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J. Biol. Chem., Vol. 277, Issue 41, 38571-38578, October 11, 2002
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From the Robarts Research Institute (Vascular Biology Group),
London Health Sciences Center, Departments of Medicine (Cardiology),
Biochemistry, Medical Biophysics, University of Western Ontario,
London N6A 5K8, Canada
Received for publication, July 5, 2002
Hsp47 is a heat stress protein that interacts
with procollagen in the lumen of the endoplasmic reticulum, which is
vital for collagen elaboration and embryonic viability. The precise
actions of Hsp47 remain unclear, however. To evaluate the effects of
Hsp47 on collagen production we infected human vascular smooth muscle cells (SMCs) with a retrovirus containing Hsp47 cDNA. SMCs
overexpressing Hsp47 secreted type I procollagen faster
than SMCs transduced with empty vector, yielding a greater accumulation
of pro The production of type I procollagen is a multistep process that
requires the participation of several enzymes and chaperones to ensure
the faithful secretion of this trimeric protein. Within the endoplasmic
reticulum (ER),1 two
pro Hsp47 is a heat shock protein, present exclusively in the ER, that
plays a vital role in procollagen processing. Hsp47 is expressed only
by collagen-producing cells (8, 9) and in vitro binds to
collagens type I to V as well as gelatin (10). Within the ER, Hsp47 has
been found to interact with nascent type I procollagen chains (11, 12),
with fully translated pro The contexts in which Hsp47 expression have been identified in humans
are noteworthy. Increased levels of Hsp47 have been observed in
atherosclerotic plaque (18), keloid lesions (19), fibrotic lungs (20),
and diseased kidneys (21, 22). Furthermore, the fundamental importance
of Hsp47 in collagen biosynthesis is unequivocal: Hsp47-null mice die
before birth and the embryos display ruptured blood vessels and a
marked reduction in the amount of mature type I collagen (23). The
exact mechanism by which Hsp47 contributes to the production of
procollagen is unclear, however. Several possible roles have been
proposed including facilitating pro To examine the effects of Hsp47 on type I collagen production, we have
introduced the human Hsp47 gene into a
collagen-producing human cell. For this, we have used human
vascular smooth muscle cells (SMCs), which are critical to the
elaboration of collagen in the vessel wall (29, 30). This allowed us to
evaluate the response to elevated levels of Hsp47 in the context of a
fully functional pathway for procollagen biosynthesis. The results
indicate that Hsp47 has the capacity to drive collagen production by a mechanism that involves up-regulation of pro Cell Culture--
A human vascular SMC clonal line, designated
HITB5, was generated from the human internal thoracic artery, as
described previously (31). This line bears remarkable similarity to
SMCs in the vessel wall, including the expression of smooth muscle
Overexpression of Hsp47 in Human SMCs--
A retroviral gene
delivery system was utilized to generate human SMCs expressing Hsp47
under the control of the PCMV promoter. The full-length
cDNA encoding human Hsp47, together with 87 bp of the
5'-untranslated region and 707 bp of the 3'-untranslated region, was
excised with HindIII and SmaI from the plasmid
cln9-33 (kindly provided by Dr. K. Nagata, Kyoto University, Kyoto,
Japan) (32). This fragment was inserted into HindIII and
StuI sites of the retroviral expression vector, pLNCX2
(CLONTECH, Palo Alto, CA). Orientation and sequence
accuracy of the pLNCX2.Hsp47 construct was verified by cDNA
sequencing using a ABI 377-XL stretch DNA sequencer and ABI sequence
navigator software (PE Applied Biosystems, Foster City, CA).
Retrovirus containing pLNCX2.Hsp47 or pLNCX2 was generated by calcium
phosphate-mediated transfection of the Phoenix-amphotrophic retrovirus
packaging cell line (kindly provided by Dr. G. P. Nolan, Stanford
University Medical School, Stanford, CA, distributed by ATCC, Manassas,
VA) (33). The virus-containing supernatant was harvested 48-72 h later
and, after centrifugation and filtration (0.45-µm pore size), was
incubated with proliferating HITB5 SMCs for 48 h in the presence
of 20% fetal bovine serum and 5 µg/ml Sequa-breneTM (Sigma-Aldrich).
Infection efficiency was estimated at 40% based on infection of
parallel cultures with pLNCX2.EGFP. Cells stably expressing Hsp47 and
control cells infected with pLNCX2 alone were selected with 500 µg/ml
G418 for 2 weeks. Overexpression of Hsp47 was confirmed before every
experiment by Western blot analysis.
Western Blot Analysis of Hsp47 and Type I Collagen
Expression--
Analysis of type I collagen and Hsp47 expression was
determined by Western blot analysis, as described (29). Briefly, cells and conditioned media were harvested in the presence of 0.1 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin.
