Signal

transduction MEREDITH

in erythropoiesis

MASON-GARCIA

Department

of Pharmacology,

AND BARBARA Tulane

University

S. BECKMAN’

School

of Medicine,

New

Orleans,

Louisiana

70112,

USA

The polypeptide hormone erythropoietin (Ep) is a growth factor whose actions on the erythroid progenitor cell induce proliferation and differentiation. The signal transduction system activated by Ep to mediate these cellular processes remains largely uncharacterized despite many years of research devoted to its elucidation. It is clear that an Ep receptor-mediated activation of adenylate cyclase or guanylate cyclase does not occur, although CAMP and cGMP may play modulatory roles. The role of calcium in the action of Ep is less clear. Although the presence of extracellular calcium seems to be an abso-

dent on Ep for growth (1). CFU-E are committed progenitor cells that respond to Ep by undergoing

lute requirement

sociated of events

ABSTRACT

for Ep-induced

proliferation,

the posi-

tive changes induced by Ep in intracellular calcium occur with a time course suggestive of influx through ion channels opening within the cell membrane rather than release of intracellular stores by inositol trisphosphate. There is good evidence for the involvement of phospholipases A2 and C in the actions of Ep, including an early rise in lipoxygenase metabolites of arachidonic acid. Activation of phospholipase C can also result in the activation of protein kinase C in response to Ep. We present a model for the signal transduction pathway of Ep that is consistent with current knowledge and provides a framework for the coordinate actions of several intracellular mechanisms in the mediation of Ep-induced proliferation. Mason-Garcia, M.; Beckman, B. S. Signal transduction in erythropoiesis. FASEBJ. 5: 2958-2964; 1991. Key

Words:

signal

transduction

ERYTHROPOIETIN Hematopoiesis

erythropoietin

phospholipases

gressive

to renew

is the

increase

and

process

replace

in

the

through

which

the

organism

the

number

reflecting an increasing dependence This maturation culminates in the tor cell known as the colony-forming

of

cellular

Ep

on Ep to sustain formation of the unit-erythroid

receptors, growth. progeni-

(CFUE); of all the erythroid progenitor cells, the CFU-E possesses the greatest number of Ep receptors and is absolutely depen-

2958

of proliferation

followed

by

terminal

differentiation

and the loss of further proliferative capacity. In vitro, CFU-E respond to Ep by forming clonal colonies of eight or more hemoglobinized cells after 48 h in culture. The most mature erythroid progenitor cells, those of the proerythroblast/ erythroblast series, are committed progenitors that no longer require the presence of Ep to complete the program of erythroid differentiation. Erythropoietin is clearly the growth factor most closely as-

of the tor

with the initiation and completion collectively known as erythropoiesis.

intracellular

cells

events

changes

is less might

that

evident.

A

Ep

better

evokes

of the sequence The nature in these

progeni-

understanding

also have implications extending system, since an understanding

of

these

beyond the of the mecha-

hematopoietic nisms controlling proliferation and differentiation is central to our understanding of how these normal cellular processes are subverted in neoplastic diseases. The material presented here is intended to provide an overview of our current understanding of the intracellular signaling pathways that are activated by Ep, and to propose a model for the ways in which these pathways act and interact to regulate the proliferation of erythroid progenitor cells. Erythropoietin Produced stress, weight

and

primarily

the erythropoietin in the

kidney

receptor

in response

to hypoxic

Ep is a 166-amino-acid glycoprotein with a molecular of 34-38 kDa (2). It is heavily glycosylated, with ap-

40%

proximately saccharides.

AND

cells and formed elements of blood. This complex and vital process represents the net result of many interactions among various growth factors and progenitor cells which give rise to the mature cells of the hematopoietic system. Erythropoiesis is a subset of this larger scheme, including only the events that lead from the appearance of the committed erythroid progenitor cell through the formation of mature red blood cells. The early progenitor cells of the erythroid series are functionally characterized based on their response to two growth factors, interleukin 3 (IL 3)2 and erythropoietin (Ep). The most primitive progenitor, the burst-forming unit-erythroid (BFU-E), is characterized by an absolute requirement for IL 3 for continued growth. That these cells are also responsive to Ep is shown by the formation in vitro of large clusters (bursts) of as many as 10 hemoglobinized cells in response to Ep. The maturation of BFU-E is accompanied by a proacts

rounds

erythroid several

of its molecular

Erythropoietin

binding

to

a specific

mass

exerts

cell-surface cells (BFU-E

erythroid progenitor The biochemical nature (EpR) has been a subject

of the of intense

its

contributed biological

receptor on and CFU-E). erythropoietin investigation

by oligoeffects

its

by

target

receptor in recent

years, culminating in the cloning of the murine EpR in 1989 (3) and of the human EpR in 1990 (4). The EpR exists in both low- and high-affinity states, and in studies where Ep is cross-linked to EpR the ligand-receptor complex appears as two bands (5). When a correction is made for the contri-

‘To whom correspondence should be addressed, at: Department of Pharmacology, 1430 Tulane Ave., New Orleans, LA 70112, USA. 2Abbreviations: BFU-E, bunt-forming unit-erythroid; BHA, butylated hydroxyanisole; BW755c, 3-amino-l[M-(tnifluoromethyl)phenyl]-2-pyrazoline; [Ca],, intracellular calcium concentration;

CFU-E,

colony-forming

unit-erythroid;

Ep, erythropoietin;

H-7,

l-(S-isoquinolinesulfonyl)-2-methylpiperazine; 12-HETE, 12-hydroxyeicosatetraenoic acid; 12- HPETE, 12-hydroperoxyeicosatetraenoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; 15-HPETE, 15acid; IL, intenleukin; IP3, inositol LT, leukotniene; NDGA, nordihydroguaiaretic acid; NGF, nerve growth factor; PKC, protein kinase C; PMA, phorbol myristate acetate; R, receptor; ST 638, a-cyano-3-ethoxy-4-

hydroperoxyeicosatetraenoic tnisphosphate;

hydroxy-5-phenylthiomethylcinnamamide.

