From the
To assess the mechanisms controlling the developmental
regulation of tropoelastin expression in vivo, we developed a
reverse-transcription-polymerase chain reaction (RT-PCR) assay to
detect tropoelastin pre-mRNA as an indicator of ongoing transcription
in intact tissue. RNA was isolated from mid-fetal (early-elastogenic),
neonatal (peak tropoelastin expression), and adult (very low
tropoelastin expression) rat lungs and reverse transcribed, and the
cDNA was amplified with intron specific primers. A weak hybridization
signal for tropoelastin pre-mRNA was seen in mid-fetal samples, and
paralleling the increase in steady-state mRNA levels, a strong signal
for pre-mRNA was detected in neonatal samples, indicating
transcriptional regulation. Stimulation of fetal lung tropoelastin
expression by maternal administration of dexamethasone also led to an
increase in pre-mRNA levels. However, signal for tropoelastin pre-mRNA
in adult samples was equal to that detected in neonatal samples, even
though mRNA levels had dropped about 80-fold. Persistence of
tropoelastin transcription in adult tissue was also seen in cell
culture models and was verified by nuclear runoff assay. In addition,
an RT-PCR assay for
Elastin is a resilient connective tissue protein that is present
in the extracellular matrix of most vertebrate tissues, but it is
especially abundant in the interstitium of tissues that undergo
repeated physical deformations, such as lungs, large blood vessels, and
skin(1) . Elastic fibers are protein polymers which are
assembled extracellularly and are comprised mostly of elastin, which is
the product of enzymatically cross-linked tropoelastin monomers, and
microfibrillar proteins(2) . In most mammalian tissues, the bulk
of elastogenesis occurs during late fetal and early neonatal periods,
and by maturity production is complete and synthesis of new
tropoelastin ceases(1, 3) . Because elastin is an
extremely durable polymer and essentially does not turn over in healthy
tissues(4, 5, 6) , fiber function and tissue
integrity are not compromised by this limited pattern of production.
Certain diseases and conditions, however, such as pulmonary
hypertension and dermal elastosis(7, 8, 9) , are
associated with continued or a renewed and excessive accumulation of
elastin. The tissue-specific and temporally precise production
indicates that tropoelastin expression is tightly controlled, but the
mechanisms that regulate tropoelastin expression in vivo are
not known.
We reported that the marked down-regulation of
tropoelastin expression mediated by 1,25-dihydroxyvitamin D
To circumvent these problems, we developed a rapid and
sensitive reverse transcription-polymerase chain reaction
(RT-PCR)
The 5` forward and 3`
reverse-complement PCR primers for amplification of rat
Initially, we developed the RT-PCR assay with
primers specific to sequences in adjacent exons, 35 and 36, to allow us
to detect cDNAs of both the pre-mRNA and the fully processed transcript (Fig. 2). After 25 cycles, we could detect the 369-bp cDNA by
ethidium bromide staining in the neonatal sample but not the 1023-bp
pre-mRNA product (Fig. 1A). By Southern
hybridization with exon-specific oligo B, the signal for 369-bp cDNA
was seen in all lung samples and was about 14-fold stronger in the
3-day neonatal sample than that produced by fetal or adult RNA (Fig. 1, B and C). The relative difference in
the signal for the cDNAs produced with exon primers was identical to
that determined by Northern hybridization of the same samples (compare Fig. 1C to 1D) and demonstrated the sensitivity
and accuracy of the RT-PCR assay. However, when we hybridized this blot
with probe i35 for pre-mRNA cDNA (1023 bp), we detected little to no
signal for tropoelastin pre-mRNA in the 3-day neonatal sample but a
strong signal in the fetal and adult samples (data not shown). Since we
used exon primers to amplify both mRNA and pre-mRNA products, these
results were likely erroneous and caused by competition for the RT-PCR
reagents by the highly abundant mRNA template in the neonatal sample.
This apparently was the problem, since a different, yet reproducible
and internally consistent pattern of pre-mRNA cDNA signal was detected
when we switched to intron-specific primers, thereby amplifying solely
pre-mRNA.
To optimize the RT-PCR assay with intron primers, we
determined the relationship of signal strength to cycle number and to
the amount of RNA added (Fig. 3). Because of the low
concentration of pre-mRNA, intron-specific cDNAs were not seen by
ethidium bromide staining but were easily detected by Southern
hybridization. With less than 30 cycles, only the 478-bp tropoelastin
pre-mRNA cDNA was produced with intron 35 primers, and the signal for
this product increased exponentially between 21 and 29 cycles (Fig. 3). In addition, the efficiency of product amplification
between 21 and 29 cycles was essentially 100%. With 31 or more cycles
the specific tropoelastin signal increased only slightly and
nonspecific bands appeared.
In this study, we report that distinct cellular mechanisms
control tropoelastin expression at different stages of development. In
all vertebrates, the tissue-specific induction of tropoelastin
expression occurs during mid to late fetal or embryonic
periods(1) , and our results indicate that gene transcription
controls production at these early ages. Elastin production continues
throughout postnatal growth but is particularly active in many tissues
during the neonatal period, and we found that tropoelastin mRNA and
pre-mRNA levels increase proportionately as rat lung morphogenesis
progressed from fetal to early and late neonatal stages (Fig. 1,
4, and 5). In addition, maternal administration of glucocorticoid,
which accelerates fetal lung development and stimulates elastin
production(25) , mediated a proportional increase in
tropoelastin pre-mRNA levels (Fig. 9). The close correlation
between mRNA and pre-mRNA levels indicates that the induction and
up-regulation of tropoelastin expression during the early phases of
lung development is controlled primarily at the level of transcription.