Equal amounts of protein were resolved on 6 and 12% polyacrylamide
gels for type I collagen and Hsp47, respectively. Type I collagen was
detected using a polyclonal rabbit antiserum to the C-telopeptide
region of the Analysis of Procollagen Synthesis and Secretion Rates by
Metabolic Labeling and Immunoprecipitation--
Duplicate cultures of
SMCs incubated for 1 h in methionine-free Dulbecco's Modified
Eagle's Medium (Invitrogen) were then pulsed for 1 h in
methionine-free Dulbecco's modified Eagle's medium supplemented with
100 µCi/ml [35S]methionine (ICN). Following three
washes with Dulbecco's phosphate-buffered saline, the cultures were
chased with Dulbecco's modified Eagle's medium containing 4 mM methionine (Invitrogen). At designated times, the medium
was harvested with 0.1 mM phenylmethylsulfonyl fluoride and
10 µg/ml leupeptin, and washed cells were solubilized in radioimmune
precipitation assay buffer (1% Igepal CA-630 (Sigma-Aldrich), 0.5%
sodium deoxycholate, 0.1% SDS in phosphate-buffered saline) containing
protease inhibitors. The media and cell lysates were clarified by
centrifugation and precleared with 5 µl of protein A-agarose (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min. Equal amounts of
protein were immunoprecipitated using LF67 (1:500) and 15 µl of
protein A-agarose overnight at 4 °C. Immunoprecipitates were washed
extensively with radioimmune precipitation assay buffer and resolved on
a 6% polyacrylamide gel. Quantification of radioactive bands in dried
gels was performed using a phosphorimager screen and Image-Quant
software (Amersham Biosciences).
Northern Blot Analysis--
HITB5 SMCs transduced with pLNCX2 or
pLNCX2.Hsp47 were harvested using TrizolTM reagent (Invitrogen). Total
RNA was separated on a 1.2% agarose-formaldehyde gel and transferred
to Zetaprobe GT membrane (Bio-Rad Laboratories). Membranes were
incubated overnight in hybridization solution containing 100 ng of
cDNA probe labeled by random-hexamer priming with
[ Immunohistochemistry--
Transduced SMCs were grown on glass
coverslips until confluent and fixed for 20 min in ice-cold methanol.
Cells were rehydrated for 10 min in phosphate-buffered saline, blocked
for 30 min in 10% normal goat serum, and incubated with LF67 (1:200)
for 1 h. After washing, bound primary antibody was detected with
an fluorescein isothiocyanate-labeled goat-antirabbit IgG (1:500) that
was applied for 1 h. Nuclei were stained with 2.5 µg/ml Hoechst
33258 in phosphate-buffered saline for 5 min. Following three 10-min
washes in phosphate-buffered saline, coverslips were mounted on glass
slides with PermaFlour (ImmunonTM, Pittsburgh, PA). Collagen
fibrils were visualized by confocal microscopy using a Zeiss LSM 410 microscope and a krypton/argon laser emitting at 488 nm. An argon ion
ultraviolet laser emitting at 351 nm for the detection of Hoechst 33258 was used to image the nuclei.
Identification of CBP2 Promoter Single Nucleotide Polymorphisms
in Humans--
Genomic DNA was prepared from leukocytes isolated from
peripheral blood samples of 26 control subjects of various ethnic
groups. Two sets of sequencing primers (P1,
5'-CCACTGTCGCCCAGATTATTTA-3' and 3'-CAGTGCCCTTCTCCATACTTGT-5'; P2,
5'-CAGGTACCGGGTCTGGTCT-3' and 3'-GTCTCCCGCCCCTCACCT-5') were designed
to cover a 1-kb region upstream from the CBP2 gene, as
reported by Ikegawa et al. (37). Amplification conditions
were as follows: 94 °C for 5 min, 30 cycles of 30 s incubations
at 94 °C, 58 °C and 72 °C, and a final 10 min extension step
at 72 °C. The expected fragment sizes were 624 and 640 bp for the P1
and P2 reaction, respectively. DyeNamic ET Terminator sequencing kit
was used according to the manufacturer's instructions and samples
loaded onto ABI 377-XL stretch DNA sequencer. ABI Sequence Navigator
software (PE Applied Biosystems) was used to align and compare DNA
fragments for sequence differences.
The CBP2 promoter [
SAS version 6.12 (SAS Institute, Cary, NC) was used for statistical
analyses. Allele frequencies were determined from electrophoretogram tracings of genomic DNA sequence, except for the [ In Vitro Analysis of Promoter Function--
The effect of the
[
Embryonic dermal fibroblasts were transfected with reporter constructs
using calcium phosphate precipitation. The fibroblasts were
serum-deprived for 48 h and incubated with or without
all-trans retinoic acid for 20 h. Luciferase activity
was measured according to the manufacturer's instructions (Promega
Corp.) on a lumat LB9507 luminometer (Berthold Technologies, Oak Ridge,
TN) and expressed relative to total protein content (BCA protein assay kit, Pierce).
