The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wajeman-Chao, S. A.
Right arrow Articles by Chambers, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wajeman-Chao, S. A.
Right arrow Articles by Chambers, D. A.
The Journal of Immunology, 1998, 161: 4825-4833.
Copyright © 1998 by The American Association of Immunologists

Mechanism of Catecholamine-Mediated Destabilization of Messenger RNA Encoding Thy-1 Protein in T-Lineage Cells1

Sophie A. Wajeman-Chao*, Susan A. Lancaster*, Lloyd H. Graf Jr.{dagger},{ddagger} and Donald A. Chambers2,*,{dagger}

* Department of Biochemistry and Molecular Biology, {dagger} Center for Molecular Biology of Oral Diseases, and {ddagger} Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ig superfamily cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans is hypothesized to function in cell adhesion and signal transduction in T cell differentiation, proliferation, and apoptosis. This study analyzes effects of cAMP and catecholamines on transcriptional Thy-1 gene expression. Incubation of murine thymocytes or S49 mouse thymoma cells with dibutyryl-cAMP, 8-bromo-cAMP, cholera toxin, norepinephrine, or isoproterenol caused time- and concentration-dependent decreases in levels of Thy-1 mRNA assayed by Northern hybridization or T2 nuclease protection. After 4 h of treatment with 500 µM dibutyryl-cAMP or 8-bromo-cAMP, 1 nM cholera toxin, 100 µM norepinephrine, or 100 µM isoproterenol, Thy-1 mRNA levels were 60 to 96% lower than those of controls. Norepinephrine-mediated decreases in Thy-1 mRNA levels were prevented by the ß-adrenergic receptor antagonist propranolol (10 µM). Dibutyryl-cAMP and norepinephrine decreased the apparent half-life of S49 cell Thy-1 mRNA from >>6 h to 2 to 3 h, whereas nuclear run-on assays showed no cAMP or norepinephrine effect on de novo transcription of the Thy-1 gene. In mutant S49 cells lacking cAMP-dependent protein kinase A, neither dibutyryl cAMP nor norepinephrine affected Thy-1 mRNA levels. These observations show that exogenous cAMP and norepinephrine can induce decreases in steady state Thy-1 mRNA levels in T-lineage cells through posttranscriptional destabilization of Thy-1 mRNA, associated with protein kinase A-mediated protein phosphorylation. Catecholamine-mediated ß-adrenergic protein kinase A-dependent Thy-1 mRNA destabilization may be an example of a more general mRNA decay system regulating cellular responses to stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hypothalamic-pituitary-adrenal axis and efferent branches of the human sympathetic nervous system are activated in response to psychogenic stress, resulting in increased release of immunomodulatory neurosteroids and catecholamines (CAs)3 (1, 2, 3, 4). The adrenergic CA neurotransmitter, norepinephrine (NE), is discharged locally from sympathetic nerve terminals in synapse-like junctions with immune cells in spleen at concentrations as high as 1 µM (5); close associations have also been identified in thymus and lymph nodes (6). In response to stress, NE and the related sympathetic CA epinephrine are also released into the bloodstream, where they act hormonally on circulating immune cells. Recent reports have noted that human and murine lymphocytes synthesize and internalize CAs (7, 8, 9) and can contain intracellular concentrations of CAs as high 10-4 M (8). Thus, during a stress response, it is likely that immune cells are exposed to CAs at supraphysiologic concentrations as much as three to four orders of magnitude greater than found normally in the circulation.

Work in this and other laboratories has revealed that the modulation of immune function by CAs is pleiotypic and affects a variety of cells of the immune system, including T cells, B cells, and NK cells (reviewed in Refs. 1, 2, 4, and 10). Our previous studies have shown that CAs and cAMP inhibit activation of T cells (10, 11, 12, 13) and the generation of antitumor immunity (14). The studies reported in this paper extend this work from the level of the cell to that of the gene by investigating mechanisms of CA-mediated regulation of the genetic expression of the cell surface protein, Thy-1, the parent member of the Ig supergene family (15).

We chose to study the Thy-1 gene for a number of reasons. 1) The Thy-1 gene and its product protein are widely conserved throughout evolution, are found in mice and humans, and are expressed in both the nervous and immune systems (16, 17). In mice, Thy-1 protein is prominently expressed on the surfaces of thymocytes, is expressed basally on various T-lineage cell lines, and can be induced on B cells (15, 16, 17, 18, 19). 2) Expression of Thy-1 protein by thymocytes and T cells is associated with multiple roles for Thy-1 in immune cell function including cell-cell recognition, adhesion, differentiation of thymocytes into T cells, T cell activation (18, 20, 21, 22, 23, 24, 25, 26, 27), T cell apoptosis (28, 29), and the hyperplasia of B lymphocytes that express an introduced Thy-1 gene in transgenic mice (30, 31). 3) Thy-1 serves as a prototype for receptors and other similar surface proteins common to both the nervous and the immune systems; domains of the Thy-1 molecule resemble Ig-like domains of other prominent lymphocyte cell surface proteins including CD2, CD3, CD4, CD7, CD54, and the neuronal cell adhesion protein/NK cell antigen, N-CAM (CD56). 4) Thy-1 protein is linked to membranes through a glycophosphoinositol (GPI) linkage (32, 33, 34) in close proximity in T cells and thymocytes to other important GPI-linked cell surface molecules such as LFA-3 and CD59, and also to a number of membrane-spanning and intracellular membrane-associated molecules involved in T cell activation, including LFA-1, the TCR complex, CD4, CD8, Src kinases, and others (32, 33, 34). 5) The Thy-1 gene has been cloned and sequenced, and appropriate probes for studying its regulation can be constructed or obtained (35, 36, 37). In this report, we show that levels of Thy-1 mRNA in mouse thymocytes can be regulated posttranscriptionally at the level of mRNA stability by CAs acting though a cAMP/protein kinase A (PKA)-dependent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of murine thymocytes and spleen cells

Specific pathogen-free male BALB/c mice (Harlan Laboratories, Indianapolis, IN, or The Jackson Laboratory, Bar Harbor, ME), 4 to 6 wk old, were killed by cervical dislocation, and thymuses or spleens were surgically removed and minced. Thymocytes or splenocytes were isolated by passing the minced preparations though nylon mesh into RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 50 µM 2-ME (Sigma Chemical, St. Louis, MO). Cells were washed in PBS and counted in a hemocytometer. Cell viabilities, determined by trypan blue exclusion, were at least 95% at the initiation of experiments.

