Regulation of Type I Collagen mRNA by Amino Acid Deprivation in Human Lung Fibroblasts*

(Received for publication, August 22, 1996, and in revised form, January 7, 1997)

Meir Krupsky Dagger , Ping-Ping Kuang and Ronald H. Goldstein §

From the Pulmonary Center and the Department of Biochemistry, Boston University School of Medicine and the Boston Veterans Affairs Medical Center, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The steady state levels of alpha 1(I) collagen mRNA are decreased by retinoic acid and prostaglandin E2. These effector substances decrease the uptake of A system amino acids. We examined the effect of amino acid deprivation on the steady state levels of alpha 1(I) collagen in human lung fibroblasts. Maintenance of fibroblasts in amino acid-free medium decreased alpha 1(I) collagen mRNA levels by 29% at 24 h and 78% at 72 h. Frequent refeeding of cultures with amino acid-free medium resulted in more rapid decreases in intracellular amino acids and in alpha 1(I) collagen mRNA levels. The decrease in alpha 1(I) collagen mRNA levels was mediated by decreases in mRNA stability as assessed by a half-life determination using actinomycin D and by decreases in the rate of transcription as assessed by nuclear run-on assay. Treatment of fibroblasts with medium containing amino acids resulted in rapid restoration of alpha 1(I) collagen mRNA levels. This increase in alpha 1(I) collagen mRNA expression required protein synthesis as determined by cycloheximide sensitivity and was inhibited by prostaglandin E2. These data indicate that alpha 1(I) collagen mRNA levels are sensitive to alterations in the amount of intracellular amino acids and suggest a potential mechanism whereby alpha 1(I) collagen accumulation may be regulated independent of inflammatory mediators following lung injury.


INTRODUCTION

The biosynthesis of type I collagen is complex and involves both intracellular and extracellular sites of regulation. The accumulation of type I collagen by lung fibroblasts is increased by transforming growth factor-beta and insulin and is decreased by prostaglandin E2 (PGE2),1 retinoic acid, and interferon-gamma (1-7). We and others previously reported that both PGE2 and retinoic acid induce large decreases in the expression of alpha 1(I) collagen mRNA by these cells (2-4). These effects require protein synthesis and are mediated by decreases in the rate of transcription of the alpha 1(I) collagen gene as well as by decreases in the stability of the alpha 1(I) collagen mRNA.

PGE2 and retinoic acid decrease the uptake of neutral amino acids transported by A system amino acids in lung fibroblasts (7, 8). The activity of the A system is Na+-dependent and is responsive to hormones, extracellular amino acids, and other effector substances (9, 10). The decreases in amino acid transport were rapid and occurred within 1 h of exposure to the effector substance. In contrast, the decreases in the steady state levels of alpha 1(I) collagen mRNA occurred between 8 and 12 h following exposure. This kinetic relation whereby decreases in amino acid uptake preceded decreases in collagen mRNA levels suggests that depletion of amino acid levels are associated with decreases in alpha 1(I) mRNA levels. Certain other mRNAs are regulated by amino acid availability. For example, asparagine synthetase mRNA is up-regulated by amino acid starvation, whereas beta -actin and glyceraldehyde 3-phosphate are decreased in abundance (11-13).

In these studies, we examined the relation between amino acid depletion and alpha 1(I) collagen mRNA levels in human lung fibroblasts. We find that amino acid depletion caused marked decreases in alpha 1(I) collagen mRNA levels that rapidly reaccumulate following the addition of amino acids. This decrease in alpha 1(I) collagen mRNA was mediated by decreasing the rate of transcription of the alpha 1(I) collagen gene and by decreasing the stability of the mRNA.


MATERIALS AND METHODS

Cells and Tissue Cultures

Human embryonic lung fibroblasts (IMR-90, Institute for Medical Research, Camden, NJ) were grown in Dulbecco's modified Eagle's medium with 0.37 g of sodium bicarbonate/100 ml, 10% fetal bovine serum, 100 units/ml penicillin, 10 µg/ml streptomycin, and 0.1 mM nonessential amino acids. After confluence was reached, the serum content of the medium was reduced to 0.4%. The numbers of cells were determined by triplicate cell counts with an electronic particle counter (Coulter Counter ZM).

