©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thrombin, Phorbol Ester, and cAMP Regulate Thrombin Receptor Protein and mRNA Expression by Different Pathways (*)

(Received for publication, August 25, 1994; and in revised form, November 4, 1994)

Ute Zacharias (1)(§) Yichun Xu (1) Jacqueline Hagège (1) Jean-Daniel Sraer (1) Lawrence F. Brass (2) Eric Rondeau (1)

From the  (1)From INSERM U 64 and Association Claude Bernard, Hôpital Tenon, 75020 Paris, France and the (2)Hematology-Oncology Section, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways.

Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation.

Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression.

The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation.

The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor.

These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.


INTRODUCTION

Apart from its critical role in thrombosis and hemostasis, thrombin exhibits a variety of cell-activating functions(1, 2) probably mediated by its guanine nucleotide-binding protein (G-protein)^1 coupled receptor(3, 4) . Receptor activation occurs by cleavage of the amino-terminal extracellular domain by thrombin, exposing a new amino terminus that acts as a tethered peptide ligand and activates the receptor. Accordingly, thrombin receptor agonist peptides (TRAP), corresponding to the new amino terminus exposed, mimic receptor activation by thrombin(5) .

G-protein coupled receptors undergo rapid desensitization after short-time activation by an agonist that is reversible. However, after continuous exposure to agonist for several hours, long-term desensitization becomes evident as a down-regulation of total receptor number(6) . Down-regulation may occur in a homologous or heterologous way, that is, after chronic activation of the receptor by its agonist (7) or after chronic activation of another receptor or process(8) . Examples on the regulation of G-protein coupled receptors during cross-talk of signal transduction pathways are limited to adrenergic and muscarinic receptors(8, 9, 10) .

Two major signal transduction pathways activated by G-protein coupled receptors are stimulation of phospholipase C and adenylylcyclase, mediating cellular effects by activation of the effector kinases protein kinase C and protein kinase A, respectively. Many reports have indicated cross-talk between these two signaling pathways. Activation of one pathway has been shown to be stimulatory or inhibitory to the other pathway(11) .

Thrombin has been shown to regulate at least two second messenger pathways in a number of cell types. Thrombin stimulates phospholipase C and inhibits adenylylcyclase probably by activating G(q) and G(i), respectively(12, 13) . Additionally, thrombin interacts indirectly with other signal transduction pathways that may be involved in thrombin receptor regulation in a positive or negative feedback. Thrombin induces the synthesis of platelet-derived growth factor in vascular smooth muscle cells (14) and endothelial cells(15) , of nerve growth factor in glial cells(16) , of endothelin and vasopressin in epithelial cells(17) , and the release of arachidonic acid followed by prostacyclin production in endothelial cells(18) .

Less is known about the regulation of thrombin receptor expression. Basic fibroblast growth factor has been shown to increase thrombin receptor mRNA in rat vascular smooth muscle cells (19) and a combination of phytohemagglutinin and PMA to decrease thrombin receptor mRNA in Jurkat T cells(20) . Nelken et al.(21) described altered thrombin receptor expression in atherosclerotic plaques in vivo.

It has been difficult to study thrombin receptor protein expression by classical ligand binding techniques because thrombin binds with high affinity to cell surface proteins other than its receptor, and TRAP, the functional agonist, has only a low affinity for thrombin receptor (22) . To overcome these problems, a thrombin receptor-specific antibody has been used to establish a quantitative binding assay for thrombin receptor expression.

Many experimental and human glomerulonephritides are associated with fibrin deposition, infiltration by inflammatory cells, and proliferation of intrinsic glomerular cells, possibly mediated by thrombin(23) . The presence of thrombin receptor protein and mRNA on human mesangial cells has recently been established, and the mechanisms of thrombin receptor internalization induced by thrombin, TRAP, and phorbol ester have been compared(24) .

The purpose of this study was to examine whether prolonged activation of protein kinase C or protein kinase A signaling pathways could regulate thrombin receptor mRNA and protein expression and to compare the mechanism of heterologous regulation to that of homologous regulation induced by thrombin and TRAP.

We found that long-term activation of thrombin receptor by its agonists thrombin and TRAP results in homologous down-regulation of receptor protein after 24 h that is not dependent on protein kinase C and not accompanied by a change in thrombin receptor mRNA expression. Heterologous down-regulation of thrombin receptor protein is observed after treatment with PMA or after elevation of intracellular cAMP levels for 24 h. PMA-induced down-regulation is dependent on protein kinase C and paralleled by a partial inhibition of thrombin receptor mRNA expression. Down-regulation induced by increasing the intracellular cAMP level, that is probably mediated by protein kinase A, leads to a dramatic inhibition of thrombin receptor mRNA expression whereas it seems not to require previous internalization of thrombin receptor.