0892-6638/91/0005-2958/$01.50.

© FASEB

ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

bution of the cross-linked Ep molecule, these bands represent proteins with apparent molecular masses of 85 and 100 kDa. As the gene for EpR encodes a single protein with a predicted molecular weight of 55 (unglycosylated) or 65 kDa (glycosylated), the difference in apparent size seen in the cross-linking studies cannot be accounted for by receptor dimerization. It has been suggested that this difference may be attributed to the interaction between the EpR and a second, uncharacterized intracellular protein. Such an interaction has recently been proposed between the receptor for nerve growth factor (NOF) and the protein product of the proto-oncogene trk (6). Like the EpR, the NGFR lacks a characterized signal transduction mechanism, and exists in high- and low-affinity states. Trk is a tyrosine kinase, which could account for the increase in tyrosine phosphoproteins that occurs in response to NGF, despite the absence of an intrinsic tyrosine kinase activity in the NGFR (6). It has recently been proposed that the EpR receptor and the receptors for prolactin, growth hormone, IL 2, IL 3, IL 4, IL 6, IL 7, and granulocyte/macrophage colony-stimulating factor constitute a new family of receptors (7). Whereas the major area of homology among these receptors is in the extracellular domain, there is also homology in the cytoplasmic domain among the EpR, IL 2R, and IL 3R, as seen in Fig. 1 (5). Like the members of the epidermal growth factor receptor family, these receptors all possess a single membranespanning domain, but they lack the intrinsic tyrosine kinase activity of the epidermal growth factorlike receptors. Indeed, the receptors of this newly proposed family are interesting in that their structures provide no obvious clue to the signal transduction system to which they are coupled, nor are the signal transduction systems well characterized in the cells that express these receptors.

SIGNAL Elucidation the binding many

TRANSDUCTION

AND

of the signal transduction of Ep to EpR has been

researchers,

yet

the

discovery

ERYTHROPOIESIS pathway a subject of a clear

activated of interest paradigm

by to has

proved elusive. Studies in this field are also complicated by the fact that Ep activates both proliferation and differentiation in its target cells, raising the possibility that multiple signaling pathways may be required to act and interact in response to Ep. Most of the classical intracellular signaling molecules studied seem to exert a modulatory effect rather than being directly coupled to the actions of the Ep molecule. A review of the early literature in this field is nevertheless useful in establishing which pathways may not be immediately linked to the actions of Ep, whereas a review of more recent studies suggests that novel intracellular signaling systems may be activated to regulate the complex of intracellular events mediated by Ep. Each signal transduction system implicated in the actions of Ep will be discussed in turn, followed by a model we are proposing to explain how some of these systems may interact to regulate proliferation. The

role

of cyclic

nucleotides

During the early 1970s, cAMP and cOMP were proposed as second messengers for Ep. Although experimental evidence suggests that these molecules may play a modulatory role in erythropoiesis, neither cAMP nor cGMP appears to be directly linked to the actions of Ep. The earliest studies linking the actions of Ep with cAMP were based on the observation that cAMP or its analogs

I

EPO-R

IL-2R

IL-3R

Figure 1. Schematic representations of the receptors for erythropoietin receptor (EPO-R), the /3 subunit of the interleukin 2 receptOr (IL 2Rj3) and the interleukin 3 receptor (IL 3R). The homology

in the extracellular domain includes four cysteine moieties (Cl-C4), a feature common to all members of the growth hormone/prolactin/cytokine

superfamily.

domain (shaded area) IL 3R (after D’Andrea

exists and

The

homology

only among Zon,

in

the

the EpR,

intracellular

IL 2R, and

ref 5).

would increase erythrocyte numbers in vivo (Gidari et al., see ref I). However, in studies of the effects of cAMP on erythropoiesis in vitro, cAMP and its analogs did not potentiate Ep-induced heme synthesis in rat bone marrow cells (Oraber et al., 1972; see ref 1). This lack of involvement of cAMP was substantiated by further studies showing that Ep did not increase cellular levels of cAMP in rat CFU-E (Graber et al. 1974; see ref 1). The involvement of cGMP as an intracellular signal activated by Ep was proposed by Rodgers et al. (see ref 1), who found that Ep induced an elevation in cOMP in cultured rabbit bone marrow cells. Graber et al. (1977; see ref 1) reported a similar increase in cGMP in Ep-stimulated rat fetal liver cells. Neither group, however, found a significant increase in cGMP until 4 h after treatment of the cells with Ep, suggesting that activation of guanylate cyclase is not directly coupled to activation of the Ep receptor. However, cOMP may modulate proliferation of the erythroid progenitor cell, as White et al. (8) found the maximum increase in cGMP to appear immediately before the onset of the mitotic (M) phase of the cell cycle; a second but smaller peak also occurred during the S phase, when DNA is synthesized. The

role

of calcium

Calcium ion is another cellular signaling molecule that has been linked to the actions of Ep. Changes in intracellular calcium concentrations ([Ca21) are known to be important in regulating many cellular processes, including the activation of enzymes such as phospholipase A2 and protein kinase C. Elevations in [Ca2]1 are also required for the initiation of DNA synthesis during cellular proliferation. Thus, it is reasonable to explore the relationship between Ep and changes in [Ca21j. Changes in [Ca2]1 may be caused by the influx of extracellular Ca2 through the opening of calcium channels in the cell membrane, or by the release of Ca2 from intracellular stores through the actions of inositol tnisphosphate (1P3) or 12-hydroxyeicosatetraenoic acid (12-HETE).

