60 s
at 94 °C, 90 s at 48 °C, and 60 s at 72 °C in an Eppendorf
Microcycler E (Eppendorf, Fremont, CA)
Subcloning and Sequencing of PCR
Product
EcoRI and BamHI (40 units) were added
to the reaction mixture containing the PCR product and incubated at 37
°C for 2.5 h. After electrophoresis in 1% agarose and treatment
with ethidium bromide, an amplification product of approximately 600 bp
was observed and excised from the gel. The excised DNA was purified and
then ligated between the EcoRI and BamHI sites of the
polylinker region of pBluescript SK- (Stratagene, La Jolla, CA).
Xl-1 Blue bacteria (Stratagene) were transformed with pBluescript
containing the insert and incubated overnight at 37 °C on LB plates
containing ampicillin.
S]dATP
(Amersham) and a Sequenase 2.0 DNA Sequencing Kit (U. S. Biochemical
Corp.) and double stranded plasmid DNA that was prepared by the
alkaline lysis procedure
(45) .
cDNA Library Screening
A Uni-Zap XR cDNA library
was prepared using poly(A) RNA isolated from nodular
SMC
(20) . XL-1 Blue Escherichia coli (Stratagene) cells
were infected with 10
plaque forming units of the Uni-Zap
cDNA library and plated onto 100
15-mm LB Agar plates. The
plates were incubated at 37 °C for 8 h.
Pipes buffer, pH 6.5, and 100
mg/ml denatured sonicated salmon sperm DNA. A cDNA probe was prepared
using the 600-bp EcoRI/BamHI restriction fragment of
pBS600 excised from a low melting point agarose gel. The probe was
labeled with [
P]dCTP (Amersham) using a
MultiPrime DNA Labeling System (Amersham). Hybridization was
accomplished using 1
10
counts per filter at 42
°C for 16 h. To identify positive clones, filters were washed, and
then exposed to Kodak X-Omat film overnight at -70 °C in a
cassette including an intensifying screen. Positive clones were
isolated, replated, and prescreened until single isolates were
obtained.
In Vivo Excision of Potential Clones
Clonal
isolates were excised in vivo, from the Uni-Zap vector
following the procedures described by the manufacturer and used to
transform XL-1 Blue bacteria and plated onto LB plates containing 50
µg/ml ampicillin coated with
isopropyl-1-thio--D-galactopyranoside and
5-bromo-4-chloro-3-indoyl
-D-galactoside. Plates were
grown overnight at 37 °C. Single white colonies were picked and
grown overnight at 37 °C in LB broth containing 50 µg/ml
ampicillin.
Southern Blotting
Genomic DNA was isolated from
nodular porcine SMC using TRI Reagent, according to the
manufacturer's instructions. Genomic DNA (12 µg) was digested
with 40 units of BamHI, EcoRI, or HindIII
for 20 h at 37 °C. Each sample was loaded onto a 0.7% agarose gel
containing 1 mg/ml EtBr and run at 60 V until the markers (1 kilobase
pair molecular weight ladder; Life Technologies, Inc., Grand Island,
NY) were well separated (approximately 5 h).
SSC, 5
Denhardt's solution, 1% SDS, and 100 µg/ml denatured
salmon sperm DNA at 65 °C for 4 h. A 600-bp cDNA (excised from
pBS600) was random primer labeled with [
P]dCTP
and added to the prehybridization solution at an activity of 1.2
10
counts/ml. Hybridization occurred at 65 °C
for 20 h. Subsequently the membrane was washed 4 times for 30 min each
at 65 °C in 0.5
SSC, 0.1% SDS before exposure to X-Omat
film.