Chemicals

NE, isoproterenol (ISO), propranolol, dibutyryl cAMP (DBcAMP), 8-bromo-cAMP (8-BrcAMP), CT, Con A, actinomycin D, 2-ME, diethyl pyrocarbonate, ethidium bromide, proteinase XI, RNase A, and the sodium salt of dextran sulfate were obtained from Sigma. T7 RNA polymerase was obtained from Boehringer Mannheim, Indianapolis, IN. RNase T2, the large fragment of Escherichia coli DNA polymerase 1, and restriction endonucleases PstI, XbaI, and KpnI were obtained from Life Technologies. [methyl-3H]Thymidine, [{alpha}-32P]dCTP, [{alpha}-32P]UTP, and [{alpha}-33P]UTP were obtained from Amersham, Arlington Heights, IL.

Cell culture

All cell cultures were maintained in a humidified atmosphere containing 5% CO2 at 37°C. Thymocytes and spleen cells were cultured for indicated times at initial densities of 5 x 106 cells/ml in 100-mm plates (Corning, Elmira, NY) in serum- and protein-free RPMI 1640 medium containing 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Life Technologies). BALB/c mouse S49 T lymphoma wild-type cells (wt) (38), adenylyl cyclase (AdCy)-associated stimulatory G protein (Gs)-deficient (cyc-) (39), and PKA-deficient (kin-) (40) mutant S49 cell clones (University of California, San Francisco Cell Culture Facility, San Francisco, CA) were grown in high glucose DMEM (Life Technologies) supplemented with 10% heat-inactivated horse serum or FBS (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate; they exhibited viabilities of at least 92% under these culture conditions. When thymocytes or S49 cells were treated for the standard time (4 h) or indicated times from 1 to 12 h with DBcAMP or 8-bromo cyclic AMP (8BrcAMP) (500 µM), with 10-9 M CT (Sigma), with 100 to 400 µM NE or ISO, wwith 10 µM propranolol, or with 2 to 10 µg/ml {alpha}-amanitin (Boehringer Mannheim), their viabilities remained at 80% or greater. NE and ISO were added to cells from freshly prepared 100 mM stocks in 10 mM HCl. Propranolol was added to cultures from aliquot-frozen 10 mM stocks in serum-free RPMI 1640 medium.

RNA purification

Preparatory to RNA isolation, thymocytes and spleen cells were cultured for indicated times at 5 x 106 cells/ml in RPMI 1640 medium. S49 cells were cultured for indicated times at an initial density of 1 x 106 cells/ml in DMEM containing horse serum or FBS and antibiotics.

Total cellular RNA was extracted either by the guanidinium thiocyanate/cesium chloride centrifugation method for tissues with high RNase content (41) or by the acid guanidinium thiocyanate-phenol-chloroform method (42); the two methods provided similar yields and high purities and integrities of nucleic acids indicated by OD260/280 nm, DNase digestion, and ethidium bromide staining and UV transillumination of electrophoretically fractionated RNAs.

Northern hybridization

Purified RNAs were denatured and subjected to electrophoresis in formaldehyde-containing 1.0% agarose minigels (43). Gels were then rinsed in water and either stained with ethidium bromide (0.9 µg/ml in water) and visualized under UV transillumination (305 nm) to determine m.w. and integrity of RNA samples or transferred with 20x SSC to nylon membranes (Biotrans; ICN, Irvine, CA) overnight and baked at 80°C at a vacuum of 15 in Hg in a vacuum oven (National Appliance, Portland, OR) for 1 to 2 h. Baked filters were prehybridized and hybridized at 42°C according to the recommendations of the supplier (ICN) in 8- and 4- to 8-ml volumes, respectively, with the addition of 10% sodium salt of dextran sulfate. From 1 to 2 x 107 cpm of an 0.98-kb 32P-labeled PstI fragment of a BALB/c Thy-1.2 genomic clone in M13 mp8 phage (encompassing translated exon 3, and part of exon 4, as well as the intron that separates these exons, a gift from Dr. J. Buxbaum, New York VA Medical Center, New York, NY (37), was present as the hybridization probe. Probes were labeled to specific activities of 1 to 10 x 108 cpm/µg by the random oligonucleotide primer method (44). Hybridizations were conducted at 42°C overnight. Filters were washed and exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) with intensifying screens at -70°C for 1 to 7 days. To test hybridization with a control probe, Thy-1 probe was eluted from the filters, which were then rehybridized with ~107 cpm of a 32P-labeled XbaI-KpnI fragment of a mouse ß-actin (cytoplasmic actin) genomic clone (45) or with 5–8 x 106 cpm of a 32P-labeled EcoRI fragment of a human 18S rRNA genomic clone (46), a gift from Dr. Geula Gibori (Department of Physiology and Biophysics, University of Illinois Chicago, IL). All experiments were performed at least three times.

RNase T2 protection assay

To construct a vector for synthesis of Thy-1 protection probes, the 0.98-kb PstI fragment of the Thy-1.2 genomic clone described above (37) was isolated and subcloned into the PstI site of pGEM-1, and plasmids from isolated colonies were sequenced to determine orientation of inserts. Plasmid DNA was cut with SacI and religated to remove a 612-bp SacI fragment comprised mainly of intervening sequences. The resulting 374-bp fragment contains 317 bp of exon 3 coding sequence and 57 bp of the intron immediately upstream (35). The Thy-1 plasmid was linearized with PstI, extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8) at a final concentration of 1 mg/ml, and used to direct synthesis of [32P]UTP-labeled negative strand RNA probes by T7 RNA polymerase. These procedures, as well as digestion of template with RNase-free DNase, were performed as described by Costa et al. (47). Total cellular RNA (20 µg), enriched where indicated in the figure legends for poly(A)+ sequences using the poly(A) Tract mRNA isolation system of Promega (Madison, WI), was hybridized with 5 µl (about 2 x 105 dpm) of negative strand RNA probe (47). Digestion of the hybridization product with RNase T2 (18 units) and electrophoresis on 8% polyacrylamide denaturing gel were as described (47). The 317 exon 3-derived bases of labeled negative strand Thy-1 transcript were protected in these studies.

An 0.3-kb fraction of mouse ß-tubulin cDNA, provided by Dr. S. Ross (Department of Microbiology, University of Pennsylvania, Philadelphia, PA), was used as a control probe in protection assays to analyze mRNA stability. A 64 base fragment of the mouse {gamma}-actin gene (45, 48) was used as the control probe for other protection assays. All experiments were performed at least three times.

