©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Erythropoietin-dependent Inhibition of Apoptosis Is Supported by Carboxyl-truncated Receptor Forms and Blocked by Dominant-negative Forms of Jak2 (*)

Hongming Zhuang (1), Zhutian Niu (2), Tong-Chuan He (1), Sunil V. Patel (2), Don M. Wojchowski (1) (2)(§)

From the (1)Departments of Biochemistry and Molecular Biology and (2)Veterinary Science and the Center for Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Apoptosis, or programmed cell death (PCD), recently has emerged as an important homeostatic mechanism within several hematopoietic lineages. This process is subject to both positive and negative modulation by cytokines and within the erythroid lineage is inhibited by interleukin-3, stem cell factor, and erythropoietin (Epo). Through the expression of carboxyl-truncated Epo receptor mutants in FDC-P1 cells, a receptor form possessing 80 membrane-proximal cytoplasmic residues is shown to efficiently mediate Epo-dependent inhibition of PCD. This is in contrast to previous studies that attributed this activity to a distal carboxyl-terminal receptor subdomain (and/or heterodimerization of wild type Epo receptors with a truncated non-functional receptor form). Epo-dependent inhibition of PCD also is shown to be blocked by ectopic expression of kinase-deficient dominant-negative forms of Jak2 (Jak2VIII and Jak2-829), further underlining a role of this membrane-proximal subdomain of the Epo receptor in the inhibition of PCD. To our knowledge, this comprises the first direct evidence for an essential role for a Jak tyrosine kinase (Jak2) in this apoptotic response pathway.


INTRODUCTION

During the development of hematopoietic cells, an apparently constitutively poised program of apoptosis (or programmed cell death (PCD))()has been shown to significantly affect progenitor cell survival and homeostasis(1, 2) . Among at least certain lineages, commitment to this program is prevented through exposure to select cytokines including IL-2, IL-3, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and cillary neurotropic factor(3, 4, 5, 6, 7, 8, 9, 10) . Within the erythroid lineage, PCD has been shown to be attenuated by IL-3, stem cell factor, insulin-like growth factor-1(11) , and erythropoietin (Epo)(12, 13, 14) . Notably, this inhibited PCD can be contrasted with and is perhaps balanced by factors that induce PCD, including IL-4(15) , tumor necrosis factor(16) , corticosteroids versus early thymic lymphocytes(17, 18) , and the Fas antigen(19, 20, 21) .

With regard to molecular effectors of PCD, several classes of factors have been defined. These include Bcl-2 and related factors (Bcl-x, Bcl-x-s, Bax, Mcl-1, A1, and Bhrf-1; see Ref. 22 for review), Myc (23, 24, 25) and Myb (26) as immediate early gene products that enforce apoptosis when expressed at elevated levels, the tumor suppresser gene p53(27, 28) , and several families of proteins whose expression has been associated specifically with apoptosis (i.e. Myd, Gadd, and Gas gene products)(22) . In hematopoietic systems, the ability of Bcl-2 to block PCD upon withdrawal of IL-3 has been demonstrated in both lymphoid (29) and myeloid cells(7) . The forced expression of Myc and Myb has been shown to advance PCD(25, 26) , and apoptosis has been shown in Epo-dependent murine FVA cells to proceed from both G1 and S phases of the cell cycle(30) . However, little is presently understood concerning how these effectors of PCD might be modulated upon cytokine exposure.

An important advance toward understanding cytokine action is constituted by the recently defined association between cytokine receptors of the type I/II superfamilies and members of the Janus family of protein-tyrosine kinases (i.e. Jak1, Jak2, Jak3, and Tyk-2)(31, 32, 33, 34, 35, 36, 37, 38) . In the Epo receptor system, for example, Epo activates Jak2, and this cytosolic protein-tyrosine kinase has been evidenced to associate with the Epo receptor within a conserved box 1/box 2-encoding membrane-proximal receptor subdomain (39). Recently, through the development of a dominant-negative form of Jak2, we have shown that activation of this receptor-associated kinase is required for Epo-induced mitogenesis(40, 41) . In the present study we have used factor-dependent murine FDC-ER and DAER cell lines to assess whether Jak2 (as a receptor-associated effector) might also be involved in the effects of Epo on PCD. First, experiments using Epo receptor cytoplasmic domain mutants delineate a membrane-proximal receptor domain that supports Epo-inhibited PCD. Notably, this is in contrast to recent studies by Nakamura et al.(42) , who have implicated a role for a carboxyl-terminal domain of the Epo receptor in inhibited PCD. Second, evidence that Epo-induced activation of Jak2 is essential for Epo-dependent inhibition of PCD is provided by studies using ectopically expressed dominant-negative forms of this Janus kinase. To our knowledge, this is the first direct evidence among type I cytokine receptor systems for a critical role for a specific protein-tyrosine kinase in this signaling pathway.


