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Transcriptional activation of the ╨Ю┬▒11 procollagen gene in systemic sclerosis dermal fibroblasts. Role of intronic sequences

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ARTIIRITIS & RHEUMATISM
Vol. 39, No. 8, August 1996, pp 1847-1354
Q 1996, American College of Rhcumatology
1347
TRANSCRIPTIONAL ACTIVATION OF
THE al(1) PROCOLLAGEN GENE IN
SYSTEMIC SCLEROSIS DERMAL FIBROBLASTS
Role of Intronic Sequences
ELENA G. HITRAYA and SERGIO A. JIMENEZ
Objective. To investigate the transcriptional regulation of the d ( 1 ) procollagen gene (COLlAl) in
cultured dermal fibroblasts from patients with diffuse
systemic sclerosis (SSc) of recent onset and to evaluate
the role that intronic sequences may play in the upregulated expression of COLlAl in SSc dermal fibroblasts.
Methods. Dermal fibroblasts from 6 patients with
diffuse SSc of recent onset and from 3 healthy individuals were studied. The steady-state levels of cwl(1)
procollagen messenger RNA were evaluated by Northern hybridization analysis, and the transcriptional regulation of COLlAl was examined by transient transfection experiments with deletion constructs containing
portions of COLlAl promoter (with 5’ end points at
-5.3 WO, -23 kb, and -804 bp and 3‘ end point at +42
bp) ligated to the chloramphenicol acetyltransferase
(CAT) reporter gene. To examine the role of intronic
sequences, constructs containing, in addition to the
COLlAl promoter, a portion of the first intron (+380
bp to +1,440 bp) cloned in front of the CAT gene were
transfected. The efficiency of transfections was normalized relative to the net amount of CAT plasmid actually
transfected into recipient cells, determined by a modified Southern hybridization procedure.
Results. Maximal CAT activity was observed with
constructs extending from -804 bp to +42 bp in both
Supported by NIH grant AM-19616. Dr. Hitrayd’s work was
supported by a fcllowship from the Arthritis Foundation.
Elena G. Hitrayd, MD, PhD, Scrgio A. Jimtnez, MD: Jefferson Medical College, Thomas Jcffcrson University, Philadelphia,
Pennsylvania.
Address reprint requests to Sergio A. Jimenez, MD, Thomas
Jefferson Univcrsity, Room 509 Bluemle Life Sciences Building, 233
South 10th Street. Philadclphia, PA 19107-5541.
Submittcd for publication September 1, 1995; acccptcd in
revised form March 13, 1996.
normal and SSc fibroblasts. However, the activity driven
by this construct was 80-110% higher in SSc fibroblasts. The CAT activity driven by a construct with a 5’
end point at -5.3 kb was only 1520% higher in SSc
cells, and the CAT activity driven by a construct with a
5’ end point -2.3 kb was 3 5 4 5 % higher in SSc
fibroblasts. The CAT activity driven by the -804-bp
promoter construct was increased up to 4-fold in SSc
fibroblasts in comparison with normal cells when the
intronic segment spanning +380 bp to +1,440 bp was
included in the transfected construct.
Conclusion. The results directly demonstrate the
transcriptional activation of COLlAl in dermal fibroblasts from SSc patients. The data also indicate that
first-intron sequences of COLlAl are required for maximal transcriptional activity of the collagen gene and
may play an important role in the up-regulation of its
expression in SSc fibroblasts.
Systemic sclerosis (SSc; scleroderma) is a connective tissue disease of unknown etiology characterized by
deposition of excessive amounts of collagen and other
connective tissue macromolecules in the skin and various internal organs (1,2). It was previously demonstrated
that fibroblasts from affected SSc skin cultured in vitro
maintain their in vivo phenotype for several passages
and produce excessive amounts of extracellular matrix
proteins (3,4). The exaggerated extracellular matrix production by SSc fibroblasts is the result of increased gene
cxpression, as demonstrated by higher transcription
rates of types I and 111 collagen and fibronectin genes
and elevated steady-state levels of their corresponding
transcripts (5-8). Furthermore, a chimeric construct
consisting of thc human a2(I) procollagen promoter
linked to the chloramphenicol acetyltransferase ( C A T )
1348
HITRAYA AND JIMENEZ
Table 1. Clinical and dcmographic fcatures of the patients with diffuse systemic sclcrosis (SSc), at the time of presentation*
Patient
Agelracclscx
1
2
3
4
62iwrF
5
35iwlE
46iwr~
36IBF
56iwlF
6
22iwF
Duration
of ssc,
months?