Nuclear run-on assays

Nuclear extracts were prepared using a standard Dounce homogenization protocol (49) and were stored frozen at -70°C in 200-µl aliquots. Thy-1.2 genomic gene plasmid (a gift from Dr. F. Grosveld, National Institute for Medical Research, London, U.K.), control probe (a genomic clone for the human 28S rRNA gene supplied by Dr. M. Cullum, Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago), and control plasmid (pBluescript; Stratagene Cloning Systems, La Jolla, CA) were purified using spin columns (Qiagen, Chatsworth, CA), digested with RNase A and proteinase XI, extracted twice with phenol-chloroform, precipitated with ethanol, rinsed, and linearized. The probes were bound to nitrocellulose membranes as described (49). Extension of nuclear transcripts with incorporation of [33P]UTP, hybridization of purified transcripts with membrane-bound plasmids, and washing and RNase digestion of the hybridized membranes were as described (49), with the exception that phenol-chloroform-extracted RNAs were purified using Sephadex G-25 spin columns for RNA (5 Prime->3 Prime, Boulder, CO). All experiments were performed at least twice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cAMP on Thy-1 mRNA levels in S49 cells and thymocytes

Previous studies in our laboratory revealed that cAMP can act as a second messenger to affect expression of Thy-1 protein in mouse T-lineage cells (50). Thus, we questioned whether cAMP and/or CAs also affect the steady state levels of Thy-1 mRNA in thymocytes and S49 T lymphoma cells. RNase protection assays performed on poly(A)+ RNAs from S49 cells incubated in the presence or absence of the cAMP analogue, DBcAMP, showed that 4-h treatments of S49 cells or BALB/c thymocytes with 500 µM DBcAMP can lead to decreases in cellular Thy-1 mRNA levels of 90% or more (Fig. 1GoA). In contrast, in S49 cells DBcAMP did not affect levels of mRNA for the "housekeeping" {gamma}-actin gene (Fig. 1GoB). Although butyrate can have cAMP-independent effects on cellular differentiation (51, 52), we found that treatment of S49 cells with 1 mM sodium butyrate for 4 h did not diminish Thy-1 mRNA levels (results not shown). Addition of 500 µM 8BrcAMP to S49 cells for 4 h also decreased Thy-1 mRNA levels by 80% (results not shown). We routinely used 500 µM DBcAMP (a concentration standard for studies of this kind) which maximally decreased S49 cell and BALB/c thymocyte Thy-1 mRNA in 4 h, while not affecting cellular viability or incorporation of [14C]leucine into cellular protein (50) to assay effects of exogenous cAMP on Thy-1 mRNA levels. Raising endogenous levels of cAMP by incubating S49 cells with CT (10-9 M for 4 h), which activates AdCy leading to increased endogenous cAMP (53), decreased Thy-1 mRNA levels but did not significantly decrease ß-actin mRNA (Fig. 2Go). Similar responses to CT and 8BrcAMP were observed with BALB/c thymocytes (not shown).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Effects of DBcAMP on Thy-1 mRNA concentration in S49 cells and BALB/c thymocytes. S49 cells and BALB/c thymocytes were incubated in medium alone or with DBcAMP (500 µM) for 4 h, and poly(A)+ RNAs (from 20 µg of total cellular RNA) were tested for their ability to protect the 317 exon-derived bases of the Thy-1.2 and 64 bases of the {gamma}-actin negative strand probes. The symbols (-) and (+) indicate the absence or presence of cAMP, respectively. A, Thy-1 probe protected by RNAs in one representative experiment of three yielding similar results. Autoradiographic bands were scanned with an Ambis recording densitometer. Bars representing relative amounts of antisense Thy-1 probe protected by RNAs from DBcAMP-treated cells as percentages of protected probe in DBcAMP-untreated cells (100%) are shown directly below the corresponding lanes of the autoradiogram. B, protection of {gamma}-actin probe by the same S49 poly(A)+ RNA analyzed in A.

 


View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of CT on Thy-1 mRNA levels in S49 cells. S49 cells were cultured for 4 h in the presence or absence of 10-9 M CT. Total cellular RNAs were electrophoretically fractionated on formaldehyde-agarose gels (20 µg of RNA/lane) and transferred to nylon membranes. Transfer membranes were hybridized to 32P-labeled 0.98-kb Thy-1.2 probe, 1 to 10 x 108 cpm/µg by autoradiography. After scanning and image analysis, membranes were stripped and rehybridized with ß-actin probe of similar specific activity. Amounts of Thy-1 or ß-actin mRNA in CT-treated cells are plotted as percentages of the amount of mRNA from untreated cells (100%) as bars beneath the corresponding bands. Data are from one representative experiment of three yielding similar results.

 
To examine the time course of cAMP effects on Thy-1 mRNA levels, S49 cells were grown in the presence or absence of 500 µM DBcAMP for periods up to 4 h, and Northern blot analyses were performed on total RNAs. Thy-1 mRNA levels in S49 cells were decreased by 50% after 2 h of incubation with 500 µM DBcAMP, and after 3 to 4 h they approached an inhibited steady state level lower by ~90% than that of untreated S49 cells (Fig. 3GoA). ß-Actin mRNA levels were not affected (Fig. 3GoB), suggesting specificity of this cAMP-mediated regulation.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. Time course of DBcAMP effects on Thy-1 mRNA levels in S49 cells. S49 cells were grown in medium alone or with 500 µM DBcAMP for 1 to 4 h, and total RNA (20 µg/lane) was electrophoretically fractionated on formaldehyde-agarose gels. Northern blots were hybridized to randomly primed 32P-labeled 0.98-kb Thy-1.2 probe (A), probe was eluted, and the blots were rehybridized to 32P-labeled ß-actin probe (B). Normalized data from densitometric scans are presented as percentages of the mRNAs in untreated cells (100%) beneath the corresponding bands.

 
Effects of CAs on Thy-1 mRNA levels in S49 cells and thymocytes

Since stress-associated CAs are adrenergic agonists capable of stimulating ß-adrenergic receptors (ß-ARs) of T-lineage cells activating cAMP signal transduction pathways and since we had demonstrated that CAs down-regulate thymocyte and S49 cell expression of Thy-1 protein, we asked whether CAs affect steady state levels of Thy-1 mRNA. Exposure of BALB/c thymocytes to 100 µM NE for 4 h was accompanied by a 65% decrease in Thy-1 mRNA concentration in BALB/c thymocytes (Fig. 4GoA) with no significant effect on {gamma}-actin mRNA (Fig. 4GoB), consistent with specificity of the NE effect on Thy-1 expression. In parallel experiments, S49 cells were incubated for 4 h with 100 to 400 µM concentrations of the sympathetic CA, NE, or the synthetic ß-adrenergic agonist CA, ISO, and levels of Thy-1 mRNA were determined by RNase protection assays (Fig. 5Go). Densitometric analyses showed that 100 µM concentrations of either NE or ISO mediated as much as 40% (NE) or >60% (ISO) reduction of Thy-1 mRNA and that 200 to 400 µM concentrations of either CA caused an approximate 80% reduction of Thy-1 mRNA, as compared with the level of the mRNA in untreated S49 cells. As before, NE did not affect the levels of mRNA for {gamma}-actin mRNA at any concentration tested (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. Effects of NE on the level of Thy-1 mRNA in BALB/c thymocytes. Thymocytes were cultured for 4 h in serum-free RPMI medium in the presence or the absence of 100 µM NE. Total RNAs (20 µg) were analyzed by RNase protection using 32P-labeled negative strand probes for Thy-1 (A) or {gamma}-actin (B). Relative amounts of Thy-1 and of {gamma}-actin probe protected in NE-treated ({blacksquare}) as compared with untreated ({square}, 100%) thymocytes, estimated by densitometry and image analysis are presented.