SMCs Overexpressing Hsp47 Generate Increased Levels of
Intracellular and Extracellular pro
Infection of HITB5 SMCs with retrovirus containing Hsp47 cDNA
yielded SMCs that expressed, on average, 10-12-fold greater levels of
Hsp47 protein than SMCs infected with vector alone (Fig. 1). To determine the consequences of this
for type I procollagen abundance, cells and conditioned media were
immunoblotted using LF67, a polyclonal antibody that detects pro
It was also apparent that the effect of Hsp47 overexpression was
distinct from that of ascorbate. Incubation of vector-transduced SMCs
with 100 µM ascorbate resulted in a decline in the level of intracellular pro
Taken together therefore, overexpression of Hsp47 in SMCs has a
profound effect on the elaboration of type I procollagen, but this
effect cannot be categorized as one that acts solely by enhancing, or
delaying, procollagen transport.
Overexpression of Hsp47 in SMCs Increases the Rate of Procollagen
Secretion--
To clarify the basis of the observed effects of Hsp47
overexpression, type I procollagen secretion kinetics were assessed by
pulse-chase experiments. Vector-infected and Hsp47-overexpressing HITB5
SMCs were incubated for 4 days with or without 100 µM
ascorbate and labeled for 1 h with [35S]methionine,
and secretion of newly synthesized type I procollagen was tracked by
immunoprecipitation of procollagen and collagen in the culture media
using LF67, followed by phosphorimaging. As shown in Fig.
2, type I procollagen was secreted from
cells overexpressing Hsp47 at a substantially greater rate than from vector-transduced SMCs. As expected, the addition of ascorbate to
control vector-transduced SMCs also increased the procollagen secretion
rate from these SMCs (Fig. 2B). Addition of ascorbate to
HITB5-Hsp47 cells resulted in an even greater rate of secretion, i.e. greater than from HITB5-Hsp47 cells not incubated with
ascorbate and also greater than from HITB5-vector SMCs incubated with
ascorbate.
Overexpression of Hsp47 in SMCs Increases the Intracellular
Procollagen Transport Kinetics but Also Enlarges the Pool of Newly
Synthesized Procollagen Chains--
The intracellular transport
kinetics were also determined by pulse-chase analysis. As illustrated
in Fig. 3A, the clearance rate
of type I procollagen chains was increased in Hsp47-overexpressing cells. The profiles followed monoexponential decays, which when fitted
with first order rate constants yielded a t1/2 of 56 min for HITB5-vector SMCs, 28 min for HITB5-Hsp47 cells, and 35 min for
HITB5-vector SMCs incubated with ascorbate (Fig. 3B).
Addition of ascorbate to Hsp47-overexpressing SMCs further increased
the type I procollagen clearance rate, with kinetics best described by
two exponential components corresponding to t1/2
values of 11 and 106 min for the fast and slow components,
respectively. Addition of the lysosomal inhibitor, chloroquine (25 µM) or the proteosomal inhibitor, ALLN (100 µM), had minimal effect on the clearance rates of type I procollagen under basal circumstances or in SMCs overexpressing Hsp47
(data not shown), indicating that intracellular degradation is a
relatively minor pathway for procollagen in these cells. The increased
rate of intracellular disappearance of procollagen by Hsp47 was thus
primarily accounted for, and in keeping with, the augmented
secretion kinetics described above.
In addition to the effect on transport kinetics, inspection of the
radio-immunoblots revealed that immediately following the pulse, the
pool size of labeled pro Hsp47 Overexpression Increases pro Hsp47 Overexpression Augments Type I Collagen Fibril
Formation--
To determine if the augmentation of Hsp47 expression
influenced the formation of a type I collagen fibril matrix, confluent HITB5 SMCs were incubated for 9 days in the presence or absence of 100 µM ascorbate, added freshly every 3 days, and cultures were then immunostained using LF67. As shown in Fig.