10
11
8
12
6
8
Visccral involvement$
Raynaud’s
phcnomcnon
-
+
+
+
+
+
GI
Pulmonary
Cardiac
Renal
-
-
+
-
-
-
-
-t
-
+
+
-
+
-
-
* Criteria for definition of diffusc SSc werc those described by LeRoy et al (44).
1- From diagnosis of SSc by a rheumatologist.
$ Defined according to Lally et al (45). GI = gastrointestinal.
gene was shown to cxhibit 3-5-fold higher transcriptional activity when transfcctcd into SSc fibroblasts
comparcd with normal fibroblasts, thus providing further
evidence of incrcased transcription of type I collagen
genes in SSc fibroblasts (9).
The exact mcchanisms responsible for the transcriptional activation of collagen gcnc cxprcssion under
normal or pathologic conditions are not known. Various
rcgions of the gene are likely to be involved, including
TATA and CCAAT sequcnccs, or othcr spccialized
regulatory elements (10-15). These sequences are principal targets for thc action of promoter-specific transcription factors which may affect the initiation of transcription of their target genes or control the formation of
thc transcription complex itself (16-21). It has also been
shown that the activity of some procollagcn gcnc promoters is modulated by enhancer elements which may be
located cithcr upstream or downstream of the transcription start site. For instance, somc sequcnces locatcd in
the first intron of the a l ( I ) procollagen gene (COI.lA1)
were shown to greatly cnhancc thc transcriptional activity of COLlAl promoter in transiently transfected normal fibroblasts (22-27).
In the present study, we investigated the transcriptional regulation of COLl A1 in dermal fibroblasts
from SSc patients. The results demonstrated that the
transcription of the COLlAl promoter is increased in
SSc dcrmal fibroblasts in comparison with normal cells.
The results further showed that the presence of intronic
sequences in a chimcric gene construct containing -804
bp of the COLlAl promoter markedly enhanced its
transcriptional activity in SSc fibroblasts.
PA’I’IENTS AND METHODS
Patients and control subjects. Dcrmal fibroblasts from
6 patients with diffuse SSc of rcccnt onsct and from 3 agc- and
scx-matchcd hcalthy individuals were utilized for these studies.
All SSc patients fulfilled the American College of Rheumatology (formerly, the American Rhcumatism Association) criteria
for thc classification of SSc (28). Relevant demographic and
clinical characteristics of the patients are presented in Table 1.
Cell cultures. Dermal fibroblast cultures were established from site-matchcd skin biopsy samples obtained from
the dorsal forearm of hcalthy adults and SSc patients, using
standard tissue culturc tcchniques as described previously (29).
Cells were grown in Eaglc’s minimal essential medium supplcmented with 10% fetal bovine serum (Gibco, Grand Island,
NY), 1% vitamins, and 2 mM L-glutamine. The cells were
maintained at 37°C in a moist atmosphere of 5% C02/95% air,
and the medium was changed twice wcckly. For all studies,
only carly-passage fibroblasts (passages 3-5) wcre utilized, to
avoid the possibility that the cells may undergo changes in their
original phenotype during extended subculture.