SIGNAL TRANSDUCTION IN ERYTHROPOIESIS 2959 ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

The possible of Ep will be Misiti and requirement finding

that

enhance progenitors,

role of each pathway in mediating the actions discussed separately. Spivak (see ref 1) extensively characterized the for extracellular calcium in erythropoiesis,

the

calcium

Ep-induced whereas

ionophore, colony the

A23187,

formation calcium

would

modestly

in murine erythroid chelator EGTA would

markedly inhibit colony formation. The inhibition induced by the addition of EGTA could be reversed by restoring calcium ion to the culture medium through the addition of calcium chloride, thus demonstrating that the inhibitory effect of EGTA was calcium related. Bonanou-Tzedaki et al. (9) further studied the effect of modulators of calcium and reported that either the calcium chelator EGTA, the calcium channel blocker verapamil, or the inhibitor of intracellular calcium mobilization TMB-8 would inhibit Ep-induced proliferation (as

reflected

by

erythroid found that

the

incorporation

progenitors the ionophore

of tritiated

from rabbit bone A23187 did not

thymidine)

marrow. enhance

in

They also Ep-induced

nor did it induce proliferation in the absence findings suggest that the presence of extracellular calcium is necessary for the proliferation of erythroid progenitors but that it is not sufficient to induce this process in the absence of Ep. proliferation,

of Ep. These

The

signaling

molecule

that

has

been

most

extensively

studied as a mediator of the release of intracellular calcium is IP3. Activation of the phosphatidylinositol-specific phospholipase C results in the generation of IP3, which can release calcium from intracellular stores in the endoplasmic reticulum. The time course for generation of these molecules is very rapid: 1P3 levels may peak within 2-5 s of the activation of phospholipase C, with a rise in intracellular calcium occurring within seconds of the release of 1P3. In a preliminary report, Thompson et al. (10) studied the effect of Ep on 1P3 levels in munine erythroid progenitors and reported that Ep does not cause an increase in inositol phosphates, which suggests that Ep does not act via this intracellular signaling pathway. We also have found no increase in IP3 in response to Ep (M. Mason-Garcia, J. -S. Tou, and B. S. Beckman, un-

hematopoietic

system.

Metabolites

of

the

cyclooxygenase

pathway include prostaglandins and thromboxane. Three major lipoxygenase pathways exist (5-, 12-, and l5-lipoxygenase), leading to the formation of the leukotrienes (LT5), 12-hydroperoxyeicosatetraenoic acid (12-HPETE), and 15hydroperoxyeicosatetraenoic acid (l5-HPETE), respectively. Metabolites of both the cyclooxygenase and lipoxygenase pathways

have

been

studied,

but

those

of the

lipoxygenase

important in the regulation of proliferation. For example, the leukotriene LTB4 is known to induce mitogenesis in some cell types, including myeloid progenitors (13) and leukemic cell lines of myelogenous, promyelocytic, and histiocytic origin (14). Similarly, 5lipoxygenase inhibitors have been shown to be potent inhibitors of proliferation in leukemic cells of several types (15) and in myeloid and erythroid progenitor cells (16). The rate-limiting step in the formation of both cyclooxygenase and lipoxygenase metabolites is the release of arachidonic acid from membrane phospholipids through the action of a phospholipase. Arachidonic acid at the sn-2 position of the phospholipid may be released directly by the actions of a phospholipase A2, or indirectly by the sequential actions of a phospholipase C and a diacylglycerol lipase. In regard to the actions of Ep, Beckman and Seferynska (17) have shown that the phospholipase inhibitors quinacrine, pbromphenacyl bromide, or 7,7-dimethyleicosadienoic acid will inhibit Ep-induced colony formation in erythroid progenitor cells from munine fetal liver. The use of these relatively nonselective inhibitors, however, did not permit a distinction between the possible involvement of a phospholipase A2 vs. a phospholipase C. Further studies by Beckman et al. (18) and Mason-Garcia et al. (unpublished results) have provided insight into this problem. In the former study, the phospholipid pool of erythroid progenitor cells was prepathways

labeled

appear

with

to be more

3H arachidonic

acid, followed by treatment

of

pension of erythroid precursor cells and analyzing the calcium response with a fluorometer, found a significant increase in [Ca2]1 only after 30 s of exposure of the cells to Ep, with the maximum response at 4-6 mm. The amplitude of the response was also dose dependent, increasing with the amount of Ep added, and it could be blocked by preincuba-

Ep for 30 s. Ep was shown to release labeled arachidonate from all major phospholipids present, with 55% of the total coming from phosphatidylcholine, followed by phosphatidylethanolamine (24%), phosphatidylinositol (18%), and phosphatidylserine (3%). Although these results suggest that Ep activates a phospholipase in these cells, it is still not clear whether the phospholipase is A2 or C. The work of Mason-Garcia et al. (unpublished results) indicates that a phospholipase C may be involved, because an inhibitor of diacylglycerol lipase is shown to inhibit Ep-induced proliferation and differentiation. These results suggest that the sequential phospholipase C/diacylglycerol lipase pathway is used, at least in part, to release arachidonic acid in these cells. Snyder and Desforges (16) first investigated the role of the lipoxygenase pathway of arachidonic acid metabolism in hematopoiesis. They found that several compounds that can act as inhibitors of both the cyclooxygenase and lipoxygenase pathways (3-amino-1[M-(trifluoromethyl)-phenyl-2-pyrazoline [BW755c]; nordihydroguaiaretic acid [NDOA]; and butylated

tion

hydroxyanisole

published

The

results).

recent

fluors

such

Ep-induced al. (11) used

changes

at 3

mm.

of cell-permeant, has

in [Ca2]1

progenitors, The

to baseline

with

made

possible

calcium-activated the

at the cellular

investigation

level.

of

Miller

et

fluorescence microscopy to analyze changes in calcium in response to Ep in individual human

intracellular erythroid

availability as fura-2

response

by 10 mi

finding had

a significant peaked

increase

by 5

mm and

and

Kay

Mladenovic

in [Ca2]1 had

(12),

returned

using

a sus-

antierythropoietin.