Enhancement of other regulatory mechanisms, such as mRNA stability and
translational efficiency, may also contribute to the abundant
biosynthesis of elastin during active periods of growth.
As tissue
development and growth are completed, elastin production is turned off,
and except in response to injury or disease, no new elastin is made
over the lifetime of the organism(4, 5, 6) . We
found, however, that tropoelastin pre-mRNA levels in matured adult lung
remained elevated, although the steady-state mRNA level decreased
sharply from peak levels attained in neonatal lung (Fig. 5).
Furthermore, we detected the same persistence of tropoelastin pre-mRNA
levels in fibroblasts isolated from adult lung (Fig. 8) and in
lung fibroblasts that were aged in culture (Fig. 7). Thus, the
discrepancy between pre-mRNA and steady-state mRNA levels in fully
grown tissues indicates that a post-transcriptional mechanism controls
the down-regulation of tropoelastin expression.
Our conclusions are
based on the detection of intron sequences in newly transcribed
pre-mRNA. As stated, we likely detected only nuclear pre-mRNA molecules
since intron sequences are rapidly degraded once they are spliced from
the primary transcript (15, 28) and because pre-mRNAs
are retained in the nucleus until splicing is completed(14) .
The nuclear processing of pre-mRNA, which includes splicing, capping,
polyadenylation, and transport into the cytosol, occurs relatively
quickly, and the speed of this process is proportional to the length of
the primary transcript. The tropoelastin gene codes for a 45-kb
pre-mRNA (37) and based on kinetic data from other
systems(13, 38) , synthesis and processing of this
transcript would require about 2-3 min. Compared to the half-life
of the mature mRNA, which is about 24-48 h during periods of
active elastin production(11) , tropoelastin pre-mRNA molecules
are likely to be short lived during active periods of elastogenesis.
Thus, the levels of tropoelastin pre-mRNA reflect the rate of active,
ongoing transcription. Although it could be argued that tropoelastin
pre-mRNA is processed slowly in adult tissue, thereby leading to an
accumulation of prespliced transcripts, nuclear run-off assay (Fig. 7) demonstrated that the persistence of pre-mRNA expression
was due to continued transcription of the tropoelastin gene and was not
due to age-specific processing of the primary transcript.
Although
tropoelastin expression and elastin deposition are influenced by
various agents and conditions, the mechanisms controlling age-specific
production have not been identified(1) . As stated, our data
suggest that stimulation of tropoelastin expression is controlled
transcriptionally, and findings from other studies support this idea.
Mauviel et al.(39) demonstrated that
interleukin-1
A novel finding in our studies is that
transcription of the tropoelastin gene apparently does not turn off
after biosynthesis of the protein has ceased. This observation,
however, is opposed to conclusions reached in other studies. Using
transiently transfected cells, Kähäri et al.(42) reported that tumor necrosis factor-
Although multiple
mechanisms can participate in the control of gene
expression(45) , production of structural proteins is primarily
regulated at the level of transcription. There are, however, numerous
examples of proteins whose production is primarily regulated by a
post-transcriptional mechanism(46) . Many of these products,
such as cytokines, iron-metabolism proteins, oncogenes, and
cytoskeletal proteins, are expressed during physiologic transitions or
for brief periods during developmental processes, and changes in the
stability of the mRNA provide a mechanism to rapidly govern protein
synthesis and activity. In contrast, once the growth of elastic tissue
is complete, new elastin production is not needed, and thus, the
post-transcriptional control we describe is a novel mechanism to
control the expression of a stable structural protein. As stated, the
5`-flanking region of the elastin gene resembles that of a typical
housekeeping gene in that it contains GC-rich islands, has no consensus
TATA box, and uses multiple start sites(37) . In addition, the
elastin gene has developed relatively recently, having evolved along
with high pressure circulatory systems and lungs(47) . For a
structural protein, the primary sequence of elastin differs markedly
among vertebrates. For example, elastin is not found below the bony
fish, and that expressed by fish is markedly different from terrestrial
elastin(48, 49) . Thus, compared to more ancient
extracellular matrix proteins, such as the collagens, it is possible
that unique regulatory mechanisms have evolved in the elastin gene.
Previously, we have reported that down-regulation of tropoelastin
expression is associated with a marked reduction in the stability of
its mRNA, and it is likely that the pre-mRNA transcribed in adult
tissue is targeted for rapid degradation. The half-life of mRNA
transcripts is influenced by poly(A) tail length and regulatory
sequences located in the 5`- or 3`-untranslated
regions(46, 50, 51) , and these events are
likely mediated by specific RNA-binding proteins (52). The 3`-UTR of
tropoelastin mRNA does not contain sequences that have been associated
with regulated degradation of other transcripts, and, thus, decay of
tropoelastin mRNA may be controlled by unique cis-acting
sequences. The 3`-UTR of tropoelastin mRNA is similar in size and
sequence among mammals, and portions of the 3`-UTR of tropoelastin mRNA
have extensive identity among human, bovine, and rat
sequences(20, 53) . Thus, cis elements in
tropoelastin mRNA may be involved in the regulation of tropoelastin
gene expression, and we are currently attempting to identify such
elements.