Extraction of RNA and Northern blot analysis. Fibroblasts wcre grown to confluence in 75-cm2 flasks. Total RNA
was isolated by acid guanidinium isothiocyanate-phenolchloroform extraction (30). The amount of RNA was quantitated by spcctrophotometric absorbance at 260/280 nm. Samples containing equal amounts of RNA (10 p,g) were subjected
to Northern hybridization analysis utilizing 0.8% agarosc gels
under denaturing conditions. The quality of the RNA was
evaluated by staining with cthidium bromide to visualize the
18s and 28s ribosomal RNA subunits under ultraviolet (UV)
light. The RNA was then transferred in a vacuum blotting
system (Vacugcnc 2016; LKB, Bromma, Sweden) to nitrocellulose filters (Nitroplus; MSI, Westboro, MA). RNA bound to
nitrocellulose was UV crosslinked (UV Stratalinker 2400;
Stratagcnc, La Jolla, CA). Equal transfer of RNA was assessed
visually by UV illumination of ribosomal RNA bands after
transfcr to the filters.
Hybridizations were performed with complementary
DNAs (cDNAs) radiolabeled with a3’P-d(3TP by nick translation to a specific activity of >los counts per minute/pg DNA
(31). Prehybridizations and hybridizations were performed in a
solution containing 50% formamide, 5 X saline-sodium
phosphate-EDTA, 5 X Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), and 250 pg/ml denatured salmon sperm
DNA. Hybridizations were performed at 42°C for 18-24 hours.
The filters were washed at 68°C with a final stringcncy of 0.1X
saline-sodium citrate (SSC)/O.l% SDS, and were exposed to
x-ray film (Eastman Kodak, Rochcstcr, NY) in cassettes with
COLlAl TRANSCRIPTIONAL ACTIVATION IN SSc FIBROBLASTS
-5.3 kb
-2.3 kb
-804bP
CAT
5.3 kbal CAT
CAT
2.3 kbal CAT
qFE
804 bpal CAT
+380
-804 bp
CAT
+1440
804 bpal CAT + intron
Figure 1. Schematic rcprcsentation of the COLlAI chloramphenicol
acetyltransferase (CAT) constructs utilized for transient transfection
experiments. 5.3 kbalCAT = a contruet containing -5.3-kb to i 42-bp
sequences from COLlA1 ligated to the CAT coding fragment in
Bluescript KS + (BS) vector (34); 2.3 kba1CAT z: a construct
containing -2.3-kb to +42-bp sequences from COI.1Al ligated to the
CAT coding fragment in BS vcctor (34); 804 bpalCAT = a construct
containing -804-bp to +42-bp sequcnccs from COLlAl ligated to the
CAT coding fragment in either BS vector or pBR322 (34); 804
bpalCAT + intron = a construct containing, in addition to -804 bp
to +42-bp sequences from the promoter of COLlA1, the fragment
spanning +383 to +1,440 bp of intronic sequences cloned 3' of the
CAT gene in pBR322 vector.
intensifying screens (Kodak) at -70°C for 18-24 hours. The
autoradiograms were scanned by densitometry at 633 nm with
a laser scanner (LKB). The human cDNAs utilized for RNA
hybridizations were Hf677, a 1.8-kb proal(1) procollagcn
cDNA corresponding to the COOH-terminal propeptidc and
the carboxy-terminal portion of thc triple-helical region of
human proal(1) procollagen chain of type I procollagen (32),
and pA6, a cDNA specific for human cytoplasmic 7s RNA (33).
Transient transfection and CAT assays. For transient
transfection experiments, the COLl A1 promoter constructs
5.3-kb a1 CAT, 2.3-kb a1 CAT, 804-bp a1 CAT, and the
804-bp a1 CAT + intron construct containing in addition the
first intron region encompassing +380 bp to +1,440 bp werc
used (Figure 1). The preparation of the constructs without
intronic sequences has been described in our previous publication (34). For the constructs containing +380-bp to
+1,440-bp intronic sequences, a 1-kb Snza I from the first
intron, shown to have enhancer activity (23,26), was inserted
utilizing Barn HI linkcrs into the Barn HI site 3' of the CAT
gene to give 804-bp a1 CAT + intron. We chose to clone the
intronic sequences 3' of the CAT gene in order to mimic the
conformation of some other cellular enhancers in which thc
presence of nucleotidc strctchcs of variable length is necessary
to allow the folding of the intron enhancer toward the promoter and the interaction of the multimetric trans-acting
factors recognizing the regulatory sequences in the promoter
and in the first intron. As a control, we utilized pBL CAT 2, a
construct containing the herpes simplex virus (HSV) thymidinc
kinase promoter ligated to the CAT gene and pSV2AP, a
construct containing the simian virus 40 (SV40) promoter and
enhancer ligated to a rat alkaline phosphatasc cDNA (provided by Dr. K. Yoon, Philadelphia, PA).