 


View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 5. Effects of CAs on steady state levels of Thy-1 mRNA in S49 cells. S49 cells were cultured for 4 h in the presence of the indicated concentrations of NE or of ISO, or in the absence of CAs. Total RNAs from the cells were analyzed by RNase T2 protection. Normalized results from densitometry and image analysis of the autoradiograms are presented as bars below the corresponding bands.

 
Effects of the ß-AR antagonist propranolol on NE regulation of Thy-1 mRNA levels in S49 cells

Previous studies by us and others showed that ß-AR antagonists did not interfere with inhibitory effects of NE or other CAs on lymphocyte activation by mitogens (11, 12, 13). To determine roles of ß-ARs in CA-mediated down-regulation of Thy-1 mRNA levels, S49 cells were incubated from 2 to 4 h with 100 µM NE in the presence and absence of the ß-AR antagonist propranolol (10 µM, a concentration previously shown to inhibit CA-directed accumulation of cAMP in S49 cells while not exhibiting cytotoxic effects (48), and the concentration of Thy-1 mRNA was determined by nuclease protection assay (Fig. 6Go). Propranolol antagonized the NE-mediated reduction in Thy-1 mRNA concentration, suggesting that the CA-mediated Thy-1 mRNA decreases were ß-AR-dependent.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 6. Propranolol (Prop) sensitivity of NE regulation of Thy-1 mRNA levels in S49 cells. S49 cells were cultured for the indicated times in the presence or absence of 10 µM propranolol, 100 µM NE, or 10 µM propranolol and 100 µM NE combined. Amounts of Thy-1 mRNA in total RNAs (20 µg) from the cultured cells were measured in RNase T2 protection assays. Data shown are from one representative experiment of three yielding similar results. Normalized data from densitometric scanning and image analysis of the autoradiograms are shown as percentages of the amount of Thy-1 mRNA in untreated S49 cells ({square}, 100%), below the corresponding bands. Error bars represent SEs of means of percentages computed from the three experiments. Agt, agent.

 
Effects of NE and cAMP on Thy-1 mRNA stability in S49 cells

One explanation for the CA/cAMP effects on Thy-1 mRNA is that these agents act posttranscriptionally. To examine this possibility, de novo RNA synthesis was inhibited, and the stability of Thy-1 mRNA in S49 cells treated with 100 µM NE or 500 µM DBcAMP was compared with that of untreated cells. Cells were cultured for varying times in the presence of the drug {alpha}-amanitin (2 µg/ml; a concentration at least twofold that required for complete inhibition of mammalian RNA polymerase II and an order of magnitude lower than the threshold of inhibition of RNA polymerase III) (54), and Thy-1 mRNAs were measured by nuclease protection. In the absence of cAMP or CAs, Thy-1 mRNA exhibited an apparent half-life in excess of 6 h (Fig. 7Go). When 100 µM NE or 500 µM DBcAMP was added to S49 cells simultaneously with {alpha}-amanitin, the apparent half-life of Thy-1 mRNA decreased markedly to 2 to 3 h (Fig. 7Go).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of cAMP and NE on Thy-1 mRNA stability in S49 cells. S49 cells were cultured at a starting density of 1 x 106 cells/ml in medium containing 2 µg/ml {alpha}-amanitin, in the presence or absence of exogenous DBcAMP (500 µM) or NE (100 µM) and harvested at the indicated times. Thy-1 mRNA was assayed in the poly(A)+-enriched fraction by RNase T2 protection. Autoradiograms representing protected bands from one experiment of two yielding similar results are shown for untreated cells (A), DBcAMP-treated cells (B) and NE-treated cells (C). Autoradiograms from this representative experiment were scanned and the images were analyzed. Amounts of Thy-1 probe protected by mRNA in the presence of {alpha}-amanitin alone (•), 500 µg of DBcAMP and {alpha}-amanitin ({blacksquare}), or 100 µg of NE and {alpha}-amanitin ({blacktriangleup}) are plotted vs time, as percentages of mRNA protected at time zero (100%).

 
Effects of cAMP and NE on transcription of the Thy-1 gene

An alternative to CA/cAMP-mediated posttranscriptional regulation of Thy-1 mRNA is that these agents also affect transcription. To investigate CA and cAMP effects on the rate of transcription of the Thy-1 gene, nuclear run-on assays were conducted on nuclei prepared from S49 cells grown in the presence and absence of 100 µM NE or 500 µM DBcAMP. These assays measure the extension of transcripts that have been initiated in nuclei at the time of cell harvest, thus determining the contribution of transcription, as distinguished from posttranscriptional mechanisms, to regulation of steady state mRNA concentration. The ratio between the amount of Thy-1 probe-hybridizing nuclear transcripts and the amount of 28S rRNA transcript (an abundant RNA polymerase 1 transcript used as a loading control) was undiminished after 4 h of exposure to either NE (Fig. 8Go) or DBcAMP (data not shown). Thus, neither NE nor cAMP affects the rate of Thy-1 gene transcription.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 8. Effects of NE on Thy-1 mRNA synthesis in S49 cells. Nuclear extracts were prepared from S49 cells cultured in the absence or presence of 100 µM NE for 1 to 4 h. Nuclear run-on assays were performed as described in Materials and Methods. Amounts of {alpha}-[33P]UTP-labeled nuclear transcripts bound to immobilized Thy-1 and 28S rRNA probes were determined by autoradiography. The autoradiogram shown is from one of two similar experiments. Bands represent hybridization of 33P-labeled nuclear transcripts with membrane-bound probes for Thy-1.2 and 28S rRNA. Based on densitometric scans and image analysis, ratios of transcription of Thy-1 mRNA to 28S rRNA were determined, normalized to the ratio at T = 0 h (100%), and graphically depicted immediately below the corresponding bands.