MATERIALS AND METHODS

Cell Lines

The FDC-P1-derived murine myeloid cell lines (FDC-ER, FDCER-372, FDCER-329, and FDCER-256) (43) and DA-1-derived cell lines (DAER, DAERJK2VIII, and DAERJak2-829) (40, 41) used in this study have been described previously (also see below). Each was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10mol/liter 2-mercaptoethanol, and 3% conditioned medium from WEHI-3B cells.

DNA Fragmentation Assays

In assays of apoptosis, cells were incubated in Dulbecco's modified Eagle's medium with 1% fetal calf serum in the presence or the absence of Epo 8-9 h prior to the assay of PCD-associated DNA fragmentation. The method of genomic DNA extraction was adapted from that described by Wang et al. (44). Briefly, 8 10 cells were collected and washed twice in phosphate-buffered saline. Washed cells were lysed by vortexing (1 min) in 1% Triton X-100, 0.32 M sucrose, 5 mM MgCl, 10 mM Tris-HCl, pH 7.5. Nuclei were then isolated by centrifugation (600 g for 5 min) and were suspended in 0.2 ml of 1% SDS, 5 mM NaEDTA, 10 mM Tris-HCl, pH 8.0. Sequential incubations with RNase A (10 mg/ml for 10 min at 37 °C) and proteinase K (1 mg/ml for 1 h at 37 °C) were performed to liberate DNA. DNA was then precipitated by the addition of 0.3 ml of 7.6 M NaI, 20 mM NaEDTA, 40 mM Tris-HCl, pH 8.0, 0.5 ml of isopropanol. Precipitates were collected by centrifugation and were washed sequentially in 40% isopropanol and 70% ethanol. Air-dried samples were resuspended in 1 mM NaEDTA, 10 mM Tris, pH 8.0, and were analyzed by electrophoresis (1.2% agarose gel, ethidium bromide staining).


RESULTS AND DISCUSSION

In primary studies aimed at defining the possible involvement of Jak2 in Epo-dependent inhibition of PCD, PCD-associated DNA fragmentation was analyzed in FDC-P1 cells expressing a series of carboxyl-terminally truncated Epo receptor mutants (Fig. 1). This includes receptor forms that efficiently mediate Jak2 activation and proliferation (wild type Epo receptor and ER-372), efficiently support Jak2 activation but require high levels of Epo to support proliferation (ER-329), or fail to mediate either Jak2 activation or proliferation (ER-256). Levels of cell surface Epo receptor expression in these cell lines are highly comparable and have been assessed by equilibrium binding analyses, [I]Epo cross-linking studies, and assays of sensitivity of receptor forms to endoglycosidase H to assess intracellular processing and transport(43, 45) . Properties of these receptor forms and corresponding FDC cell lines are summarized in .


Figure 1: Epo receptor truncation mutants and derived FDC-ER cell lines used in PCD analyses.



In assays of Epo-inhibited PCD, FDC-derived cell lines expressing the wild type receptor (FDC-ER cells), the truncation mutant ER-372 (FDCER-372 cells), and the truncation mutant ER-329 (FDCER-329 cells) were observed to efficiently support inhibited PCD (Fig. 2). In this and subsequent studies, PCD was assayed based on levels of cytokine-inhibited DNA fragmentation. In contrast, in cells expressing the highly truncated Epo receptor mutant ER-256 (FDCER-256 cells), DNA fragmentation was observed in both the absence and the presence of Epo. Among these receptor forms, each either supported Epo-inhibited apoptosis (wild type ER, ER-372, ER-329) or was apparently fully deficient in this activity (ER-256). This result, together with the consideration that each receptor form is expressed at comparable levels () argues that the observed activities in inhibited PCD are attributable to form rather than to possible general differences in expression. In addition, each of the receptor forms wild type ER, ER-372, and ER-329 has been shown to efficiently mediate Epo-induced activation of Jak2 at comparable efficiencies(46) , and mitogenic properties have been shown to be essentially equivalent among at least 5 independent clones for each cell line(45) .


