Cyclic Nucleotide Regulation of Type-1 Plasminogen Activator-Inhibitor mRNA Stability in Rat Hepatoma Cells
IDENTIFICATION OF cis-ACTING SEQUENCES*

Joanne H. HeatonDagger , Maribeth Tillmann-Bogush, Nancy S. Leff, and Thomas D. Gelehrter

From the Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta -globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta -globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta -globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta -globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Eukaryotic gene expression is determined not only by the rate of gene transcription, but also by the rate at which the transcript is degraded. The steady state concentration of a particular mRNA, as well as the rate at which a new transcriptionally induced steady state is attained, is directly related to the message half-life (1, 2). Numerous recent studies have been aimed at understanding the role of specific sequences within the mRNA in determining basal transcript stability and have led to the identification of consensus sequences that confer stability or instability to a transcript (2, 3). Although it is known that many stimuli alter mRNA stability, little is known about either the cis-acting sequences in the RNA or the cellular factors involved in regulation of message decay.

Plasminogen activators (PAs)1 are serine proteases that are critical for thrombolysis and also play a key role in other physiological functions involving tissue remodeling. Plasminogen activator-inhibitors (PAIs) are specific inhibitors of PAs and are members of the serine protease inhibitor (serpin) family of proteins. Type-1 PAI (PAI-1) is a 50 kDa glycoprotein found in plasma, platelets, and a variety of cell types; its expression is regulated by growth factors, cytokines, and hormones, including agents that alter cellular cAMP levels (4).

HTC rat hepatoma cells synthesize and secrete tissue-type plasminogen activator (tPA) and PAI-1 (5). We have previously reported that incubation with the synthetic glucocorticoid, dexamethasone, decreases HTC cell tPA activity by increasing PAI-1 mRNA and protein 5-10-fold, an effect that is entirely explained by an increase in PAI-1 gene transcription (5-7). In contrast, these cells respond to cyclic nucleotides with a dramatic (>= 50-fold) increase in tPA activity that is primarily the result of a decrease in PAI-1 mRNA and protein (8). This effect is time- and concentration-dependent; a 90% decrease in PAI-1 mRNA occurs after 12 h of incubation with 1 mM 8-bromo-cAMP plus 1 mM isobutyl-1-methylxanthine (cA) (8, 9). Nuclear run-on experiments have shown that cA causes about a 60% decrease in PAI-1 transcription, not sufficient to account for the decrease in message accumulation. We have determined the half-life of HTC cell PAI-1 mRNA by following the decrease in message level either after inhibition of transcription with actinomycin D or after washing out the transcriptional inducer, dexamethasone. By either method, PAI-1 mRNA displays a half-life of 4.0-4.5 h. Incubation of cells with cA accelerates PAI-1 mRNA decay, decreasing the half-life to 1.5 h. Thus, cyclic nucleotides regulate PAI-1 gene expression at both a transcriptional and posttranscriptional level. Interestingly, although actinomycin D does not alter the basal rate of PAI-1 mRNA degradation, it prevents the cA-induced acceleration of decay; incubation with cyclic nucleotides in the presence of actinomycin D results in a decay rate identical to that seen in the presence of actinomycin D alone (7).

The aim of this study is to understand the mechanism by which cyclic nucleotides regulate PAI-1 mRNA decay by determining the sequences within the PAI-1 transcript that are required for 8-bromo-cAMP regulation. We report here that the 3'-UTR of PAI-1 mRNA can confer cyclic nucleotide regulation on the otherwise stable beta -globin mRNA. Deletion analysis of the PAI-1 3'-UTR reveals the presence of at least two cA-responsive regions; one of these is in the 3'-most 134 nt of PAI-1 mRNA. Furthermore, the 3'-most 134-nt sequence by itself is able to confer cA responsiveness on the heterologous beta -globin mRNA, suggesting that this sequence plays a major role in the hormonal regulation of PAI-1 mRNA stability.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Eagle's minimal essential medium, glutamine, fetal calf serum, calf serum, G418, restriction endonucleases (REs), Taq DNA polymerase, T4 ligase, and TRIzol® were purchased from Life Technologies, Inc. Bovine serum albumin was purchased from Intergen Co. (Purchase, NY). Cycloheximide, 8-bromo-cAMP, and isobutyl-1-methylxanthine were purchased from Sigma. T3 and T7 RNA polymerases, RNase inhibitor, RNase A, and RNase T1 were purchased from Boehringer Mannheim. alpha -32P-labeled UTP (specific activity 800 Ci/mmol) was obtained from Amersham Pharmacia Biotech. The vector pSVL was purchased from Amersham Pharmacia Biotech, and pBluescriptKS(-) and Escherichia coli AG-1 competent bacteria were purchased from Stratagene (La Jolla, CA). Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (10) was a kind gift from Dr. Rodrigo Bravo (Bristol-Myers Squibb). Oligonucleotide primers used in PCR were synthesized by the University of Michigan Biomedical Research Core Facilities.

Constructs for Transfection Analysis-- The vector pSVL, which has an SV40 late promoter upstream and an SV40 polyadenylation signal downstream from the multicloning site (MCS), was used to drive expression of the DNA constructs used in these studies. Because the SV40 late promoter does not appear to be regulated by cyclic nucleotides, this vector allowed us to examine the effects of cA on message decay independent of transcriptional effects. All DNA vectors described below were amplified in E. coli AG-1 competent bacteria by established procedures (11).

Mouse beta -globin genomic DNA from position 233 to 1669 (numbering according to Konkel et al.) (12) was amplified by PCR to include an XbaI site 5' to bp 233 and a BglII site 3' to bp 1669. This product was subcloned into the XbaI and BamHI sites of the pSVL MCS to give pSVL beta G (Fig. 1) (13).