 
Use of S49 cell mutants to define the roles of AdCy and PKA in NE/cAMP-mediated decreases in Thy-1 mRNA

The availability of mutant S49 lymphosarcoma cells lacking the ability to synthesize cAMP in response to agonists for Gs protein-linked receptors (cyc-) (39) and of S49 mutants lacking PKA activity (kin-) (40) allowed us to use a genetic approach to determine mechanistically whether CA- and cAMP-induced decreases in Thy-1 mRNA levels occur through the AdCy-PKA signaling pathway. S49 wt, cyc-, and kin- cells were grown in the presence or absence of 500 µM DBcAMP for 1 to 4 h. Northern blot analyses (Fig. 9Go) or nuclease protection assays (results not shown) revealed that in the presence of 500 µM DBcAMP, decreases of >90% in Thy-1 mRNA levels in wt and cyc-, but not kin-, cells occurred. Treatment with 100 µM NE for hourly increments up to 4 h led to a progressive decrease in Thy-1 mRNA levels reaching <30% of control level in 4 h in wt cells (Fig. 10GoA) but failed to influence the level of Thy-1 mRNA in either cyc- (Fig. 10GoB) or kin- (Fig. 10GoC).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 9. Effects of DBcAMP on the levels of Thy-1 mRNA in S49 wt, cyc-, and kin- cells. S49 wt, cyc-, and kin- cells were grown for 4 h in the presence or the absence of 500 µM DBcAMP. Total cell RNAs (20 µg) were separated according to size on formaldehyde-agarose gels, transferred to nylon membranes, and probed with 32P-labeled 0.98-kb Thy-1 probe (5 x 108 cpm/µg). RNAs from S49 wt, cyc-, and kin- cells are indicated above the lanes. Data were subjected to densitometric image analysis and amounts of Thy-1 mRNA in DBcAMP-treated wt, cyc- and kin- cells ({blacksquare}), relative to amounts of Thy-1 mRNA in untreated cells ({square}), are shown below the corresponding lanes of the autoradiogram.

 


View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 10. Effects of NE on levels of Thy-1 mRNA in S49 cyc- and kin- cells. cyc- and kin- S49 cell clones were cultured for 1 to 4 h in the presence of 100 µM NE. RNAs (20 µg) from treated cells were analyzed by RNase T2 protection, and autoradiographic bands of protected probe are shown for the wt cells (A), cyc- cells (B), and kin- cells (C). Bands were subjected to densitometric image analysis and relative amounts of protected Thy-1 probe from NE-treated cells were plotted and are shown below the corresponding autoradiographic bands.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments described in this paper examine the mechanism(s) by which CAs and cAMP decrease Thy-1 gene expression in murine thymocytes and T cells. The CAs NE and ISO, acting by propranolol-sensitive, ß-AR-dependent mechanisms, the membrane-permeating cAMP analogues DBcAMP and 8BrcAMP, and the AdCy-stimulating microbial toxin, CT, all induce 60 to 96% decreases in cellular Thy-1 mRNA levels within 4 h in both primary BALB/c murine thymocytes and S49 BALB/c T lymphoma cells. RNA stability studies demonstrated that NE and cAMP dramatically accelerate decay of Thy-1 mRNA in S49 cells in the presence of {alpha}-amanitin whereas nuclear run-on studies argue against major specific effects of NE or DBcAMP on the rate of de novo transcription of the Thy-1 gene (Figs. 7Go and 8Go).

The high concentrations of CAs necessary for the inhibitory response have raised concerns that such effects may be nonphysiologic or may result from oxidation products of CAs. We believe that such explanations are unlikely for a number of reasons: 1) the concentrations of CAs found in the environment of the lymphocyte during the "stress response" are likely to approach those that mediate the in vitro response, suggesting that these increased CA levels used in our studies are representative of a "pathophysiologic state." Indeed, the basal level of CAs in the spleen has been reported to be micromolar (5). Thus, local concentrations of CAs in immune organs could well exceed this level and in spleen could reach the concentrations we used in these studies. In addition, in an in vitro system studying the generation of antitumor immunity (14), we found that as CA concentrations are increased toward "pathophysiologic" levels, the cellular response to the tumor is inhibited; 2) the inhibitory effects of CAs are reversible by ß-adrenergic antagonists and mimicked by addition of standard concentrations of cAMP analogues, arguing that such responses are dependent on normal ß-adrenergic ligand-receptor occupancy and subsequent cAMP-dependent signal transduction; 3) that oxidative products of CAs are unlikely to mediate the effects described herein is suggested by our observation that during a 4-h incubation period, under conditions identical with those we have used, little oxidation of CAs is seen. Additionally, when cells are incubated with oxidation products of CAs, e.g., adrenochrome, no inhibitory effect is found.

Studies with cyc- and kin- mutant S49 cells revealed that NE-mediated reduction in Thy-1 mRNA levels is dependent both on active Gs-linked AdCy, indicating a requirement for cAMP synthesis, and on PKA activity, suggesting involvement of protein phosphorylation. DBcAMP, serving as an exogenous cAMP source, was able to depress Thy-1 mRNA levels in cyc- S49 cells by replacing the need for cellular synthesis of cAMP; however, neither DBcAMP nor NE decreased Thy-1 mRNAs in kin- cells, further illustrating that Thy-1 mRNA regulation requires PKA activity. Collectively, these results argue that adrenergic CAs can act as first messengers on ß-AR-like receptors of S49 cells and BALB/c thymocytes to activate Gs-linked AdCy and cause intracellular accumulation of cAMP; cAMP, in turn, stimulates PKA to phosphorylate an unknown substrate protein, which participates in posttranscriptional mRNA destabilization processes that decrease Thy-1 mRNA in these T-lineage cells (Fig. 11Go).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 11. Proposed mechanism for catecholamine-mediated Thy-1 mRNA destabilization.

 
Previous in vitro and in vivo studies have shown that both ß-AR-dependent and ß-AR-independent actions of CAs contribute to immunomodulation (10, 12, 14, 55). The Thy-1 regulation by CAs may serve as a model for gene regulation underlying a ß-AR-dependent component of stress-mediated immunosuppression. The association of cAMP with the down-regulation of expression of a number of genes at the level of mRNA stability in immune cells and other cell types has been reported. Such genes include those for IL-2 (56), which is also cAMP down-regulated at the transcriptional level (57), the {alpha}-subunit of {alpha}ß TCR of human thymocytes (58), the ornithine decarboxylase gene of CTL (59), as well as the ß2-ARs of T-lineage cells (60) and several other cell types (61, 62, 63). Ongoing studies in this laboratory have indicated that other mRNAs (for the cytokines TNF-{alpha}, TNF-ß, and IFN-{gamma}) are also down-regulated in Con A-stimulated primary mouse spleen cell cultures by both NE and cAMP in a manner similar to that of Thy-1.