Figure 2: Epo-dependent inhibition of apoptosis as mediated by carboxyl-truncated Epo receptor mutants in FDC-ER cell lines. Epo-dependent inhibition of PCD-associated DNA fragmentation was assessed in FDC-ER, FDCER-374, FDCER-329, and FDCER-256 cell lines as described under ``Materials and Methods.''



The above data show that a membrane-proximal cytoplasmic subdomain of the Epo receptor, which is essential for Jak2 binding and activation (36), likewise is necessary and sufficient for supporting Epo-dependent inhibition of PCD-associated DNA fragmentation. Notably, this finding is in contrast to studies of the human Epo receptor by Nakamura et al.(42) in which Epo-inhibited apoptosis was suggested to be mediated by a carboxyl-terminal receptor subdomain. These latter studies aimed to assess Epo-inhibited PCD activity for a truncated Epo receptor form (EpoR-T), which derives from the alternate splicing of transcripts in early erythroid progenitor cells. Stably transfected murine BaF3 cells were used as a model to reconstitute activity for a corresponding Epo receptor cDNA. However, it recently has been resolved that the BaF3 cell line used in these analyses expresses significant levels of endogenous full-length Epo receptors and that the receptor form studied apparently is mitogenically inactive. To explain observed apparent effects on apoptosis, an alternate model more recently has been proposed in which heterodimerization of this truncated form with full-length receptors might act to down-regulate mitogenesis and cell survival(47) . However, as noted by Schwall(48) , the further consideration that the apparent failure of EpoR-T to inhibit apoptosis depends upon Epo concentration is inconsistent with the conclusion that this receptor form lacks the domains required to inhibit PCD. Finally, it is relevant to consider that, although BaF3 cells were originally described as pro-B cells, this cell line recently has been shown to express several erythroid-specific factors (including Gata-1 and Eklf) and to support Epo-induced induction of globin gene transcription (but not hemoglobinization) following stable transfection with the wild type Epo receptor(49) . Due to this potential for erythroid differentiation, the use of BaF3 cells possibly complicates interpretations of effects of Epo on cell survival. In contrast, the models used in the present studies (FDC-P1- and DA-1-derived cell lines) do not express erythroid-specific factors at detectable levels (43)()and are not subject to this complication.

To further affirm the present findings, analyses of apoptosis of FDC-wtER versus FDCER-329 cells also were performed in the presence of Epo at decreasing concentrations (Fig. 3). Interestingly, higher concentrations of Epo were required to inhibit the apoptosis of FDCER-329 cells. This is also the case for Epo-dependent mitogenesis of FDCER-329 versus FDC-wtER cells as shown by He et al.(46) and Quelle et al.(43) . Thus, it is interesting to at least speculate that Epo-dependent mitogenesis and inhibition of PCD may, in fact, depend upon common upstream effectors.


Figure 3: Concentration-dependence of Epo-inhibited PCD in FDC-ER versus FDCER-329 cells. Exponentially growing washed cells were cultured in the presence (or the absence (-)) of Epo at the indexed concentrations for 7 h. PCD-associated DNA fragmentation was then assayed as described under ``Materials and Methods.''



The above experiments using Epo receptor carboxyl-terminal truncation mutants implicate a role for Jak2 in Epo-inhibited PCD yet do not directly test whether Jak2 activation is necessary to support the observed Epo-dependent inhibition of DNA fragmentation. This possibility was addressed using DA-1-derived cell lines, which ectopically express both the cloned murine Epo receptor and one of two kinase-deficient, dominant-negative forms of Jak2 (i.e. Jk2VIII or Jak2-829 (DAERJak2VIII and DAERJak2-829 cell lines, respectively)). Jk2VIII is a construct mutated within the type VIII phosphotransferase motif of the carboxyl-terminal protein-tyrosine kinase domain, and Jak2-829 is a carboxyl-terminal truncation mutant lacking this protein-tyrosine kinase domain (Fig. 4). Each mutant has recently been shown to efficiently inhibit Epo-induced activation of endogenous Jak2 and mitogenesis(40, 41) . The ability of DAERJak2VIII and DAERJak2-829 cells to support Epo-inhibited PCD-associated DNA fragmentation was assessed through comparison with control parental DAER cells (Fig. 4). For cells expressing each dominant-negative form of Jak2, Epo exerted little, if any, detectable protection against PCD yet efficiently inhibited DNA fragmentation in control DAER cells. Thus, these latter studies using cells specifically inhibited in Epo-induced activation of endogenous Jak2 provide direct evidence for a requisite role for Jak2 in Epo inhibition of PCD.