The chimeric construct of rat PAI-1 and mouse beta -globin diagrammed in Fig. 1 was prepared as follows. The 1200-bp coding portion of the mbeta -globin gene was amplified by PCR to include an XbaI recognition sequence 5' to bp 233 and the BglII recognition sequence 3' to bp 1436. The 1730-bp 3'-UTR of PAI-1 was PCR-amplified to generate the sequence with the BglII recognition site 5' to bp 1331 and the XbaI site 3' to bp 3060. (Nucleotides are numbered from the start site of transcription (14).) These PCR products were digested with the appropriate REs and subcloned into XbaI-digested pSVL in a one-step ligation to give the construct called pSVL G/P. The chimeric construct was verified by restriction analysis and partial DNA sequence analysis. The neomycin resistance (neo) gene under control of the cytomegalovirus promoter was subcloned into the EcoRI site of the pSVLbeta G and pSVL G/P as well as pSVL.

Constructs having deletions in the 3'-UTR of PAI-1 (Fig. 3) were prepared from pSVL G/P using convenient RE sites; additional sites in the neo gene precluded preparing the deletions from the vector containing the neo gene. All deletion constructs diagrammed in Fig. 3 were prepared in a similar manner, using the indicated RE digestion to excise a portion of the sequence, incubation with the Klenow fragment of DNA polymerase to create blunt ends, followed by self-ligation using T4 DNA ligase. Because the pSVL vector carries recognition sites for NcoI, the construct G/PDelta AU3A was prepared from pBluescriptKS(-)G/P. In addition, because mbeta -globin has an NcoI recognition site at position 307, BssHII and NcoI digestion of pBluescriptKS(-)G/P creates three fragments. The 4100-bp and 1500-bp fragments were gel-purified (leaving behind the 250-bp fragment to be deleted), incubated with the Klenow fragment of DNA polymerase to make blunt ends, and ligated. The desired deletion and the orientation of the fragments were verified by RE analysis and partial DNA sequencing. In each case, the G/P sequence with the desired deletion was excised from pSVL or from pBluescriptKS(-) using XbaI and subcloned into the XbaI site in the MCS of pSVLneo and the identity of the new construct was confirmed by RE analysis and partial DNA sequencing. Constructs are designated by the regions of the PAI-1 sequence deleted as indicated in the diagram at the top of Fig. 3.

We also generated constructs in which portions of sequence from the PAI-1 3'-UTR were inserted into mouse beta -globin 3'-UTR as illustrated in Fig. 5. To provide a site for insertion of PAI-1 sequence, a BglII recognition site was constructed in the 3'-UTR of beta -globin as follows. PCR was used to amplify the beta -globin gene from bp 233 to bp 1470, including 5' XhoI and 3' BglII sites, and a portion of the beta -globin 3'-UTR from bp 1471 to bp 1668, including 5' BglII and 3' XbaI recognition sites. The products were cloned into the XhoI and XbaI sites of pSVLneo in a one step ligation to produce pSVL G/G. Portions of the PAI-1 3'-UTR were amplified by PCR to include BglII recognition sites on both the 3' and 5' ends. Each PCR product was inserted into the pSVL G/G construct by ligation at the BglII site. Presence of a single insert and orientation were confirmed by PCR and RE analysis.

Constructs for Riboprobes-- A PCR-amplified fragment of mbeta -globin from bp 1256 to bp 1436 (which includes coding and intronic sequence) was subcloned into the PstI and BamHI sites of the pBluescriptKS(-) MCS. When linearized by digestion with HindIII (site in the pBluescriptKS(-)) and transcribed using T7 RNA polymerase, this plasmid generates an antisense riboprobe complementary to 119 nt of the mbeta -globin coding region mRNA (Fig. 1).

The 177-bp XbaI to AccI fragment of rat GAPDH cDNA (10) was subcloned into the MCS of pBluescriptKS(-). This plasmid was digested with BstEII and transcribed using T3 RNA polymerase to generate an antisense riboprobe that protects an 80-nt fragment of GAPDH mRNA.

Preparation of Radiolabeled RNA-- Riboprobes were prepared by published methods (11). Plasmid DNA (approximately 500 ng) linearized with the appropriate RE was incubated with T7 or T3 RNA polymerase in transcription buffer (Boehringer Mannheim) containing RNase inhibitor, ATP, CTP, GTP, and [32P]UTP at 37 °C for 60 min. RNase-free DNase I was added, and, after 15 min at 37 °C, the reaction was extracted with phenol/chloroform (1:1) and the RNA was precipitated in the presence of ammonium acetate and ethanol. The labeled RNA was purified by electrophoresis through a 6% polyacrylamide, 8 M urea gel, eluted from the gel, and ethanol-precipitated.

Cell Culture-- HTC cells were maintained in monolayer cultures as described previously (5). In preparation for experiments, cells were plated in 60-mm tissue culture dishes and grown to confluence. Cultures were washed in sterile phosphate-buffered saline (PBS) and incubated in serum-free medium containing 0.1% bovine serum albumin with or without 0.1 mM cycloheximide. After 10-16 h, the medium was removed, the monolayers washed twice with PBS to remove cycloheximide, and fresh medium with or without cA was added. Cells were harvested at the times indicated for each experiment. For experiments in which the RNA from the transfected genes was to be analyzed, incubation with cycloheximide provides two major advantages. First, cycloheximide significantly enhances expression from the SV40 late promoter (5-50-fold), resulting in easier detection of transcript from the transfected gene (13). Second, cycloheximide can be readily removed and its effects completely reversed by washing the monolayers with PBS and adding fresh medium (15, 16), resulting in a dramatic decrease in SV40 promoter activity. This protocol allowed us to measure mRNA degradation without using actinomycin D, which not only inhibits transcription but also blocks the cA effect on PAI-1 mRNA (7). Cycloheximide is known to alter the decay rates of some transcripts (2); however, the degradation rate of endogenous PAI-1 mRNA (determined using actinomycin D to block transcription) in cells from which cycloheximide has been removed after a 12-h incubation with the inhibitor, is nearly identical to that in cells not treated with cycloheximide. Furthermore, in cells transiently treated with cycloheximide, as in untreated cells, endogenous PAI-1 mRNA decay is accelerated after incubation of the cells with cA.