Northern Analysis
Total cellular RNA was isolated
from smooth muscle cells using TRI Reagent which is based on
guanidine-thiocyanate-phenol-chloroform extraction
procedure
(45, 46) . Each sample was denatured in 10%
glyoxal and 50% formamide by heating to 65 °C for 5 min. After
electrophoresis in 1% agarose the gels were treated for 30 min with 200
mM NaOH, vacuum transferred to GeneScreen Plus (DuPont NEN)
membranes, and neutralized. The 28 S and 18 S RNA species were examined
on each membrane to evaluate the consistency of sample loading and
transfer. After UV cross-linking the nucleic acid on the membrane
(Stratalinker; Stratagene), the membrane was prehybridized in 8%
Denhardt's solution (1% Ficoll, polyvinyl pyrrolidine, and Pentax
fraction V of bovine serum albumin), 43% formamide, 0.69 M
NaCl, 0.09% Na pyrophosphate, 0.9% SDS, 0.7% dextran sulfate, 1.74
mg/ml heparin, and 86 mg/ml denatured salmon sperm DNA for 2 h
overnight at 42 °C. Random primer P-labeled cDNA
(10
cpm/ml) was added to the same buffer, and the membrane
hybridized at 42 °C overnight. The membrane was then washed twice
in 0.2% SDS, 1
SSC for 15 min at room temperature and once in
0.2% SDS, 0.1
SSC at 50 °C. After incubation at -70
°C for 15-48 h the membrane was analyzed by autoradiography.
Quantitation of radioactive signals was achieved using a Betascope 603
(Betagen, Intelligenetics Inc., Mountain View, CA). The gp38k mRNA
signal was detected using either the full-length cDNA encoding porcine
gp38k cloned in this laboratory and described in this manuscript for
Northern assays or a 600-bp PCR product for the Southern assay. The
Northern blot was stripped and subsequently reprobed with a mouse
600-bp polymerase chain reaction product encoding human
glyceraldehyde-3-phosphate dehydrogenase and a 500-bp polymerase chain
reaction product encoding human
-actin (provided by Dr. S. Kumar,
University at Albany).
Photography
Phase-contrast microscopy was
performed using a Diaphot-TMD inverted microscope (Nikon, Tokyo, Japan)
using a 10 objective.
-actin (SM
-actin), an
antigen expressed in differentiated SMC
(18, 19) , and
observed that both antigens co-localize in the nodule indicating that
both are present in the nodular structures (data not presented).
Substrate Effects on gp38k Expression
The time required
for nodule formation is significantly reduced in SMC cultures seeded on
a reconstituted gelatinous matrix
(21) . Therefore we examined
the effects of two reconstituted matrices, Matrigel and a collagen gel
on gp38k expression. Fig. 1shows the nodules formed on each of
these substrates at 24-36 h after seeding monolayer cells.
Panels A-C show SMC, growing in medium 199 supplemented with
5% fetal bovine serum, on untreated plastic (A) or Matrigel
(B and C) substrates at 24 h after seeding. Panel
A shows that on plastic the cells are attached and spread but have
not reorganized to form nodules as shown in the photograph in panel
B. Panel C shows a lower power image recorded by a video imaging
system and reveals a density of 81 nodules/cm. Panel D shows nodule formation in cultures seeded onto collagen gel and
recorded by video imaging. This image was recorded after 36 h since, as
we previously reported, nodule formation is slower on this
substrate
(21) . The number of nodules in the collagen gel image
was 78 nodules/cm
.
Figure 1:
Effects of reconstituted matrix on SMC
nodule formation. Cultures were seeded onto plastic (A),
Matrigel (B and C), or collagen gel (D) and
incubated for 24 h (A-C) or 36 h (D). Panels A and B were photographed using a Diaphot-TMD inverted
microscope and a 10 objective. The images in panels C and D were obtained using video imaging microscopy with a
2
objective, as described under ``Experimental
Procedures.''
Cultures seeded onto collagen gel or
Matrigel were examined for gp38k expression as shown in Fig. 2.
In both cases gp38k is detected in the cultures seeded on the
reconstituted matrices. Western immunoassay was used to determine if
either Matrigel or collagen gel contained gp38k and none was detected
(data not presented).