The major finding from all these studies is that in no case was the time course for Ep-induced changes in intracellular calcium compatible with that which would be caused by an IP3-mediated release of calcium from intracellular stores. Thus, although it appears that elevations in [Ca2]1 follow the stimulation of erythroid progenitors by Ep, the source of this

calcium

endoplasmic The

role

Metabolites mitogenic

is

probably

not

the

1P3-sensitive

pool

of

the

reticulum. of arachidonic

acid

and

its metabolites

of arachidonic acid have been implicated in the response in many cell types, including cells of the

the cells with

[BHA])

would

significantly

inhibit

the

for-

mation of BFU-E derived from monocyte-depleted bone marrow, whereas the selective cyclooxygenase inhibitor indomethacin was ineffective. Beckman and Nystuen (19) studied the effects of these inhibitors in CFU-E from murine fetal liver and found no significant inhibition of colony formation by the cyclooxygenase inhibitors, sodium meclofenamate or aspirin, but a significant and dose-dependent inhibition of colony formation was induced by the combined inhibitors, BW 755c, NDOA, and BHA. Although the inhibitors used were not selective for the lipoxygenase pathway, the finding that cyclooxygenase-selective inhibitors had no effect suggests that the combined inhibitors were exerting their effects via inhibition of the lipoxygenase pathway.

2960 Vol. 5 November 1991 MASON-GARCIA AND BECKMAN The FASEB Journal ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

In another

study,

Beckman

et al. (20)

characterized

with

HPLC the lipoxygenase metabolites produced by these cells and found that Ep induced a small increase in 15-hydroxyeicosatetraenoic acid (15-HETE, the stable metabolite of 15-HPETE) and a large increase in 12-hydroxyeicosatetraenoic acid (12-HETE, the stable metabolite of 12-HPETE). These increased levels were seen as early as 15 mm after the addition of Ep to the cells and persisted for 2 h. Using radioimmunoassay, they determined that 12-HETE was the major metabolite, and that levels remained significantly elevated up to 24 h after Ep was added. It is noteworthy that 5- and 12-lipoxygenase products can activate guanylate cyclase, as this interaction could explain the later increase in cOMP seen in response to Ep (8). In a subsequent study, Beckman and Seferynska (17) demonstrated that exogenous 12-HPETE, but not 12-HETE, 15-HPETE, or 15-HETE, would significantly induce erythroid colony formation in the absence of Ep. More recent work has also shown that exogenous leukotriene B4 (a metabolite of the 5-lipoxygenase pathway) will also induce colony formation, but not leukotriene C4 or D4 (B. S. Beckman, unpublished results). Most recently it has been shown with specific radioimmunoassays that Ep induces a rapid rise in both LTB4 and 12-HETE levels. Increases in both metabolites are detectable by 30 s and are significant by 2 mm in the case of LTB4 and by 10 mm in the case of l2-HETE (Mason-Garcia et al., unpublished results). These elevations are sustained through 60 mm for 12-HETE, but LTB4 has returned to control levels by that time (18). Taken together, these data suggest that lipoxygenase metabolites of arachidonic acid metabolism, specifically LTB4 and 12-HPETE, may play an early and important role in mediating the actions of Ep. The

role

of protein

kinases

A change in protein phosphorylation is seen as an early response to many agonists, including Ep. In intact cells, it has been reported that cellular proteins are phosphorylated as rapidly as 7 mm after exposure of the cells to Ep, and that a major phosphoprotein of erythroid progenitor cells, p80, is phosphorylated within 2.5 mm of Ep exposure (21). This increased phosphorylation represents one of the earliest intracellular events that has been reported to occur in response to erythropoietin. Since the acceptor group for phosphorylation of these proteins has not been identified, tyrosine kinases and serine/threonine kinases are both candidates for activation by Ep, and indeed some evidence exists for the involvement of each type. Tyrosine kinase activity is often intrinsically associated with the receptors of growth factors: the epidermal growth factor receptor, the platelet-derived growth factor receptor, the insulin-like growth factor receptor, and the insulin receptor itself all contain tyrosine kinase domains. Members of the receptor family to which the EpR belongs lack the consensus sequence for tyrosine kinase activity. Nevertheless, activation of the IL 2R, which is also in the EpR family, has been reported to induce tyrosine phosphorylation of cellular proteins (22). Furthermore, both insulin and insulin-like growth factors are known to be positive modulators of erythroid colony formation (23), supporting a role for tyrosine phosphorylation in erythropoiesis. Inhibition of tyrosine kinase activity has also been shown to modulate the actions of erythropoietmn: inhibitors of tyrosine kinase have been found to inhibit proliferation in erythroid progenitors while permitting differentiation to occur normally. Noguchi et al. (24) reported that normal murine CFU-E treated with herbimycin formed single hemoglobinized cells in response to Ep, but did not form the

