© 1995 American Heart Association, Inc.
Articles |
Decreased Elastin Synthesis in Normal Development and in Long-term Aortic Organ and Cell Cultures Is Related to Rapid and Selective Destabilization of mRNA for Elastin
From the Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Canada, and the Departments of Biochemistry and Clinical Biochemistry, University of Toronto.
Correspondence to Dr F.W. Keeley, Division of Cardiovascular Research, Hospital for Sick Children, 555 University Ave, Toronto, Canada, M5G 1X8. E-mail fwk@resunix.ri.sickkids.on.ca.
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Abstract |
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Abstract We have previously shown that aortic organ cultures from 1- to 3-day-old chickens initially mimic the high levels of elastin production seen in vivo. However, more prolonged incubation of these tissues results in decreased synthesis of elastin. In the present study, we demonstrate that decreased production of elastin in these aortic organ cultures is selective for elastin compared with collagen and is correlated with decreased steady state levels of mRNA for elastin. These decreases in steady state levels of elastin mRNA are due at least in part to a rapid and selective destabilization of mRNA for elastin, the half-life of which falls from
25 hours in fresh aortic tissues to 15 hours
after incubation for only 8 hours. Destabilization of elastin mRNA can
be prevented by incubation in the presence of blockers of DNA
transcription (5,6-dichlorobenzimidazole riboside and actinomycin D)
and mRNA translation (cycloheximide). Furthermore, the half-life of
aortic elastin mRNA decreases from 25 hours in the 1-day-old
chicken to 7 hours in the 8-week-old chicken, demonstrating that
destabilization of mRNA is an important contributing factor in the
decline in production of aortic elastin taking place during
normal postnatal growth.
Key Words: elastin • mRNA turnover • aorta • organ culture • development
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Introduction |
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Elastin is one of the major structural proteins of large arteries, elastic ligaments, and lung parenchyma, contributing the physical properties of extensibility and elastic recoil, which are crucial for the normal physiological function of these tissues. This connective tissue protein is synthesized as a soluble monomeric precursor called tropoelastin, which is subsequently secreted from the cell and assembled into a highly stable, insoluble, branched polymeric structure in the extracellular matrix through covalent cross-links derived from lysine residues.1 2 Elastin is a major synthetic product of aortic tissues in early stages of postnatal development.3 4 5 6 7 However, synthesis of the protein generally peaks early during arterial growth, decreases rapidly with further development, and essentially ceases in the aortic tissue of adults. During the period of rapid accumulation of aortic elastin, transformation of soluble to insoluble elastin is a rapid and efficient process.3
Although organ cultures prepared from aortic tissue of 1- to 3-day-old chicks mimic the high levels of elastin synthesis, accounting for rapid in vivo accumulation,6 some time ago we noted that tropoelastin synthesis in these organ cultures decreases markedly with longer periods of incubation.8 Similarly, others have observed that smooth muscle cells cultured from aortic tissue lose much of their ability to produce elastin within a few passage numbers.9 In spite of the possible relevance for normal regulation of elastin production, this phenomenon and its mechanism have not been investigated in detail. In the present study, we show that the loss of the synthetic capacity of cultured aortic tissue is selective for elastin and that this effect is related at least in part to a rapid destabilization of mRNA for elastin in the tissue. Furthermore, with normal aortic development, the increased turnover rate of elastin mRNA contributes significantly to the decreased steady state levels of elastin mRNA and decreased elastin production.
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Materials and Methods |
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Aortic tissue from chickens of various ages was dissected as previously described.6 For experiments with a duration of 24 hours or less, organ culture methods were as reported elsewhere.6 Details of incubation conditions for specific experiments are described in the text. For organ cultures of 7 and 14 days, aortic tissue was removed under sterile conditions into medium 199 (GIBCO) containing 10% fetal calf serum, 2% penicillin/streptomycin, and 1% fungizone, gassed with 95% oxygen/5% carbon dioxide, and incubated at 41°C. The medium was changed every 2 days. Aortic smooth muscle cells were grown from explants of 2-day-old chick aortas and cultured under similar conditions in medium 199 containing 10% fetal calf serum and 2% penicillin/streptomycin. The cells were passaged when confluent and used at the second-passage stage.
