©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Inhibition of Thrombin Receptor-mediated Ca Entry by Protein Kinase C (*)

(Received for publication, July 17, 1995; and in revised form, August 17, 1995)

Yanping Xu J. Anthony Ware (§)

From the Cardiovascular Division and the Harvard-Thorndike Laboratories of the Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombin initiates many physiological processes in platelets and other megakaryocyte-lineage cells by interacting with surface receptors and generating rises in cytoplasmic Ca; these rises result from both Ca release from intracellular stores and receptor-mediated Ca entry. Regulators that limit Ca entry after its initiation by thrombin have not been identified. In this study, prevention of expression of a single protein kinase C isoenzyme (PKCbeta) by antisense cDNA overexpressed in HEL cells, a human megakaryoblastic cell line that expresses thrombin receptors, promotes thrombin receptor-mediated Ca entry without altering thrombin-induced intracellular release of Ca. The cytoplasmic Ca rise initiated by endoperoxide analogs was not affected by inhibiting PKCbeta. Overexpression of a cDNA encoding wild-type PKCbeta mutated to prevent recognition by the antisense cDNA abolished the enhancement of Ca influx following thrombin. Thus, PKCbeta appears to be a specific negative regulator of thrombin receptor-mediated Ca entry.


INTRODUCTION

The protease thrombin is generated at sites of vascular injury and is a central mediator of hemostasis, thrombosis, inflammation, and vascular proliferation(1) . Thrombin stimulates several cell types, including platelets and other cells of megakaryocytic lineage, monocytes, endothelium, and vascular smooth muscle, by triggering surface receptors to generate intracellular messengers(2, 3) . The addition of thrombin to platelets initiates a rise in cytoplasmic Ca ([Ca]) (^1)that results from release of Ca from intracellular stores (4) followed by Ca entry via a receptor-regulated cation channel(5, 6) . Several messengers, including an unidentified Ca influx factor (8) and inositol 1,3,4,5-phosphate(9) , have been postulated to initiate receptor-mediated Ca influx(10, 11) ; however, few regulators that limit receptor-mediated Ca entry, which is necessary to prevent excessive [Ca], have been characterized. Activation of the intracellular phospholipid-dependent protein kinase C (PKC) with phorbol esters prevents both entry and intracellular mobilization of Ca induced by thrombin(12, 13) . PKC can also be activated by generation of lipid regulators (14) that are mobilized following thrombin stimulation(2) ; some isoenzymes of PKC (e.g. PKCbeta) are regulated by a rise in [Ca](15) . Since limiting or negative mediators of Ca influx might be reasonably considered to be regulated by a process requiring Ca(16) , we asked whether selective inhibition of a Ca-dependent PKC isoenzyme would modify the thrombin receptor-mediated Ca influx. For these experiments, we utilized human erythroleukemic (HEL) cells(17) , a megakaryoblastic cell line that has functional thrombin receptors and shares with platelets many components of the thrombin signaling mechanism(12, 18) . HEL cells offer an additional advantage for the present studies, in that they express only one (PKCbeta) of the Ca-responsive PKC isoenzymes (12, 19) and therefore required only a single intervention to eliminate Ca-dependent PKC activity from the cells. As there are few chemical PKC inhibitors that are specific for individual isoenzymes, a strategy based on antisense DNA was utilized to reduce selectively PKCbeta in HEL cells.


MATERIALS AND METHODS

Construction, Expression, and Detection of anti-PKCbeta

To achieve selective inhibition of PKCbeta, a segment of cDNA (55 base pairs) specific to PKCbeta (5`TCCGGCTCCCCGCGCGCAAGATGGCTGACCCGGCTGCGGGGCCGCCGCCGAGCGA3`) from -20 to 35 (initiating ATG (underlined) numbered 1-3) was cloned into pcDNA1/Neo (Invitrogen) in an antisense orientation. The nucleotide sequence of the first 20 and the last 35 base pairs of the cDNA corresponded to that of the 5`-end of the untranslated and the beginning of the translated regions (respectively) of human PKCbeta cDNA.

Low passage HEL cells (a generous gift of Thalia Papayannopoulou, University of Washington, Seattle) were either transfected with the antisense PKCbeta construct or with the vector only (control cells) by electroporation. Cells that stably expressed anti-PKCbeta cDNA (anti-beta cells) were selected by limiting dilution and on the basis of cell survival in the presence of Geneticin (1.2 mg/ml). For Northern blot analysis, total RNA was prepared from the cells using the method described previously(12) . The cDNA probes of PKCbeta, PKC, and PKC are the same as described previously(19) . The cDNA probes for alpha, beta-thromboglobulin, and the thrombin receptor were generous gifts from Drs. Peter Newman (Blood Research Institute of Southeastern Wisconsin), Mortimer Poncz, and Lawrence Brass (University of Pennsylvania), respectively; the anti-thrombin receptor antibody was kindly provided by Dr. Brass.