SIGNAL

TRANSDUCTION

IN ERYTHROPOIESIS

classic erythroid colonies. Herbimycin is a selective inhibitor of the tyrosine kinase product (p6O”) of the Rous sarcoma virus gene (v-src), but other substrate inhibitors of tyrosine kinase such as genistein and ST638 (a-cyano-3-ethoxy-4hydroxy-5-phenylthiomethylcinnamamide) have also been found to induce differentiation in murine erythroid cells that have been transformed with the Friend virus (25). These data suggest the involvement of a tyrosine kinase in erythropoiesis as a positive modulator of proliferation or as a negative modulator of differentiation. In neither case, however, is there evidence for the actions of Ep itself being directly coupled to a tyrosine kinase activity. If rapid phosphorylation of intracellular proteins is an early event in the actions of Ep, then activation of a serinethreonine kinase may be induced by Ep. Such a kinase is unlikely to be a cAMPor cOMP-dependent protein kinase, as cAMP levels do not increase in cells treated with Ep and significant increases in cGMP do not occur for several hours. The likely candidates, therefore, are the calcium/calmodulindependent kinase or the calciumand phospholipid-dependent protein kinase (protein kinase C, or PKC). The role of the calcium/calmodulin-dependent kinase in erythropoiesis has not been studied. However, the role of PKC in normal erythropoiesis has been characterized by several investigators. Treatment of normal murine erythroid progenitors with the phorbol ester, phorbol myristate acetate (PMA), a nonhydrolyzable activator of PKC, has been reported to enhance erythroid colony formation (Fibach et al., 1980; see ref 1) and to suppress erythroid colony formation (Sieber et al., 1981; see ref 1). These discrepant findings may be reconciled by the fact that although acute treatment with PMA can activate PKC, chronic PMA can inhibit PKC through down-regulation. Thus, studies designed to inhibit rather than activate PKC, may provide data that are easier to interpret. Jenis et al. (26) have shown that the protein kinase inhibitors H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazmne) or staurosporine produce a dose-dependent inhibition of Ep-induced colony formation in normal murine CFU-E, suggesting that activation of PKC is part of the signal transduction pathway of Ep. We have made similar observations, with the additional finding that inhibitors of PKC will inhibit Ep-induced proliferation in normal murine erythroid progenitors, as measured by incorporation of tritiated thymidine (M. Mason-Garcia and B. S. Beckman, unpublished). Furthermore, Spangler et al. (21) have shown that the Ep-mediated increase in expression of the c-myc proto-oncogene in erythroid progenitors occurs via a PKC-dependent pathway; the association of increased c-myc expression with increased proliferation further supports the role of PKC in Ep-induced proliferation. Protein kinase C may also interact with the other signal transduction pathways that have been implicated in the actions of Ep. For example, tyrosine protein phosphorylation is required for the PKC-mediated proliferation of T lymphocytes (27). Also, inhibitors of PKC will diminish the Ep-induced elevation in LTB4 (Mason-Garcia et al., unpublished results) without affecting the increase in 12-HETE, suggesting that PKC may be involved in activation of the 5-lipoxygenase. A novel role for protein kinase C in signal transduction at the nucleus has also been reported, first for prolactin in the nuclei of hepatocytes by Buckley et al. (28), and most recently for erythropoietin in the nuclei of erythroid progenitor cells by our laboratory (29). In isolated nuclei free of membrane contamination, we have demonstrated that Ep stimulates a dose- and time-dependent phosphorylation of endogenous substrate, which can be inhibited by an antibody to erythropoietin and is also sensitive to inhibition by several inhibitors of protein kinase C (H-7, staurosporine, and 2961

ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

sphingosmne).

Dose-dependent

activation

of PKC

in these

isolated nuclei can also be achieved with phorbol myristate acetate, phorbol dibutyrate, and oleolyacetylglycerol, all known to be activators of protein kinase C. Further support for this nuclear protein kinase C activity is provided by the demonstration nuclei of these

Taken role

together,

PKC

for

vated

by

PKC

of immunoreactive cells (29).

these

in

the

studies

support

intracellular

/3 II

j3 I and the

signaling

in the

existence

of a

pathways

acti-

erythropoietin.

A PROPOSED

MODEL

FOR

THE

ACTIONS

OF

ERYTHROPOIETIN

The

information

gained

that lipids represent naling molecules arachidonic

from

these

an important in erythropoiesis:

acid,

either

studies

makes

it clear

group of intracellular sigphospholipids provide

directly

or via

a diacylglycerol

inter-

mediate; arachidonic acid is further metabolized to leukotriene B or 12-HPETE; diacylglycerol may provide arachidonic acid and/or serve as an activator of protein kinase C. A proposed model act and interact

for the way to regulate

presented

in Fig.

In

proliferation

the

in which these molecules Ep-induced proliferation

may is

2. pathway,

it is proposed

that

Ep

binds

to its cell-surface receptor, which activates both a phospholipase A2 and a phospholipase C; these enzymes cause the release from membrane phospholipids of arachidonic acid or diacylglycerol. glycerol lipase

tein kinase arachidonic formation nase also metabolites,

Diacylglycerol can to release arachidonate

be

acted on by diacylor it can activate pro-

C. Protein kinase C mediates the activation of the acid 5-lipoxygenase enzyme, which results in the of leukotriene B4. Arachidonic acid 12-lipoxygeacts on arachidonate to form 12-HPETE. These along

with

protein

C,

kinase

tracellular calcium and intracellular ing ion channels and transporters

act

to increase

in-

pH, possibly by activatin the cell membrane. This

increase in both intracellular calcium and the initiation of DNA synthesis. Activation

pH is required

C

and

can

induce

expression

oncogenes, which actions of Ep (21,

of

is also 30).

the

known

of protein

c-fos

to be

an

proto-

c-myc

early

event

in the

The first event hypothesized to occur in the induction proliferative response is the activation of phospholipases and C, which release arachidonate from membrane pholipids by cleavage of specific bonds: phospholipase cleaves the sn-2-acyl bond of phospholipids to yield phospholipid acid);

and

arachidonic

phospholipase

acid

C cleaves

acylglycerol and the phosphorylated systems, the coupling of a single both

phospholipase

ported. bradykinin pholipase similar The

A2

For example, receptor A2 and observations

and

Burch

(or

at the

other

head

receptor

A2 are

a G protein

a family

activation

et al.

a time tracellular tion of

course

nucleotide-binding cell, release

that

be positively

which

precedes

calcium, phospholipase

making A2

that

proteins of arachidonate of the

reported

it unlikely that the is calcium-mediated.

phos-

(32) made hormone.

increases cellular calcium levels, PKC-mediated inactivation dogenous phospholipase A2 inhibitor lipocortin, of guanine progenitor

of

that the

to both

of enzymes

in a variety of tissues; their activity can lated by different mechanisms, including

actions erythroid

di-

In some

(31) reported

phospholipase C; Chang for gonadotropin-releasing

phospholipases

group.

