(Received for publication, August 16, 1995; and in revised form, November 30, 1995)
From the
The immature erythroid J2E cell line proliferates and terminally
differentiates following erythropoietin stimulation. In contrast, the
mutant J2E-NR clone does not respond to erythropoietin by either
proliferating or differentiating. Here we show that erythropoietin can
act as a viability factor for both the J2E and J2E-NR lines, indicating
that erythropoietin-initiated maturation is separable from the
prevention of cell death. The inability of J2E-NR cells to mature in
response to erythropoietin was not due to a defect in the
erythropoietin receptor sequence, although surface receptor numbers
were reduced. Both the receptor and Janus kinase 2 were phosphorylated
after erythropoietin stimulation of J2E-NR cells. However, protein
interactions with the erythropoietin receptor and Grb2 were restricted
in the mutant cells. Subsequent investigation of several other
signaling molecules exposed numerous alterations in J2E-NR cells;
phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase
C, p120 GAP, and mitogen-activated protein kinases (p42 and p44)
observed in erythropoietin-stimulated J2E cells were not seen in the
J2E-NR line. These data indicate that some pathways activated during
erythropoietin-induced differentiation may not be essential for the
prevention of apoptosis.
Erythropoietin (Epo) ()is the principal regulator of
red blood cell development, which acts primarily on immature erythroid
precursors to maintain cell viability and promote proliferation and
terminal differentiation(1, 2, 3) . There are
generally fewer than 1000 Epo receptors (Epo-R) on the surface of
erythroid precursors cells (4) and the intracellular signaling
events following the receptor/ligand interaction are rapidly being
deciphered. The Epo-R and its associated protein Janus kinase 2 (JAK2)
are phosphorylated (5, 6, 7, 8) ,
while the membrane protein, pp43 is dephosphorylated(9) . Shc
is also phosphorylated after Epo stimulation and binds to
Grb2(10) . Activation of Ras, increased phosphorylation of
GTPase-activating protein (GAP), and enhanced kinase activity of Raf-1
have also been documented(11, 12) . In addition,
mitogen-activated protein (MAP) kinases are stimulated in TF-1 and
Ba/F3 cells, although their role in Epo-stimulated proliferation is
unclear(13, 14, 15) . Epo also initiates
phosphorylation of
p92
(16) , and
phosphatidylinositol (PI) 3-kinase associates with the phosphorylated
Epo receptor via its Src homology 2
domains(17, 18, 19) . Down-regulation of the
receptor is achieved via association of the Src homology-PTP1
phosphatase and subsequent dephosphorylation(20) .
The J2E
cell line was generated by transforming immature erythroid cells with
the raf/myc-containing J2 retrovirus(21) . These cells
proliferate and differentiate in response to Epo; hemoglobin is
synthesized and the cells undergo morphological alterations, which
culminate in a proportion of cells
enucleating(21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
However, a mutant subclone J2E-NR, which arose spontaneously from a J2E
culture, fails to replicate faster, or mature, in response to Epo
despite binding and internalizing the hormone(22) . Unlike J2E
cells, GATA-1 or globin transcripts do not rise in J2E-NR cells after
Epo stimulation (27, 28) ; nevertheless, the
hemoglobin-producing machinery is intact since exogenous hemin is able
to initiate synthesis of a modest amount of the oxygen carrier. ()These observations indicated that a signaling defect may
exist in the J2E-NR cells which prevents Epo-induced terminal
differentiation(22) .
In this study we examined the capacity of Epo to (i) support the viability of J2E and J2E-NR cells and (ii) activate a number of signaling molecules in these cells. We report that Epo was able to enhance the survival of both J2E and J2E-NR cells in the absence of serum. Furthermore, following ligand binding, the Epo-R and JAK2 were phosphorylated in both lines, but significant differences were observed with several other signal transduction proteins. It was concluded that, in this system, distinct pathways may be involved in Epo-induced differentiation and cell survival.
Figure 1:
Epo maintains cell viability in J2E and
J2E-NR cells. Cells were cultured for up to 48 h in the presence of 10%
serum () or the absence of serum, with (
) or without
(
) 5 units/ml Epo. Cell viability was determined by eosin
exclusion. Each value represents the mean ± standard deviation (n = 3).
Figure 2: Epo maintains morphological integrity of J2E and J2E-NR cells. Cells cultured for 12 h in the presence of serum (A), in the absence of serum (B), or in the absence of serum but with 5 units/ml Epo (C). They were then cytocentrifuged on to glass slides and placed in May-Grunwald Giemsa stain. The bar represents 20 µm.