EXPERIMENTAL PROCEDURES

Materials

The following materials have been obtained as indicated: amastatin, hirudin, diisopropyl fluorophosphate, PMA, collagenase type IV, 3-isobutyl-1-methylxanthine (IBMX), 8-bromo-cAMP (8-Br-cAMP), prostaglandin E 1 (PGE1), forskolin, and purified human alpha-thrombin (3000 units/mg, 1 unit/ml = 10M) from Sigma; I-sodium (17 Ci/mg) from Dupont de Nemours (France); protein assay regent from Bio-Rad (Germany); and Sephadex G-25 from Pharmacia (Schweden).

TRAP and GF 109 203 X were kindly provided by Dr. S. Seiler (Bristol-Myers Squibb) and by Dr. J. Kirilovsky (Glaxo, France), respectively.

Ascites and purified monoclonal anti-human thrombin receptor antibody ATAP2, made against a peptide sequence of the tethered ligand domain (25) , was used for antibody binding assay.

Methods

Protein Labeling

Purified antibody ATAP2 was radioiodinated by the IODOGEN method(26) . Briefly, 1 mCi of I-sodium was allowed to react with 50 µg of IODOGEN, before addition of 30 µg of antibody ATAP2, and the mixture was kept on ice for 30 min. Free I was separated from labeled antibody on a Sephadex G-25 column saturated with bovine serum albumin (BSA) and extensively washed with 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 0.1% Tween 20.

Cell Culture

Glomerular mesangial cells were obtained by collagenase digestion of isolated glomeruli derived from human kidneys judged to be unsuitable for transplantation. They were purified and identified by their morphologic and functional features as described previously(27) .

Incubations were performed between the third and the fifth subculture of mesangial cells grown in multidish wells (Nunclon, Nunc) in normally defined medium as described(28) . When reaching confluence, cells were washed, growth-arrested by serum starvation for 24 h, and incubated with the compound to be tested. To avoid degradation of TRAP, all experiments using TRAP were performed in the presence of 10 µM amastatin(29) .

RNA Extraction and Northern Blot Analysis

Serum-starved mesangial cells were exposed to various agonists for 24 h at 37 °C. Total RNA was extracted by the phenol-chloroform method as described (30) and precipitated by 3 M LiCl. Total RNA (5 µg/lane) was separated by electrophoresis in 0.9% agarose gel containing 20% formaldehyde and transferred to a nylon membrane (GeneScreen Plus). Hybridization was performed using a P-labeled human 872-base pair cDNA probe specific for thrombin receptor that has been prepared by polymerase chain reaction according to Vu et al.(3) . Autoradiographs were developed after 24 to 72 h. After dehybridization by boiling, the filter was rehybridized with a P-labeled 412-base pair glyceraldehyde-3-phosphate dehydrogenase probe obtained by polymerase chain reaction according to Tokunaga et al.(31) .

Antibody Binding Assay

Human mesangial cells, cultivated in 96-well plates and serum-starved, were exposed to various agonists for the indicated times at 37 °C and immediately transferred on ice to terminate reactions. The following steps were performed on ice. Cells were washed with ice-cold phosphate-buffered saline containing 1 mM CaCl(2) and, for some experiments, permeabilized by treatment with methanol for 1 min. Plates were incubated with radioiodinated antibody ATAP2 (35,000 cpm/well corresponding to 2.5 ng of ATAP2/well) in minimally defined medium containing 0.1% BSA for 1 h. After washing three times with phosphate-buffered saline containing 0.1% BSA, cells were solubilized by incubation with 1 N NaOH at room temperature for 10 min. Cell-associated radioactivity was measured in a -counter. An aliquot was used for protein determination using protein assay reagent from Bio-Rad (32) and BSA as a standard. Antibody binding data were expressed as specific binding (total minus nonspecific binding). Typically, binding of untreated cells was about 1250 cpm/well corresponding to 105,000 cpm/mg of protein and was set at 100%. Nonspecific binding in the presence of 1 µM TRAP 14 was less than 5%.


RESULTS

Homologous Down-regulation of Thrombin Receptor Protein

Antibody ATAP2, that is directed against the tethered ligand domain of thrombin receptor amino terminus and recognizes intact and thrombin-cleaved receptor(25, 33) , has been used to establish a quantitative binding assay for thrombin receptor expression (see ``Experimental Procedures''). Permeabilization of cells with methanol prior to antibody binding allowed differentiation between surface-expressed and total thrombin receptor antigen.