Figure 2:
Western analysis of cultures seeded on
reconstituted matrix. Cultures were seeded onto plastic (A and
C), collagen gel (B), or Matrigel (D) and
incubated for 24 h. Conditioned medium was collected from each culture,
fractionated by SDS-polyacrylamide gel electrophoresis, and probed with
anti-gp38k antibody (38 kDa
protein).
gp38k Full-length Sequence
To identify the
complete amino acid sequence of gp38k, gp38k protein was isolated from
nodular cell conditioned medium via heparin affinity chromatography
followed by elution with a step gradient of NaCl
(27) . The
gp38k-containing fractions were further purified by polyacrylamide gel
electrophoresis (48). Partial amino acid sequence was obtained from
non-digested and Lys-C-digested gp38k yielding the underlined sequences
shown in Fig. 3.
Figure 3:
cDNA sequence of pBS38kDa and conceptual
translation of the amino acid sequence. A 1733-bp cDNA insert encodes
an open reading frame of 383 amino acids beginning with an ATG codon at
nucleotide position 66. The conceptual translation indicates a putative
signal sequence including amino acid residues 1-21 (bold type).
Single underlined regions mark sequences identified by direct
amino acid sequencing of purified SMC gp38k. The position of a putative
N-linked glycosylation site is indicated by a vertical
arrow and a consensus heparin binding site by a double
underline (residues 144-149). The sequences used to design
degenerate oligonucleotides are bracketed and in bold type at
residues 132-137 and 325-330.
A cDNA clone encoding gp38k was selected from
a nodular SMC cDNA library using a PCR product described under
``Experimental Procedures.'' As shown in Fig. 3, the
gp38k cDNA insert is 1733 bp, contains a single open reading frame
beginning with the ATG at base pair 67, and can encode a polypeptide
sequence 383 amino acids in length with a calculated molecular mass of
42,440 Da. That this sequence corresponds correctly to the 38-kDa
heparin-binding protein secreted by nodular SMC cultures is confirmed
by sequence identity with five internal peptide sequences which were
obtained by direct amino acid sequencing of the purified protein. The
sequences of these 5 polypeptides, generated by digesting the purified
secreted protein with Lys-C, and their positions within the cloned cDNA
are shown in Fig. 3. Previous reports
(26, 27) indicated that the gp38k protein radiolabels when SMC are
metabolically labeled with [H]glucosamine, is
sensitive to endoglycosidase F treatment, and is synthesized with a
reduced apparent M
when SMCs are grown in the
presence of tunicamycin, an inhibitor of N-linked
glycosylation. Consistent with these properties is the occurrence of a
consensus sequence for asparagine-linked glycosylation which occurs at
amino acid 60 encoded by the cDNA (Fig. 3).
gp38k Is Homologous to Proteins in Other Species
A
comparison of the cDNA encoded sequence with the SwissProt, GenBank,
and Protein Inquiry Resource (PIR) data bases, using the internet BLAST
and FASTA e-mail servers (described under ``Experimental
Procedures''), revealed sequence homology with cDNAs isolated from
human cartilage, bovine oviduct, and mouse
macrophage
(36, 37, 38, 39) as shown in
Fig. 4
. The human and porcine homologues are 84% similar over the
length of the 383-amino acid polypeptide. The bovine and mouse
homologues show less homology over the length of the proteins, but
there are regions that are highly conserved between all of the
homologues. The mouse homologue is 401 amino acids and the bovine
homologue 546 amino acids in length. Recently a homologous sequence was
discovered in Drosophila and that sequence encodes a 455-amino
acid protein
(40) .
Figure 4:
Alignment of gp38k with homologous
sequences from human, murine, Drosophila, and bovine species.
Amino acid sequences of gp38k, human HC gp39K (GenBank M80927), mouse
macrophage (GenBank M94584), bovine oviduct glycoprotein (D16639), and
Drosophila are compared with chitinase (SwissProt P29030).
Alignment was achieved using the Waterman algorithm in the MacDNASIS
Pro-analysis system. Consensus positions are highlighted and
insertions indicated by dashed
lines.
gp38k Is Encoded by a Single Gene
In order to
estimate the number of gene copies in porcine genomic DNA, Southern
blot analysis was performed with samples restricted with
BamHI, EcoRI, or HindIII enzymes as shown in
Fig. 5
. Using stringent hybridization conditions we detected a
single hybridization band in the HindIII restricted sample
suggesting that gp38k is encoded as a single copy gene. The multiple
bands present in the BamHI and EcoRI restricted
samples likely resulted from internal restriction sites.