We thank Drs. Robert P. Mecham and Anders Persson for
helpful discussion and Drs. David Rowe and Alex Lichtler for providing
sequence information of the rat
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (I) procollagen pre-mRNA accurately revealed
the known transcriptional regulation of this gene. Our results
demonstrate that the induction and maintenance of elastogenesis is
controlled by a transcriptional mechanism, whereas, the cessation of
tropoelastin expression is controlled by a post-transcriptional
mechanism.
or phorbol ester is controlled primarily by a
post-transcriptional mechanism leading to accelerated degradation of
the mRNA(10, 11) . These findings suggested that the
normal cessation of tropoelastin expression is controlled
post-transcriptionally, but because these studies were carried out in
cell culture, we did not know if this mechanism represented an actual in vivo regulatory process or a cell culture artifact. To
assess the regulation of tropoelastin expression in vivo, we
could have attempted nuclear runoff assays on nuclei isolated from lung
or aorta, but it would be difficult to obtain viable nuclei from these
fibrous-rich tissues. Transgenic animals containing reporter constructs
under control of the tropoelastin promoter would seem to be an
appropriate model to study regulation, but the tropoelastin promoter,
at least the first 5 kb,
(
)is effective in
elastogenic and non-elastogenic cells (12) indicating that
temporal- and tissue-specific regulatory elements have not yet been
identified.
assay to quantify tropoelastin pre-mRNA levels as
an indicator of ongoing transcription. Since pre-mRNA (or hnRNA) is
rapidly processed to mRNA and transported from the
nucleus(13, 14, 15, 16) , assessment of
the relative levels of preprocessed mRNA should estimate the rate of
active transcription. Our results demonstrate that the induction and
up-regulation of tropoelastin expression is under transcriptional
control, whereas in mature tissue, expression of tropoelastin pre-mRNA
is maintained at high levels, even though steady-state mRNA levels are
low. Thus, the cessation of tropoelastin expression is governed by a
post-transcriptional mechanism, which is a unique regulatory mechanism
to control production of an age-specific structural protein.
Animals
Timed-pregnant and older Sprague-Dawley
female rats were obtained from Charles River Laboratories (Cambridge,
MA). Animals were killed with sodium pentobarbital, and lungs were
removed from 19-day gestation fetuses, 3- and 11-day-old neonates,
6-month-old mothers, and 8-, 12-, and 18-month-old adults. For
dexamethasone studies, pregnant rats, at 16 days of gestation, were
treated daily with an intramuscular injection of normal saline or 1
mg/kg dexamethasone-21-phosphate for 3 consecutive days before
sacrificing at 19 days of gestation.
Cell Culture
Lung fibroblasts were isolated by
explant culture from 3-day-old neonates and from 6-month-old adult
mothers as described(17) . Cells were grown to visual
confluence, divided 1:4, and used at passage 2. Other cells used in
these studies were RFL-6, an elastogenic fibroblast cell line derived
from lung tissue of normal 18-day gestation Sprague-Dawley rat fetus
(American Type Culture Collection, Rockville, MD), extended passage
(>25) RFL-6 cells, supplied by Dr. James Grant, Department of
Pediatrics, Washington University, and PC-12, a rat pheochromocytoma
cell line (ATCC).
RNA Isolation
Total RNA was isolated from lungs of
individual animals and from cultured cells by guanidine-phenol
extraction(18) . For the RT-PCR assay, RNA samples were
aliquoted and stored at -70 °C. To remove any contaminating
DNA, total RNA (50 µg) was incubated for 1 h at 37 °C in 1
digestion buffer (40 mM Tris-HCl, pH 7.9, 10
mM NaCl, 6 mM MgCl
, 0.1 mM CaCl
) containing 5 units of RQ1 RNase-free DNase and 1
unit of RNasin (all reagents from Promega, Madison, WI), and RNA was
extracted with phenol-chloroform and ethanol precipitated. As
determined by UV spectrophotometry, ethidium bromide staining, and
Northern hybridization, RQ1-DNase treatment did not affect total RNA
mass, RNA integrity, or the relative amount of tropoelastin or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs (data not
shown). DNase-treated RNA was used for all Northern and RT-PCR
experiments presented in this report. Northern hybridization and washes
were done under stringent conditions as described(19) .
Gel-purified cDNA fragments for rat tropoelastin, REL 115 or REL 124D
(20), rat
1(I) procollagen cDNA, and GAPDH were labeled by random
priming with
[
P]dCTP. Autoradiographic
signal was quantified by densitometry and normalized to the relative
amounts of GAPDH mRNA.
Nuclear Run-off Assay
Transcription rate was
measured using isolated nuclei as described in detail(10) .
Briefly, 2.5 10
intact nuclei from RFL-6 cells were
incubated at 30 °C for 15 min in transcription buffer containing
250 µCi of [
-
P]GTP (>3000 Ci/mmol).
Total RNA was isolated and hybridized to denatured, gel-purified cDNAs
slotted onto nitrocellulose. Probes used were a rat tropoelastin cDNA,
124D, a bovine tropoelastin cDNA, 12-1(21) , and a human
-actin cDNA. As an indicator of total transcription, nascent
radiolabeled RNA was hybridized to a 2.5-kb human AluI repeat
fragment derived from the
-globin gene. Background signal was
determined by hybridization to linearized parental plasmid DNA.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)
DNase-treated total RNA (0.1 µg) was reverse
transcribed with random hexamers using kit reagents and under the
manufacturer's recommended conditions (GeneAmp RNA PCR kit,
Perkin Elmer Cetus). For each sample, a parallel reaction was run with
no reverse transcriptase. The RT reaction was placed at room
temperature for 10 min, then incubated in a thermal cycler at 42 °C
for 15 min, 99 °C for 5 min, and then cooled at 5 °C for 5 min.