Figure 3:
Epo
prevents DNA degradation in J2E and J2E-NR cells. Cells were cultured
for 24 h in the presence or absence of 10% serum, with or without 5
units/ml Epo. DNA was extracted and separated on 0.8% agarose gel.
/HindIII markers (m) are shown at left.
Significantly, the data presented in Fig. 1show that Epo was able to inhibit the death of J2E cells, although not as efficiently as serum. The protective effect of Epo was dose-dependent, with maximum protection from apoptosis occurring between 0.1 and 10 units/ml (data not shown). This is the same concentration range that maximally stimulates proliferation and differentiation(23) . Cytocentrifuge preparations (Fig. 2) revealed that fewer apoptotic cells were present in the cultures and that the cells appeared much healthier; membrane ruffling was reduced, cells were rounded, and nuclei intact. Moreover, Epo was able to restrict the breakdown of DNA evident in serum-deprived cultures (Fig. 3). Others have also reported that Epo can reduce, but not completely prevent, DNA damage and programmed cell death of immature erythroid cells(36, 37) . Thus, Epo not only induced the maturation of J2E cells in the presence of serum(21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) , but also restricted apoptosis when serum was withdrawn.
The capacity of Epo to act as a viability factor was also tested with J2E-NR cells. Despite the inability of the hormone to promote terminal differentiation(22) , Epo was able to maintain the viability of the J2E-NR clone (Fig. 1). Cellular morphology was also protected (Fig. 2) and degradation of DNA suppressed (Fig. 3). These unexpected results demonstrated that Epo could still affect J2E-NR cells; although the hormone failed to elicit a proliferative/differentiative response, it was able to restrict cell death.
Figure 4: Epo induces fewer tyrosine phosphorylations in J2E-NR cells. J2E (A) and J2E-NR (B) cells were serum-starved for 5 h (0) and then stimulated with 5 units/ml Epo for 1-60 min. Lysates (100 µg) were separated on 8.5% SDS-PAGE gels and immunoblotted with antiphosphotyrosine antibody. Dashes (-) at right indicate Epo-induced tyrosine phosphorylation events. Molecular mass markers (46-200 kDa) are shown at left. Time (min) after Epo stimulation is shown above each lane.
Figure 5: Epo induces rapid tyrosine phosphorylation of the Epo-R and JAK2. A, lysates (100 µg) from unstimulated cells were immunoblotted with anti-Epo-R (187) antibody. = indicates multiple forms of the Epo-R. NIH3T3 cell lysate was included as a negative control. B and C, cells were serum-starved for 5 h (0) and lysates prepared from cells stimulated for 5-30 min with 5 units/ml Epo. Time (min) is shown above each lane. In panel B, lysates (1 mg) were immunoprecipitated with anti-Epo-R (187) antibodies and immunoblotted with antiphosphotyrosine antibodies. In panel C, lysates (5 mg) were immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted with anti-JAK2 antibodies.
Figure 6: Proteins associated with the Epo-R in J2E and J2E-NR cells. J2E cells (A) or J2E-NR cells (B) were serum-starved for 5 h (0) and then stimulated for 5-60 min with 5 units/ml Epo. Lysates (3 mg) were immunoprecipitated with anti-Epo-R(187) antibodies and immunoblotted with anti-phosphotyrosine antibodies. - indicates unique phosphoproteins for each line, while -* indicates phosphorylated proteins common to both cell lines. Molecular mass markers (30-220 kDa) are shown at left. Time (min) after Epo stimulation is shown above each lane.
Grb2 associates with the Epo-R(42) ; hence, proteins linked with this molecule were studied in J2E and J2E-NR cells after Epo stimulation. Fig. 7A shows that shortly after addition of Epo to J2E cells, phosphoproteins with approximate molecular masses of 53, 55, 56, 72, 75, 145, and 150 kDa were associated with Grb2. The 72- and 75-kDa proteins were most probably phosphorylated forms of the Epo-R, and Grb2 binding to a 145-kDa protein has been reported in Epo-stimulated DA-3 cells(42) . Of all the protein interactions involving Grb2 identified in differentiating J2E cells (Fig. 7A), only the 55-kDa molecule and the Epo-R associated with Grb2 in J2E-NR cells (Fig. 7B). Increasing the amount of protein in the immunoprecipitation did not reveal any further molecules in J2E-NR extracts. It was concluded from these experiments that differences existed between J2E and J2E-NR lines with respect to the proteins associating with the Epo-R complex.