Normal and SSc fibroblasts were plated and grown to
1349
70% confluence in 60-mm dishes. The media were changed the
next day, and 3 hours later the cells were transfected with 20 pg
of each plasmid, utilizing thc calcium phosphate/DNA coprecipitation method followed 4 hours later by 10% glycerol shock
for 1 minute, as described previously (35). Fresh medium was
then added and the cells harvcstcd 48 hours after transfection
and fractured by sonication. Total protein of the cytoplasmic
extracts was determined with a protein assay kit (Bio-Rad,
Hercules, CA). Identical amounts of protein from each cell
cxtract (20 pg/assay) were used for parallel determination of
CAT activity using ''C-chloramphcnicol as substrate (36).
Acetylated and nonacetylated forms of radiolabeled chloramphenicol were separated by thin-layer chromatography and
visualized by autoradiography. The percentage of I4Cchloramphcnicol converted to the acetylated forms was determined by cutting out appropriate areas from the thin-layer
chromatography sheets and assaying for ''C-radioactivity by
scintillation counting. The level of CAT activity was normalized to the amount of CAT plasmid DNA transfected into
recipient cells measured by dot-blot hybridization as described
below.
Dot-blot hybridization analysis of transfected CAT
plasmids. To standardize for the efficiency of transfections, we
did not perform cotransfection with a control plasmid such as
that encoding alkaline phosphatase or human growth hormone, because it was recently reported that expression of the
control plasmid can be modulated by warn-acting enhancer or
silencer DNA elements (37), thus failing to provide an accurate control. The amount of CAT plasmid DNA transfected
into recipient cells was determined by dot-blot hybridization as
described by Abken and Rcifenrath (38). Briefly, 15 pI of the
lysate was incubated with RNase A (100 pg/ml) and subsequently with proteinase K (100 pglml), each for 30 minutes at
37°C. Two volumes of 20X SSC was added, and DNA was
dotted onto a membrane in a serics of dilutions (1 pl, 3 pl, 10
p1, 30 p1 of diluted cell cxtract), and hybridized to 32P-labclcd
pCAT DNA as probe. Thc intcnsitics of the hybridization
signals indicated the amount of transfected plasmid DNA
present in the crude cell extracts.
RESULTS
To assure that a hornogcnous group of dysregulated SSc cells which are clearly overproducers of collagen was studied, the steady-state levels of COLlAl
transcripts were examined in normal and SSc fibroblasts
by Northern hybridization analysis. Semiquantitative
assessment of the results demonstrated that the SSc cell
lines displayed >Zfold higher levels of COLlAl messenger RNA in comparison with normal fibroblasts
matched for donor age, sex, and site of biopsy (Figure 2).
The levels of 7s cytoplasmic RNA werc also simultaneously assessed in normal and SSc fibroblasts to control
for any variability introduced during loading and transfer of nucleic acids.
To study the transcriptional regulation of
COL1A l , deletion constructs containing portions of
HITRAYA AND JIMENEZ
1350
NI N2 N3 S1 S2 S3 S4 S5 S6
A
T
YW
2
-1
a
z
U
E
g
w
w
a:
N1 N2 N3
Figure 2. A, Northern hybridization analysis of total RNA from normal (N) and systemic sclerosis ( S ) dermal fibroblasts. The blots
were hybridized with specific complcmcntary DNAs for d ( I ) procollagen and 7s RNA. B, Densitometric analysis of Northern
blots after correction for 7s KNA in the same samples. Values are thc mcan and SD of the results from 2 independent cxpcrimcnts.
mRNA = rncsscnger KNA.