Synthesis of insoluble elastin and collagen was measured by incorporation of [U-14C]proline (Amersham Canada) into these matrix proteins. After preincubation for varying periods in unlabeled medium, fresh medium containing [14C]proline (0.5 µCi/mL) was added, and the tissues were incubated for a further 3 hours. Tissues were then processed as described previously.10 Briefly, after incubation was complete, the vessels were washed well with unlabeled medium, blotted dry, placed into screw-capped tubes containing CNBr (50 mg/mL in 70% formic acid bubbled with nitrogen), and incubated at room temperature for 24 hours. The insoluble residue remaining after CNBr treatment was washed with boiling distilled water, and the washings were combined with the extract. This residue was solubilized by partial hydrolysis in 5.7 mol/L HCl for 0.5 hour at 110°C, and the hydrolyzate was added to scintillation fluid and counted. We have previously shown that the residue after extraction with CNBr under these conditions is essentially pure elastin.10 Elastin synthesis was expressed as counts per minute incorporated into this elastin residue per milligram of aortic tissue incubated.
The CNBr extract and hot water washings were evaporated to dryness, reconstituted in 5.7 mol/L HCl, and hydrolyzed for 24 hours at 110°C. Collagen synthesis was measured as [14C]hydroxyproline incorporated into proteins in the CNBr extract10 by using a modification of the method of Blumenkrantz and Asboe-Hansen11 and expressed as counts per minute per milligram aortic tissue incubated. Although the CNBr extract contains many proteins, collagen is the only protein in this extract that contains substantial quantities of [14C]hydroxyproline formed by the posttranslational hydroxylation of [14C]proline.
Total RNA was prepared from aortic tissues and cells by extraction with
guanidine thiocyanate and centrifugation through cesium
chloride by using previously published methods.12 Northern
blots of total RNA were sequentially hybridized to cDNA probes for
chicken elastin (supplied by Dr J. Foster, Department of Biochemistry,
Boston University School of Medicine), chicken 
2(I) procollagen
(supplied by Dr Louis C. Gerstenfeld, Harvard Medical School, Boston,
Mass), and human GADPH (American Type Culture Collection), each labeled
with [32P]CTP by random priming. Steady state mRNA levels
were determined by scanning of films with an LKB-Ultragel scanner or a
UVP gel documentation system with NIH Image software. For determination
of half-lives of mRNAs, tissue or cells were incubated in the
presence of DRB (60 µm/L). Total RNA was extracted at the times
indicated, and specific mRNAs were estimated as before by Northern
blotting using cDNA probes for elastin and GAPDH sequentially on the
same blot. Residual mRNA after various incubation times was normalized
to mRNA levels for tissue before DRB addition (time zero).
Half-lives were calculated from the slope of the best-fit
straight line to a semilogarithmic plot of density versus time,
assuming first-order kinetics for decay of mRNAs
(half-life=0.693/slope). Equal loading of lanes for Northern
blotting was confirmed by ribosomal RNA bands visualized by ethidium
bromide staining.
Differences between groups were assessed by ANOVA followed by Duncan's new multiple-range test or by ANCOVA to assess significance of differences between data sets.
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Results |
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Measurements of insoluble elastin production by [14C]proline incorporation over an incubation period of 3 hours as a function of duration of aortic organ culture (Fig 1A
) showed a decreasing capacity of the tissue to
produce insoluble elastin with time. Normalized to fresh tissue (0
hours), elastin production fell to 10% of initial values by
21 hours and was maintained at that level of synthesis for up to 14
days in organ culture. Although the rate of elastin synthesis was
greatly diminished by 21 hours of organ culture, incorporation of
[14C]proline into insoluble elastin remained linear over
a 3-hour labeling period (Fig 1B). This decline in elastin
production was not related to a general deterioration of the
cultured aortic tissue, since production of collagen, another
major connective tissue protein of the vessel wall, was unaffected by
prolonged incubation (Fig 1A).
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This decrease in synthetic capacity for insoluble elastin in the cultured aortic tissue was due neither to depletion of medium components nor to accumulation in the incubation medium of inhibitory factors, since (as we had previously shown for tropoelastin synthesis8 ) neither labeling in fresh medium of tissue previously incubated for 21 hours nor labeling of fresh tissue in conditioned medium from 21-hour incubations had any significant effect on synthesis rates. We have previously shown that efficient elastin production and assembly in fresh aortic organ cultures do not require the addition of fetal calf serum to the incubation medium.3 6 Addition to medium of fetal calf serum up to levels of 7.5% also had no significant influence on the decline in elastin production in these aortic cultures. Furthermore, incubation of aortic tissues in 20% serum prepared freshly from chick blood had no effect.
Both IGF-I and TGF-ß have been reported to increase synthesis of
elastin in cultured aortic smooth muscle cells.13 14 15 16 17 18 19 20 The
possible effects of these growth factors to preserve production
rates of insoluble elastin in aortic organ culture were therefore
investigated. Although small enhancements of collagen and elastin
production were seen in fresh aortic organ cultures (Fig 2), particularly with TGF-ß, neither growth factor
prevented the selective decline in elastin production with
prolonged incubation.
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Since aortic tissues from 1- to 7-day-old chicks are normally incubated intact,6 oxygenation and nutrient supplies to the cultured tissue are dependent on passive diffusion, aided by vigorous shaking during incubation. In order to determine whether decreasing elastin production was due to inadequate diffusion of nutrients, aortic tissues were subdivided before incubation in order to increase surface area for diffusion, and the tissues were cultured in the presence of 95% O2 while vigorous shaking was maintained. The decline in elastin production with incubation time was not altered under these conditions. We have previously shown that careful attention to control of pH in long-term aortic cultures is important, presumably because of high levels of lactate production during in vitro incubation.6 21 However, addition of 5 mmol/L lactate to the culture medium did not affect the synthesis of elastin in freshly isolated tissues.
Northern analysis of total mRNA prepared from aortic organ
cultures after various periods of in vitro incubation showed decreasing
steady state levels of mRNA for elastin (Fig 3). Since
total RNA loading of the gel was approximately equivalent in all lanes,
as determined by staining with ethidium bromide before hybridization,
the proportion of mRNA for elastin appeared to be rapidly declining
during culture to levels 20% to 30% of the steady state levels
detected in fresh aortic tissue. Consistent with elastin and
collagen synthesis data (Fig 1
), stripping and rehybridization of
the same blots with a cDNA probe for collagen showed no substantial
decrease in steady state mRNA levels for this protein over the same
period of incubation (Fig 3). However, reprobing of the blots with cDNA
for GAPDH, a glycolytic pathway enzyme commonly used as a
"housekeeping" message for Northern analysis, showed a
fourfold to sixfold increase in GAPDH mRNA over this period of
incubation (Fig 3).
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Steady state levels of mRNA for elastin were also compared in freshly
isolated aortic tissue, in aortic tissue from 2-day-old chicks
previously cultured for 40 hours, and in second-passage smooth
muscle cells cultured from 2-day-old chick aortas (Fig 4). Again, for equivalent total RNA
loadings, the proportion of mRNA for elastin was severalfold higher in
the fresh tissue compared with aortic tissue cultured for 40 hours or
with second-passage aortic smooth muscle cells. Steady state mRNA
levels for elastin in cultured aortic tissue and cells appeared to be
approximately equal.
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Since steady state mRNA levels are determined by rates of transcription
and rates of decay of message, the effect of organ culture on the
half-life of elastin mRNA was determined. Aortic tissue was used
either freshly isolated from chicks or after a culture period of 8
hours. At that point, DRB (60 µmol/L) was added to the organ culture,
and incubation continued. Tissues were taken at various time points
after the addition of DRB, and total RNA was extracted and probed with
cDNAs for elastin and GAPDH. mRNA was quantified by densitometry, and
mRNA levels were plotted as a function of incubation time after
addition of DRB (Fig 5). The half-life of elastin
mRNA in fresh aortic tissue was 26 hours. In contrast, in aortic
tissue that had been previously cultured for 8 hours, the half-life
of the mRNA for elastin had fallen to 15 hours. The half-life of
elastin mRNA isolated from second-passage smooth muscle cells
cultured from aortas of 2-day-old chickens was <10 hours. In
contrast, the half-life of mRNA for GAPDH was unaffected by time in
organ culture (Fig 5). Similar results were seen when actinomycin D (5
µg/mL) was used as an inhibitor of transcription (Fig 6).
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The fact that the rate of decline of aortic steady state elastin mRNA
with incubation was slower in the presence of DRB or actinomycin D than
in control tissues incubated in the absence of these agents (Fig 6)
suggested that as well as blocking transcription of new elastin mRNA
and thus allowing message decay rates to be estimated, these
inhibitors might also be preventing the transcription of
factors responsible for destabilizing the elastin mRNA.