Transfections-- HTC cells were transfected using the Ca2PO4 DNA precipitation technique, and stably transfected cells were selected in media containing 1 mg/ml G418 as described previously (17). Colonies of stably transfected cells were pooled to minimize the possible effects of different integration sites.

Ribonuclease Protection Analysis-- Total cellular RNA was isolated either by the method of Chomczynski and Sacchi (7, 18) or by the TRIzol® reagent method as described by Life Technologies, Inc. Ribonuclease protection analysis was carried out essentially as described (11). Total cellular RNA (20-40 µg/sample) was precipitated and resuspended in 30 µl of hybridization buffer (40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide) containing 1-5 × 105 cpm of 32P-labeled riboprobe. After 5 min at 85 °C, the samples were placed at 50 °C for 16-20 h. RNase digestion buffer (350 µl of 10 mM Tris-Cl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 20 µg/ml RNase A, and 100 units/ml RNase T1) was added to each reaction. After 30 min at 30 °C, SDS (0.5% final concentration) and proteinase K (125 µg/ml) were added and incubation continued at 37 °C for 30 min. The samples were then extracted with phenol/chloroform and ethanol-precipitated. The RNA pellets were washed with 80% ethanol, allowed to air-dry, and resuspended in an 80% formamide gel-loading buffer. Samples were subjected to electrophoresis through 6% polyacrylamide, 8 M urea gels and the gels were dried under vacuum. The amount of radioactivity in each protected fragment was determined by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). For each sample the signal in the specific band of interest was normalized to the signal in the 80-nt protected fragment of GAPDH.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The 3'-UTR of PAI-1 mRNA Confers Cyclic Nucleotide Responsiveness onto the beta -Globin Transcript-- To determine the sequences in the PAI-1 mRNA involved in the cA regulation of message stability, we stably transfected HTC cells with DNA constructs carrying portions of the PAI-1 sequence and examined the degradation rates of the transcribed mRNAs. The pSVL vector was chosen because the SV40-late promoter, unlike some other viral promoters, does not appear to be regulated by 8-bromo-cAMP.2 To confirm that the cA regulation can be faithfully reproduced in a transfection system, we first prepared a PAI-1 mini-gene using the SstI recognition site to delete 530 bp of the coding region, and subcloned it into the vector pSVLneo. The mRNA transcribed from the PAI mini-gene can be distinguished from the endogenous PAI-1 mRNA in ribonuclease protection analysis using a PAI-1 riboprobe that overlaps the deleted sequence. The half-life of the transcript from the PAI-1 mini-gene is 4.0 h and is decreased to 2.0 h in cells treated with cA (data not shown). These results are nearly identical to those observed for endogenous PAI-1 mRNA (7) and confirm that the cyclic nucleotide regulation of the transfected mini-gene faithfully reflects the regulation of the endogenous gene. These results also confirm that under these conditions cA regulation of message stability can be reproduced in cells that have been transiently treated with cycloheximide.

To determine which regions of PAI-1 transcript are involved in the cA regulation, we prepared chimeric genes of rat PAI-1 and mouse beta -globin. The beta -globin transcript is normally very stable, and its decay is not regulated by cyclic nucleotides. As a first step, we compared degradation rates of transcripts from the beta -globin gene and a chimeric gene containing globin coding region and the full 3'-UTR of PAI-1 using the constructs illustrated in Fig. 1. As shown in Fig. 2, transcripts from the stably transfected pSVL beta G or pSVL G/P gene can be readily detected in ribonuclease protection analysis using the 5'G riboprobe. Because there is no endogenous globin, only the protected 119-nt fragment representing the beta -globin coding region and the 80-nt fragment from the GAPDH control are seen. The 119-nt fragment is not present in the RNA from non-transfected cells (data not shown).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic representation of DNA constructs. Mouse beta -globin genomic DNA from bp 233 to bp 1669 was amplified by PCR, and the 1430-bp product was subcloned into the pSVL MCS to give pSVL beta G. The mbeta -globin coding sequence (1200 bp including intronic sequences, solid bar) and the PAI-1 3'-UTR (1730 bp, open bar) were amplified by PCR to include the restriction endonuclease sites shown. The PCR products were ligated into the pSVL plasmid as indicated to give pSVL G/P. A template for a mbeta -globin riboprobe that will recognize 119 bp of the coding region (5'G riboprobe) was prepared by subcloning a PCR-amplified fragment from bp 1256 to bp 1436 into the MCS of pBluescriptKS(-). triangle , poly(A) signal.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of cA on decay rates of transcripts from chimeric constructs. Following a 10-h incubation in serum-free medium containing 0.1 mM cycloheximide, duplicate monolayer cultures of HTC cells stably transfected with pSVL beta G (A) or pSVL G/P (B) were washed in PBS and incubated in serum-free medium with or without 1 mM 8-bromo-cAMP and 1 mM isobutyl-1-methylxanthine. At the times indicated, the cells were harvested, total RNA isolated, and ribonuclease protection analysis carried out on 20 µg (pSVL beta G) or 40 µg (pSVL G/P) RNA per sample using 32P-labeled riboprobes for 5' globin and for GAPDH (GAP). The sizes of the protected fragments are: globin, 119 nt; GAPDH, 80 nt. Size markers in lane M are DNA from HpaII-digested and 32P-labeled pBR322. Duplicate lanes represent duplicate cultures. C and D, the experiments shown in panels A and B were analyzed using the PhosphorImager. For each sample the signal in the globin protected fragment was normalized to that in the GAPDH fragment, and the data are presented as a percentage of the amount at time zero. bullet , control; triangle , cA.