RNA Isolation and Northern Analysis

Total cellular RNA was isolated by the single-step method employing guanidinium thiocyanate/phenol/chloroform extraction as described by Chomczynski and Sacchi (14). RNA was quantitated by absorbance at 260 nm. Purity was determined by absorbance at 280 and 310 nm. RNA (10 µg) was electrophoresed on a 1% agarose, 6% formaldehyde gel and transferred to a nitrocellulose filter. RNA loading was assessed by ethidium bromide staining of ribosomal bands fractionated on agarose-formaldehyde gels and by co-hybridization with Gs, which is a constitutively expressed mRNA that codes for a GTP-binding protein (15). Hybridization was performed using 0.5-1.0 × 106 cpm/lane labeled probe (specific activity, 4-10 × 108 cpm/µg), and the filter was washed according to methods described by Thomas (16). The filter was exposed to x-ray film for autoradiography at several different times to ensure that the bands could be quantified by densitometry within the linear range. The loading was verified by probing with an oligonucleotide that specifically identifies the 18 S ribosomal subunit (17). The probes utilized in these experiments were a rat cDNA alpha 1(1) collagen clone (18) that specifically identifies corresponding human alpha 1(1) mRNA, Gs, a GTP-binding protein (kindly provided by Dr. R. Reed, John Hopkins University School of Medicine) (15), and cyclooxygenase-1 (19).

Assessment of Free Amino Acid Pools

Quiescent fibroblast cultures were incubated with fresh medium containing no amino acids at 37 °C for the indicated time periods. Following the incubation, the medium or the cellular material was extracted with 1 ml of 10% trichloroacetic acid. The amount of free amino acids in the supernatant was determined using an automatic amino acid analyzer as described previously (7, 8).

Nuclear Run-on Assay

Medium was removed from 150-mm dishes, and the cells were washed twice with Puck's saline and scraped into a Nonidet P-40 lysis buffer. Following two low speed spins, the pellet was reconstituted in a glycerol buffer. In vitro labeling of nascent RNA and hybridization with cDNAs immobilized on nitrocellulose filters were performed according to the methods outlined by Greenberg and Ziff (20) and Groudine et al. (21). No hybridization occurred to filters containing plasmids without inserts.

Statistics

A Student's t test was used for means of unequal size (22). Probability values <0.05 were considered significant.


RESULTS

Quiescent confluent lung fibroblasts were maintained in medium without amino acids to deplete intracellular amino acids. After varying periods of time, we determined the steady state levels of alpha 1(I) collagen mRNA. Gel loading was assessed by ethidium bromide staining and by probing the filter with an oligonucleotide that recognizes the 18 S ribosomal subunit. The levels of alpha 1(I) collagen mRNA were slightly decreased (29 ± 7%, mean ± S.E., n = 3) in fibroblasts maintained in amino acid-deficient medium for 24 h. Maintenance in amino acid-free medium for 72 h resulted in a large decrease in alpha 1(I) collagen mRNA levels (Fig. 1). The results of five such experiments revealed that amino acid deprivation for 72 h decreased alpha 1(I) collagen mRNA levels by 78% ± 5 (mean ± S.E.).


Fig. 1. The effects of amino acid deficiency on alpha 1(I) collagen mRNA levels. Quiescent fibroblast cultures were untreated or cultured in amino acid-deficient medium for 48 h. The medium was removed and replaced with medium without amino acids (AAF) or medium containing the full complement of 15 amino acids (AAR). After 24 h, the RNA was extracted, electrophoresed, and transferred to nitrocellulose. The filter was probed with a cDNA for alpha 1(I) collagen and an oligonucleotide that identifies the 18 S ribosomal subunit. After probing, an autoradiogram was obtained, and densitometry was performed.
[View Larger Version of this Image (47K GIF file)]

The addition of the usual complement of 15 amino acids present in complete medium restored alpha 1(I) collagen mRNA levels, indicating that the absence of added amino acids was not toxic to the cells (Fig. 1). The availability of amino acids markedly affected alpha 1(I) collagen mRNA levels, but only minimally affected the steady state levels of Gs mRNA (which encodes a GTP-binding protein) and cyclooxygenase-1 mRNA (Fig. 2). The amino acid-mediated induction of alpha 1(I) collagen mRNA levels required protein synthesis. Cycloheximide alone at 5 µM inhibits protein synthesis by greater than 90% (23) and does not affect alpha 1(I) collagen mRNA levels in these cells ((24) and data not shown). However, the presence of cycloheximide blocked the amino acid-induced increase in alpha 1(I) collagen mRNA levels (Fig. 3).