The first strand cDNA was diluted to 100 µl with 1 PCR
buffer II, as described in the kit manual, containing 2 mM
MgCl
and 100 pmol each of 5` and 3` specific oligomers, and
the reaction mixture was overlaid with 100 µl of mineral oil. For
experiments with amplification of GAPDH mRNA, the reaction mixture was
equally split into two tubes before adding specific primers and PCR
reagents. Before adding 2.5 units of AmpliTaq DNA polymerase, the
mixture was denatured at 95 °C for 4 min. Samples were denatured at
95 °C for 1 min, annealed at 55 °C for 1 min, and extended at
72 °C for 1 min. After 25 cycles, the PCR mixture was incubated at
72 °C for 7 min. RT-PCR products were extracted from the mineral
oil with chloroform-isoamyl alcohol, and 40 µl of each sample was
directly resolved by electrophoresis through 2% SeaKem
ME agarose (FMC, Rockland, ME). After transfer onto nylon
membranes, blots were hybridized with
P-labeled oligomer
probes as described(20) . To confirm the identity of the
amplified cDNAs, intron 35 products were directly sequenced using a
fMol Sequencing kit (Promega) or were asymmetrically cut with XbaI.
PCR Primers and Hybridization Probes
PCR primers
and oligomer probes were commercially synthesized and crude-column
purified (Bio-Synthesis, Inc., Lewisville, TX), were based on unique
sequences of the rat tropoelastin gene, and avoided exons known to be
alternatively spliced(20, 22, 23) . The 5`
forward and 3` reverse-complement PCR primers for amplification of
intron 21 sequences in tropoelastin pre-mRNA were CAGTGGAATCCAGGCTCTGA
(i21F, see Fig. 2) and ATCACCATCCACTCACCCAC (i21RC),
respectively, and the oligomeric probe to detect the 260-bp RT-PCR
product was CATACATACATACATACATAC (i21). For intron 35, the 5` and 3`
PCR primers were GTCAGAGGTCAAGGTCTAGG (i35F) and TCAGTCTAGACATGCAACAC
(i35RC), respectively, and intron 35-specific probe to detect the
478-bp RT-PCR product was GACATACCACCAGGTGGCGC (i35). The 5` forward
and 3` reverse-complement primers for amplification of exon-specific
sequences in tropoelastin mRNA were TGGAGCCCTGGGATATCAAG (e35F), which
is defined by sequences in the 5` end of exon 35, and
GAAGCACCAACATGTAGCAC (e36RC), which is complementary to sequences
within the 3`-UTR, and the oligomer probe for this 369-bp PCR product
was TCACTTTCTCTTCCGGCCACAAGATTTCCCAAAGCAG (oligo B).
Figure 2:
RT-PCR strategy. Total RNA was reverse
transcribed with random hexamers, and sequences in introns 21 and 35 of
tropoelastin pre-mRNA were selectively amplified using specific primers (F, forward primers; RC, reverse complementary
primers; prefix i indicates intron-specific primers or
probes). These primers generated cDNAs of 260 bp for intron 21 and 478
bp for intron 35, and products were detected by Southern hybridization
using intron-specific oligomeric probes (i21 or i35).
For RT-PCR of mRNA (see Fig. 3), forward (e35F) and reverse
complementary (e36RC) exon-specific primers were used to
generate a 369-bp cDNA that spanned the 5` end of exon 35 to within the
3`-untranslated region (UTR) of exon 36, and the product was
detected with an exon-36 specific oligonucleotide (probe B).
These same primers were designed to produce a 1023-bp cDNA from
tropoelastin pre-mRNA. Exon and intron sizes are drawn to scale. The
3`-UTR of rat tropoelastin mRNA is about 1.5 kb, and hence, extends
further than what is shown here.
A mutant of the
intron 35 region was made by amplifying genomic DNA with i35F and with
a 40-nt primer (i35RCI-RC) which contains the sequences of i35RC at its
3` end and 20 bases of intron sequences, which begin 108 bp 5` of
i35RC, at its 5` end. As shown by Celi et al.(24) ,
initial extension is accomplished only with the i35RCI portion of the
primer, and during the second round of amplification, the 3` RC end is
filled in. When amplified with i35F and i35RC primers, this mutant
template creates a PCR product that is 88 bp less than that produced
from tropoelastin pre-mRNA. RT-PCR reactions of total lung RNA were
spiked with 0.1 pg of mutant DNA to assess any differences in
amplification efficiency among samples. In separate reactions, the
identity of the internal standard was confirmed by direct sequencing
and by restriction enzyme analysis.
1(I)
procollagen pre-mRNA were TTCAAGCATCTATCTGTAGC and
CTAAAGTTAGATACACCCTA, respectively, and the intron 1-specific probe to
detect the 704-bp RT-PCR product was GTCAGAGTACGTCGAAGATC. Oligomeric
probes were labeled with [
-
P]ATP using T4
polynucleotide kinase(19) . GAPDH mRNA was amplified using
CTGAGAACGGGAAGCTTGTCATCAA(250-275) and GCCTGCTTCACCACCTTCTTGATG
(836-860) for the 5` forward and 3` reverse-complement PCR
primers, respectively, and the 610-bp product was detected by
hybridization with CTCATGACCACAGTCCAT(580-598).