Elucidation of the mechanism of CA-mediated RNA destabilization will entail identifying the target sites in the mRNA that dictate stability or instability. Studies reviewed by Sachs (65) have established that mRNA destabilization may involve sites in diverse regions of susceptible mRNAs. A favored site often is a repeat of an AUUUA motif embedded in an adenosine nucleotide- and uridine nucleotide-rich region (AUR) within the 3'-untranslated region (3'-UTR) of the mRNA (66). The 3'-UTR of the Thy-1 mRNA contains two AUUUA sequences in an uridine-rich region which closely resembles AURs of stability-regulated mRNAs (5, 66). A major result from our studies using S49 cells and their mutants revealed that a CA/cAMP-dependent protein phosphorylation event is required for Thy-1 mRNA destabilization. Present work in our laboratory is directed toward identifying specific protein(s) that participate in the destabilization process. Initial isotope transfer experiments have identified several S49 cell proteins that bind prominently to a 116-base region of the Thy-1 3'-UTR resembling specific AURs present in the 3'-UTRs of other stability-regulated mRNAs (35). Binding activity of three of those proteins was markedly higher in extracts obtained from wt than from kin- S49 cells and in extracts from cAMP-treated than from those of untreated wt S49 cells, suggesting that cAMP, acting though a PKA-dependent mechanism, increases the amounts or specific binding activities of the three proteins (64). These findings are consistent with a hypothesis that one or more of the proteins play a role in CA/cAMP/PKA-dependent Thy-1 mRNA destabilization targeted to the 3'-AUR. The three Thy-1 AUR-binding proteins also adhere to the AU-rich 3'-UTR of the stability-regulated ß2AR mRNA, a sequence bound by a 35-kDa protein hypothesized to have a role in CA/cAMP-mediated destabilization of ß2-AR mRNA (62, 63), raising the possibility that Thy-1 AUR-binding protein(s) and the ß-AR mRNA-binding protein are members of a family of CA/cAMP-responsive mRNA stability-regulatory factors.

Collectively, our studies and the work of others support the concept that CAs can be immunosuppressive and function in part though cAMP-dependent pathways. Other studies in our laboratory have suggested cAMP-independent CA-mediated effects as well (10, 12, 14). The overall hypothesis guiding these studies is that the nervous system can interact with the immune system by regulating ligands and receptors necessary to immune function. The local and systemic release of CAs that accompanies psychogenic stress is likely to play a role in generating immune dysfunction through mechanisms involving altered expression of cell surface molecules that affect signal transduction and cell-cell interaction in immune cells. This hypothesis is consistent with our general model of stress-related mRNA regulation proposing that the interactions of environmental biologic response modifiers with cell surface ligands activate signal transduction processes, leading to down-regulation of the mRNAs specifying these ligands, thereby generating cellular nonresponsivity (Fig. 11Go). The question has been raised as to the biologic value of inhibiting the immune system in response to stress. Although there is no definitive answer, it can be argued that temporary blunting of the immune response during a stress reaction ensures that the body does not overreact by activating long term responses that are not needed, require energy, and may even prove detrimental, e.g., lead to autoimmunity. Regardless of the biologic reasons or potential evolutionary advantage of depressed immune function during situations of stress, the biologic reality is that stress has pathophysiologic consequences that require explanation in molecular terms.


   Acknowledgments
 
We thank Drs. Rhonna Cohen, Robert Costa, Thomas Henderson, and Mariano Tao for helpful discussion.


   Footnotes
 
1 These studies were supported by the Office of Naval Research and the Council for Tobacco Research. Back

2 Address correspondence and reprint requests to Dr. Donald A. Chambers, Department of Biochemistry and Molecular Biology (MC 536), University of Illinois, 1819 West Polk Street, Chicago IL 60612-7334. Back

3 Abbreviations used in this paper: CA, catecholamine; DBcAMP, dibutyryl cyclic AMP; 8BrcAMP, 8-bromo cyclic AMP; NE, norepinephrine; ISO, isoproterenol; CT, cholera toxin, Vibrio cholerae enterotoxin; AdCy, adenylyl cyclase; PKA, protein kinase A; ß-AR, ß-adrenergic receptor; 3'-UTR, 3'-untranslated region; wt, wild-type; cyc-, lacking adenylyl cyclase activity due to mutation in adenylyl cyclase-associated Gs protein; kin-, lacking protein kinase A activity; GPI, glycosylphosphatidylinositol; Gs, stimulatory G protein; AUR, adenosine nucleotide- and uridine nucleotide-rich region. Back