Figure 4: Epo-dependent inhibition of PCD-associated DNA fragmentation is blocked in DAER cell lines expressing dominant-negative Jak2 mutants Jk2VIII and Jak2-829. A, kinase-deficient dominant-negative forms of Jak2 expressed ectopically in DAER cells. B, PCD-associated DNA fragmentation in DAER versus DAERJk2VIII (left) and DAERJak2-829 cells (right) in the absence (-) or the presence of Epo (12 units/ml).



As indicated above, the conclusion that the Epo-dependent activation of Jak2 is required for inhibition of PCD by this cytokine is also supported indirectly by the observed ability of the receptor form ER-329 to protect against PCD-associated DNA fragmentation in FDCER-329 cells. Specifically, this receptor form, although somewhat deficient in mitogenesis, contains a cytoplasmic region that is necessary and sufficient for Epo-induced Jak2 activation(43, 46) . Whereas this conclusion regarding the apparent requirement for Jak2 in Epo-inhibited PCD derives from studies of two independent sets of cell lines that cannot be equated directly with regard to molecular mechanisms of cytokine-mediated inhibition of apoptosis, these findings are of interest in several contexts. This includes potential roles of known apoptotic effectors in cytokine-inhibited PCD, effects of mitogenic factors on apoptosis, and the nature of downstream effectors of Epo- and Jak2-inhibited PCD.

In several systems, bcl-2 has been implicated as a mediator of IL-3-dependent inhibition of PCD. This includes studies in lymphoid cells derived from IL-3-dependent pro-B lymphocyte line FL5.12 (29) as well as murine myeloid 32D cells(50) . This implied role for Bcl-2 depends largely upon its observed capacity to attenuate PCD upon forced expression. In studies by Baffy et al.(50) , evidence is also provided for an IL-3/Bcl-2-dependent redistribution of cellular calcium, whereas studies by May et al.(51, 52) suggest a possible role for the protein kinase C-dependent, IL-3- and Epo-induced phosphorylation of Bcl-2. However, these events presently are only associated with Bcl-2 action, and further studies are required to establish possible direct roles for Bcl-2 and related factors (Bcl-x, Bcl-x-s, Bax, Mcl-1, and A1) in cytokine-inhibited PCD. Notably, this includes the delineation of downstream effectors that mediate the presently established role for Jak2 in this process.

In the context of pathways to inhibition of PCD versus mitogenesis, positive roles for both Myc and Myb in PCD have been evidenced. Specifically, PCD is activated by over-expression of either factor(25, 26) . For Myc, this apparently depends upon cooperativity with Max (53) and is associated with a specific increase in cyclin A (54). Also, in transgenic mice harboring a c-Fos promoter/-galactosidase receptor construct, sustained transcriptional activation at the Fos promoter has been observed penultimate to commitment to PCD(55) . Together, these observations raise important questions regarding the extent to which overlap might exist in sets of effectors of mitogenesis versus inhibition of PCD. In this context, the receptor form ER-329 (Fig. 1) lacks carboxyl-terminal sites recently suggested by Miura et al. (56) to recruit p85/p110 phosphatidylinositol 3-kinase to receptor complexes. Thus, Epo-inhibited PCD may occur independently of this effector.

Finally, studies by Otani et al.(7) in IL-3/IL-2-dependent 32D-derived b53 cells have provided indirect evidence (using herbimycin A) for an essential role for tyrosine phosphorylation events in IL-3-inhibited PCD. The present study extends these analyses by defining Jak2 kinase as at least one requisite factor in this pathway. To our knowledge, this is the first identification of a role for a specific, cytokine-regulated protein-tyrosine kinase in PCD. The nature of additional factors that transduce Jak2-dependent signals to PCD effectors comprises a presently unresolved problem of significant interest.

  
Table: Properties of Epo receptor forms and derived FDC cell lines



FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL44491 and DK40242 and Research and Career Development Award HL03042 (to D. M. W.). 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 should be addressed: 115 Henning Bldg., University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140.

The abbreviations used are: PCD, programmed cell death; Epo, erythropoietin; IL, interleukin.

D. M. Wojchowski, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Steven Elliot of Amgen, Inc. for the generous provision of the recombinant human Epo used in these studies.


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