Figure 7: Proteins associated with Grb2 in J2E and J2E-NR cells. J2E cells (A) or J2E-NR cells (B) were serum-starved for 5 h (0) and then stimulated for 2-60 min with 5 units/ml Epo. Lysates (3 mg) were immunoprecipitated with anti Grb2 antibodies and immunoblotted with antiphosphotyrosine (PY) or anti-Grb2 (Grb2) antibodies. - indicates unique phosphoproteins, while -* indicates phosphoproteins common to both cell lines. Molecular mass markers (46-200 kDa) are shown at left. Time (min) after Epo stimulation is shown above each lane.
Figure 8:
Epo
induces tyrosine phosphorylation of GAP, PI 3-kinase and PLC in
J2E cells. Cells were serum-starved for 5 h (0) and then stimulated for
2-60 min with 5 units/ml Epo. Lysates (3 mg) were
immunoprecipitated with anti-phosphotyrosine (A and B) or anti-PLC
(C) antibodies and immunoblotted
with anti-GAP (A), PI 3-kinase (B), or
phosphotyrosine (C) followed by PLC
(C)
antibodies. Time (min) after Epo stimulation is shown above each
lane.
Figure 9:
Epo
signaling is disrupted in J2E-NR cells. Cells were serum-starved for 5
h (0) and then stimulated for 2-60 min with 5 units/ml Epo.
Lysates (3 mg) were immunoprecipitated with anti-phosphotyrosine (A and B) or anti-PLC (C) antibodies and
immunoblotted with anti-GAP (A), PI 3-kinase (B),
phosphotyrosine (C), followed by PLC
(C)
antibodies. Time (min) after Epo stimulation is shown above each
lane.
Figure 10: p42 and p44 MAP kinase are activated by Epo and sodium butyrate in J2E cells. J2E cells were serum-starved for 0-300 min (A), then after 5 h were restimulated with either 5 units/ml Epo (B and D) or 500 µM sodium butyrate (C). Lysates (100 µg) in panels A-C were immunoblotted with anti-ERK1 antibodies. Lysates (1 mg) in panel D were immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted with anti-ERK1 antibodies. Panel E shows an in-gel kinase assay for lysates (20 µg) from serum-starved unstimulated control (C) cells and cells stimulated with 5 units/ml Epo (E) or 10% serum (F) for 10 min. The position of the unphosphorylated, inactive p42 and p44 MAP kinases are indicated at right in panels A-C, while the phosphorylated and active forms are indicated in panels D and E. The time (min) after serum starvation (panel A) or stimulation (panels B-D) is indicated above each lane. p42 and p44 MAP kinase protein levels remained constant during these experiments, and any differences were due to loading errors.
To assess the importance of the MAP kinase pathway in maintaining cell viability, the phosphorylation status of p42 and p44 MAP kinases was determined in Epo-stimulated J2E-NR cells. Following dephosphorylation of MAP kinase with serum deprivation (Fig. 11A), the cells were exposed to Epo. Unlike J2E cells, no activation of the p42 and p44 MAP kinases was observed when Epo or sodium butyrate were added to J2E-NR cells (Fig. 11, B and C). Immunoprecipitation with anti-phosphotyrosine antibodies also failed to detect an increase in the phosphorylation status of the MAP kinases (Fig. 11D). Since Epo inhibits the death of J2E-NR cells and the MAP kinase pathway was not activated by the hormone, we suggest that this pathway is not involved in maintaining the viability of these cells.
Figure 11: p42 and p44 MAP kinase are not activated in J2E-NR cells. J2E-NR cells were serum-starved for 0-300 min (A), then after 5 h were restimulated with either 5 units/ml Epo (B and D) or 500 µM sodium butyrate (C). Lysates (100 µg) in panels A-C were immunoblotted with anti-ERK1 antibodies. Lysates (1 mg) in panel D were immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted with anti-ERK1 antibodies. The position of the unphosphorylated, inactive p42 and p44 MAP kinases are indicated on the right in panels A-C, while the position of phosphorylated forms are indicated on the right in panel D. The times (min) after serum starvation (panel A) or stimulation (panels B-D) are indicated above each lane.
The mutant J2E clone J2E-NR binds and internalizes Epo, but does not proliferate or differentiate in response to the hormone(22) . We show here that Epo still has an effect on the J2E-NR cells by supporting viability in the absence of serum. Therefore, some signals from the Epo-R must be transmitted within these cells to prevent apoptosis. As no faults were identified in the receptor and the Epo-R was phosphorylated with Epo stimulation, it appeared that some essential signaling protein was defective, thereby preventing the maturation message from being transduced.