COLlAl promoter were transiently transfected into
normal and SSc fibroblasts in order to determine
whether the transcriptional activity of COLlAl is increased in SSc fibroblasts and, if so, to identify the
region of the promoter that may be responsible for this
alteration. The results showed that among the constructs
tested, maximal CAT activity was observed with the
A
1
2
3
1
2
construct extending from -804 bp to +42 bp in both
normal and SSc fibroblasts (Figure 3A). However, the
activity driven by this construct was 80-1 10% higher in
SSc fibroblasts than in normal fibroblasts (Figure 3B).
The activity driven by a construct containing further
upstream regions of the promoter with 5' end points at
-5.3 kb and -2.3 kb was substantially lower than that
3
10
A@
c
8
2
6
NORMAL
SCLERODERMA
E
G
0
w
4
C
(02, l l l l l l l l l l L l d l J
-r
L I ' Z I I J l C C L C U
W1111J.J-n"
UI L L X l
(lll'\rJ
A,,
L.J-n"
U I
.1-1
\L"'CJ
A,, LlllU
uu-vvy
U I L n I
(I111c.J 2, C I I I I J L I U L L J .
lllC
aLcryrdrru
(nL,
dllU
nonacetylated (C) forms of "C-chloramphcnicol separated by thin layer chromatography are shown. B, Quantitative representation of CAT activity.
Pcrccntagc of acetylation was determined from quantitation of thc acetylated and nonacctylatcd forms of 14C-chloramphcnicolcorrcctcd for thc
amount of CAT plasmid DNA introduced into cells, determined by DNA dot-blot hybridization. Values are the mean and SD of the results from
3 independent experiments with 3 normal and 6 SSc cell lines. See Figure 1 for other definitions.
COLlAl TRANSCRIPTIONAL ACTIVATION IN SSc FIBROBLASTS
A
H
1
2
1
2
Ac
1 2 1 2
8
16
1351
NORMAL
SCLERODERMA
T
2
bU
Ac
6-
s
0
5
w
C
C
4-
T
w
2-
T
N l
N2
uu
s3 ss
0
1
804 bpal CAT
804 bpal CAT + intron
Figure 4. A, Representative autoradiograrn of CAT assay from transient transfection experiments with normal (N1 and N2) and systemic
sclerosis (SSc) ( S 3 and S5) fibroblasts transfected with 804-bp a1 CAT (lines 1) and 804-bp a1 CAT t intron (lines 2) constructs. The
acetylated (Ac) and nonacetylated (C) forms of ''C-chloramphcnicol separated by thin layer chromatography are shown. B, Quantitative
representation of CAT activity. Percentage of acetylation was determined from quantitation of the acetylated and nonacctylatcd forms of
'"C-chloramphenicol corrected for the amount of CAT plasmid DNA introduced into the cells, determined by DNA dot-blot hybridization
(shown in Figure 5). Values are the mean and SD of the results from 3 independent experiments with 3 normal and 6 SSc cell lines. See Figure
1 for other definitions.
driven by the -804-bp construct. Furthermorc, the CAT
activity was only modestly higher in SSc fibroblasts than
in normal cells (15-20% higher for 5.3-kb a1 CAT and
35-4596 higher for 2.3-kb a1 CAT) (Figure 3B). To
ensure that the obscrved effects were not due simply to
a generalized metabolic activation of SSc fibroblasts in
comparison with normal fibroblasts, we set out to show
that increased transcription was specifically driven by the
collagen promoter. For this purpose, control plasmids
containing SV40 and HSV thymidinc kinase strong
constitutive promoters were transiently transfected into
normal and SSc fibroblasts. The results did not revcal
any differences in the transcriptional activity driven by
these 2 control plasmids in normal and SSc fibroblasts
(data not shown).