5A, vector-infected SMCs in
ascorbate-deficient media produced collagen fibrils that accumulated in
a heterogeneous pattern, which included relatively thick and amorphous
aggregations. In SMCs overexpressing Hsp47, the amorphous appearance of
the thicker fibril bundles could still be appreciated. However, there
was a striking increase in the overall number of collagen fibrils
produced. Vector-transduced SMCs incubated with ascorbate also yielded
more fibrils than vector-transduced SMCs in ascorbate-deficient
conditions. However these fibrils were also finer and organized in a
more intricate network. These features likely reflect a fibril
structure arising from more complete hydroxylation, consistent with the
clearer delineation of the pro Human CBP2 Promoter Region Shows Genetic Polymorphism--
The
amplified production of type I collagen fibrils by increased Hsp47
represents a novel corollary to previous data showing that selectively
decreasing the level of Hsp47 leads to reduced production of type I
collagen (23). Although the mechanistic basis of these two responses
may differ, the data together point to the actions of Hsp47 as a
primary regulator of collagen production. This raises the question of
whether collagen production in the population might be impacted by
interindividual variation in Hsp47 production. To assess the
possibility that Hsp47 expression is variable in the population, we
screened for the presence of SNPs in the 1-kb upstream promoter region
of CBP2, the gene that encodes Hsp47 (37, 42). Genomic DNA
sequence analysis in 26 unrelated individuals demonstrated the presence
of 5 common SNPs (Fig. 6A). The allele frequencies in subjects from various ethnic groups are
summarized in Table I. All 5 SNPs were
within a region containing several DNA motifs, and the [ The [ Hsp47 has a number of attributes that distinguish it from other
ER-resident chaperones. These include its specificity for procollagens
as well as an atypical binding profile whereby it interacts with both
folded and unfolded conformations of its target protein (11, 13, 14,
16). The results of the current study indicate that Hsp47 can act as a
primary stimulator of type I collagen production. Selectively
increasing the amount of Hsp47 within SMCs reconfigured the collagen
production phenotype such that both intracellular and extracellular
steady-state levels of type I procollagen were increased. This profile
was associated with up-regulated pro The pool of intracellular procollagen that formed in SMCs within 1 h of adding labeled methionine was substantially increased in
Hsp47-overexpressing SMCs compared with control SMCs. Expansion of this
particular pool of procollagen could be due either to an increase in
the procollagen chain synthesis rate or a decrease in intracellular
degradation of nascent procollagen chains. The contribution of the
latter pathway is likely small, given that significant co-translational
degradation of pro Increased Hsp47 also influenced the kinetics of procollagen secretion,
with an increased intracellular clearance rate and an increased rate of
secretion out of the cell. Because these changes were in the context of
increased procollagen synthesis, it is difficult to determine if they
were a direct result of the procollagen-Hsp47 interaction in the ER or
a more integrated response of several ER proteins ensuring that
procollagen was efficiently transported out of the cell, in the face of
increased procollagen synthesis. Importantly however, the effect of
augmented expression of Hsp47 on transport kinetics did not depend on
ascorbate concentration. Under conditions of ascorbate depletion and
supplementation, procollagen secretion increased by a similar extent,
and the enhanced secretion induced by ascorbate was additive to that
induced by Hsp47 alone. In addition, unlike ascorbate, Hsp47
overexpression did not abrogate the production of procollagen forms
that are presumably imperfectly structured, suggested by the banding
pattern on Western blot analysis. Similarly, the morphology of the
assembled collagen fibrils in cultures of Hsp47-overexpressing cells
suggested that the collagen structure was not optimized, in contrast to
that in ascorbate-supplemented cultures. Although Hsp47 has been found
to bind poorly hydroxylated procollagen (13, 46), the current
observations suggest that Hsp47 does not have a corrective role in the
context of suboptimal procollagen hydroxylation.
Because SMCs overexpressing Hsp47 elaborated a greater extracellular
network of type I collagen fibrils, it is possible that the enhanced
procollagen expression and synthesis was a secondary consequence of the
modified external milieu. However, extracellular collagen fibrils have
been well established to down-regulate, not increase, expression of
pro The augmentation of collagen production by increased Hsp47 levels has
interesting implications. It is well established that an absence or
reduction of Hsp47 leads to reduced collagen production (12, 23). Taken
together with the current findings therefore, any perturbation that
selectively alters the amount of Hsp47 in the ER seems to reset the
amount of collagen that is produced. It is noteworthy therefore that
within the human population there are common nucleotide polymorphisms
in the promoter region of the CBP2 gene encoding Hsp47. The
CBP2 [ The current findings may also have therapeutic implications. There may
be circumstances in which augmented or accelerated production of
collagen would be beneficial. An important example of this is the
production of a structurally sound fibrous cap of on the surface of
atherosclerotic plaques. Caps with insufficient fibrillar collagen are
prone to rupture which can lead to thrombus formation and heart attacks
(30). Surprisingly, few defined stimuli are known to drive collagen
synthesis, and these are typically multifunctional proteins, such as
TGF- We thank G. Nolan for the Pheonix cell
line, K. Nagata for the full-length Hsp47 cDNA clone, B. Sanwal for
the anti-Hsp47 antibody, and L. Fisher for the anti-type I collagen antibody.
*
This work was supported by grants from the Canadian
Institutes of Health Research (MT11715) and Heart and Stroke Foundation of Canada (T4458).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.
§
Career Investigators of the Heart and Stroke Foundation of Ontario.
¶
To whom correspondence should be addressed: London Health
Sciences Center, 339 Windermere Rd., London, Ontario N6A 5A5. Tel.: 519-663-3973; Fax: 519-434-3278; E-mail: gpickering@robarts.ca.