JAK2 was
considered as a candidate molecule that may be defective in the J2E-NR
cells. We have shown elsewhere that this protein is important for
transmitting both proliferation and differentiation signals. ()However, JAK2 was phosphorylated in J2E-NR cells and also
appeared unlikely to be responsible for the defect. Interestingly, it
has recently been shown that phosphorylation of JAK2 is not sufficient
for Epo-induced mitogenesis as mutation of the ``extended box
2'' domain of the Epo-R can prevent a growth signal from being
transmitted, despite JAK2 phosphorylation(45) .
Significantly, differences were observed in the Epo-R complex of J2E
and J2E-NR cells. Several proteins consistently co-precipitated with
the Epo-R in J2E cells, and these appeared similar to previous reports (7, 46, 47) . However, distinct differences
were reproducibly seen in the J2E-NR receptor complex. These changes to
the Epo-R complex in J2E-NR cells appear to have initiated numerous
downstream signaling alterations. Whereas J2E cells underwent the
anticipated phosphorylation of GAP, PI 3-kinase, and MAP kinases with
Epo(11, 13, 14, 15, 17, 18, 19) ,
similar modifications did not occur in J2E-NR cells. An additional
change was observed with PLC; although the phosphorylation status
of this protein rose steadily in J2E cells with hormonal stimulation,
it was hyperphosphorylated in J2E-NR cells prior to exposure to Epo.
Thus, major changes in intracellular signaling were observed in J2E-NR
cells. These alterations clearly affected the ability of the J2E-NR
cells to proliferate and differentiate, but had no impact on the
cells' capacity to survive in the presence of Epo.
It has been suggested that cytokine-mediated protection from apoptosis is not necessarily linked to the ability to promote growth(48, 49) . Moreover, it has been proposed that the full-length Epo-R, and not a truncated form of the molecule, can transmit a signal to prevent apoptosis that is distinct from a mitogenic signal(50, 51) . These observations corroborate the suggestion of Koury and Bondurant (3) that ``the survival effect of Epo during terminal differentiation appears to be separate from any mitotic signal.'' Our results support, and extend, these hypotheses to show that it is possible for Epo to maintain the viability of erythroid cells, in the absence of proliferation and differentiation. Independent signaling pathways probably exist within cells for survival, mitosis, and maturation. It is noteworthy that Epo alone did not completely protect the cells from DNA damage and death, as reported previously(36, 37) , and other factors are probably required to fully maintain viability.
The MAP kinase family of enzymes are obviously very important in cellular signaling. MAP kinase activation has been associated with enhanced mitogenic activity(43, 52) , but some recent publications have indicated that these proteins are also involved in cellular differentiation(53, 54) . Although Epo-induced activation of MAP kinases has been shown previously(13, 14, 15) , the results shown here indicate that MAP kinase activation may be linked with differentiation, rather than proliferation. Significantly, the J2E-NR cells remained viable with Epo without MAP kinase activation, indicating that this pathway is not essential for maintaining cell survival. This result supports the observation that suppression of apoptosis by v-abl is not associated MAP kinase activation(55) .
PI 3-kinase has been shown to be necessary for a signaling pathway that prevents cell death in neurons(56) . The data presented in this report demonstrate that PI 3-kinase was not activated in J2E-NR cells, yet the cells remained viable. It appears, therefore, that phosphorylation of the p85 subunit of PI 3-kinase is not essential for Epo-induced protection from apoptosis. Several groups have shown an association between PI 3-kinase and the activated Epo-R(17, 18, 19) , but it has been suggested that this association is not linked with proliferation(19) . Thus, the precise role of PI 3-kinase in Epo signaling requires further elucidation.
Two other explanations
were entertained for the inability of J2E-NR cells to proliferate and
differentiate after Epo stimulation. One possibility was that J2E-NR
cells have reduced Epo-R numbers and the levels are too low to transmit
the maturation signal. This suggests that signal transduction may be a
quantitative, and not necessarily a qualitative,
phenomenon(57) . However, based on the observation that
phosphorylation of only 15% of Epo-R was sufficient to induce maximum
differentiation of J2E cells, this proposition appears
unlikely. Another possibility was that the J2E-NR cells may have
reverted to a less mature form of erythroid precursor. J2E cells
occasionally mutate and can display an altered phenotype(58) .
If so, the J2E-NR cells may only respond to Epo as a viability factor,
and not as a maturation agent. In support of this notion, the loss of
the erythroid-specific surface marker, Ter 119, by J2E-NR cells (57) indicates these cells may have acquired a more primitive
phenotype (59) . Nevertheless, the J2E-NR clone together with
the parental J2E cells provide us with an opportunity to dissect
signaling pathways within erythroid cells leading to viability, mitosis
and differentiation.