In ordcr to examine the role that intronic sequences may play in the up-regulated expression of
COLlAl, a construct containing a portion of the first
intron (+380 bp to +1,440 bp) in addition to the
-804-bp promoter sequences (804-bp a1 CAT + intron) was transiently transfected into normal and SSc
fibroblasts. Thc rcsults demonstrated marked increases
in CAT activity driven by the 804-bp a1CAT + intron
construct in comparison with the construct containing
only sequences from the promoter, in both normal and
SSc fibroblasts (Figure 4A). Inclusion of the intronic
sequences increased the activity of the -804-bp promoter by 40-50% in normal fibroblasts; however, in SSc
fibroblasts, the activity driven by the 804-bp a1 CAT +
intron construct was 280-360% higher than the activity
driven by constructs without intronic sequenccs. The
increases in the transcriptional activity of the -804-bp
promoter induced by thc inclusion of intronic sequences
were statistically significant for both normal ( P < 0.02)
and SSc fibroblasts ( P < 0.001) (Figure 4B). To correct
for the efficiency of the transfection in cach experiment,
the amount of plasmid DNA actually transfccted into
the cells was assessed by dot-blot analysis (Figure 5).
Thus, the results shown in Figures 3B and 4B represent
1 2 1 2
1 2 1 2
uu
N1 N 2
uu
s3 s5
Figure 5. DNA dot-blot analysis of transfected 804-bp a1 CAT (lines
1) and 804-bp a1 CAT t intron (lines 2) CAT plasmid DNA into
normal (N1 and N2) and systemic sclerosis (S3 and S5) fibroblasts from
the transient transfection experiment shown in Figurc 4. DNA was
dotted in a series of dilutions onto a nitrocellulose mcmbranc and
hybridized to 3'P-labeled pCAT DNA. See Figure 1 for definitions.
1352
HITRAYA AND JIMENEZ
the relative CAT activity corrected for the efficiency of
plasmid DNA transfection into the cells.
DISCUSSION
Several studies have examined the mechanisms
that regulate the expression of various collagen genes
under normal conditions, during development and differentiation, and under the influence of cytokines and
growth factors (for review, sec rcfs. 10-12). Most studies
have focused on the regulation of the genes encoding the
2 polypeptide chains of type I collagen, which is the most
abundant collagen in the body and the protein that
seems to play the most important role in the fibrotic
process of SSc. Numerous structural regulatory elements
have been identified and mapped in these genes (13-15).
The 5' flanking region of the human COLlAl contains
a TATA box at -23 bp and a CCAAT motif at - 125 bp
(14). Additional upstream sequences include a
pyrimidine-rich sequence (- 136 bp to - 167 bp), a viral
core enhancer motif (-277 bp to -285 bp), and an
adenovirus E1A enhancer-like clement (-307 bp to
-316 bp) (10-12).
Studies by Rossouw (23) using transient transfections in Xenopus laevis oocytes showed that cxtcnsion of
a COLlAl construct spanning -253 bp to +117 bp to 5'
end points at -950 bp or -2,400 bp increased the
transcription of the reporter gene 2-fold and 12-fold,
respectively, suggesting the presence of positive regulatory elements upstream of -253 bp. In a study by Roast
et a1 (39), transfcctions of fetal fibroblasts with constructs of the human COLlAl promoter containing
-2,500 bp to +123 bp showed that sequenccs from
-2,500 bp to -331 bp had inhibitory transcriptional
effects. Our studics in NIH-3T3 cells transfected with
plasmids containing various dclctions of a -5.3-kb promotcr fragment (34) showed that maximal transcriptional activity resided within the proximal promoter
region encompassing -804 bp, although they also suggested the presence of negative elements between -5.3
kb and -804 bp. It is possible that the different cells
utilized in these studies may account for the contradictory results.
Similar to other cukaryotic gencs, COLlAl contains rcgulatory clements within the first intron, and it
has been suggested that interactions between upstream
promoter sequences and intronic regulatory elements
may be essential for the transcriptional regulation of its
expression (22-27). Sequence analysis of the first intron
revealed the prcscncc of 2 high-affinity Spl sitcs (+927
and +1,207), 2 medium-afinity Spl sites in reverse
orientation (+731 and +1,241), a viral core cnhancer
motif (+ l,OOO), a pyrimidine-rich region (+ 1,258 to
+1,296), and 4 GC boxes (24).
The modulatory regions located in the first intron
o f COLlAl have been studied cxtensively (22-27,40),
but the results have not been conclusive and have often
been contradictory. However, the findings of Bornstein
ct a1 (22,24) strongly suggestcd that transcription is
regulated by the interplay of positivc and negative
intronic elements, and showed the occurrencc of complex interactions of nuclear factors with promoter and
intronic elements (25,41,42). Indeed, an intronic APl
binding site has bccn shown to restore the enhancement
provided by the full intron in an intronless construct
(40). Although most studies demonstrated that strong
positive elements are present in the first intron and are
rcquired for optimal transcription of COLl A1 , studies
by Olsen et a1 in mouse NII-I-3T3 cells stably transfected
with a human COLlAl minigcne driven by a -2.3-kb
promoter showed no differences in the cxpression of the
gene with deletions of intronic sequences either from
+380 to +1,440 bp or from +380 to +1,610 bp (43). The
contradictory results reported in the literature may
reflect intrinsic differences in the stage of differentiation
or tissue of origin of the cclls utilized for transfections.
Alternatively, these discrepancies may be related to
more fundamental species-specific responses.
Despite the recent advanccs in the understanding
of the regulation of collagen gene expression under
normal conditions, very little has been learned regarding
the intimate mechanisms responsible for the pathologic
increase in the expression of collagen genes in SSc. It
was previously demonstrated that fibroblasts from affected SSc skin exhibit exaggerated extracellular matrix
production which is the result of increased cxpression of
the corresponding genes (6,7). Increased transcriptional
activity of the a2(I) procollagen gene (COLlA2) in SSc
fibroblasts was documented by transient transfections of
constructs containing the human COLlA2 gene promoter ligated to the CAT reporter gene into normal and
SSc fibroblasts (9). The results showed that the constructs containing thc normal gene promoter displayed
3-5-fold higher transcriptional activity when transfected
into SSc fibroblasts compared with normal fibroblasts,
thus providing further cvidence of an intrinsic upregulation of the transcription of type I collagen genes in
SSc fibroblasts.
The goal of our study was to examine the transcriptional regulation of COLl A1 gene expression in
SSc fibroblasts and to evaluate the role that intronic
sequences may play in the up-regulated cxprcssion of
COLl A1 TRANSCRIPTIONAL ACTIVATION IN SSc FIBROBLASTS
this gene in dermal fibroblasts from SSc patients. Our
results demonstrated that a construct containing -804
bp of the COLlAl promoter showcd maximal transcriptional activity and that the activity driven by this promoter region was -2-fold highcr when transfected into
SSc fibroblasts than when transfectcd into matched
normal cclls. Furthermore, we found that inclusion of
sequences from the first intron resulted in much higher
transcriptional activity in both normal and SSc cells, and
augmcnted by up to 4-fold the differences in transcriptional activity between normal and SSc cclls. Thus, the
results directly demonstrated transcriptional activation
of COLlAl in dermal fibroblasts from SSc patients.
These data also indicate that scquences located in the
first intron of COLlAl may be requircd for maximal
transcriptional activity of the collagen gene and may play
an important role in the up-regulation of its expression
in SSc. Precise identification of promoter and intronic
elements involved in the abnormal transcriptional regulation of COLlAl in SSc fibroblasts will be necessary to
further understand the complex mcchanisms responsible
for the up-regulated collagen gene cxpression in SSc.
ACKNOWLEDGMENT
The assistancc of C. Smith in the preparation of the
manuscript is gratefully acknowledged.
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2.
3.
4.
5.
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sequence, procollagen, systemic, activation, role, transcription, genes, sclerosis, dermal, intronic, fibroblasts
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