Quantitative analysis of this experiment is shown in Fig. 2 (C and D). As expected, the beta -globin transcript has a long half-life that is not regulated by cA. In this experiment, the half-life of the transcript from the G/P construct is 8.5 h. Although not as short as PAI-1, this half-life is much shorter than that of beta -globin and is decreased to 3.5 h when cells are incubated with cA, a 2.4-fold cA-induced increase in decay rate. This is nearly identical to the cA effect on the PAI-1 mini-gene mRNA. These results confirm that cA regulation can be reproduced in the transfection system and demonstrate that the 3'-UTR of PAI-1 mRNA can confer this regulation onto the beta -globin transcript. Determinants of mRNA stability have been described in the coding regions of some transcripts (19-21). However, because it is clear from our results that the 3'-UTR of PAI-1 mRNA is sufficient to confer both an increase in degradation rate and cyclic nucleotide responsiveness on a heterologous message, we focused our studies on this region.

Deletion Analysis of the PAI-1 3'-UTR-- In an effort to locate the region within the PAI-1 3'-UTR responsible for cyclic nucleotide responsiveness, we used convenient restriction endonuclease sites to delete selected portions of the PAI-1 cDNA in the context of the G/P construct. The 3'-UTR of PAI-1 mRNA has several sequences of potential interest (14) illustrated in the top portion of Fig. 3. AU-rich elements (AREs) that include AUUUA motifs flanked with U-rich stretches have been implicated in the instability of mRNAs of cytokines and oncogenes (3). The PAI-1 3'-UTR has three AUUUA motifs (ATTTA in the cDNA), two in close proximity at nt 2053 and 2117, and a third farther downstream at position 2728, close to the two 70-nt U-rich stretches (T-rich in the cDNA) (nt 2821-3023). PAI-1 has a highly conserved region in its 3'-UTR; the 127-nt sequence in rat PAI-1 from nt 2506 to nt 2632 is 80% identical in mink, murine, bovine, porcine, and human PAI-1 and shares 85-97% identity when compared pairwise with each of these species (14, 22-26). Finally, PAI-1 has an unusual stretch of 39 GpA dinucleotides between nt 2363 and 2442. The location of these regions within the 3'-UTR, the RE sites that flank them and the designations given them are shown in the top of Fig. 3. Regions "A" and "C" are so named for lack of putative regulatory sequences.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Schematic representation of deletion constructs and effect of 8-bromo-cAMP on mRNA half-lives. The chimeric gene pSVL G/P described in Fig. 1 was the parent plasmid used to prepare constructs with deletions in the PAI-1 3'-UTR. The positions of several unique restriction sites and of certain potentially interesting sequences in the 3'-UTR of PAI-1 are shown at the top of the figure. The closed triangles (black-triangle) indicate the location of ATTTA pentamers, and the closed bar (black-square) represents the 127-bp region that is 80% identical in murine, rat, bovine, mink, porcine, and human PAI-1. GpA and TTTs show the locations of the 39 GpA-repeat and the T-rich regions, respectively. The G/P deletion constructs shown on the lower left side of the figure were prepared using the RE sites as described under "Experimental Procedures" and are designated by the PAI-1 sequence deleted as indicated below the schematic in the top portion of the figure. HTC cells stably transfected with each of the deletion constructs were examined for the effect of cA on the degradation rates of the transcripts as described in the legend to Fig. 2. The mRNA half-lives calculated from several experiments are summarized. The means ± S.E. (for the Globin and G/P constructs, n = 8) or ± S.D. (for the deletion constructs, n = 2 or 3) are shown. The ratio of the message half-life in untreated and cA-treated cells was calculated separately for each experiment and the mean of these ratios is reported in column Control/cA. triangle , poly(A) signal.

Deletion constructs of pSVL G/P were made by restriction enzyme digestion and blunt end ligation as described under "Experimental Procedures" and shown in Fig. 3. HTC cells were stably transfected with each deletion construct, incubated with or without cA, and analyzed by ribonuclease protection analysis using the beta -globin riboprobe as described above. Fig. 3 summarizes the results from experiments with all of the deletion constructs tested, and Fig. 4 shows examples of data from representative individual experiments with three of these deletion constructs. The full-length beta -globin message decays slowly and shows no cA regulation, whereas the transcript from the G/P construct, which has the PAI-1 3'-UTR, is more labile and its degradation is accelerated in cA-treated cells. Interestingly, all of the deletion constructs tested exhibit some degree of cyclic nucleotide regulation, suggesting that two, or possibly more, regions in the 3'-UTR can confer cA regulation. Of particular interest is the construct G/PDelta 1600 (Delta 1331-2926) (Figs. 3 and 4B) that retains only the 3'-most 134 nt of the PAI-1 3'-UTR. The half-life of this transcript was decreased 2.3-fold in cyclic nucleotide-treated cells, demonstrating that the 3'-most 134-nt sequence of PAI-1 is sufficient to confer cA responsiveness. However, the construct G/PDelta U II (Delta 2923-3060), in which only this sequence is deleted, also displays a 2-fold increase in the rate of degradation in the presence of cA, consistent with the presence of another upstream regulatory region (Figs. 3 and 4A). Examination of the other deletion constructs, such as the G/PDelta U I (Delta 2718-2926) (Fig. 4C), failed to locate the upstream element because they all contain the 3'-most 134-nt element, which by itself is sufficient to mediate cyclic nucleotide responsiveness. The deletion construct G/PDelta U I + U II also retains cA regulation, suggesting that the other functional element is located upstream from bp 2714. 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of 8-bromo-cAMP on decay rates of transcripts from deletion constructs. HTC cells transfected with pSVL G/PDelta U II (nt 2923-3060, panel A), pSVL G/PDelta 1600 (nt 1331-2926, panel B), or pSVL G/PDelta U I (nt 2718-2926, panel C) were analyzed for cyclic nucleotide regulation of mRNA degradation as described in the legend to Fig. 2. Ribonuclease protection analysis was carried out on 40 µg of RNA per sample. In each graph duplicate cultures from a single experiment are shown. bullet , control; triangle , cA.

Insertion Analysis: The 3'-most 134 nt of PAI-1 Is Sufficient to Confer Cyclic Nucleotide Regulation-- To more clearly define the cis-acting regions involved in the cA regulation, we prepared constructs in which selected portions of the PAI-1 3'-UTR sequence were inserted into the 3'-UTR of the beta -globin gene (Fig. 5). These constructs were then stably transfected into HTC cells and experiments performed as described above for the deletion constructs. Results from experiments with four of these constructs are shown in Fig. 6. The mRNA transcribed from pSVL G/G, like the beta -globin transcript, is stable and not regulated by cyclic nucleotides (Fig. 6A). However, when the 3'-most 130 nt of PAI-1 sequence (lacking the final 6 A nucleotides; see Fig. 8) was inserted into the globin 3'-UTR (pSVL G/G + U II), the resultant construct was regulated by cA. Fig. 6B shows a composite of three such experiments; the mean half-life of 9 h was decreased to 4 h upon incubation with cA, a 2.2-fold change. The construct pSVL G/G + 1400 nt has an insertion of 1378 nt of the 5' end of the PAI-1 3'-UTR. The transcript from this construct displayed a half-life of 4.7 h, which was decreased to 2.4 h upon incubation with cA (Fig. 6D). In contrast, the message transcribed from pSVL G/G+U I, which has the upstream U-rich region, is not cA-regulated (Fig. 6C). These results confirm that the 3'-most 134-nt region of PAI-1 3'-UTR is sufficient to confer cyclic nucleotide regulation and that there is at least one other regulatory element in the upstream region of the 3'-UTR. The PAI-1 sequence between bp 2770 and bp 2923, containing the upstream U-rich stretch (U I), does not appear to confer cyclic nucleotide regulation.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Schematic representation of PAI-1 insertion constructs. The mbeta -globin sequence from bp 233 to bp 1470 was prepared by PCR to include a 5' XhoI recognition site and a BglII site 3' to bp 1470 (solid bar). The mbeta -globin 3'-UTR from bp 1471 to bp 1669 that includes the polyadenylation signal (triangle ) and a 5' BglII site and a 3' XbaI site was amplified by PCR (hatched bar). The products were cloned into the XhoI and XbaI sites of pSVLneo to give the construct pSVL G/G that has a BglII recognition site 25 bp downstream from the translation stop codon. PAI-1 fragments of interest were prepared by PCR to include BglII recognition sites on both the 5' and 3' ends and were ligated into the BglII site of pSVL G/G. The resultant constructs are designated by the region of the PAI-1 sequence inserted.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of 8-bromo-cAMP on decay rates of transcripts from insertion constructs. Experiments were carried out as described in the legend to Fig. 2 using HTC cells transfected with pSVL G/G (panel A), pSVL G/G + UII (bp 2925-3054, panel B), pSVL G/G + U I (bp 2714-2926, panel C) or pSVL G/G + 1400 nt (1331-2709, panel D). Ribonuclease protection analysis was carried out on 40 µg of RNA per sample. In panel A the average ± S.E. of five experiments (10 cultures) and in panel B the average ± S.E. of three experiments (6 cultures) are shown. In panels C and D, duplicate cultures from a single experiment are shown. bullet , control; triangle  or open circle , cA.

Because the functional cyclic nucleotide-responsive element includes one of the two U-rich regions, we attempted to determine whether this sequence alone was sufficient. Fig. 7 shows the results of experiments carried out with HTC cells stably transfected with constructs that have inserts of either one or both of the U-rich regions, as well as two other upstream portions of the PAI-1 3'-UTR. These results show that neither of the U-rich regions alone (bp 2770-2923 or bp 2925-3024) nor both together (bp 2710-3024), can confer cA responsiveness, and suggest that the final, A-rich, 30 nt of the 3'-most 134-nt sequence is required. Fig. 8 shows the 134-nt cA-responsive sequence with the U-rich and A-rich regions highlighted.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of 8-bromo-cAMP on decay rates of transcripts from insertion constructs. PAI-1 insertion constructs were prepared as described in Fig. 5. The left side of the figure shows the PAI-1 sequence numbers and regions of potential interest (as defined in the legend to Fig. 3) included in each insertion. The ability of the insert to confer cA-induced destabilization of the transcript is shown on the right, where a "+" indicates a control/cA ratio of mRNA half-lives greater than 2.2 and a "-" indicates a ratio of less than 1.4.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Sequence of the 3'-most 134 nt of the rat PAI-1 3'-UTR. The sequence to PAI-1 mRNA from nt 2927 to nt 3060 as reported by Zeheb et al. (14) is shown. The U-rich region is underlined, and the A-rich region is designated by a bold underline.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we have examined the mechanism by which cyclic nucleotides accelerate the rate of rat HTC cell PAI-1 mRNA degradation. We have shown that sequences in the 3'-UTR of PAI-1 can confer both instability and cA regulation of degradation on the otherwise stable and non-regulated mouse beta -globin mRNA. The PAI-1 3'-UTR has at least two cA-responsive elements; one of these elements is in the 3'-most 134 nt, which by itself is able to confer cA regulation on the globin message.

Studies from several laboratories have defined specific elements in the 3'-UTR of transcripts that are responsible for their relative stability. With a few exceptions (2, 27), these have all been instability determinants. The most extensively studied instability determinants are the AU-rich elements, or AREs, found in the 3'-UTR of many highly labile transcripts including those of proto-oncogenes (28, 29), growth factors (30), and cytokines (31, 32). AREs may have either multiple AUUUA motifs resulting in the nonomer UUAUUUA(U/A)(U/A) (28), AUUUA motifs scattered in a generally U-rich sequence, or an AU-rich region without an AUUUA motif (3). In addition, two U-rich domains in the yeast MFA2 3'-UTR appear to be involved in regulation of poly(A) nuclease activity (33) and a 29-nt U-rich sequence in human amyloid precursor protein mRNA has been implicated in regulation of its degradation (34).

Rat PAI-1 has two AUUUAs in close proximity at nt positions 2053 and 2117, but these are not in an otherwise AU-rich region. A third AUUUA sequence is near the two 70-nt U-rich stretches (nt 2821-2892 is 57% U and nt 2952-3023 is 51% U, separated by a 60-nt sequence that is only 20% U), making this region a candidate as an instability determinant. Interestingly, one of these U-rich regions is included in the 3'-most 134-nt fragment that is able to confer both an increase in basal degradation rate and cA regulation of decay on the beta -globin transcript. Although the U-rich region may be involved in the cyclic nucleotide responsiveness, it is not by itself sufficient as seen by the results shown in Fig. 7. Decay rates of transcripts from G/G constructs carrying either one or both of the U-rich sequences, but lacking the last 30 nt, are not regulated by cA. In addition, the upstream functional element(s), is not simply the upstream U-rich sequence, as evidenced by the fact that constructs lacking both U I and U II (G/PDelta U I + U II (Fig. 3) and G/G + 1400 nt (Figs. 6 and 7)) display cA regulation of mRNA decay.

The growing number of reports of hormone, growth factor, and cytokine regulation of mRNA stability reflects the growing realization of the importance of this process in overall regulation of gene expression. Rapid changes in the abundance of a short-lived transcript can be effected by a small change in the half-life (2). There are now several studies that have identified cis-acting sequences and/or trans-acting factors potentially involved in hormonal regulation of degradation. The dramatic stabilization of the frog liver vitellogenin mRNA is mediated by binding of an estrogen-inducible protein, recently identified as the KH domain protein, vigilin, to a 27-nt sequence within the 3'-UTR (35, 36). Tumor necrosis factor-alpha induces a 4-fold stabilization of GLUT1 mRNA in 3T3-L1 pre-adipocytes, which appears to be mediated by a 105-nt GC-rich portion of the 3'-UTR and is correlated with increased protein binding to the 3'-UTR (37). Stabilization of amyloid precursor protein mRNA in activated peripheral blood mononuclear cells is accompanied by an increase in binding of a protein to a 29-nt U-rich element in the 3'-UTR (38). Finally, in rat pituitary cultures, thyroid hormone accelerates degradation of thyrotropin beta  message and causes an increased binding of a cellular factor to a 41-nt portion of the 3'-UTR of the transcript (39). This 41 nt includes a 12-nt consensus sequence found in several unstable mRNAs, including the 3'-most 134 nt of PAI-1 mRNA (11 of the 12-nt consensus at nt 3010-3020).

Agents that elevate cellular cAMP levels also have been shown to regulate mRNA degradation, in some cases stabilizing message (40-42), but more often causing down-regulation of mRNAs. Treatment of rat Sertoli cells with follicle-stimulating hormone causes destabilization of androgen receptor, follicle-stimulating hormone receptor, and G-protein alpha -subunit mRNAs (43-45). In both rat ovary and human endometrial stromal cells, luteinizing hormone/human chorionic gonadotropin receptor mRNA is down-regulated by human chorionic gonadotropin, apparently through accelerated degradation (46, 47). Cyclic nucleotide analogues also destabilize asialoglycoprotein receptor mRNA in HepG2 cells (48), tyrosine aminotransferase in H4 rat hepatoma cells (49), angiotensin type I and type II receptor mRNA (50, 51) and the kidney-specific transcription factor, LFB3, mRNA in porcine kidney cells in culture (52). In two cases, cA regulation of message stability has been associated with binding of proteins to the 3'-UTR of the transcript. Chlorophenylthio-cAMP both stabilizes rat hepatoma cell phosphoenolpyruvate carboxykinase mRNA 10-fold and decreases the binding of a 100-kDa protein to the 3'-UTR of this transcript (53). In hamster smooth muscle cells, isoproterenol destabilizes beta 2-adrenergic receptor mRNA and increases the binding of both an AUF1 related protein and a more specific beta ARB protein to the 3'-UTR of beta 2-adrenergic receptor mRNA (54, 55). Sequences in human and hamster beta 2-adrenergic receptor mRNAs that are A+U-rich have been implicated in both protein binding and regulation of message stability (56, 57). The 134-nt region that we find to confer cA regulation of PAI-1 mRNA stability has both a U-rich and an A-rich region (Fig. 8), similar but not identical to those in beta 2-adrenergic receptor mRNA.

The cyclic nucleotide regulation of PAI-1 mRNA accumulation, first observed in HTC cells (8), has also been demonstrated in rat testicular peritubular cells (58), astrocytes (59) and osteoblasts (60), mink lung epithelial cells (61), human fibrosarcoma cells (62), umbilical vein endothelial cells (63), and synovial cells (64). Our previous experiments have shown that in rat HTC cells, whereas transcriptional regulation plays a role in the cA-induced decrease in PAI-1, the major effect is on message stability. The studies reported here demonstrate that at least two elements in the 3'-UTR of PAI-1 mRNA are involved in regulation of PAI-1 message decay. One of these regulatory elements is the 3'-most 134 nt. We have found that a number of cellular proteins interact with the same PAI-1 sequence that can confer cA regulation,3 suggesting that one or more of these factors may play a role in cyclic nucleotide regulation. Our current studies are aimed at delineating the exact sequences responsible for the cyclic nucleotide regulation of mRNA degradation and defining the binding proteins involved in this regulation, as well as identifying the other cis-acting element(s) in the PAI-1 3'-UTR.

    ACKNOWLEDGEMENT

We thank Erin Janssen for technical assistance during her tenure on a Summer Student Research Program Fellowship from the Michigan Diabetes Research and Training Center.

    FOOTNOTES

* This work was supported by Public Health Service Grant CA22729 from the National Cancer Institute (to T. D. G.) and National Research Service Award DK09437 from the National Institutes of Health (to M. T.-B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Human Genetics, M4708 MSII/0618, University of Michigan Medical School, 1301 E. Catherine St., Ann Arbor, MI 48109-0618. Tel.: 734-763-3460 or 734-764-5491; Fax: 734-763-5831; E-mail: heatonj{at}umich.edu.

1 The abbreviations used are: PA, plasminogen activator; PAI, plasminogen activator-inhibitor; PAI-1, type-1 PAI; UTR, untranslated region; tPA, tissue-type plasminogen activator; PCR, polymerase chain reaction; nt, nucleotide(s); bp, base pair(s); Pipes, 1,4-piperazinediethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; MCS, multicloning site; cA, 8-bromo-cAMP plus isobutyl-1-methylxanthine; ARE, AU-rich element; RE, restriction endonuclease.

2 J. H. Heaton and T. D. Gelehrter, unpublished observation.

3 M. Tillmann-Bogush, J. H. Heaton, and T. D. Gelehrter, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Hargrove, J. L., Hulsey, M. G., and Beale, E. G. (1991) BioEssays 13, 667-674[Medline] [Order article via Infotrieve]
  2. Ross, J. (1995) Microbiol. Rev. 59, 423-450[Abstract]
  3. Chen, C.-Y., and Shyu, A.-B. (1995) Trends Biochem. Sci. 20, 465-470[CrossRef][Medline] [Order article via Infotrieve]
  4. Andreasen, P. A, Georg, B., Lund, L. R., Riccio, A., and Stacey, S. N. (1990) Mol. Cell. Endocrinol. 68, 1-19[CrossRef][Medline] [Order article via Infotrieve]
  5. Gelehrter, T. D., Sznycer-Laszuk, R., Zeheb, R., and Cwikel, B. J. (1987) Mol. Endocrinol. 1, 97-101[Abstract]
  6. Heaton, J. H., and Gelehrter, T. D. (1989) Mol. Endocrinol. 3, 349-355[Abstract]
  7. Heaton, J. H., Kathju, S., and Gelehrter, T. D. (1992) Mol. Endocrinol. 6, 53-60[Abstract]
  8. Heaton, J. H., and Gelehrter, T. D. (1990) Mol. Endocrinol. 4, 171-178[Abstract]
  9. Barouski-Miller, P. A., and Gelehrter, T. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2319-2322[Abstract]
  10. Fort, Ph, Marty, L., Piechaczyk, M., El Sabrouty, S., Dani, Ch, Jeanteur, Ph, and Blanchard, J. M. (1985) Nucleic Acids Res. 13, 1431-1442[Abstract]
  11. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1989) Current Protocols in Molecular Biology, pp. 1.1.1-1.8.9 and 4.7.1-4.7.8, Greene and Wiley Interscience, New York
  12. Konkel, D. A., Maizel, J. V., Jr., and Leder, P. (1979) Cell 18, 865-873[Medline] [Order article via Infotrieve]
  13. Kathju, S. (1994) Regulation of Tissue Type Plasminogen Activator Gene Expression by Glucocorticoids and Cyclic Nucleotides in Rat Hepatoma Cells.Ph.D. dissertation, University of Michigan, Ann Arbor
  14. Zeheb, R., and Gelehrter, T. D. (1988) Gene (Amst.) 73, 459-468[CrossRef][Medline] [Order article via Infotrieve]
  15. Peterkofsky, B., and Tomkins, G. M. (1968) Proc. Natl. Acad. Sci. U. S. A. 60, 222-228[Medline] [Order article via Infotrieve]
  16. Gelehrter, T. D., and Tomkins, G. M. (1970) Proc. Natl. Acad. Sci. U. S. A. 66, 390-397[Abstract]
  17. Kathju, S., Heaton, J. H., Bruzdzinski, C. J., and Gelehrter, T. D. (1994) Endocrinology 135, 1195-1204[Abstract]
  18. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  19. Caponigro, G., Muhlrad, D., and Parker, R. (1993) Mol. Cell. Biol. 13, 5141-5148[Abstract]
  20. Schiavi, S. C., Wellington, C. L., Shyu, A.-B., Chen, C.-Y. A., Greenberg, M. E., and Belasco, J. G. (1994) J. Biol. Chem. 269, 3441-3448[Abstract/Free Full Text]
  21. Wellington, C. L., Greenberg, M. E., and Belasco, J. G. (1993) Mol. Cell. Biol. 13, 5034-5042[Abstract]
  22. Bijnens, A. P., Knockaert, I., Cousin, E., Kruithof, E. K. O., and Declerck, P. J. (1997) Thromb. Haemostasis 77, 350-356[Medline] [Order article via Infotrieve]
  23. Chuang, T.-H., Hamilton, R. T., and Nilsen-Hamilton, M. (1995) Gene (Amst.) 162, 303-308[CrossRef][Medline] [Order article via Infotrieve]
  24. Ginsburg, D., Zeheb, R., Yang, A. Y., Rafferty, U. M., Andreasen, P. A., Nielsen, L., Dano, K., Lebo, R. V., and Gelehrter, T. D. (1986) J. Clin. Invest. 78, 1673-1680[Medline] [Order article via Infotrieve]
  25. Mimuro, J., Sawdey, M., Hattori, M., and Luskutoff, D. J. (1989) Nucleic Acids Res. 17, 8872[Medline] [Order article via Infotrieve]
  26. Prendergast, G. C., Diamond, L. E., Dahl, D., and Cole, M. D. (1990) Mol. Cell. Biol. 10, 1265-1269[Medline] [Order article via Infotrieve]