Developmental Expression of Tropoelastin
We
isolated total lung RNA from 19-day fetal, 3-day-old neonatal, and
6-month-old adult rats. Based on previous studies on developmental
changes in the rat lung(25, 26, 27) , we
expected that these ages would represent distinct stages of
tropoelastin expression, namely the onset, maximal production, and
cessation. Indeed, as demonstrated by Northern hybridization,
steady-state levels of tropoelastin mRNA, relative to the levels of
GAPDH mRNA, were low in the 19-day fetal lung, shortly after
tropoelastin expression begins in the rat(26) , were about
23-fold higher in the 3-day neonatal lung, and were markedly repressed
in the adult, when expression is low to non-existent (Fig. 1D, also see Fig. 5A).
Figure 1:
RT-PCR of tropoelastin (TE) mRNA with exon-specific primers. DNase-treated RNA (0.1
µg) from 19-day fetal (F19d), 3-day neonatal (N3d), and 6-month adult rat lungs was reverse transcribed and
amplified with tropoelastin exon-specific primers (e35F and e36RC). A, by ethidium bromide staining, the 369-bp cDNA product (arrow, also see Fig. 2), representing processed mRNA, was
detected only in the 3-day neonatal sample. B, as demonstrated
by Southern hybridization with exon-specific probe B (see Fig. 2),
RT-PCR of each RNA sample produced the specific cDNA. Autoradiographic
exposure was 3 h. C, densitometric quantification of the cDNA
bands revealed that the RT-PCR assay accurately determined the
age-related differences in the relative levels of tropoelastin mRNA
(compare with panel D). Values for the neonate and adult
signals are expressed relative to that for the fetal, which was set to
2 to match the number for the fetal samples in panel D. D, the developmental pattern of tropoelastin mRNA
expression was determined by Northern hybridization. Total RNA was
isolated from lungs of four individual 19-day rat fetuses, 3-day-old
neonates, and 6-month-old adult rats, and 5 µg/lane was analyzed by
Northern hybridization for tropoelastin and GAPDH mRNAs. The resultant
autoradiogram was scanned, and the signal density of tropoelastin mRNA
was expressed relative to that of GAPDH mRNA. As seen, tropoelastin
steady-state mRNA levels are low in fetal lung, are markedly elevated
during neonatal growth, and returned to low levels by
maturity.
Figure 5:
Tropoelastin pre-mRNA expression persists
in late adult tissue. A, total lung RNA (5 µg/lane) from a
19-day rat fetus, from a 3-day neonate, and from 6-, 8-, 12-, and
18-month adult rats was analyzed by Northern hybridization for
tropoelastin (TE) and GAPDH mRNAs. As seen, tropoelastin
steady-state mRNA levels are markedly reduced in the older adults (8
months and older), whereas GAPDH mRNA levels remain relatively
constant. Autoradiographic exposure was 5 days. B, total RNA
(0.1 µg) from the same animals was amplified by RT-PCR using
intron-35 primers. Products corresponding to tropoelastin pre-mRNA were
detected by hybridization with P-labeled i35, and
autoradiographic exposure was 2 h. C, after reverse
transcription, half of the RT-PCR reactions was removed, added to a new
tube, and amplified with GAPDH-specific primers. Shown is the
autoradiograph of the Southern blot hybridized for the 610-bp cDNA
product, which, along with the Northern data, demonstrates constitutive
expression of GAPDH and nearly equivalent loading among
samples.
RT-PCR Assay
To demonstrate the in vivo regulatory mode of tropoelastin expression at different stages of
development, we designed an RT-PCR assay to assess tropoelastin
pre-mRNA as an indicator of ongoing transcription in intact tissues.
Total lung RNA was treated with RNase-free DNase to remove any
contaminating DNA and was reverse transcribed with random hexamers. The
resulting single-stranded cDNA was amplified by PCR using primers
specific to intron or exon sequences of the tropoelastin gene, and PCR
products were detected by Southern hybridization with specific probes
(see Fig. 2).
Figure 3:
Conditions for RT-PCR of tropoelastin
pre-mRNA. Upper panels, total RNA (0.1 µg) from 3-day
neonatal rat lung was reverse transcribed, and separate reactions were
amplified for 21-35 cycles with intron-35 primers (i35F and
i35RC). The PCR products were resolved by agarose-gel electrophoresis
and detected by Southern hybridization with P-labeled i35.
Signal for the 478-bp cDNA of tropoelastin pre-mRNA increased
exponentially between 21 and 29 cycles. Additional amplification (31 or
35 cycles) produced artifacts and provided only a small increase in
specific signal. No products were detected after 35 cycles in reactions
without the initial addition of reverse transcriptase
(-RT). Bottom panels, various amounts of RNA
(0.0125-0.4 µg) from 3-day neonatal rat lung were reverse
transcribed and amplified for 25 cycles with intron-35 primers.