Fig. 2. The effects of alterations in amino acid availability on expression of alpha 1(I) collagen, Gs, and cyclooxygenase-1 mRNAs. In two separate experiments, quiescent fibroblast cultures were maintained in amino acid-deficient medium for 48 h. The medium was removed and replaced with medium without amino acids (AAF) or medium containing the full complement of 15 amino acids (AAR). After 24 h, the RNA was extracted. A, the filter was probed with a cDNA for Gs. The filter was stripped and reprobed for alpha 1(I) collagen mRNA and finally reprobed with an oligonucleotide that identifies the 18 S ribosomal subunit. B, the filter was probed with a cDNA for alpha 1(I) collagen. The filter was stripped and reprobed for cyclooxygenase-1 (Cox 1) mRNA and finally reprobed with an oligonucleotide that identifies the 18 S ribosomal subunit.
[View Larger Version of this Image (42K GIF file)]


Fig. 3. The effect of cycloheximide on amino acid-induced increases in alpha 1(I) collagen mRNA levels. Quiescent fibroblast cultures were cultured in amino acid-deficient medium for 48 h. The medium was replaced with either amino acid-free medium (AAF) or medium containing amino acids (AAR) with or without cycloheximide (CHX). After 24 h, the RNA was isolated, and the expression of alpha 1(I) mRNA was assessed. After probing, an autoradiogram was obtained, and densitometry was performed.
[View Larger Version of this Image (46K GIF file)]

We examined the kinetic relation between the addition of amino acids and the reexpression of alpha 1(I) mRNA. Quiescent fibroblasts were maintained in medium with or without amino acids for 48 h. Northern analysis was performed on amino acid-deficient cells that were refed with serum-free medium containing amino acids. The steady state level of alpha 1(I) collagen mRNA was increased at 8 h (approximately 4-fold as assessed by densitometry of the two major alpha 1(I) collagen mRNA signals) and further increased at 24 h following refeeding with medium containing amino acids (Fig. 4).


Fig. 4. The time course for activation of alpha 1(I) mRNA expression after readdition of amino acids. Quiescent fibroblast cultures were maintained in amino acid-deficient medium without serum for 48 h. For control dishes (time 0), the medium was removed, and RNA was extracted. In other dishes, the medium was replaced by medium containing amino acids, and RNA was extracted at the indicated time interval.
[View Larger Version of this Image (39K GIF file)]

To determine whether frequent refeeding with amino acid-free medium increased the depletion of intracellular amino acids by increasing efflux and preventing reuptake, we determined the amount of free amino acids in the medium and in the intracellular compartment. The fibroblasts were placed in amino acid-deficient medium, and the amount of A system amino acids in the medium was determined after 6 h. We found that glycine, alanine, and lesser amounts of proline accumulated in the medium by efflux (Fig. 5). Small amounts of leucine transported by the L system were also detected in the medium.


Fig. 5. The accumulation of amino acids in the medium after addition of amino acid-free medium. The medium was removed, cells were rinsed three times with Puck's saline, and medium without amino acids was added. After 6 h, the medium was removed, and the levels of glycine, alanine, proline, and leucine in the medium were determined by amino acid analysis.
[View Larger Version of this Image (14K GIF file)]

The intracellular concentration of the A system amino acids glycine, proline, and alanine and the L system amino acid leucine was determined in cells incubated in medium with or without amino acids. The intracellular levels of the amino acids were variably decreased. Glycine decreased by 17%, proline by 64%, alanine by 71%, and leucine by 88% after 12 h (Fig. 6). When the medium was changed at 4 and 8 h to remove amino acids accumulating in the extracellular space from efflux (and therefore potentially available for reuptake), the intracellular levels of glycine, proline, and alanine decreased further, whereas leucine levels were unchanged.