Received for publication February 25, 1998. Accepted for publication July 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ader, R., N. Cohen, D. Felten. 1995. Psychoneuroimmunology: interactions between the nervous system and the immune system. Lancet 345:99.[Medline]
  2. Madden, K. S., V. M. Sanders, D. L. Felten. 1995. Catecholamine influences and sympathetic neural modulation of immune responsiveness. Annu. Rev. Pharmacol. Toxicol. 35:417.[Medline]
  3. Buckingham, J. C., H. D. Loxley, H. C. Christian, J.G. Philip. 1996. Activation of the HPA axis by immune insults: roles and interactions of cytokines, eicosanoids and glucocorticoids. Pharmacol. Biochem. Behav. 54:285.[Medline]
  4. Besodovsky, H. O., A. Del Rey. 1996. Immune-neuro-endocrine interactions: facts and hypotheses. Endocr. Rev. 17:64.[Abstract/Free Full Text]
  5. Felten, S. Y., J. Housel, D. L. Felten. 1986. Use of in vitro dialysis for evaluation of splenic norepinephrine and serotonin. Soc. Neurosci. Abs. 12:1065.
  6. Felten, S. Y., D. L. Felten. 1991. Innervation of lymphoid tissue. R. Ader, and D. L. Felten, and N. Cohen, eds. Psychoneuroimmunology 27. Academic Press, New York.
  7. Bergquist, J., A. Tarkowski, R. Ekman, A. Ewing. 1994. Discovery of endogenous catecholamines in lymphocytes and evidence for catecholamine regulation of lymphocyte function via an autocrine loop. Proc. Natl. Acad. Sci. USA 91:12912.[Abstract/Free Full Text]
  8. Josefsson, E., J. Bergquist, R. Ekman, A. Tarkowski. 1996. Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis. Immunology 88:140.[Medline]
  9. Faraj, B.A., Z. L. Olkowski, R. T. Jackson. 1994. Expression of a high affinity serotonin transporter in human lymphocytes. Int. J. Immunopharmacol. 16:561.[Medline]
  10. Chambers, D. A., R. L. Cohen, R. L. Perlman. 1993. Neuroimmune modulation: signal transduction and catecholamines. Neurochem. Int. 22:95.[Medline]
  11. Chambers, D. A., Jr D. W. Martin, Y. Weinstein. 1974. Effect of cyclic nucleotides on purine biosynthesis and induction of PRPP synthetase during lymphocyte activation. Cell 3:375.[Medline]
  12. Cook-Mills, J. M., R. L. Cohen, R. L. Perlman, D. A. Chambers. 1995. Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action. Immunology 85:544.[Medline]
  13. Cook-Mills, J. M., H. G. Munshi, R. L. Perlman, D. A. Chambers. 1992. Mouse hepatitis virus infection suppresses modulation of mouse spleen cell activation. Immunology 75:542.[Medline]
  14. Cook-Mills, J. M., M. M. Mokyr, R. L. Cohen, R. L. Perlman, D. A. Chambers. 1995. Neurotransmitter suppression of the in vitro generation of a cytotoxic T-lymphocyte response against the syngeneic MOPC-315 plasmacytoma. Cancer Immunol. Immunother. 40:79.[Medline]
  15. Williams, A. F., J. Gagnon. 1982. Neuronal cell Thy-1 glycoprotein: homology with immunoglobulin. Science 216:696.[Abstract/Free Full Text]
  16. Crawford, J. M., R. W. Barton. 1986. Thy-1 glycoprotein: structure, distribution, and ontogeny. Lab. Invest. 54:122.[Medline]
  17. Raff, M. C.. 1971. Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6:52.[Medline]
  18. Muller-Sieburg, C. E., G. F. Tidmarsh, I. L. Weissman, G. J. Spangrude. 1989. Maturation of hematolymphoid cells that express Thy-1. A. E. Reif, and M. Schlesinger, eds. Cell Surface Antigen Thy-1: Immunology, Neurology and Therapeutic Application 289. Marcel Dekker, New York.
  19. Louie, S. W., L. M. Ramirez, A. M. Krieg, C. R. Maliszewski, G. A. Bishop. 1993. Endogenous secretion of IL-4 maintains growth and Thy-1 expression of a transformed B cell line. J. Immunol. 150:399.[Abstract]
  20. Firer, M. A., B.-Z. Zacharia, M. Kostikov, Y. Irlin. 1995. The Thy-1 molecule: its properties and functions. Isr. J. Med. Sci. 31:382.[Medline]
  21. Schlesinger, M.. 1989. The function of Thy-1. A. E. Reif, and M. Schlesinger, eds. Cell Surface Antigen Thy-1: Immunology, Neurology and Therapeutic Application 589. Marcel Dekker, New York.
  22. Norcross, M. A., R. T. Smith. 1989. Mitotic triggering of T-cells by anti-Thy-1 antibodies. A. E. Reif, and M. Schlesinger, eds. Cell Surface Antigen Thy-1: Immunology, Neurology and Therapeutic Application 397. Marcel Dekker, New York.
  23. Carlsson, S. R., T. I. Stigbrand. 1983. Alterations in expression and glycosylation pattern of Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes. J. Immunol. 130:1837.[Abstract]
  24. Gunter, K. C., T. R. Malek, E. M. Shevach. 1984. T cell-activating properties of an anti-Thy-1 monoclonal antibody: possible analogy to OKT3/LEU-4. J. Exp. Med. 159:716.[Abstract/Free Full Text]
  25. MacDonald, H. R., C. Bron, M. Rousseaux, C. Horvath, J. C. Cerottini. 1985. Production and characterization of monoclonal anti-Thy-1 antibodies that stimulate lymphokine production by cytolytic T cells. Eur. J. Immunol. 15:495.[Medline]
  26. Hueber, A. O., M. Pierres, H. T. He. 1994. Thy-1 functions as a recognition/signaling molecule during mouse T lymphocyte development. Braz. J. Med Biol. Res. 27:275.[Medline]
  27. Killeen, N.. 1997. T-cell regulation: Thy-1—hiding in full view. Curr. Biol. 7:R774.[Medline]
  28. McConkey, D. J., M. Jendal, S. Orrenius. 1992. Cellular signaling in thymocyte apoptosis. Semin. Immunol. 4:371.[Medline]
  29. Hueber, A. O., G. Raposo, M. Pierres, H. T. He. 1994. Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism. J. Exp. Med. 179:785.[Abstract/Free Full Text]
  30. Chen, S., F. Botteri, H. vander Putten, C. P. Landel, G. A. Evans. 1987. A lymphoproliferative abnormality associated with inappropriate expression of the Thy-1 antigen in transgenic mice. Cell 51:7.[Medline]
  31. Evans, G. A., S. Chen. 1989. Expression of the Thy-1 gene and Thy-1-derived fusion genes in transgenic mice. Mol. Biol. Med. 6:549.[Medline]
  32. Low, M. G.. 1989. Anchoring of Thy-1 to the cell membrane. A. E. Reif, and M. Schlesinger, eds. Cell Surface Antigen Thy-1: Immunology, Neurology and Therapeutic Application 71. Marcel Dekker, New York.
  33. Aurrand-Lions, M., M. Pierres, P. Naquet. 1997. Thy-3, a developmentally regulated T cell glycoprotein associated to Thy-1 in detergent resistant membrane microdomains. Cell. Immunol. 176:173.[Medline]
  34. Brown, D.. 1993. The tyrosine kinase connection: how GPI-anchored proteins activate T cells. Curr. Opin. Immunol. 5:349.[Medline]
  35. Giguere, B., K. I. Isobe, F. Grosveld. 1985. Structure of the murine Thy-1 gene. EMBO J. 4:2017.[Medline]
  36. Moriuchi, T., H. C. Chang, R. Denome, J. Silver. 1983. Thy-1 cDNA sequence suggests a novel regulatory mechanism. Nature 301:80.[Medline]
  37. Teng, M.-H., S. Hedayat, A. A. Alexander, R. Barkin, R. S. Basch, J. N. Buxbaum. 1989. A Thy-1-negative lymphoma cell variant defective in the formation of glycosyl-phosphatidylinositol membrane protein anchors. Mol. Immunol. 4:391.
  38. Horibata, K., A. W. Harris. 1970. Mouse myelomas and lymphomas in culture. Exp. Cell Res. 60:61.[Medline]
  39. Bourne, H. R., P. Coffino, G. M. Tomkins. 1975. Selection of a variant lymphoma cell deficient in adenylate cyclase. Science 187:750.[Abstract/Free Full Text]
  40. Coffino, P., H. R. Bourne, G. M. Tomkins. 1975. Somatic genetic analysis of cyclic AMP action-selection of unresponsive mutants. J. Cell. Physiol. 85:603.[Medline]
  41. MacDonald, R. J., G. H. Swift, A. E. Przybyla, J. M. Chirgwin. 1987. Isolation of RNA using guanidinium salts. Methods Enzymol. 152:219.[Medline]
  42. Chomczynski, P., N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.[Medline]
  43. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Electrophoresis of RNA through gels containing formaldehyde. Molecular Cloning: A Laboratory Manual 2nd ed.7.43. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  44. Feinberg, A. P., B. Vogelstein. 1984. A technique for radiolabeling DNA restriction fragments to high specific activity. Anal. Biochem. 137:266.[Medline]
  45. Alonso, S., A. Minty, Y. Bourlet, M. Buckingham. 1986. Comparison of three actin-coding sequences in the mouse: evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23:11.[Medline]
  46. Schmickel, R. D., I. L. Gonzalez, J. M. Erickson. 1985. Nucleolus organizing genes on chromosome 21: recombination and nondisjunction. Ann. NY Acad. Sci. 450:121.[Medline]
  47. Costa, R. H., E. Lai, J. E. Darnell. 1986. Transcriptional control of the mouse prealbumin (transerythrin) gene: both promoter sequences and a distinct enhancer are cell-specific. Mol. Cell. Biol. 6:4697.[Abstract/Free Full Text]
  48. Wajeman-Chao, S. A. 1995. Neuro-immune interactions: regulation of Thy-1 mRNA in immune cells. Doctoral dissertation, University of Illinois at Chicago, Chicago, IL.
  49. Ausubel, F. M., R. Brent, R.E. Kingston, D. D. Moore, J. G. Seideman, J. A. Smith, and K. Struhl, eds. 1993. Identification of newly transcribed RNA. In Current Protocols in Molecular Biology. Vol. 1. Wiley Interscience, New York, p. 4.10.1.
  50. Lancaster, S. A. 1992. Regulation of Thy-1 Gene Expression by cAMP in Lymphoid Cells. M.S. Thesis, University of Illinois at Chicago, Chicago, IL, p. 34.
  51. Leder, A., P. Leder. 1975. Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells. Cell 5:319.[Medline]
  52. Prasad, K. N., P. K. Sinha. 1976. Effect of sodium buyrate on mammalian cells in culture: a review. In Vitro 12:125.[Medline]
  53. Zhong, W. W., S. R. Chavali, and R. A. Forse. 1994. Effect of prostaglandin E2 and of intracellular cyclic AMP elevating agents on the mitogen induced mouse splenocyte proliferation in a serum free culture condition. Life Sci. 55:PL 193.
  54. Jaehning, J. A., R. Weinmann, T. G. Brendler, H. J. Raskas, R. G. Roeder. 1976. Function and regulation of RNA polymerases II and III in adenovirus-infected KB cells. R. Losick, and M. Chamberlain, eds. RNA Polymerase 823. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  55. Felten, D. L., S. L. Felten, D. L. Bellinger, K. S. Madden. 1993. Fundamental aspects of neural-immune signaling. Psychother. Psychosom. 60:46.[Medline]
  56. Anastassiou, E. D., F. J. P. Pagliogianni, H. Balow, H. Yamada, D. T. Boumpas. 1992. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R{alpha} gene expression at multiple levels J. Immunol. 148:2845.
  57. Jain, J., C. Loh, A. Rao. 1995. Transcriptional regulation of the IL-2 gene. Curr. Opin. Immunol. 7:333.[Medline]
  58. Martinez-Valdez, H., E. Thompson, A. Cohen. 1988. Coordinate transcriptional regulation of {alpha} and {delta} chains of T-cell antigen receptors by phorbol esters and cyclic adenosine monophosphate in human thymocytes. J. Biol. Chem. 263:9561.[Abstract/Free Full Text]
  59. Farrar, W. L., M. Vinocour, J. I. Cleveland, A. Harel-Bellan. 1988. Regulation of ornithine decarboxylase activity by IL-2 and cAMP. J. Immunol. 141:967.[Abstract]
  60. Post, S. R., O. Aguila-Buhain, P. A. Insel. 1996. A key role for protein kinase A in homologous desensitization of the ß2-adrenergic receptor pathway in S49 lymphoma cells. J. Biol. Chem. 271:895.[Abstract/Free Full Text]
  61. Port, J. D., L. Y. Huang, C. C. Malbon. 1992. ß-Adrenergic agonists that down-regulate receptor mRNA up-regulate a Mr 35,000 protein(s) that selectively binds to ß-adrenergic receptor mRNAs. J. Biol. Chem. 267:24103.[Abstract/Free Full Text]
  62. Huang, L.-Y., B. G. Tholanikunnel, E. Vakalopoulou, C. C. Malbon. 1993. The Mr 35,000 ß-adrenergic receptor messenger RNA binding protein induced by agonists requires both AUUUA pentamers and U-rich domains for RNA regulation. J. Biol. Chem. 268:25769.[Abstract/Free Full Text]
  63. Dangel, V., J. Giray, D. Ratge, H. Wisser. 1996. Regulation of ß-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2. Biochem. J. 317:925.
  64. Jr Graf, L. H., I. I. Kuzin, D. A. Chambers. 1997. Catecholamine-mediated Thy-1 degradation: mRNA binding proteins. FASEB J. 11:A1381.
  65. Sachs, A. B.. 1993. Messenger RNA degradation in eukaryotes. Cell 74:413.[Medline]
  66. Greenberg, M. E., J. G. Belasco. 1993. Control of the decay of labile protooncogene and cytokine mRNAs. J. G. Belasco, and G. Brawerman, eds. Control of Messenger RNA Stability 199. Academic Press, New York.