Construction and Expression of mut-PKCbeta

To create a mutated PKCbeta that was not inhibited by the antisense PKCbeta construct, the 5`-end of the untranslated and the beginning of the translated region of the cDNA encoding rat PKCbeta (a generous gift of Dr. I. B. Weinstein, Columbia University) was mutated to reduce the complementarity to anti-beta cDNA. The mutant cDNA was cloned into pREP4 vector (Invitrogen) carrying the hygromycin resistance gene. Anti-beta cells were transfected with the mut-PKCbeta construct by electroporation. Stable transfectants were selected by limiting dilution and on the basis of cell survival in the presence of 100 µg/ml hygromycin.

[Ca] Measurements

Cytoplasmic ionized Ca ([Ca]) was measured in Fura-2 loaded HEL cells as described previously(12) . Briefly, HEL cells were washed and resuspended in HEPES-Tyrode's buffer(12) . Fura-2/AM was added in a final concentration of 2 µM to the cells, which were incubated for 30 min at 37 °C. Fluorescence measurements were obtained using a dual excitation wavelength spectrofluorometer (SPEX Fluorolog-2, Edison, NJ). Fura-2 signals were calibrated as described previously(20) . All measurements were performed on cells suspended in HEPES-Tyrode's buffer containing 1 mM Ca; in some experiments, NiCl(2), EGTA, or MnCl(2) was added just before stimulation with thrombin.


RESULTS AND DISCUSSION

Expression of both RNA and protein of PKCbeta was significantly reduced in anti-beta cells compared with that in either wild-type HEL cells or the control cells (Fig. 1, a and b). In addition to PKCbeta, the predominant isoenzymes expressed by HEL cells are PKC and PKC, which are also expressed in platelets(12, 19) ; neither PKC nor PKC was inhibited by anti-PKCbeta cDNA (Fig. 1a). Inhibition of PKCbeta provoked no obvious alteration of differentiation, as shown by the similar expression of integrin alpha, beta-thromboglobulin, and thrombin receptor (Fig. 1c).


Figure 1: Inhibition of PKCbeta expression in megakaryocyte-lineage cells. a, Northern transfer analysis of RNAs extracted from wild-type (HEL), control, and anti-beta HEL cells. The blot was hybridized with cDNA probes complementary to PKCbeta, PKC, PKC, or glyceraldehyde-phosphate dehydrogenase (GAP-DH) as a control for RNA loading, as indicated. Approximate mRNA sizes are: 2.6 kb for PKCbeta; 2.2 kb for PKC; and 2.7 kb for PKC. b, immunoblot of total protein extracted from wild-type, control, and anti-beta HEL cells with a PKCbeta-specific monoclonal antibody (Seikagaku America, Inc.). c, Northern transfer analysis of RNA extracted from wild-type (HEL), control, and anti-beta cells. The blot was hybridized with cDNA probes encoding portions of alpha, beta-thromboglobulin, and the thrombin receptor, as indicated. Approximate mRNA sizes are: 3.5 kb for the thrombin receptor; 2.5 kb for alpha; and 1.6 kb for beta-thromboglobulin (betaTG).



Because of the effect that overall PKC activation exerts on [Ca] homeostasis, we asked whether this sharp reduction in PKCbeta would alter [Ca] following thrombin. The thrombin-induced [Ca] in clonal populations of anti-beta HEL cells loaded with the Ca-sensitive fluorophore Fura-2 was significantly enhanced when compared with that of the control cells (Fig. 2, a and b). This result suggests that thrombin-induced elevation of [Ca] in HEL cells is normally inhibited by PKCbeta; this effect appeared to be specific for thrombin, because [Ca] induced by the endoperoxide analog U46619, which activates the thromboxane A(2) receptor, is not affected in anti-beta cells (Fig. 2, c and d). Enhanced [Ca] does not result from altering the expression of thrombin receptors on anti-beta cells, as assessed by flow cytometry utilizing a fluorescently labeled anti-thrombin receptor antibody (data not shown) or by expression of mRNA encoding the thrombin receptor (Fig. 1c).


Figure 2: Intracellular Ca concentration measured by Fura-2 fluorescence in both control and anti-beta HEL cells. a, representative tracing showing changes in [Ca] following addition of thrombin (0.5 unit/ml at time shown by arrow) in both control and anti-beta cells. b, bar graph showing mean ± S.E. of the differences between peak and basal [Ca] in both control (n = 13) and anti-beta cells (n = 23); p < 0.01, two-tail Student's t test. c, changes in [Ca] following addition of U46619 (1 µM) in control and anti-beta cells. d, bar graph showing mean ± S.E. of the concentration difference between peak values and basal level of [Ca] in control (n = 8) and anti-beta cells (n = 5).