Southern hybridization demonstrated that the signal for the 478-bp cDNA
increased proportionately with the amount of input RNA. No signal was
detected in reactions containing 0.1 µg of RNA without reverse
transcriptase (-RT). The migration of size standards is listed
on the left side of both blots. An additional size standard (Std) of 1023 bp was amplified using exon primers (e35F and
e36RC) and a 4.8-kb fragment of the rat tropoelastin gene, which
includes exon 35, intron 35, and exon 36. Autoradiographic exposure was
2 h for both blots.
We also demonstrated that the production
of pre-mRNA cDNA was proportional to the amount of input RNA. With 25
cycles of amplification, the signal for intron-35 cDNA increased
linearly between 0.0125 and 0.4 µg of 3-day-old neonatal rat lung
RNA added to the RT-PCR reaction (Fig. 3). These findings show
that the RT-PCR assay accurately reflects relative differences in the
levels of pre-mRNA over at least a 32-fold range in template
concentration. No product was detected in samples without reverse
transcriptase indicating the specificity of the assay and that no
significant amount of cellular DNA was present in the RNA samples (Fig. 3). Based on these results, we used 0.1 µg of
DNase-treated RNA and 25 cycles for all subsequent reactions.
Restriction enzyme digestion and direct sequence analyses of RT-PCR
products confirmed their identity as components of rat tropoelastin
intron 35 (data not shown). As an additional control, we detected no
products in RT-PCR reactions containing exon or intron primers with RNA
isolated from PC12 cells, a rat pheochromocytoma cell line that does
not express tropoelastin mRNA (data not shown), demonstrating that
nonspecific products were not produced in this assay.
Tropoelastin Pre-mRNA Levels Persist in Adult
Tissues
Total lung RNA from the same animals used for Northern
hybridization was analyzed by RT-PCR with intron 35-specific primers (Fig. 4). Low levels of tropoelastin pre-mRNA were detected in
the 19-day fetal samples, but about 24-fold higher levels were produced
from the 3-day neonatal RNA samples (Fig. 4, A and B). The magnitude in the increase of signal for tropoelastin
pre-mRNA in the neonatal sample was essentially identical to that
determined for the steady-state mRNA (Fig. 1D), and this
tight correlation indicates that gene transcription controls
tropoelastin production during early lung development. In contrast, the
levels of tropoelastin pre-mRNA remained elevated in all adult lung
samples (Fig. 4, A and B) even though
steady-state mRNA levels were reduced at least 20-fold in the mature
tissue (Fig. 1D). An essentially identical pattern of
tropoelastin pre-mRNA levels was detected using primers specific for
intron 21 (Fig. 4C) indicating that our findings were
not due to artifacts created by intron structure or with a given pair
of primers. Amplification of GAPDH mRNA generated an equal signal among
samples (Fig. 4C), thereby demonstrating that our
findings for tropoelastin pre-mRNA were not due to a loading artifact.
In another set of reactions, we added a fixed amount of the i35RCI-RC
mutant to each RNA sample and amplified using intron 35-specific
primers. The same pattern of tropoelastin pre-mRNA expression was seen
as in other experiments, but the internal standard was amplified
equally among RNA samples (Fig. 4D). These data indicate
that the measured differences in tropoelastin pre-mRNA levels were not
due to sample-specific variations in PCR efficiency. In addition, the
cDNA products detected are likely not due to spliced intron fragments
since these sequences are rapidly degraded once removed from the
pre-mRNA(15, 28) . Thus, these findings indicate that
tropoelastin transcription does not turn off at the end of elastin
production and that a post-transcriptional mechanism regulates the low
levels of tropoelastin mRNA in the mature tissue.
Figure 4:
Tropoelastin (TE) pre-mRNA
expression persists in adult tissue. A, total RNA (0.1 µg)
from three 19-day fetal, 3-day neonatal, and 6-month adult rat lungs
was amplified by RT-PCR with (+RT) or without
(-RT) reverse transcriptase using intron-35 primers.
Products were detected by hybridization with P-labeled
i35, and autoradiographic exposure was 2 h or 6 h for the two
representative blots shown. B, autoradiographic signal for the
478-bp cDNA of tropoelastin pre-mRNA was quantified by densitometry and
normalized to the signal of the 3-day neonatal sample, which was
arbitrarily set at 25. The data presented are the means ± S.D.
of results from four separate sets of age-specific lung RNA. C, identical results were obtained when samples were amplified
with intron-21 or intron-35 primers. Autoradiographic exposure was 24
h. Amplification of GAPDH mRNA demonstrated equivalent loading and
efficiency. D, to assess potential differences in PCR
efficiency among RNA samples, tropoelastin pre-mRNA and an internal
standard mutant (0.1 pg/reaction) were amplified together with
intron-35 primers. Products were detected by hybridization with
P-labeled i35. As seen, the signal strength for the mutant
was equal among samples.
The persistence of
tropoelastin pre-mRNA expression in 6-month-old rat lung may reflect
continued transcription during the switch from high to low elastin
production, and in much older animals, when elastin production has
ceased completely, tropoelastin transcription may eventually shut off.
Our findings, however, indicate that tropoelastin transcription
continues at high rates long after elastin production is completed (Fig. 5). As shown by Northern hybridization, only trace levels
of tropoelastin mRNA are detected in rat lung RNA in animals older than
6 months (Fig. 5A). Relative to that in 3-day neonate
lung, the steady-state levels of tropoelastin mRNA were about
70-80-fold less in the 8-, 12-, and 18-month lungs. However,
tropoelastin pre-mRNA levels remained at nearly equivalent levels in
all neonate and adult tissues (Fig. 5B) suggesting that
post-transcriptional control of tropoelastin expression in the mature
tissue is an active and predominant regulatory mechanism throughout
postnatal life.