Published, JBC Papers in Press, August 5, 2002, DOI 10.1074/jbc.M206689200
The abbreviations used are:
ER, endoplasmic
reticulum;
SMC, smooth muscle cell;
SNP, single nucleotide
polymorphism.
Functional Linkage between the Endoplasmic Reticulum Protein
Hsp47 and Procollagen Expression in Human Vascular Smooth Muscle
Cells*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1(I) collagen in the extracellular milieu. Interestingly, the
amount of intracellular pro1(I) collagen was also increased. This
was associated with an unexpected increase in the rate of pro1(I) collagen chain synthesis and 2.5-fold increase in pro1(I) collagen mRNA expression, without a change in fibronectin expression. This amplification of procollagen expression, synthesis, and secretion by
Hsp47 imparted SMCs with an enhanced capacity to elaborate a fibrillar
collagen network. The effects of Hsp47 were qualitatively distinct
from, and independent of, those of ascorbate and the combination of
both factors yielded an even more intricate fibril network. Given the
in vitro impact of altered Hsp47 expression on procollagen
production, we sought evidence for interindividual variability in Hsp47
expression and identified a common, single nucleotide polymorphism in
the Hsp47 gene promoter among African Americans that significantly
reduced promoter activity. Together, these findings indicate a
novel means by which type I collagen production is regulated by the
endoplasmic reticulum constituent, Hsp47, and suggest a potential basis
for inherent differences in collagen production within the population.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) collagen chains and one pro2(I) collagen chain first
associate at their globular carboxyl termini (1). This interaction is
facilitated by protein-disulfide isomerase and stabilized by interchain
disulfide bonds (2, 3). Winding of the long triple helical domain then
proceeds in the carboxyl to amino direction. The fidelity of this
helix-folding reaction is dependent on hydroxylation of proline
residues by prolyl-4-hydroxlase (4). If the activity of
prolyl-4-hydroxlase is impaired, as in ascorbate deficiency, the
improperly folded procollagen chains are retained within the ER and
secreted at a slower rate or targeted for degradation (5, 6). Once the
triple helix is formed, the protein is transported to the Golgi
apparatus and brought to the cell surface after a period of Golgi
cisternal maturation (7).
collagen chains (13), with non-helical and
poorly hydroxylated procollagen trimers (14) and with
well-hydroxylated, triple helical procollagen (15, 16). Once the
procollagen-Hsp47 complex reaches the cis-Golgi, Hsp47
dissociates and is recycled back to the ER (13, 17).
collagen chain elongation,
preventing improper association of unassembled and possibly
underhydroxylated pro collagen chains (11, 13, 14), winding of the
triple helix (24), maintaining thermal stability of the triple helix
once formed (16, 25), and diverting assembled procollagen into the
cisternal maturation transport pathway (15, 26). Although there is
experimental support for each of these potential roles, a cohesive
model has yet to emerge and not all of the data are consistent. For
example, it remains unclear whether the interaction between Hsp47 and
procollagen slows or accelerates procollagen transport. The former
might be expected if Hsp47 serves to maintain the folded conformation
during stress, whereas the latter would be more likely if Hsp47 plays a
direct role in winding of the nascent pro collagen chains (24). Evidence for both speeding (27) and slowing (28) of procollagen secretion by Hsp47 has been reported.
1(I) collagen gene expression and stimulation of procollagen chain synthesis, in concert
with enhanced procollagen secretion. We also provide evidence for
interindividual genomic variants associated with Hsp47, which, together
with the identified effects on procollagen production, implicate Hsp47
as a determinant of collagen elaboration in the population.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-actin, smooth muscle-myosin heavy chain isoforms SM1 and SM2,
calponin, heavy caldesmon, and metavinculin, as well as the capacity to
contract. HITB5 SMCs were maintained in M199 (Invitrogen) supplemented
with 10% fetal bovine serum. Mouse embryonic dermal fibroblasts were kindly provided by Dr. L. Dagnino (University of Western Ontario, London, ON) and were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum.
1(I) chain of human type I collagen (LF67, 1:8000
dilution, gift of Dr. L. W. Fisher, National Institute of Dental
Research, Bethesda, MD) (34, 35). Hsp47 was detected using a mouse
monoclonal antibody to rat Hsp47 (1:4000 dilution, gift of Dr. B. D.
Sanwal, University of Western Ontario, London, ON). Membranes were
incubated with antibodies overnight at 4 °C. An antirabbit
peroxidase-conjugated IgG or antimouse peroxidase-conjugated Fab
fragment was used to detect bound antibody by chemiluminescence
(Promega Corp., Madison, WI). Washed membranes were exposed to x-ray
film (Kodak XAR-5, Kodak, Toronto, ON).
-32P]dCTP. Hsp47 mRNA was detected using a
plasmid containing a cDNA clone for rat Hsp47 (pIP1) (36).