To determine whether enhanced [Ca] resulted specifically from the reduction of PKCbeta expression by the antisense construct, we restored the expression of PKCbeta by stably transfecting cDNA encoding the full-length PKCbeta into clonal populations of anti-beta cells. To prevent inhibition of expression of the transfected PKCbeta by the constitutively expressed anti-beta cDNA, the degeneracy of the genetic code was exploited to generate a mutant PKCbeta cDNA (mut-PKCbeta) that had minimal complementarity with the anti-beta cDNA but still encoded the same amino acid sequence as native PKCbeta (Fig. 3a). Expression of the mut-PKCbeta was not inhibited by the anti-beta construct, as verified by cotransfection of the two constructs in COS7 cells, which do not normally express PKCbeta, followed by Northern transfer analysis (data not shown). Transfection of mut-PKCbeta into the anti-beta cells restored the expression of PKCbeta, as assessed by immunoblotting (Fig. 3b), and abolished the enhancement of thrombin-induced [Ca] in the anti-beta cells (Fig. 3, c and d). Thus, the reversal of the changes induced by antisense restoration of PKCbeta in the same clone of cells strengthens the argument that PKCbeta specifically inhibits the thrombin-induced increase in [Ca].


Figure 3: Restoration of PKCbeta expression with mutant PKCbeta. a, wild-type human PKCbeta sequence against which the antisense construct was targeted (top) and the sequence of the corresponding portion of the mutated full-length rat PKCbeta that was overexpressed in the anti-beta cells. b, immunoblot of PKCbeta expression in control (leftlane) and two clones of anti-beta HEL cells transfected with a mutant PKCbeta cDNA (mut-PKCbeta). Immunoblotting was performed with a polyclonal anti-PKCbeta antibody (Santa Cruz). c, changes in [Ca] upon adding thrombin (0.2 unit/ml) to control and mutant PKCbeta cells. d, bar graph shows the mean ± S.E. of the differences between peak and basal levels of [Ca] in control (n = 3) and mut-PKCbeta (n = 10) cells.



This inhibition of the thrombin-induced increase in [Ca] by PKCbeta might result from an effect on Ca entry, on Ca release from intracellular stores, or on both. To distinguish among these possibilities, the contribution of Ca entry from extracellular medium to [Ca] was eliminated by either briefly chelating extracellular Ca with EGTA or adding Ni, which blocks the Ca entry via receptor-operated cation channels(5, 7) . Following these interventions, [Ca] was not significantly different between anti-beta and control cells (Fig. 4, a and b). Furthermore, the divalent cation entry, assessed by measuring the Mn quench of intracellular Fura-2 fluorescence (6) after addition of thrombin, was less in the control cells than in the anti-beta cells (Fig. 4c). Thus, PKCbeta reduces the magnitude of thrombin-induced Ca entry but has little effect on release of Ca from intracellular stores. These results also demonstrate that some aspects of thrombin receptor function were not affected by inhibition of PKCbeta; additionally, PKC-mediated inhibition of thrombin-induced mobilization of Ca from intracellular storage sites (12, 13) does not require the presence of known Ca-regulated PKC isoenzymes in HEL cells.


Figure 4: Determination of the component of [Ca] regulated by PKCbeta. a, bar graphs of the mean ± S.E. of the difference between peak thrombin-induced and basal levels of [Ca] in control and anti-beta cells suspended in buffer to which 2 mM EGTA has been added (n = 3). b, bar graphs of the mean ± S.E. of the differences between peak thrombin-induced and basal levels of [Ca] in control and anti-beta cells suspended in buffer to which 2 mM NiCl(2) has been added (n = 3). c, representative tracing of Fura-2 fluorescence changes (360 nM excitation wavelength) of control and anti-beta cells suspended in medium containing Mn (0.1 mM) shortly after stimulation with thrombin (0.5 unit/ml).



After addition of thrombin (but not ADP) to platelets(6, 7) , a measurable delay precedes Ca entry, suggesting that the Ca channel is not directly linked to the receptor but instead is activated by intracellular mediators, generated perhaps by depletion of intracellular Ca stores (10, 11) or by phospholipid hydrolysis(21) . Thus, PKCbeta might interfere with a mediator that initiates or potentiates Ca influx; such a mediator would presumably be generated by thrombin but not endoperoxide analogs such as U46619. An alternative possibility is that PKCbeta, once activated, might phosphorylate a receptor-mediated Ca channel specifically associated with the thrombin receptor. Either model has implications for the specificity of agonist effect. Thus, although other mechanisms for limiting Ca entry may exist for other agonists, this reduction in thrombin receptor-mediated Ca influx by PKCbeta, a Ca-regulated PKC, represents a novel and selective cross-regulatory mechanism that could prevent excessive accumulation of cytoplasmic Ca following thrombin stimulation.


FOOTNOTES

*
This work was supported by Grants HL38820 and HL47032 from the National Institutes of Health (to J. A. W.) and a fellowship (to Y. X.) from the Massachusetts Affiliate of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by Research Career Award HL02271 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Medicine, RW453, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-3584; Fax: 617-667-4833; jaware{at}bih.harvard.edu.

(^1)
The abbreviations used are: [Ca], cytoplasmic Ca; PKC, protein kinase C; HEL, human erythroleukemic; mut-PKCbeta, mutant PKCbeta cDNA; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Drs. J. D. Chang, M. Yukawa, E. O. Harrington, S. Tang, and L. von Andrian of the Ware laboratory, Drs. Michael Simons and Shaun Coughlin for assistance and/or helpful discussion, and John Jaster for administrative assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.