Using this binding assay, the effects of thrombin and TRAP on thrombin receptor expression on mesangial cells have been studied. After persistent activation of thrombin receptor for 24 h, thrombin (Fig. 1A) and TRAP (Fig. 1B) induced a dose-dependent loss of cell surface binding sites for antibody ATAP2 to 27% and 29% of control, respectively. In parallel, the total number of ATAP2 binding sites as revealed after cell permeabilization was reduced to 39% and 37% of control at maximal doses of thrombin and TRAP, respectively. Chemically inactivated thrombin by treatment with diisopropyl fluorophosphate (28) or thrombin in the presence of hirudin, a known inhibitor of thrombin(34) , failed to elicit receptor down-regulation. Binding of an antibody directed against the plasma membrane-associated urokinase receptor (35) was unaffected (not shown).


Figure 1: Effect of thrombin and TRAP on thrombin receptor protein expression in mesangial cells. Cells were exposed to the indicated doses of thrombin (A) and TRAP (B) for 24 h at 37 °C. Cell surface (bullet) and total (circle) thrombin receptor antigen was measured as binding of radioiodinated antibody ATAP2 to intact and permeabilized cells, respectively. Means ± S.E. of five different experiments made in duplicate are presented.



Heterologous Down-regulation of Thrombin Receptor Protein: Effect of Protein Kinase C

PMA, a known activator of protein kinase C (36) , has recently been shown to induce heterologous internalization of thrombin receptor after short-time exposure to mesangial cells(24) . After persistent treatment for 24 h, PMA induced a dose-dependent decrease in antibody ATAP2 binding sites on intact and permeabilized cells to 20% and 32% of control, respectively (Fig. 2), suggesting down-regulation of total thrombin receptor protein.


Figure 2: Effect of PMA on thrombin receptor protein expression in mesangial cells. Cells were exposed to the indicated doses of PMA for 24 h at 37 °C. Cell surface (bullet) and total (circle) thrombin receptor antigen was measured as binding of radioiodinated antibody ATAP2 to intact and permeabilized cells, respectively. Means ± S.E. of five different experiments made in duplicate are presented.



Thrombin receptor down-regulation by PMA points to a possible role of protein kinase C in this process. A specific inhibitor of protein kinase C, GF 109 203X(37) , has been used to test this hypothesis. As shown in Fig. 3, thrombin receptor down-regulation induced by PMA was inhibited by GF 109 203X in a dose-dependent manner. Almost complete inhibition was observed with 5 µM GF 109 203 X. In contrast, the protein kinase C inhibitor did not antagonize thrombin receptor down-regulation induced by thrombin (Fig. 3) and TRAP (not shown), suggesting that protein kinase C-dependent and protein kinase C-independent mechanisms are involved in thrombin receptor down-regulation induced by PMA and thrombin, respectively.


Figure 3: Effect of a protein kinase C inhibitor on thrombin receptor protein expression after stimulation with thrombin and PMA. Mesangial cells were preincubated with GF 109 203 X for 30 min before treatment with 10 nM thrombin and 20 ng/ml PMA for 24 h at 37 °C, respectively. The presence of cell surface thrombin receptor antigen was measured as binding of antibody ATAP2 to intact cells. Means ± S.E. of four different experiences made in duplicate are presented.



Heterologous Down-regulation of Thrombin Receptor Protein: Effect of cAMP

PGE1 increased the intracellular level of cAMP in mesangial cells by activating adenylylcyclase via its receptor coupled to G(s). In contrast to thrombin, TRAP, and PMA, that induced rapid internalization of thrombin receptor within 15 min and 1 h(24) , respectively, short-time exposure of mesangial cells to PGE1 did not change cell surface expression of thrombin receptor, indicating that PGE1 did not induce thrombin receptor internalization (Fig. 4A). However, after exposure for more than 12 h to PGE1, cell-surface thrombin receptor protein declined, suggesting down-regulation of thrombin receptor protein by PGE1.


Figure 4: Effect of PGE1 on thrombin receptor protein expression in mesangial cells. Cells were exposed for the indicated times to 1 µM PGE1 (A) and to the indicated doses of PGE1 for 24 h (B) at 37 °C in the presence of 0.01 mM IBMX. The presence of cell surface (bullet) and total (circle) thrombin receptor antigen was measured as binding of radioiodinated antibody ATAP2 to intact and permeabilized cells, respectively. Means ± S.E. of five different experiments made in duplicate are presented.



After 24 h, PGE1 reduced the expression of cell surface and total thrombin receptor protein in a dose-dependent manner to 41% and 54% of control, respectively (Fig. 4B).

To determine the mechanism of PGE1-induced down-regulation of thrombin receptor, the effect of other agents known to elevate intracellular cAMP has been compared to PGE1. Forskolin, a direct stimulator of adenylylcyclase, IBMX, an inhibitor of phosphodiesterase, and 8-Br-cAMP, a cell-permeable cAMP analogue, induced a dose-dependent down-regulation of thrombin receptor protein after 24 h (Fig. 5), suggesting that thrombin receptor down-regulation induced by PGE1 was mediated by cAMP and occurred probably via activation of cAMP-dependent protein kinase A.


Figure 5: Effect of agents known to elevate intracellular cAMP on thrombin receptor protein expression. Mesangial cells were incubated either without stimulus (bar 1) or treated with 1 mM 8-Br-cAMP (bar 2), 0.1 mM 8-Br-cAMP (bar 3), 50 µM forskolin (bar 4), 10 µM forskolin (bar 5), and 0.1 mM IBMX (bar 6), respectively, for 24 h at 37 °C. The presence of cell surface thrombin receptor antigen was measured as binding of antibody ATAP2 to intact cells. Means ± S.E. of four different experiments made in duplicate are presented.



Regulation of Thrombin Receptor mRNA

The presence of specific mRNA encoding the functional thrombin receptor in mesangial cells has recently been demonstrated. In order to determine whether down-regulation of thrombin receptor occurs at the protein and/or mRNA level, the effect of thrombin, PMA, and PGE1 on thrombin receptor mRNA expression after stimulation of mesangial cells for 24 h has been compared by Northern blot analysis.

As shown in Fig. 6, exposure to thrombin did not change thrombin receptor mRNA expression, suggesting that increased degradation of thrombin receptor protein was responsible for homologous down-regulation.


Figure 6: Expression of thrombin receptor mRNA after stimulation of mesangial cells with thrombin, PMA, and PGE1. Cells were incubated for 24 h at 37 °C either without stimulus (lane 1) or with 1 µM PGE1 (lane 2), 10 nM thrombin (lane 3), and 20 ng/ml PMA (lane 4), respectively. Total RNA (5 µg/lane) was separated, transferred to nylon membrane, and hybridized with a P-labeled human thrombin receptor cDNA probe (A, upper panel). After dehybridization, the filter was rehybridized with a P-labeled glyceraldehyde-3-phosphate dehydrogenase probe (A, lower panel). A, Northern blot; B, densitometric analysis of the corresponding blot.



In contrast, PMA decreased thrombin receptor mRNA expression partially to 51% of control, indicating that PMA-induced heterologous down-regulation was in part due to reduced thrombin receptor mRNA expression and in part to increased degradation of thrombin receptor protein.

PGE1, however, led to a dramatic decrease in thrombin receptor mRNA expression to 24% of control, suggesting that heterologous down-regulation by elevation of intracellular cAMP was mediated by a strong inhibition of thrombin receptor mRNA expression rather than by affecting thrombin receptor protein degradation.

Whether the reduction in thrombin receptor mRNA by PMA and PGE1 was due to reduced transcription rate or decreased mRNA stability remains to be elucidated.


DISCUSSION

Reduction in total receptor number after continuous exposure to agonist, termed down-regulation, is one mechanism of regulating the function of G-protein-coupled receptors by inducing long-term desensitization. Down-regulation is a slow process that occurs over many hours and may not be maximal before 24 h of continuous agonist exposure. Down-regulation is possible at the level of transcription, mRNA stability, and protein degradation(6) .

Homologous down-regulation has been studied most extensively for adrenergic receptors and seems to be independent of receptor phosphorylation, rapid desensitization, and receptor internalization (38, 39) . Domains, crucial for receptor down-regulation, have been identified in the third intracellular loop (40) and the cytoplasmic carboxyl-terminal tail (41) of beta(2)-adrenergic receptor. Additionally, proteins binding specifically to and destabilizing beta(2)-adrenergic receptor mRNA have recently been identified (42) .

Less well characterized is the down-regulation of G-protein-coupled receptors during cross-talk between different signal transduction pathways. Tumor-promoting phorbol esters like PMA directly activate protein kinase C and have been shown to down-regulate adrenergic receptor mRNA and protein (43, 44) and to destabilize muscarinic receptor mRNA(45) . Persistent activation of the adenylylcyclase pathway leads to heterologous down-regulation of muscarinic receptor protein(8) .