Pro1(I) collagen mRNA was detected using a human pro1(I)
collagen cDNA derived from Hf677 (ATCC). Fibronectin mRNA was
detected using mouse fibronectin cDNA (pC9912, gift of Dr. G. Kidder, University of Western Ontario, London, ON) and a cDNA probe
for human glyceraldehyde-3-phosphate dehydrogenase (pHcGAP,
ATCC) was used as a control for RNA loading. Blots were washed, and
mRNA bands were quantified after phosphorimaging.
656]C>T SNP was genotyped in
an additional 200 people by amplifying genomic DNA using the P1 primers
and the above amplification protocol. The 624-bp product was digested with endonuclease ApaL1, which yielded one fragment for the
[656]C>T SNP and two fragments with sizes of 446 and 178 bp for
the wild-type allele. The fragments were resolved in 2% agarose gels.
656]C>T SNP, which was assayed using restriction digestion. Chi-square analysis tested the deviation of genotype frequencies from Hardy-Weinberg predictions, with the nominal p < 0.05.
656]C>T SNP on promoter function was evaluated by transfecting
embryonic dermal fibroblasts with luciferase reporter constructs.
A genomic DNA fragment containing a 1 kb region upstream of the first
CBP2 exon with the [656]C>T SNP was generated using a two-primer
pair method. The mutagenesis primer pairs were:
5'-GGAGGAGTACACAGGAAGGAAAACCTG-3' and
3'-GCCAGGTTTTCCTTCCTGTGTACTCCT-5' (underlined nucleotide
indicates the mutated position). Sequencing primer sets P1 and P2 were
used to amplify the remaining regions. The DNA fragment containing the
mutation, a wild-type fragment, and an antisense fragment were
subcloned into the PGL3 vector (Promega Corp.) and appropriate
sequences verified by DNA sequence analysis.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I) Collagen--
To evaluate
the consequences of augmented Hsp47 expression, we introduced Hsp47
cDNA into a vascular SMC line, HITB5. This line was chosen for
study because of its human origin, the importance of collagen
production by vascular SMCs, and because HITB5 SMCs express copious
type I collagen (38). We thus could evaluate the effect of Hsp47
overexpression in the context of a functionally intact pathway for
collagen biosynthesis, as well as determine its effect when procollagen
hydroxylation is suboptimal, as with ascorbate depletion.
1(I)
collagen, partially processed pro1(I) collagen, and mature 1(I)
collagen. As shown in Fig. 1, cells transduced with vector alone and
cultured in the absence of ascorbate produced pro1(I) collagen that
was detectable within the cell and also in media that had been
conditioned for 48 h. Most of the collagen in the media was in the
form of procollagen or partially cleaved procollagen with much less
fully processed collagen, consistent with previous in vitro
studies (29, 39, 40). Compared with vector-transduced cells
(HITB5-vector), SMCs overexpressing Hsp47 (HITB5-Hsp47) contained a
substantially greater amount of intracellular pro1(I) collagen.
However, this elevated level of procollagen could not necessarily be
interpreted as a result of intracellular retention of target protein by
Hsp47, as observed after overexpression other ER-resident chaperones (41), because the amount of pro1(I) collagen that accumulated on the
outside of the cell was also substantially increased (Fig. 1,
bottom panel).
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Fig. 1.
SMCs overexpressing Hsp47 manifest increased
levels of intracellular and extracellular
pro 1(I) collagen. HITB5 SMCs were stably
infected with retrovirus containing empty vector
(HITB5-vector) or vector containing cDNA encoding Hsp47
(HITB5-Hsp47). Cell lysates and medium conditioned for 4 days were analyzed by Western blot analysis using a monoclonal antibody
to Hsp47 and a polyclonal antibody to type I collagen, LF67. The
consequences of Hsp47 overexpression on ascorbate-deficient and
ascorbate-supplemented SMC cultures were assessed by addition of 100 µM ascorbate 4 days prior to harvesting.
1(I) collagen and an increase in the level of
the extracellular procollagen. This profile is consistent with more
efficient secretion of procollagen out of the cell but contrasted with
the parallel increases in intra- and extracellular pro1(I) collagen
observed when Hsp47 was overexpressed. As well, the procollagen species
in ascorbate-supplemented cultures migrated through SDS-PAGE faster and
with clearer delineation of the procollagen cleavage intermediates
(Npro1(I) collagen, Cpro1(I) collagen), consistent with
structural optimization imparted by greater hydroxylation of
procollagen. The combination of Hsp47 overexpression and ascorbate treatment was additive, producing a substantially increased amount of
extracellular pro1(I) collagen, and a level of intracellular pro1(I) collagen below that of HITB5-Hsp47 SMCs in
ascorbate-deficient media but above that of HITB5-vector SMCs incubated
with ascorbate.