Consistent with this, the addition of 100 µmol/L
cycloheximide, an inhibitor of translation, to the
incubation medium had a similar effect, essentially completely blocking
the fall in elastin mRNA with incubation time (Fig 6).
These data indicated that destabilization of elastin mRNA was
responsible at least in part for the rapid decline in steady state
elastin mRNA levels in tissue and organ culture. However, it was
not clear whether this effect had any physiological
or developmental significance. Therefore, the half-life of elastin
mRNA isolated from fresh aortic tissue was compared for 18-day embryo
chicks, 2-day-old chicks, and 4- and 8-week-old chickens (Fig 7), a developmental period over which
there is a substantial decline in aortic elastin synthesis and in
steady state levels of elastin mRNA.4 5 The half-life
of aortic elastin mRNA for 18-day-old embryos and 2-day-old
chicks was determined to be 22 to 25 hours, in good agreement with our
previous data for 1-day-old chicks (Fig 5). However, the
half-life of this mRNA had fallen to 10 hours and 7 hours,
respectively, in 4- and 8-week-old chickens, clearly indicating
that decreasing stability of elastin mRNA contributes to decreased
aortic elastin production in growing chickens.
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Discussion |
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The production of aortic elastin is closely regulated with development. Synthesis and accumulation of this matrix protein are very rapid during postnatal growth of arterial vessels but essentially cease after this period of development has been completed. For example, deposition of insoluble elastin in chicken aorta reaches a peak at
2 weeks after hatching, at which time the vessel is
accumulating 3.2 mg of insoluble elastin per day per gram of aortic
tissue.6 Thereafter, production of elastin falls
rapidly, until synthesis is barely detectable by 10 to 12 weeks of
age.4 5 Similar developmental patterns have also been
reported for aortic elastin production in other
species.7 22 23 24 This pattern of synthesis is
consistent with considerable evidence that aortic elastin, once
firmly cross-linked into the extracellular matrix, turns over only
very slowly if at all.25 26 Because of this persistence of
insoluble elastin, it is particularly important that shutdown of
elastin production upon arterial maturation be
complete, since even small residual levels of synthesis would result in
the accumulation of substantial quantities of elastin over the adult
life of the animal. Although it has been reported that mRNA levels for
elastin in aortic tissue generally parallel synthesis of the protein
during aortic development and growth,4 7 27 28 little is
known of the mechanisms for regulation of synthesis of this
protein. The data in the present study demonstrate that there is a rapid decrease in the capacity of chick aortic tissue to produce elastin during organ culture. This decline begins within a few hours of culture and appears to be correlated with a fall in steady state mRNA levels for elastin. This phenomenon appears to be selective for elastin, since message levels and synthesis of type I collagen, another major aortic connective tissue protein, are essentially unchanged, and mRNA levels for GAPDH increase severalfold over the same period of organ culture. Having been established over the first 24 hours of organ culture, these new lower levels of elastin mRNA and production of elastin appear to remain stable for at least 14 days of organ culture, also arguing against a continuing deterioration of the aortic tissue. Furthermore, the proportion of elastin mRNA represented in total RNA extracted from aortic tissue after 40 hours of organ culture appears to be similar to that in second-passage smooth muscle cells cultured from chick aortas of the same age. Decreased steady state mRNA levels for elastin in cell cultures compared with organ cultures of fresh aortic tissue of the same age have also been observed by others.9
Although steady state levels of mRNA are the product of both
transcription and turnover rates, the potential contribution of message
stability to mRNA levels for proteins has only recently become
appreciated. Changes in mRNA turnover have been suggested as a
potential mechanism for the regulation of synthesis for several
proteins, including elastin28 29 30 and
collagen.31 32 Indeed, phorbol ester treatment has been
reported to have a major destabilizing effect on elastin mRNA in ear
cartilage.33 Data from the present study demonstrate
that although the half-life of elastin mRNA is 25 hours in
freshly dissected aortic tissue of 1- to 3-day-old chicks, after
organ culture periods of only 8 hours, the half-life of this
message has decreased to 15 hours. Similarly, the half-life of
elastin mRNA in cultured aortic smooth muscle cells is <10 hours. This
latter value is consistent with the half-life of elastin
mRNA reported by others for aortic smooth muscle cell
cultures.29 In contrast, the half-life of GAPDH mRNA
was unaffected by organ culture. Although our data cannot discount
contributions of alterations in transcription rates, the decline in
steady state mRNA levels for elastin and consequent decreased elastin
production in organ culture is due at least in part to rapid
and selective destabilization of elastin mRNA. Furthermore, elastin
mRNA in smooth muscle cell culture appears to be chronically
destabilized when compared with the in vivo state.