  27. Weiss, I. M., and Liebhaber, S. A. (1995) Mol. Cell. Biol. 15, 2457-2465[Abstract]
  28. Herrick, D. J., and Ross, J. (1994) Mol. Cell. Biol. 14, 2119-2128[Abstract]
  29. Zubiaga, A. M., Belasco, J. G., and Greenberg, M. E. (1995) Mol. Cell. Biol. 15, 2219-2230[Abstract]
  30. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667[Medline] [Order article via Infotrieve]
  31. Gorospe, M., and Baglioni, C. (1994) J. Biol. Chem. 269, 11845-11851[Abstract/Free Full Text]
  32. Stoecklin, G., Hahn, S., and Moroni, C. (1994) J. Biol. Chem. 269, 28591-28597[Abstract/Free Full Text]
  33. Muhlrad, D., and Parker, R. (1992) Genes Dev. 6, 2100-2111[Abstract]
  34. Zaidi, S. H. E., and Malter, J. S. (1994) J. Biol. Chem. 269, 24007-24013[Abstract/Free Full Text]
  35. Dodson, R. E., and Shapiro, D. J. (1994) Mol. Cell. Biol. 14, 3130-3138[Abstract]
  36. Dodson, R. E., and Shapiro, D. J. (1997) J. Biol. Chem. 272, 12249-12252[Abstract/Free Full Text]
  37. McGowan, K. M., Police, S., Winslow, J. B., and Pekala, P. H. (1997) J. Biol. Chem. 272, 1331-1337[Abstract/Free Full Text]
  38. Zaidi, S. H. E., Denman, R., and Malter, J. S. (1994) J. Biol. Chem. 269, 24000-24006[Abstract/Free Full Text]
  39. Leedman, P. J., Stein, A. R., and Chin, W. W. (1995) Mol. Endocrinol. 9, 375-387[Abstract]
  40. Chedrese, P. J., Kay, T. W. H., and Jameson, J. L. (1994) Endocrinology 134, 2475-2481[Abstract]
  41. Chen, M., Schnermann, J., Smart, A. M., Brosius, F. C., Killen, P. D., and Briggs, J. P. (1993) J. Biol. Chem. 268, 24138-24144[Abstract/Free Full Text]
  42. Huang, D., Hubbard, C. J., and Jungmann, R. A. (1995) Mol. Endocrinol. 9, 994-1004[Abstract]
  43. Blok, L. J., Hoogerbrugge, J. W., Themmen, A. P., Baarends, W. M., Post, M., and Grootegoed, J. A. (1992) Endocrinology 131, 1343-1349[Abstract]
  44. Themmen, A. P. N., Blok, L. J., Post, M., Baarends, W. M., Hoogerbrugge, J. W., Parmentier, M., Vassart, G., and Grootegoed, J. A. (1991) Mol. Cell. Endocrinol. 78, R7-R13[CrossRef][Medline] [Order article via Infotrieve]
  45. Loganzo, F., Jr., and Fletcher, P. W. (1993) Mol. Endocrinol. 7, 434-440[Abstract]
  46. Lu, D. L., and Menon, K. M. J. (1996) Biochemistry 35, 12347-12353[CrossRef][Medline] [Order article via Infotrieve]
  47. Han, S. W., Lei, Z. M., and Rao, C. V. (1997) Biol. Reprod. 57, 158-164[Abstract]
  48. Stockert, R. J. (1993) J. Biol. Chem. 268, 19540-19544[Abstract/Free Full Text]
  49. Smith, J. D., and Liu, A. Y.-C. (1988) EMBO J. 7, 3711-1716[Abstract]
  50. Wang, X., Nickenig, G., and Murphy, T. J. (1997) Mol. Pharmacol. 52, 781-787[Abstract/Free Full Text]
  51. Murasawa, S., Matsubara, H., Kijima, K., Maruyama, K., Ohkubo, N., Mori, Y., Iwasaka, T., and Inada, M. (1996) Hypertens. Res. 19, 271-279[Medline] [Order article via Infotrieve]
  52. Marksitzer, R., Stief, A., Menoud, P.-A., and Nagamine, Y. (1995) J. Biol. Chem. 270, 21833-21838[Abstract/Free Full Text]
  53. Nachaliel, N., Jain, D., and Hod, Y. (1993) J. Biol. Chem. 268, 24203-24209[Abstract/Free Full Text]
  54. Hadcock, J. R., Wang, H., and Malbon, C. C. (1989) J. Biol. Chem. 264, 19928-19933[Abstract/Free Full Text]
  55. Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W. A., Sherman, J. A., Bisognano, J. D., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501[Abstract/Free Full Text]
  56. Danner, S., Frank, M., and Lohse, M. J. (1998) J. Biol. Chem. 273, 3223-3229[Abstract/Free Full Text]
  57. Tholanikunnel, B. G., and Malbon, C. C. (1997) J. Biol. Chem. 272, 11471-11478[Abstract/Free Full Text]
  58. Nargolwalla, C., McCabe, D., and Fritz, I. B. (1990) Mol. Cell. Endocrinol. 70, 73-80[CrossRef][Medline] [Order article via Infotrieve]
  59. Tranque, P., Robbins, R., Naftolin, F., and Andrade-Gordon, P. (1992) Glia 6, 163-171[Medline] [Order article via Infotrieve]
  60. Fukumoto, S., Allan, E. H., Yee, J. A., Gelehrter, T. D., and Martin, T. J. (1992) J. Cell. Physiol. 152, 345-355
  61. Thalacker, F. W., and Nilsen-Hamilton, M. (1992) Biochem. J. 287, 855-862[Medline] [Order article via Infotrieve]
  62. Georg, B., Riccio, A., and Andreasen, P. (1990) Mol. Cell. Endocrinol. 72, 103-110[CrossRef][Medline] [Order article via Infotrieve]
  63. Konkle, B. A., Kollros, P. R., and Kelly, M. D. (1990) J. Biol. Chem. 265, 21867-21873[Abstract/Free Full Text]
  64. DiBattista, J. A., Martel-Pelletier, J., Morin, N., and Jolicoeur, F. C. (1994) Mol. Cell. Endocrinol. 103, 139-148[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.