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Fig. 2.
Hsp47 overexpression stimulates type I
procollagen secretion by human SMCs. SMCs treated without
(A) or with (B) 100 µM ascorbate
for 4 days were pulse-labeled with [35S]methionine for
1 h and then washed and incubated with 4 mM unlabeled
methionine. Conditioned media was harvested at designated times during
the chase period and immunoprecipitated using a polyclonal antibody to
type I collagen, LF67. Precipitates were resolved on a 6%
polyacrylamide gel, and intensity of the bands for the pro 1 (1)
collagen chain and the pro2(I) collagen chain, with which it
immunoprecipitates, were quantified. Curves were fitted using least
squares regression analysis (r > 0.98 for all plots),
and data are the mean of duplicate culture wells. Two other experiments
gave similar results.
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Fig. 3.
Hsp47 overexpression stimulates intracellular
trafficking of type I procollagen and augments the pool of newly
produced procollagen chains. Control and Hsp47-overexpressing
HITB5 SMCs were incubated for 4 days with or without 100 µM ascorbate and then pulse-labeled with
[35S]methionine for 1 h. Samples were harvested at
designated times after washing and reintroducing media with 4 mM unlabeled methionine and immunoprecipitated using LF67.
A, phosphorimages of immunoprecipitated pro 1(I) collagen
and co-immunoprecipitated pro2(I) collagen resolved on a 6%
polyacrylamide. B, band intensities of labeled procollagen
chains quantified by phosphorimaging and normalized to the baseline
signal immediately following the pulse. Curves were fitted using least
squares regression analysis (r 
0.93 for all plots).
Inset, half-life of type I procollagen was calculated using
the equation for the linear regression line following natural logarithm
transformation. C, pool sizes of newly synthesized
pro1(I) collagen and co-precipitating pro2(I) collagen chains
immediately following the 1 h incubation with
[35S]methionine. Data are the mean of three separate
experiments, each of which was performed in duplicate. *,
p < 0.05 versus HITB5-vector SMCs in the
presence or absence of ascorbate.
1(I) collagen, and the pro2(I)collagen chain with which it co-immunoprecipitates, was greater in
Hsp47-overexpressing SMCs than in vector-transduced cultures. This
surprising observation was replicated three times using SMCs from
different virus-infected cultures. On average, the pool of newly
produced pro collagen chains was 2.6-fold greater in
Hsp47-overexpressing cells than control vector-transduced cells (Fig.
3C). This increase was observed in both ascorbate-deficient
and ascorbate-supplemented cultures, and it raised the possibility that
the production rate of individual pro collagen chains was increased
by Hsp47.
1(I) Collagen mRNA
Expression in HITB5 SMCs--
To further explore the basis for the
increased pool size of newly produced type I procollagen chains in
Hsp47-overexpressing SMCs, pro1(I) collagen transcript expression
was evaluated by Northern blot analysis. As shown in Fig.
4, there was a 2.5-fold increased in
pro1(I) collagen mRNA in Hsp47-overexpressing SMCs. This
response appeared to be selective for procollagen, as expression of
fibronectin mRNA was unchanged. Therefore, the enlarged pool of
newly synthesized type I procollagen appears to be due to increased procollagen expression and chain synthesis. Moreover, these data reconcile the apparent paradox of a chaperone elevating both
intracellular and extracellular levels of the target protein, because
they establish that in Hsp47-overexpressing cells procollagen
production is increased in addition to procollagen
secretion.
View larger version (36K):
[in a new window]
Fig. 4.
Hsp47 overexpression stimulates
pro 1(I) collagen gene expression by human
SMCs. Northern blots of total RNA from SMCs transduced with pLNCX2
or pLNCX2.Hsp47 were probed for Hsp47, pro1(I) collagen,
fibronectin, and glyceraldehyde-3-phosphate dehydrogenase. Procollagen
transcript abundance was increased in SMCs overexpressing Hsp47.
1(I) collagen forms on SDS-PAGE. As
shown in Fig. 5D, SMCs that overexpressed Hsp47 and were
incubated with ascorbate produced an even more extensive network than
observed with either manipulation alone, with a dense mat of
fibrils.
View larger version (116K):
[in a new window]
Fig. 5.
Overexpression of Hsp47 in human SMCs induces
the formation of a robust collagen fibril network. Control HITB5
SMCs and SMCs overexpressing Hsp47 were plated on glass coverslips and
incubated with or without 100 µM ascorbate for 9 days. Methanol-fixed SMCs were then immunostained with a polyclonal
antibody to type I collagen, LF67 and visualized using a fluorescein
isothiocyanate-labeled secondary antibody. Nuclei were visualized with
Hoechst 33258. Amorphous aggregations of fibrils (arrows)
were apparent in cultures of HITB5-vector SMCs that were incubated in
the absence of ascorbate (A). This morphology was also
present in ascorbate-deficient HITB5-Hsp47 SMC cultures (B);
however, collagen fibril abundance was substantially greater. Addition
of 100 µM ascorbate to HITB5-vector cultures yielded
finer fibrils (C), and the combination of ascorbate and
Hsp47 overexpression resulted in the elaboration of an even more
extensive network of fine fibrils (D).