C has been re-

phospholipase via

fatty

to yield

to the

and Axelrod

is coupled

of the A2 phosA2 a lyso-

substituent

3-position

polar

for

kinase

exist modu-

in intraof the enor the

(33). In occurs rise early

Figure 2. A proposed model for the actions of erythropoietin. The binding of Ep to its receptor on the cell membrane causes the activation of phospholipases A2 and C, possibly through the actions of a G protein. Arachidonic acid that is enzymatically cleaved from membrane phospholipids is metabolized by lipoxygenases to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or leukotriene B4 (LTB4). 12-HPETE and LTB4 may facilitate the mitogenic response by inducing changes in intracellular pH and calcium concentration or by activating transcription of the nuclear proto-oncogene c-fos. Diacylglycerol (DAG) that is released by the actions of phospholipase C can be hydrolyzed to release arachidonic acid or can activate protein kinase C (PKC). The activation of PKC may result in the opening of membrane calcium channels or activation of the sodium-hydrogen antiport mechanism, to increase intracellular calcium and pH. Activation of PKC in the nucleus may stimulate transcription of the c-fos and c-myc proto-oncogenes. All these events are associated with the proliferative response.

the on

in in-

activaIf PKC-

mediated

inactivation

tion of phospholipase PKC would result

of lipocortin

were

required

for

activa-

A2 in these cells, then the inhibition in a decrease in both LTB4 and 12-HETE

of

as the amount of substrate arachidonic acid would be decreased. Our finding that only the production of LTB4 is decreased argues against the involvement of lipocortin. The third possibility, that phospholipase A2 is directly activated by the interaction between the EpR and a guanine nucleotide binding protein (G protein), is the most intriguing. The

predicted

of the classical transmembrane growth factor

conformation

of the

EpR

differs

from

that

0

protein-coupled receptor (one vs. seven domains). However, another nonclassical receptor, that of IL 6, has been reported to

contain amino acid sequences with homology to the ODP/ GTP-binding and GTPase regions of G proteins. Both the IL 6 receptor and the Ep receptor are members of the same receptor family (7). The receptor for platelet-derived growth factor, which also contains a single membrane-spanning domain,

has

also

been

reported

to be

coupled

to

an

initial

early release of arachidonic acid which precedes increases in calcium or diacylglycerol (34). Along with earlier studies by Beckman (unpublished results) that show an enhancement of Ep-induced colony formation by nonhydrolyzable analogs of GTP, these data suggest that activation of phospholipase A2 by Ep in erythroid progenitor cells could occur via a receptor-G protein interaction.

MASON-GARCIA AND BECKMAN The FASEB Journal 2962 Vol. 5 November 1991 ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

Like the phospholipases

A2, the phospholipases

represent a family of enzymes. A2, however, the phospholipases

Unlike C show

C also

the phospholipases substrate specificity

among be the

their subtypes. The best known phospholipase phosphatidylinositol-specific phospholipase

C may C, which

causes

the

molecules

release

of

two

intracellular

signaling

both

(diacylglycerol and 1P3) from phosphatidylinositol bisphosphate. As the erythroid progenitor cells do not show the early rise in 1P3 that is characteristic of the activation of this enzyme, it is more likely that the activation of a phosphatidylcholineor phosphatidylethanolamine-specific phospholipase C is involved.

specific event

The

in

the

(35),

lular

of a phosphatidylethanolamine-

C has

mitogenic

phoma cells choline-specific number zymes

activation

phospholipase

been

response

whereas

reported to

the existence

phospholipase

C

to be an early

prolactin

in

NB2

lym-

of a phosphatidyl-

has

been

reported

in

a

of cell types, including lymphocytes (36). These enalso seem to lack a requirement for increased intracelcalcium for activation (37), and have been suggested as

candidates

for

activation

by

receptor-coupled,

pertussis

toxin-sensitive G proteins (38). The inhibitory effects of a diacylglycerol lipase inhibitor on Ep-induced proliferation in the erythroid progenitor cell (Mason-Garcia et al., unpublished results) suggests that the release of arachidonic acid in these cells is mediated in part by the pathway.

activation of a phospholipase The release of arachidonate

been described The activation

in other cell types. of phospholipase

C/diacylglycerol lipase via this pathway has C and

of diacylglycerol from phospholipids tivation of protein kinase C. Support

subsequent

release

can also result in the for a role for PKC

acin

proliferation is provided by studies that show an inhibition of proliferation by inhibitors of PKC in IL 2 stimulated T lymphocytes (39) as well as Ep-stimulated erythroid progenitor cells

(26;

Mason-Garcia

Protein activation crease

and

Beckman,

unpublished

kinase C is known to stimulate of the Na/H exchanger and

in intracellular

pH.

PKC

can

also

results).

mitogenesis by the the subsequent inactivate

L-type

cal-

cium channels in a number of cell types. This represents a mechanism by which PKC could increase intracellular calcium, another requisite event in the onset of DNA synthesis. Also, PKC is known to phosphorylate DNA topoisomerase II, which is believed to be an important regulatory enzyme for DNA replication. Furthermore, the recent report from Spangler et al. (21) on the PKC-mediated activation of c-myc by Ep suggests another pathway for the involvement of PKC in the mitogenic response, as c-myc expression is associated with proliferation. Nuclear protein kinase C activity in the erythroid progenitor cell (29) may mediate the activation of both the c-myc and the c-fos proto-oncogene; both are responsive to PKC-mediated phosphorylation, although the substrates that mediate this responsiveness are not well characterized. Although the actions of PKC may play an important role in the mitogenic response, that this activation of PKC is not sufficient to activate mitogenesis in CFU-E is shown by the inhibitory effects of lipoxygenase inhibitors, suggesting an independent role for the metabolites of arachidonic acid in the

regulation

of proliferation.