The mechanism for the selective destabilization of elastin mRNA is not clear. Neither accumulation of inhibitory factors nor depletion of nutrients in the medium appears to be important. Although TGF-ß has been reported to increase the half-life of elastin mRNA in cultured cells29 and IGF-I has been suggested to be an important regulator of aortic elastin production,15 18 20 34 neither of these growth factors prevented or attenuated the decline in aortic elastin synthesis in organ culture.
The rapid increase in mRNA for GAPDH suggests a substantial shift of the tissue to glycolysis during organ culture. This effect of culture on GAPDH mRNA adds a further cautionary note to the uncritical use of this mRNA as a reference or housekeeping message.35 Unusually high rates of aerobic glycolysis have been reported in arterial tissues, and we have previously noted that organ cultures of aortic tissue are prolific producers of lactate, requiring careful attention to control of pH in long-term cultures.6 However, the addition of lactate to the organ cultures had no direct effect (ie, a decrease in elastin production) as long as pH was maintained. Although the wall thickness of chick aortas at this age is <1 mm, the rise in GAPDH mRNA suggested that diffusion of oxygen into the tissue might be inadequate for longer-term culture. However, neither increasing surface area for diffusion nor incubation in the presence of 95% oxygen affected the decline in elastin production. Decreased Po2 has been reported to suppress tropoelastin synthesis by smooth muscle cells cultured from neonatal calf pulmonary artery.36 However, the time course and magnitude of this effect make it unlikely that it can explain the decrease in elastin production observed by us in aortic organ cultures. Moreover, the lack of any effect of culture on the production of collagen, measured in this case as radioactive hydroxyproline, suggests that oxygen levels in the tissue are adequate, since hydroxylation of proline in collagen uses molecular oxygen.37 However, we cannot discount the possibility that elastin production is particularly sensitive to tissue oxygen levels through some other mechanism. Indeed, oxygen-sensing mechanisms, which have the ability to affect protein synthesis at least in part through effects on mRNA stability, have been identified in vascular and other tissues.38 39
The fact that both actinomycin D, which blocks transcription, and cycloheximide, which blocks mRNA translation, slow or prevent the decline in steady state elastin mRNA levels in cultured aortic tissue suggests that the production of a protein factor early in incubation may be required for destabilization of elastin mRNA. Alternatively, the effect of cycloheximide to preserve elastin mRNA may also be related to a coupling of mRNA decay to translation, as has been reported for other proteins.40 41
Although these data indicated the potential for control of arterial elastin synthesis at the level of mRNA stability, direct evidence for regulation by this mechanism in physiological or pathological situations was lacking. Aortic elastin synthesis and elastin mRNA levels decline rapidly over the first 8 to 12 weeks of postnatal development and growth in the chicken.4 5 Moreover, multiple mechanisms of regulation of elastin production including both transcriptional and posttranscriptional sites would be expected in order to minimize residual synthesis and accumulation of arterial elastin after development and growth was complete. Therefore, postnatal development might be expected to be a period over which decreased message stability could contribute to decreased elastin production. Indeed, the present data demonstrate that the half-life of elastin mRNA falls substantially over the first 8 weeks of development in the chicken aorta.
Investigations of the relative contributions of transcriptional rates and stability of aortic elastin mRNA to normal developmental patterns of synthesis are presently under way. In this respect, it is interesting to note that transcriptional activity in aortic tissue of transgenic mice of a construct containing 5 kb of the human elastin promoter linked to CAT has recently been reported to remain essentially constant between 5 days and 3 months of life,42 a period over which mouse aortic elastin synthesis is declining rapidly. Although the possibility that elements controlling developmental expression of this gene may be more remotely located cannot be excluded, these data may also provide a further indication of the importance of regulation at the level of mRNA stability in determining changes in elastin production during this postnatal period in both arterial and other elastic tissues.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by operating grants from the Heart and Stroke Foundation of Ontario and the Medical Research Council of Canada. Y. Hew is the recipient of a Studentship Award from the Medical Research Council of Canada.
Received March 7, 1995; accepted August 10, 1995.
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