656]C>T
SNP was especially noteworthy as it fell within a retinoic
acid-responsive element. Screening of an additional 200 subjects, 50 from each ethnic group, using ApaL1 digestion for genotyping
revealed that the [656]C>T allele was unique to the African
population. Genotype frequency did not deviate from Hardy-Weinberg
expectations. In a larger sample of African patients (n = 162), the frequency of the [656]T allele was 0.11.
View larger version (24K):
[in a new window]
Fig. 6.
Genomic DNA analysis of the human
CBP2 promoter. A, schematic indicating
the presence of 5 SNPs identified in the 1-kb upstream CBP2
promoter in 26 individuals. B, activity of the
CBP2 promoter region containing the [ 656] C>T SNP.
Embryonic dermal fibroblasts were transfected with reporter constructs
containing luciferase cDNA under the control of wild-type
CBP2 promoter, promoter in antisense orientation, and
promoter containing the [656]C>T SNP. Following serum starvation,
fibroblasts were treated for 20 h with 10 nM
all-trans retinoic acid. Luciferase activity was
standardized to protein content. *, p < 0.01 versus [656]C>T SNP; 
, p < 0.02 versus wild-type construct in the absence of retinoic acid
and the [656]C>T SNP construct in the presence of retinoic
acid.
CBP2 promoter SNP allele frequencies
656]C>T SNP Is Functionally Different from the Wild-type
CBP2 Promoter--
The [656]C>T SNP was further studied to
determine its effect on promoter activity. A reporter plasmid was
constructed with the 1-kb promoter region of CBP2 containing
the [656]C>T SNP placed upstream of cDNA encoding luciferase.
Constructs containing wild-type and antisense promoter regions were
similarly constructed. As shown in Fig. 6B, luciferase
activity in embryonic dermal fibroblasts transfected with the
[656]C>T SNP-containing construct was 0.23 that of cells
transfected with the wild-type promoter (p < 0.01). Stimulation of cells transfected with the wild-type promoter construct with all-trans retinoic acid significantly increased
luciferase activity. All-trans retinoic acid also increased
promoter activity of the [656]C>T SNP construct, suggesting a role
for additional promoter sites in retinoic acid responsiveness. However,
total luciferase activity remained strikingly suppressed, suggesting that this SNP has global consequences for CBP2 promoter activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(I)collagen gene expression, an
increased rate of type I procollagen chain synthesis, and faster
procollagen secretion. The net effect was the elaboration of an
extensive network of type I collagen fibrils. These findings point to a novel pathway for controlling type I collagen production and suggest a
linkage between an ER-resident protein and the machinery for procollagen expression.
collagen chains has not been identified, and the
proportion of the entire intracellular procollagen pool that is
degraded within the cell is reported to be only 10- 20% (43-45).
Furthermore, addition of lysosomal and proteosome inhibitors had little
effect on the procollagen transport kinetics in SMCs. In addition, the
2.6-fold increase in the pool of newly produced procollagen was
associated with a 2.5-fold increase in pro1(I) collagen transcript
abundance. Taken together therefore, the expanded pool of newly
synthesized procollagen in Hsp47-overexpressing SMCs reflected an
increase in the expression and production rate of procollagen chains.
1(I) collagen (47-49) and this, therefore, is an unlikely
explanation. There is, however, precedent for linking events within the
ER to activation of genome. The unfolded protein response, for example,
entails the stimulated expression of ER-resident proteins consequent to
the release of the chaperone BiP from its ER receptor. This is followed
by receptor clustering and autophosphorylation and activation of JNKs
(50). Recently, overexpression of calreticulin in myoblasts was
associated with up-regulation of protein phosphatase 2A, further
highlighting the potential for ER events to be transduced outside that
compartment (51).
656]C>T SNP is a mutation that was private to
African Americans and resulted in a substantial reduction in
CBP2 promoter activity. The functional consequences of
depressed CBP2 promoter function in this population warrant further study, but the finding suggests a genomic basis for
interindividual differences in collagen production within the
population and may, in part, underlie individual susceptibility to
conditions characterized by aberrant healing. Phenotypes to be studied
for possible association with their functional differences in the
CBP2 promoter would include cutaneous wound healing, keloid
formation, and vascular phenotypes.
, which influence multiple cell types diversely. The increase in
collagen production by Hsp47 observed in these studies thus raises a
potential strategy for accelerating collagen production by vascular
cells, possibly in a specific manner.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a Heart and Stroke Foundation Studentship Award.
ABBREVIATIONS
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