Although the mechanisms that mediate the production of LTB4 and 12-HPETE are fairly well characterized, their intracellular actions are less well understood. LTB4 has been associated with the mitogenic response in some hematopoietic cell types, including myeloid progenitors (13) and leukemic cell lines of myelogenous, promyelocytic, and histiocytic origin (14). Inhibitors of the 5-lipoxygenase are known to be potent inhibitors of proliferation in leukemic cells of several types

(15)

and

in myeloid

and

erythroid

progenitor

cells

(16).

Furthermore, tumor necrosis factor-a, which can act as a mitogen, has been shown to induce c-fos expression by a lipoxygenase-mediated pathway (40). LTB4 is known to stimulate calcium and sodium influx in neutrophils (41); of these

events

are

positive

modulators

of DNA

synthe-

sis. Similarly, l2-HPETE has been reported to release calcium directly from mitochondria (42). Taken together, the findings reviewed here support the hypothesis that the actions of Ep are mediated by the Epinduced generation of intracellular lipid molecules. The lipoxygenase metabolites of arachidonic acid, LTB4 and 12HPETE, may play an important role in regulating the proliferative response to Ep, as may the diacylglycerol-mediated activation of protein kinase C. In addition, there seems to exist the interesting possibility for the activation of a tyrosine kinase. These findings provide a framework on which future experiments can be based, permitting a deeper understanding of the mechanism of action of erythropoietin. This

work

was

supported

by American

Cancer

CH-345 (to B. S. B.). M. M.-G. was supported lowship from the National Science Foundation is currently a Pharmaceutical Manufacturers tion Postdoctoral Fellow.

Society

grant

by a predoctoral fel(RCD-8758119) and Association Founda-

REFERENCES 1. Spivak, J. L. (1986) The mechanism of action of erythropoietin. j Cell Cloning 4, 139-166 2. Hirth, P., Wieczorek, L., and Scigalla, P. (1988) Molecular biology of erythropoietin. Contr. Nephrol. 66, 38-53 3. D’Andrea, A. D., Lodish, H. F., and Wong, G. G. (1989) Expression cloning of the murine ezythroleukemia receptor. Cell

mt.

57, 277-285 4. Jones, S. S., D’Andrea, A. D. Haines, L. L., and Wong, G. G. (1990) Human erythropoietin receptor: cloning, expression, and biological characteristics. Blood 76, 31-35 5. D’Andrea, A. D., and Zon, L. I. (1990) Erythropoietin receptor: subunit structure and activation. j Gun. Invest. 86, 681-687 6. Kaplan, D. R., Martin-Zanca, D., and Parada, L. F. (1991) Tyrosine phosphorylation and tyrosine kinase of the trk protooncogene product induced by NGE Nature (London) 350, 158-160

7. Cosman,

D., Lyman,

S. D., Idzerda,

Park, L. S., Goodwin, R. G., cytokine receptor superfamily.

R. L., Beckmann,

M. P.,

and March, C. J. (1990) A new Trends Biochem. Sci. 15, 265-270

8. White, L., Fisher, J. W., and George, W. J. (1980) Role of erythropoietin and cyclic nucleotides in erythroid cell proliferation in fetal liver. Exp. Hematol. 8, 168-181. 9. Bonanou-Tzedaki, S. A., Sohi, M. K., and Arnstein, H. R. V. (1987) The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin. Exp. Cell Res. 170, 2 76-289 10. Thompson, L. P., Sawyer, S. T., Blackmore, P. F., and Krantz, S. B. (1988) A search for the second messenger of erythropoietin. FASEBJ 2, A813 11. Miller, B. A., Scaduto, R. C., Tillotson, D. L., Botti, J. J., and Cheung, J. Y. (1988) Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors. j Gun. Invest. 82, 309-315 12. Miadenovic, J., and Kay, N. E. (1988) Erythropoietin induces rapid increases in intracellular free calcium in human bone marrow cells. J. Lab. C/in. Med. 112, 23-27 13. Claesson, H. -E., Dahlberg, N., and Gahrton, G. (1985) Stimulation of human myelopoiesis by leukotriene B4. Biochem. Biophys. Res. Commun. 131, 579-585. 14. Snyder, D. S., Castro, R., and Desforges, J. (1989) Antiproliferative effects of lipoxygenase inhibitors on malignant human hematopoietic cell lines. Exp. Hematol. 17, 6-9 15. Tsukada, T., Nakashima, K., and Shirkawa, S. (1986) Arachidonate 5-lipoxygenase inhibitors show potent antiproliferative

SIGNAL TRANSDUCTION IN ERYTHROPOIESIS 2963 ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29

16.

17.

18.

19.

20.

21.

22.

23.