There appear to be distinct and independent pathways underlying thrombin receptor down-regulation. Homologous and heterologous down-regulation induced by thrombin, PMA, and PGE1, respectively, have been studied in detail.

Homologous down-regulation of thrombin receptor by TRAP occurs similar to thrombin and suggests that activation rather than proteolysis of the receptor is the signal for down-regulation. Homologous down-regulation of thrombin receptor protein is not accompanied by a change in thrombin receptor mRNA expression, suggesting that increased degradation of thrombin receptor protein is responsible for down-regulation of total receptor number.

One pathway of heterologous down-regulation, evoked by PMA, is dependent on activation of protein kinase C as shown by sensitivity to protein kinase C inhibitor GF 109 203 X and is mediated at least in part by inhibition of thrombin receptor mRNA expression. In parallel, direct activation of protein kinase C by PMA leads to heterologous phosphorylation(46) , desensitization(47) , and internalization (24) of thrombin receptor in different cells. Homologous down-regulation by receptor agonists thrombin and TRAP, however, is not dependent on protein kinase C. Likewise, homologous thrombin receptor desensitization and internalization have been shown to occur independent of activation of protein kinase C and endothelin and vasopressin, which share common second messenger pathways with thrombin and activate protein kinase C, do not induce down-regulation of thrombin receptor protein (not shown). Activation of protein kinase C by thrombin via phosphoinositide hydrolysis and diacylglycerol formation, however, is necessary for thrombin-induced cellular effects in human mesangial cells(28) . Additionally, growth factors like platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor did not affect thrombin receptor protein expression in human mesangial cells (not shown).

It should be noted that long-term effects of PMA are not restricted to down-regulation of protein kinase C and experiments using PMA with this purpose should be interpreted with caution. Reduced cellular sensitivity to thrombin after a 24-h pretreatment with PMA may be due to down-regulation of protein kinase C and/or to a reduction in thrombin receptor number.

Heterologous down-regulation of thrombin receptor by PGE1 is mediated by elevation of intracellular cAMP because it can be mimicked by 8-Br-cAMP, a cell-permeable cAMP analogue, by forskolin, a direct activator of adenylylcyclase, and by IBMX, an inhibitor of phosphodiesterase, and is probably mediated via cAMP-dependent protein kinase A. Heterologous down-regulation of thrombin receptor protein by PGE1 is caused by a dramatic inhibition of thrombin receptor mRNA expression.

In contrast to thrombin, TRAP, and PMA, elevation of cAMP does not induce thrombin receptor internalization, confirming that receptor internalization is not a prerequisite for receptor down-regulation. Likewise, mutations in different regions of the cytoplasmic carboxyl-terminal domain of beta(2)-adrenergic receptor result in mutants that are abolished in their sequestration but down-regulate normally(38, 39) .

Down-regulation of thrombin receptor by elevation of cAMP may be an explanation for the observed inhibitory effects of long-term treatment with 8-Br-cAMP and forskolin on thrombin-induced cellular effects like proliferation and synthesis of proteins of the fibrinolytic system in glomerular epithelial cells(48) , demonstrating that the lack of thrombin receptor expression is accompanied by a loss of thrombin responsiveness. This could also indicate a new therapeutical approach for fibrin-associated glomerular diseases in which pharmacological modulation of thrombin receptor on glomerular cells could suppress the effects of thrombin on cell proliferation and matrix remodeling.

In conclusion, our results demonstrate that thrombin receptor undergoes homologous and heterologous down-regulation in response to thrombin, PMA, and PGE1, respectively. Different mechanisms are involved in down-regulation including activation of effector kinases, inhibition of thrombin receptor mRNA expression, and increased degradation of thrombin receptor protein. The present work provides new insights for studying the underlying mechanisms of regulation of thrombin receptor. The diverse cellular actions of thrombin and the potential for modulation of thrombin receptor expression in various tissues underline the importance of defining the mechanisms by which thrombin receptor is regulated.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Present address: Max-Delbrück-Center for Molecular Medicine, R. Rössle Str.10, Berlin 13125, Germany. Tel.: 49-30-9406-3245; Fax: 49-30-9406-3730.

(^1)
The abbreviations used are: TRAP, thrombin receptor agonist peptide (TRAP 7 = SFLLRNP); PMA, phorbol 12-myristate 13-acetate; G-protein, guanine nucleotide-binding protein; BSA, bovine serum albumin; PGE1, prostaglandin E1; IBMX, 3-isobutyl-1-methylxanthine; 8-Br-cAMP, 8-bromo-cAMP.


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