Fig. 6. The intracellular levels of amino acids after addition of amino acid-free medium. The medium was removed, and cells were rinsed three times with Puck's saline and replaced with complete medium (CM) or medium without amino acids (AAF). In additional dishes, the medium without amino acids was replaced after 4 and 8 h (AAFR). After 12 h, the medium was removed, and the intracellular levels of glycine, alanine, proline, and leucine were determined by amino acid analysis.
[View Larger Version of this Image (22K GIF file)]

We examined the effect of rapidly altering the levels of amino acids by frequent refeeding with amino acid-deficient medium on alpha 1(I) collagen mRNA levels. Rapid depletion of intracellular amino acids was accomplished by refeeding the cells every 5 h (four changes) during the 24-h period with amino acid-deficient medium. We assessed the steady state levels for alpha 1(I) collagen and Gs. After 24 h, we found that alpha 1(I) mRNA levels fell by 71% in cells frequently refed with amino acid-free medium (Fig. 7). In contrast, the level of alpha 1(I) mRNA decreased only 39% in cells cultured in amino acid-deficient medium but not frequently refed.


Fig. 7. The effect of refeeding with amino acid-free medium on alpha 1(I) collagen mRNA levels. The medium was removed from cultures of quiescent confluent fibroblasts. The cells were rinsed three times with Puck's saline, and medium without amino acids was added to the dishes (AAF). In additional dishes, the amino acid-free medium was replaced every 5 h (AAFR). After 24 h, the medium was removed, and alpha 1(I) collagen mRNA levels were determined.
[View Larger Version of this Image (54K GIF file)]

To examine the mechanism whereby amino acid depletion decreased collagen mRNA levels, we determined the half-life for alpha 1(I) collagen mRNA and the rate of transcription of the alpha 1(I) gene. We found that amino acid depletion decreased the stability of the alpha 1(I) mRNA (Fig. 8). The half-life of the mRNA was assessed by measuring the decay of the mRNA after the addition of actinomycin D. Linear regression analysis using the results of three such experiments employing a variety of time points (2.5, 5, 7, and 9 h) revealed that amino acid depletion decreased the half-life of the alpha 1(I) mRNA from 9.7 to 4.3 h. Because this decrease in half-life cannot account entirely for the decreases in alpha 1(I) mRNA following amino acid deprivation, we assessed the rate of transcription by a nuclear run-on assay. Nuclei were isolated from fibroblasts maintained in medium with or without amino acids for 48 h. The rate of transcription of the alpha 1(I) gene was reduced by 73% (mean of two experiments) in nuclei obtained from fibroblasts maintained without amino acids. In contrast, the rate of transcription for Gs was unchanged. The addition of amino acids restored the transcription rate for alpha 1(I) collagen (Fig. 9).


Fig. 8. The stability of alpha 1(I) collagen mRNA in fibroblasts maintained in amino acid-deficient medium. Fibroblast cultures were untreated (Control) or cultured in amino acid-deficient medium (AAF) for 48 h. Following the addition of 5 µM actinomycin D, the cells were harvested at various time intervals, and the level of alpha 1(I) collagen mRNA was determined.
[View Larger Version of this Image (41K GIF file)]


Fig. 9. The rate of transcription of the alpha 1(I) collagen gene. Quiescent cultures were maintained in medium with (lane 1) or without amino acids (lane 2). After 48 h, in selected dishes maintained in amino acid-free medium, the medium was replaced with medium containing amino acids (lane 3). After 16 h, the nuclei were harvested, and the level of transcription was assessed. No signal was detected for plasmids without insert.
[View Larger Version of this Image (25K GIF file)]

We examined the effect of PGE2 on levels of intracellular amino acids and on alpha 1(I) collagen levels in cells deprived of amino acids. The addition of PGE2 at 10-7 M to cultures maintained in amino acid-free medium for 8 h further decreased the intracellular levels of glycine by 35% to 75.4 ± 3.5 nmol/106 cells and proline by 20% to 29.5 ± 1.1 nmol/106 cells as compared with cells maintained in amino acid-free medium. PGE2 at 10-7 M also induced a small additional decrease in alpha 1(I) mRNA levels in cells maintained in amino acid-deficient medium (Fig. 10). When fibroblasts were reexposed to amino acids, PGE2 inhibited the restoration of alpha 1(I) mRNA levels by 76 ± 7% (mean ± S.E., n = 3).