24.

effects on human leukemia cell lines. Biochem. Biophys. Res. Commun. 140, 832-836 Snyder, D. S., and Desforges, J. F. (1986) Lipoxygenase metabolites of arachidonic acid modulate hematopoiesis. Blood 67, 1675-1679 Beckman, B. S., and Seferynska, I. (1989) Possible involvement of phospholipase activation in erythroid progenitor cell proliferation. Exp. Heniatol. 17, 309-312. Beckman, B. S., Tou,J.-S., and Mason-Garcia, M. (1990) Phospholipase A2 activation is an early event in erythropoietin stimulated murine fetal liver cell proliferation-differentiation. Adv. Prostaglandin, Thromboxane, and Leukotriene Res. 21, 831-834 Beckman, B., and Nystuen, L. (1988) Comparative effects of inhibitors of arachidonic acid metabolism on erythropoiesis. Prostaglandins Leukotrienes Essent. Fatty Acids 31, 23-26 Beckman, B. S., Mason-Garcia, M., Nystuen, L., King, L., and Fisher, J. W. (1987) The action of erythropoietin is mediated by lipoxygenase metabolites in murine fetal liver cells. Biochein. Biophys. Res. Commun. 147, 392-398 Spangler, R., Bailey, S. C., and Sytkowski, A. J. (1991) Erythropoietin increases c-myc mRNA by a protein kinase Cdependent pathway. j Biol. Chem. 266, 681-684 Turner, B., Rapp, U., App, H., Greene, M., Dobashi, K., and Reed, J. (1991) Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-l kinase in a T-cell line. Proc. NatI. Acad. Sci. USA 88, 1227-1231 Sawada, K., Krantz, S. B., Dessypris, E. N., Koury, S. T., and Sawyer, S. T. (1989) Human colony-forming units-erythroid to do not require accessory cells, but do require direct interaction with insulin-like growth factor-I and/or insulin for erythroid development. j Clin. Invest. 83, 1701-1709 Noguchi, T., Fukumoto, H., Mishina, Y., and Obinata, M.

Commun. 168, 490-497 30. Umemura, T, Umene, K., Takahira, H., Takeichi, N., Katsuno, M., Fukumaki, Y., Nishimura, J., Sakaki, Y., and Ibayashi, H. (1988) Hematopoietic growth factors (BPA and Epo) induce the expressions of c-myc and c-fos proto-oncogenes in normal human erythroid progenitors. Leu/thmia Res. 12, 187-194. 31. Burch, R. M., and Axelrod, J. (1987) Dissociation of brady-

(1988) Differentiation

37. Martin,

from mouse fetal (TSA8) cells without

25. Watanabe,

of erythroid liver cells proliferation.

T., Shiraishi,

T., Sasaki,

Inhibitors for protein-tyrosine duce differentiation of mouse gistic manner. Exp. Cell Res.

progenitor

(CFU-E)

cells

and murine erythroleukemia MoL Cell. Biol. 8, 2604-2609

H., and Oishi,

kinases, ST638 erythroleukemia 183, 335-342

M. (1989)

and genistein, incells in a syner-

26. Jenis, D. M., Johnson, C. S., and Furmanski, P. (1989) Effects of inhibitors and activators of protein kinase C on late erythroid progenitor (CFU-e) colony formation in vitro. j Cell Clon. 7, 190-202 27. Munoz, E., Zubiaga, A. M., and Huber, B. T. (1991) Tyrosine

mt.

protein

phosphorylation

is required

for protein

kinase

C-

mediated proliferation in T cells. FEBS Leti.279, 319-322 28. Buckley, A. R., Crowe, P. D., and Russell, D. H. (1988) Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen. Proc. Natl. Acad. Sci. USA 85, 8649-8653 29. Mason-Garcia, M., Weill, C. L., and Beckman, B. 5. (1990) Rapid activation by erythropoietin of protein kinase C in the nuclei of erythroid progenitor cells. Biochem. Biophys. Res.

2964

Vol. 5

November

1991

kinin-induced

prostaglandin

formation

from

phosphatidyl-

inositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2. Proc. NatI. Acad. Sci. USA 84, 6374-6378 32. Chang, J. P., Morgan, R. 0., and Catt, K. J. (1988) Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. j Biol. Chem. 263, 18614-18620 33. Chang, J., Musser, J. H., and McGregor, H. (1987) Phospholipase A2: function and pharmacological regulation. Biochem. Pharmacol. 36, 2429-2436 34. Gronich, J. H., Bonventre, J. V., and Nemenoff, R. A. (1988)

Identification

and characterization

of a hormonally

regulated

form of phospholipase A2 in rat renal mesangial cells. j Biol. Chem. 263, 16645-16651 35. Offenstein, J. P., and Rillema, J. A. (1987) Possible involvement of the phospholipases in the mitogenic actions of prolactin (PRL) on Nb2 node lymphoma cells. Proc. Soc. Exp. Biol. Med. 185, 147-152 36. Nishijima, J., Wright, T. M., Hoffman, R. D., Liao, S., Symer, D. E., and Shin, H. 5. (1989) Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C. Biochemistry 28, 2902-2909

T. W., Wysolmerski,

R. B., and Lagunoff,

D. (1987)

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2-independent phospholipase C. Biochim. Biophys. Acta 917, 296-307 38. Freissmuth, M., Casey, P. J., and Gilman, A. G. (1989) G proteins control diverse pathways of transmembrane signaling. FASEBJ 3, 2125-2131 39. Clark, R. B., Love, J. T., Sgroi, D., Lingenheld, E. G., and Sha’afi, R. I. (1987) The protein kinase C inhibitor, H-7, inhibits antigen and IL-2-induced proliferation of murine T cell lines. Biochem. Biophys. Res. Commun. 145, 666-672

40. Haliday,

E. M., Ramesha,

C. S., and Ringold,

G. (1991) TNF

induces c-fosvia a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite. EMBOJ. 10, 109-115 41. Sha’afi, R. I., Naccache, P. H., Molski, T. F. P., Borgeat, P., and Goetzl, E. J. (1981) Cellular regulatory role of leukotriene B4: its effects on cation homeostasis in rabbit neutrophils. Cell. PhysioL 108, 401-408.

J.

42. Richter, C., Frei, B., and Cerutti, P. A. (1987) Mobilization of mitochondrial Ca2 by hydroperoxyeicosatetraenoic acid. Biochem. Biophys. Res. Commun. 143, 609-616

The FASEB Journal

MASON-GARCIA

AND

BECKMAN

ded from www.fasebj.org by Edinburgh University (129.215.17.190) on January 28, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 29