Transcriptional Regulation of
As a positive control for the RT-PCR assay, we
assessed the expression of 1(I) Procollagen
Expression
1(I) procollagen pre-mRNA. Similar to
the developmental pattern of tropoelastin expression (Fig. 1D), procollagen mRNA levels were low in fetal
tissue, peaked during neonatal development, and were reduced in
maturing lung (Fig. 6). The developmental expression of
1(I)
procollagen is controlled at the level of
transcription(29, 30, 31, 32) , and
thus, its pre-mRNA levels should parallel the levels of steady-state
mRNA. Indeed, as assessed by RT-PCR, expression of
1(I)
procollagen pre-mRNA was low in fetal lung, high in neonatal tissue,
and returned to low levels in the adult lung (Fig. 6). Thus,
amplification of pre-mRNA reflected the known transcriptional
regulation of
1(I) procollagen.
Figure 6:
RT-PCR demonstrates the developmental
regulation of 1(I) procollagen transcription. A, total
RNA (5 µg) from 19-day fetal (F19d), 3-day neonatal (N3d), and 6-month adult rat lungs was analyzed by Northern
hybridization for
1(I) procollagen mRNA, which had a developmental
pattern of expression similar to that for tropoelastin mRNA (see Fig.
2). The equivalency of RNA loaded per lane was confirmed by ethidium
bromide staining and by hybridization for GAPDH mRNA (data not shown).
Total RNA (0.1 µg) from the same samples was amplified by RT-PCR
with intron-1 primers. Autoradiographic signal for the 704-bp cDNA of
1 (I) procollagen pre-mRNA paralleled that for the steady-state
mRNA. Autoradiographic exposure was 16 h for the RT-PCR blot and 2 days
for the Northern. B, the autoradiographic signals for
procollagen mRNA and pre-mRNA were quantified by densitometry and were
normalized to the 3-day neonatal signals which were arbitrarily set at
25.
RT-PCR and Nuclear Run-off
Reflective of the in vivo developmental pattern of elastin production,
elastogenic cells lose their capacity to express tropoelastin as they
age in culture or are serially passed(33, 34) . To
assess the regulation of this down-regulation, we isolated total RNA
from RFL-6 cells, an elastogenic rat lung fibroblast cell line, at
passage 15 and from cells which had been passed extensively (>25).
As shown by Northern hybridization, the older cells expressed about
30-fold less tropoelastin mRNA than did the passage-15 cells, but
similar to that we detected in adult lung, the levels of tropoelastin
pre-mRNA, as assessed by RT-PCR with intron 35 primers, were equivalent
in the two cell populations (Fig. 7). To verify these findings,
we isolated nuclei from the early and extended passage cells for
nuclear run-off assay. In agreement with the RT-PCR results, the
relative transcription rate, normalized to total transcription, was the
same in both cell populations, even though tropoelastin steady-state
mRNA levels were markedly lower in the extended passage cells (Fig. 7). These findings provide additional evidence that
down-regulation of tropoelastin expression is controlled primarily by a
post-transcriptional mechanism. Furthermore, the findings with the
nuclear runoff assay, which measures nascent pre-mRNA production,
indicate that the results with RT-PCR assay were not due to
differential processing of tropoelastin mRNA.
Figure 7:
RT-PCR
and nuclear run-off assays both demonstrate that tropoelastin pre-mRNA
expression remains elevated after down-regulation of steady-state mRNA
levels. Total RNA was isolated from RFL-6 cells at passage 15 (p15) and from cells passed at least 25 times (p >
25). As shown by Northern hybridization (mRNA), tropoelastin
steady-state mRNA levels were markedly decreased in the older cells,
whereas pre-mRNA levels, as determined by RT-PCR with intron-35 primers (Pre-mRNA), were equivalent in the two samples. Tropoelastin
transcription was also assessed by nuclear run-off assay. In vitro transcribed P-labeled RNA was hybridized to parental
plasmid DNA (pBS), a genomic fragment containing AluI
repeat sequences (Alu), used as an indicator of total
transcription, and cDNAs for
-actin and bovine and rat
tropoelastin (TE). The difference in signal strength with the
bovine and rat tropoelastin cDNAs reflects the greater affinity of the
rat pre-mRNA for its homologous cDNA. Autoradiographic exposure was 2
days for the Northern, 1 day for the RT-PCR blot, and 7 days for the
run-off.
Regulation of Tropoelastin Expression in Cultured
Cells
The expression of tropoelastin in cultured cells reflects
the relative production of elastin in tissue at the time the cells were
isolated (33-36). To develop an in vitro model to study
the developmental regulation of tropoelastin expression, we isolated
interstitial fibroblasts from 3-day neonatal and adult rat lungs. Total
RNA was isolated from confluent primary cells and from the lung tissue
from which these cells were derived. As expected, tropoelastin mRNA
levels were high in both neonatal lung tissue and isolated cells and
were much lower in adult lung and fibroblasts (Fig. 8). RT-PCR
assay with intron 35 primers showed that tropoelastin pre-mRNA levels
remained elevated in adult lung and adult interstitial fibroblasts (Fig. 8). The stronger hybridization signals observed for both
the mRNA and pre-mRNA in cell samples is likely due to an enrichment of
tropoelastin-expressing cells in culture compared to the diverse cell
populations in intact lung.