Fig. 10. The effect of PGE2 on alpha 1(I) collagen mRNA levels in fibroblasts maintained in amino acid-deficient medium. Confluent quiescent fibroblasts were maintained in amino acid-free medium for 48 h. The medium was removed and replaced with medium without amino acids (AAF) or medium containing the full complement of 15 amino acids (AAR). As indicated, selected cultures also received PGE2 at 10-7 M (lanes 3 and 4) or PGE2 at 10-8 M (lane 5). After 24 h, the RNA was isolated, and the expression of alpha 1(I) collagen mRNA was assessed.
[View Larger Version of this Image (49K GIF file)]


DISCUSSION

We found that the steady state level of collagen alpha 1(I) mRNA was decreased by treatment of fibroblasts with amino acid-deficient medium. In contrast, changes in amino acid availability minimally affected the levels of the constitutively expressed cyclooxygenase-1 and Gs mRNAs. The decrease in alpha 1(I) mRNA levels was dependent on the time of exposure to amino acid-deficient medium and the rate of decrease in intracellular amino acids. Short exposures to amino acid-deficient medium resulted in a 29% decrease in mRNA levels, and prolonged exposure resulted in a 78% decrease. When the intracellular levels of amino acids were rapidly lowered by frequent refeeding with amino acid-deficient medium, the alpha 1(I) mRNA levels decreased more markedly. Frequent refeeding decreased intracellular amino acids by removing amino acids appearing in the medium by efflux and preventing reuptake. The decrease in alpha 1(I) collagen mRNA levels was not the result of toxicity because restoring amino acids to the medium resulted in a rapid restoration of mRNA levels.

Alteration of intracellular amino acid levels affects the steady state levels of several other mRNAs (9-11, 25). The steady state mRNA level for asparagine synthetase is up-regulated by amino acid derivation in baby hamster kidney cells (11, 12). A novel cis-acting element (5'-CATGATG-3') located in the proximal asparagine promoter mediates this effect and binds nuclear proteins (26). However, analysis of the promoter region of the alpha 1(I) collagen gene did not reveal a similar binding site. Other examples of amino acid-regulated mRNAs include Cu-Zn superoxide dismutase, glyceraldehyde 3-phosphate, and histone H2. These mRNAs are decreased by amino acid starvation in rat Fao hepatoma cells (13). In addition, insulin-like growth factor I mRNA levels in hepatocytes is decreased by amino acid deprivation and increased by excess (27).

Certain effector substances regulate gene expression by altering intracellular amino acid levels. Interferon-gamma decreases expression of collagenase and stromelysin by decreasing the intracellular concentration of tryptophan (25, 28). Interferon-gamma also decreases production of alpha 1(I) collagen mRNA (29). However, we found that the addition of tryptophan to amino acid-deficient medium did not restore alpha 1(I) mRNA levels2 (unpublished results) suggesting that one or more other amino acids are necessary to induce this affect.

Depletion of amino acids decreased the rate of transcription of the alpha 1(I) collagen gene and the stability of the mRNA. This process was relatively selective because the rate of transcription of Gs was unchanged by decreased amino acid availability. The molecular mechanism whereby amino acid depletion causes alterations in gene expression and mRNA stability is unknown. The sensing mechanism may involve changes in the level of aminoacylation of tRNA. For example, the rate of protein breakdown in histidine starvation of the Chinese hamster ovary cell is regulated by the levels of aminoacylation of tRNA (30). Studies employing temperature-dependent mutant cell lines suggest that regulation of asparagine synthetase activity may involve tRNA acylation (31, 32). This regulatory mechanism may also affect mRNA levels of selected genes, possibly by altering the production of a transcriptional activator under circumstances of amino acid deprivation. Our results employing cycloheximide also suggest that synthesis of one or more specific proteins (perhaps a transcription factor) is initially required for activation of alpha 1(I) collagen mRNA levels by amino acid exposure. We find that cycloheximide inhibited the induction of alpha 1(I) collagen mRNA following the addition of amino acids to amino acid-deficient cells.

Our previous results showed that PGE2 and retinoic acid decreased the transport of A system amino acids by increasing the Km of the A system transporter (7, 8). Both of these effectors decrease the rate of transcription of the alpha 1(I) collagen gene. Our results employing amino acid-deficient medium suggest that the PGE2-induced decreases in alpha 1(I) collagen mRNA levels may be mediated through decreases in intracellular amino acids. PGE2 further decreased alpha 1(I) collagen levels when fibroblasts were cultured in amino acid-deficient medium, perhaps by inhibited reuptake of amino acids accumulating in the medium by efflux. In addition, PGE2 blocked the restoration of alpha 1(I) mRNA levels following reexposure to amino acids. We previously found that PGE2 was more effective in inhibiting alpha 1(I) collagen accumulation induced by transforming growth factor-beta than that induced by insulin (33). Because insulin is a strong inducer of amino acid transport, it may inhibit the effect of PGE2 by directly stimulating uptake.

Alterations in amino acid availability may affect alpha 1(I) collagen mRNA expression by fibroblasts residing within a biomatrix and in vivo. The steady state levels of alpha 1(I) collagen mRNA decreases in fibroblasts that are placed within collagen gels, perhaps as a result of matrix-fibroblast interactions that decrease the availability of nutrients by alterations in transporter function (34-36). It is noteworthy in this regard that suspension of 3T3 fibroblasts in methylcellulose decreases the rate of transcription of the alpha 1(I) collagen gene and decreases the stability of the alpha 1(I) collagen mRNA (37). In the lung, fibroblasts that reside in the pulmonary interstitium do not express detectable levels of alpha 1(I) mRNA as assessed by in situ hybridization (38). Following lung injury, alpha 1(I) collagen levels rapidly increase, presumably from exposure to fibrogenic cytokines such as transforming growth factor-beta . However, alteration in vascular permeability and proteolytic disruption of the extracellular matrix may provide greater access for amino acids into the interstitium, resulting in fibroblast activation.


FOOTNOTES

*   This work was supported by NHLBI Grant P50HL56386 from the National Institutes of Health and the Veterans Affairs Merit Review Research Program.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    Present address: pulmonary Institute, Sheba Medical Center, Tel-Hashomer 52621 Israel.
§   To whom correspondence should be addressed: The Pulmonary Center, R 312, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4860; Fax: 617-536-8093.
1   The abbreviation used is: PGE2, prostaglandin E2.
2   M. Krupsky, P-P. Kuang, and R. H. Goldstein, unpublished results.

REFERENCES

  1. Fine, A., and Goldstein, R. H. (1987) J. Biol. Chem. 262, 3897-3902 [Abstract/Free Full Text]
  2. Redlich, C. A., Delisser, H. M., and Elias, J. A. (1995) Am. J. Respir. Cell Mol. Biol. 12, 287-295 [Abstract]
  3. Varga, J., Diaz-Perez, A., Rosenbloom, J., and Jimenez, S. A. (1987) Biochem. Biophys. Res. Commun. 147, 1282-1288 [Medline] [Order article via Infotrieve]
  4. Krupsky, M., Fine, A., Berk, J., and Goldstein, R. H. (1994) Biochim. Biophys. Acta. 1219, 335-341 [Medline] [Order article via Infotrieve]
  5. Goldstein, R. H., Poliks, C. F., Pilch, P. F., Smith, B. D., and Fine, A. (1989) Endocrinology 124, 964-970 [Abstract]
  6. Jimenez, S. A., Freundlich, B., and Rosenbloom, J. (1984) J. Clin. Invest. 74, 1112-1117 [Medline] [Order article via Infotrieve]
  7. Krupsky, M., Fine, A., Berk, J. L., and Goldstein, R. H. (1993) J. Biol. Chem. 268, 23283-23288 [Abstract/Free Full Text]
  8. Goldstein, R. H., Sakowski, S., Meeker, D., Franzblau, C., and Polgar, P. (1986) J. Biol. Chem. 261, 8734-8737 [Abstract/Free Full Text]
  9. Christensen, H. N. (1990) Physiol. Rev. 70, 43-77 [Free Full Text]
  10. Guidotti, G. G., Borghetti, A. F., and Gazzola, G. C. (1978) Biochim. Biophys. Acta. 515, 329-366 [Medline] [Order article via Infotrieve]
  11. Hutson, R. G., and Kilberg, M. S. (1994) Biochem. J. 304, 745-750 [Medline] [Order article via Infotrieve]
  12. Kilberg, M. S., Hutson, R. G., and Laine, R. O. (1994) FASEB J. 8, 13-19 [Abstract/Free Full Text]
  13. Shay, N. F., Nick, H. S., and Kilberg, M. S. (1990) J. Biol. Chem. 265, 17844-17848 [Abstract/Free Full Text]
  14. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  15. Jones, D. T., and Reed, R. R. (1987) J. Biol. Chem. 262, 14241-14249 [Abstract/Free Full Text]
  16. Thomas, P. S. (1983) Methods Enzymol. 100, 255-266 [Medline] [Order article via Infotrieve]
  17. O'Neill, L., Holbrook, N. J., Fargnoli, J., and Lakatta, E. G. (1991) Cardioscience 2, 1-5 [Medline] [Order article via Infotrieve]
  18. Genovese, C., Rowe, D., and Kream, B. (1984) Biochemistry 23, 6210-6216 [Medline] [Order article via Infotrieve]
  19. Roy, R., Polgar, P., Wang, Y., Goldstein, R. H., Taylor, L., and Kagan, H. M. (1996) J. Cell. Biochem. 62, 411-417 [CrossRef][Medline] [Order article via Infotrieve]
  20. Greenberg, M. E., and Ziff, E. B. (1984) Nature 311, 433-438 [Medline] [Order article via Infotrieve]
  21. Groudine, M., Peretz, M., and Weintraub, H. (1981) Mol. Cell. Biol. 1, 281-288 [Medline] [Order article via Infotrieve]
  22. Snedecor, E. W., and Cochran, W. G. (1967) Statistical Methods, 6th Ed., pp. 59 and 172, Iowa State University Press, Ames, IA
  23. Goldstein, R. H., Wall, M., Taylor, L., Cahill, M., and Polgar, P. (1984) Prostaglandins 28, 717-729
  24. Fine, A., Matsui, R., Zhan, X., Poliks, C. F., Smith, B. D., and Goldstein, R. H. (1992) Biochim. Biophys. Acta 1135, 67-72 [Medline] [Order article via Infotrieve]
  25. Yufit, T., Vining, V., Wang, L., Brown, R. R., and Varga, J. (1995) J. Invest. Dermatol. 105, 388-393 [Abstract]
  26. Guerrini, L., Gong, S. S., Mangasarian, K., and Basilico, C. (1993) Mol. Cell. Biol. 13, 3202-3212 [Abstract]
  27. Thissen, J. P., Pucilowska, J. B., and Underwood, L. E. (1994) Endocrinology 134, 1570-1576 [Abstract]
  28. Varga, J., Yufit, T., and Brown, R. R. (1995) J. Clin. Invest. 96, 475-481 [Medline] [Order article via Infotrieve]
  29. Czaja, M. J., Weiner, F. R., Eghbali, M., Giamborne, M-A., Eghbali, M., and Zern, M. A. (1987) J. Biol. Chem. 262, 13348-13351 [Abstract/Free Full Text]
  30. Scornik, O. A. (1984) Fed. Proc. 43, 1283-1288 [Medline] [Order article via Infotrieve]
  31. Moore, P. A., Jayme, D. W., and Oxender, D. L. (1977) J. Biol. Chem. 252, 7427-7430 [Abstract]
  32. Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633 [Medline] [Order article via Infotrieve]
  33. Fine, A., Poliks, C. F., Donahue, L. P., Smith, B. D., and Goldstein, R. H. (1989) J. Biol. Chem. 264, 16988-16991 [Abstract/Free Full Text]
  34. Grinnell, F. (1994) J. Cell Biol. 124, 401-404 [Medline] [Order article via Infotrieve]
  35. Eches, B., Mauch, C., Hüppe, G., and Krieg, T. (1993) FEBS Lett. 318, 129-133 [CrossRef][Medline] [Order article via Infotrieve]
  36. Riikonen, T., Westermarck, J., Koivisto, L., Broberg, A., Kähäri, V-M., and Heino, J. (1995) J. Biol. Chem. 270, 13548-13552 [Abstract/Free Full Text]
  37. Dhawan, J., Lichtler, A. C., Rowe, D. W., and Farmer, S. R. (1991) J. Biol. Chem. 266, 8470-8475 [Abstract/Free Full Text]
  38. Lucey, E. C., Ngo, H. Q., Agarwal, A., Smith, B. D., Snider, G. L., and Goldstein, R. H. (1996) Lab Invest. 74, 12-20 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.