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
Y. Y. Sanders, A. Pardo, M. Selman, G. J. Nuovo, T. O. Tollefsbol, G. P. Siegal, and J. S. Hagood
Thy-1 Promoter Hypermethylation: A Novel Epigenetic Pathogenic Mechanism in Pulmonary Fibrosis
Am. J. Respir. Cell Mol. Biol., November 1, 2008; 39(5): 610 - 618.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
I. J. Elenkov, R. L. Wilder, G. P. Chrousos, and E. S. Vizi
The Sympathetic Nerve---An Integrative Interface between Two Supersystems: The Brain and the Immune System
Pharmacol. Rev., December 1, 2000; 52(4): 595 - 638.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. Grima and C. Y. Cheng
Testin Induction: The Role of Cyclic 3',5'-Adenosine Monophosphate/Protein Kinase A Signaling in the Regulation of Basal and Lonidamine-Induced Testin Expression by Rat Sertoli Cells
Biol Reprod, December 1, 2000; 63(6): 1648 - 1660.
[Abstract] [Full Text]

Home page
 J. Immunol.Home page
V. V. Kalinichenko, M. B. Mokyr, L. H. Graf Jr., R. L. Cohen, and D. A. Chambers
Norepinephrine-Mediated Inhibition of Antitumor Cytotoxic T Lymphocyte Generation Involves a {beta}-Adrenergic Receptor Mechanism and Decreased TNF-{alpha} Gene Expression
J. Immunol., September 1, 1999; 163(5): 2492 - 2499.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wajeman-Chao, S. A.
Right arrow Articles by Chambers, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wajeman-Chao, S. A.
Right arrow Articles by Chambers, D. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS