©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmental Regulation of Elastin Production
EXPRESSION OF TROPOELASTIN PRE-mRNA PERSISTS AFTER DOWN-REGULATION OF STEADY-STATE mRNA LEVELS (*)

Mei H. Swee (1), William C. Parks (1)(§), Richard A. Pierce

From the (1)Departments of Medicine (Dermatology) and Cell Biology and Physiology, Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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 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.

To circumvent these problems, we developed a rapid and sensitive reverse transcription-polymerase chain reaction (RT-PCR) 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.


EXPERIMENTAL PROCEDURES

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.

The 5` forward and 3` reverse-complement PCR primers for amplification of rat 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).


RESULTS

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).

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.


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 1(I) Procollagen Expression

As a positive control for the RT-PCR assay, we assessed the expression of 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.




DISCUSSION

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 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) .

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- 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.

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.


FOOTNOTES

*
This work was supported by Grants HL-48762 and HL-29594 from the National Institutes of Health, a Genentech/American Lung Association Career Investigator Award, a Research Grant and a Career Development Award from the Dermatology Foundation, and a Grant-in-Aid from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dermatology Div., Jewish Hospital, 216 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-454-7543; Fax: 314-454-5372; E mail: bparks@imgate.wustl.edu.

The abbreviations used are: kb, kilobase(s); RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated; nt, nucleotide(s).


ACKNOWLEDGEMENTS

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 1 (I) procollagen gene.


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