Figure 5:
Southern blot analysis of porcine genomic
DNA extracted from SMC. A Southern blot containing 12 µg of
BamHI (A), EcoRI (B), and
HindIII (C) restricted genomic DNA was probed with a
random-primer labeled 600-bp PCR product. Hybridizations and washes
were done using high stringency conditions as described under
``Experimental Procedures.''
Expression of gp38k mRNA in SMC
The steady state
expression of gp38k mRNA was compared in total RNA preparations
isolated from monolayer and nodular SMC cultures. Fig. 6shows a
Northern assay of total RNA probed for gp38k, SM -actin, and
-actin mRNAs. In nodular cell cultures the expression of gp38k
mRNA is increased >10
as detected by Betagen
analysis. The Northern analysis establishes that the pBS38k cDNA insert
hybridizes with an mRNA of 1.8 kilobase, and that this mRNA is
expressed at higher levels in nodular than in monolayer SMC cultures.
The expression of a smooth muscle isoform of
-actin (SM
-actin) has been reported to be correlated with differentiated SMC
while the
-actin isoform is expressed more
broadly
(18, 19, 50, 51, 52) .
Fig. 6
shows that the gp38k mRNA level is greater in cultures that
express an increased level of SM
-actin and a decreased level of
-actin mRNA and may represent an additional marker for
differentiated SMCs.
Figure 6:
Northern assay of gp38k and actin mRNA.
Total RNA (8 µg/lane) from confluent monolayer (M) and
14-day nodular (N) cell cultures were probed with random
primer labeled cDNA to gp38k (38 kDa protein), SM -actin,
-actin, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
Time Course of gp38k Expression
Previously we
reported that nodules form in SMC culture in multilayered regions that
begin to form after 4 days in culture and continue to form after that
time
(21) . By day 14 the number of nodules has increased to
>65 nodules/cm. To examine the correlation between
nodule formation and gp38k expression we analyzed conditioned medium
and cell extracts from SMC at intervals from 2 to 14 days of culture.
The steady state levels of gp38k protein and mRNA levels were examined
by immunoblot (Fig. 7A) and Northern
(Fig. 7B) analysis, respectively, to evaluate the
correspondence between the time of nodule formation and gene
expression. The antigen is detected after 4 days of culture and the
level is increased in 12-14-day cultures (>50
nodules/cm
). Northern assays indicate that gp38k mRNA is
also detectable at days 4-6 and the level of expression increased
after day 10. These results indicate that nodulating SMC express more
mRNA for gp38k and accumulate more gp38k protein in their culture
medium as the density of nodules increases.
Figure 7:
Time course of gp38k expression.
Conditioned medium (A) was collected from SMC at 24-h
intervals during a 2-14 day time period (lanes
2-14) and equal quantities were probed with anti-gp38k
antibodies. Lane C contains 0.5 µg of heparin-purified
gp38k probed with anti-gp38k antibodies. Total RNA (B) was
extracted from SMC cultures during the same 2-14-day time period
and 8 µg loaded into each lane and probed with cDNA to gp38k and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
= 38,000
(gp38k)
(23, 26, 29, 53) . The level of
gp38k expression is 0.01 µg/ml in monolayer SMC cultures and
increases to 1.9 µg/ml in SMC cultures that have formed
nodules
(26) . Previous reports demonstrated that representative
cells in nodules are morphologically differentiated and express some of
the characteristics of medial SMC
(13, 23, 24) .
In contrast, SMC in monolayer cell culture express characteristics
representative of migrating proliferating SMC.
-actin
(18, 19) .
-actin expression.
-actin
(59) . Although the mechanism of
gp38k action is not known, the presence of heparin binding activity and
consensus sequence suggests the possibility that it may interact with
glycoprotein or proteoglycan components of the extracellular matrix or
cell surface to modify cell adhesion, cell motility, or gene
expression; and consequently influence the phenotypic state of the SMC
during the process of recovery from injury. The data presented here
represents the first report of its correlation with a specific process
important in phenotypic and morphological modulation of vascular SMC.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.