Figure 8:
Persistence of tropoelastin pre-mRNA
expression is maintained in cultured cells. Interstitial lung
fibroblasts were isolated from 3-day neonatal (N3d) and adult
rat lungs. Northern hybridization (mRNA) showed that the
age-related differences in steady-state tropoelastin mRNA levels in
intact lung (In Vivo) were duplicated in cells isolated from
the same tissues (Cell Culture). RT-PCR (0.1 µg/reaction,
25 cycles, intron-35 primers) and Southern hybridization revealed that
tropoelastin pre-mRNA levels remained elevated in adult tissues and
isolated cells. No RT-PCR product was detected in adult fibroblast RNA
samples processed without reverse transcriptase (-RT).
The autoradiographic signals for tropoelastin mRNA and pre-mRNA were
quantified by densitometry and, as presented in the histogram, were
normalized to the 3-day neonatal signals (In Vivo), which were
arbitrarily set at 10. The stronger signals detected in the cell
culture samples were likely due to an enrichment of
tropoelastin-producing cells. Autoradiographic exposure was 3 h for the
RT-PCR blot and 24 h for the Northern.
Stimulation of Tropoelastin Expression In Vivo Is
Transcriptionally Regulated
We have recently shown that
tropoelastin expression during late fetal development is up-regulated
in response to maternal administration of dexamethasone, and transient
transfection of isolated lung cells with a tropoelastin-promoter
construct suggested that this stimulation is controlled at the level of
transcription(25) . To determine if glucocorticoids up-regulate
tropoelastin transcription in vivo, we assayed total RNA from
control and dexamethasone-treated 19-day fetal rat lungs by RT-PCR with
intron 35 primers. In agreement with our previous findings,
tropoelastin mRNA levels in 19-day fetal rat lung were stimulated with
dexamethasone treatment. As demonstrated by RT-PCR, the increase in
mRNA levels was accompanied by a similar stimulation of pre-mRNA levels (Fig. 9) indicating that glucocorticoids influence tropoelastin
transcription in the developing lung.
Figure 9:
Dexamethasone stimulates tropoelastin
pre-mRNA levels in vivo. Beginning at 16 days of gestation,
pregnant rats were treated daily for 3 consecutive days with an
intramuscular injection of normal saline (Control) or 1 mg/kg
dexamethasone (+Dex). At 19 days of gestation, the
fetuses were removed, and lung RNA was isolated. As shown by Northern
hybridization (5 µg lane), tropoelastin (TE) mRNA levels
were stimulated with dexamethasone treatment, but maternal
administration of this glucocorticoid had no effect on GAPDH mRNA
levels. RT-PCR (0.1 µg/reaction, 25 cycles, intron-35 primers) and
Southern hybridization revealed that tropoelastin pre-mRNA levels were
elevated in dexamethasone-treated fetal lungs. No RT-PCR product was
detected in lung RNA samples processed without reverse transcriptase
(-RT). Autoradiographic exposure was 16 h for the RT-PCR
blot and 72 h for both Northerns.
enhances tropoelastin mRNA levels and activity from
an elastin promoter-CAT construct in both transgenic mice and
transiently transfected cells. Furthermore, tropoelastin mRNA levels
and transcription, as determined by nuclear run-off or with a
promoter-expression construct, increase proportionately in response to
insulin-like growth factor-1 or retinoic acid(17, 40) .
Recently, Bernstein et al.(9) reported that an elastin
promoter construct is more active in dermal fibroblasts from severely
photodamaged skin, which is characterized by excess elastin deposition,
than in cells derived from non-sun-exposed areas. Thus, as we show,
stimulated or continued production of tropoelastin expression is
controlled primarily at the level of transcription.
Post-transcriptional mechanisms, however, may also contribute to
up-regulation of tropoelastin in response to some
mediators(41) .
down-regulated tropoelastin expression by suppression of
transcriptional activity, even though the decrease in reporter gene
activity (about 2-fold) did not approach the marked repression of
steady-state mRNA levels (greater than 10-fold). Thus, additional
regulatory mechanisms must be involved to account for TNF
-mediated
down-regulation of tropoelastin expression. Recently, Hsu-Wong et
al.(43) reported that tropoelastin promoter activity in
transgenic mice decreased with age in various tissues suggesting that
the cessation of elastin production is controlled transcriptionally.
The discrepancy between their and our conclusion may be resolved by the
methods used. In their studies, Hsu-Wong et al. did not assess
endogenous levels of tropoelastin production nor was cell-specific
expression verified. Since they used only mice of a single founder
line, artifacts due to the insertion site of the transgene may have
affected their findings. This is an important consideration because
promoter construct activity can be influenced by chromatin structure,
methylation, and cis-acting sequences, which may be quite
distinct from those factors controlling endogenous tropoelastin
transcription. Furthermore, no tissue or temporal-specific elements
have been identified in the tropoelastin promoter, its sequence
resembles that of a ``housekeeping'' gene, and the promoter
is active in nonelastogenic cells(12, 37, 44) .
Thus, although transgenic mice with tropoelastin promoter constructs
may provide reliable reagents for study-ing the induction and
stimulation of elastin production, they may not reflect the endogenous
regulation of the cessation of elastogenesis.
1 (I) procollagen gene.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.