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and Phosphatidylinositol 3'-Kinase and Promotion of Macrophage Differentiation by Insulin-Like Growth Factor-I1
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Laboratory of Immunophysiology, Department of Animal Sciences, and
College of Medicine, Department of Pathology, University of Illinois, Urbana, IL 61801; and
Institut National de la Recherche Agronomique-INSERM, Unité de Recherches de Neurobiologie des Comportements, Bordeaux, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and overexpression of this enzyme leads to
monocytic differentiation. In this study, we cultured human HL-60
promyeloid cells with vitamin D3 and insulin-like growth
factor-I (IGF-I), a 70-amino-acid peptide that activates
phosphatidylinositol 3'-kinase (PI 3-kinase) in murine promyeloid
cells. Two days later, the proportion of cells differentiating into
macrophages in serum-free medium, as assessed by expression of the
-subunit of the ß2 integrin CD11b, increased from
5 ± 1% to 25 ± 3%. Addition of IGF-I increased the
proportion of cells differentiating into CD11b-positive macrophages to
78 ± 5%. In the absence of vitamin D3, IGF-I did not
induce expression of CD11b (6 ± 1%). The IGF-I-promoted
macrophage differentiation was blocked specifically by preincubation of
HL-60 cells with a mAb (IR3) directed against the IGF type I
receptor. Similarly, pretreatment of cells with either IR3 or an
IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b
expression when cells were cultured with vitamin D3 in
serum-containing medium. IGF-I, but not vitamin D3, caused
a sevenfold increase in the enzymatic activity of both PI 3-kinase and
atypical PKC-. Inhibition of IGF-I-inducible PI 3-kinase with either
wortmannin or LY294002 abrogated the IGF-I-induced activation of
PKC- and totally blocked the enhancement in macrophage
differentiation caused by IGF-I. These data establish that PKC- is a
putative downstream target of PI 3-kinase that is activated during
IGF-I-promoted macrophage differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a member of the
Ca2+-independent aPKC subfamily, causes monocytic
differentiation (3). These PKC--transfected monocytes displayed an
increase in the expression of the c-jun proto-oncogene as
well as enhanced AP-1-binding activity. Similarly, PKC- has recently
been suggested to mediate the actions of insulin and serum mitogenic
factors in adipogenesis (4). Moreover, platelet-derived growth factor
(PDGF) treatment of NIH-3T3 cells leads to a direct association of
PKC- with Ras-GTP protein, which serves to recruit PKC- to plasma
membrane (5). Two phosphorylation products of phosphatidylinositol
3-kinase (PI 3-kinase), phosphatidylinositol-3,4-P2 and
phosphatidylinositol-3,4,5-P3, are now known to activate
PKC-, implying that this aPKC may be a downstream target of PI
3-kinase (6). Classical growth factor receptors with intrinsic tyrosine kinase activity, such as those for insulin and insulin-like growth factor-I (IGF-I), activate PI 3-kinase in a number of cells (7, 8, 9). IGF-I acts as a survival factor to inhibit apoptosis via a PI 3-kinase-dependent pathway in both murine promyeloid cells (9, 10) and neurons (8, 11, 12). The activation of PI 3-kinase has recently been shown to be a crucial event that occurs during normal B lymphocyte differentiation (13). Indeed, inhibition of PI 3-kinase by treatment with either wortmannin or LY294002 results in a complete block of CD40 ligand-induced Ig production. Similarly, inhibition of PI 3-kinase activity blocks the differentiation of myoblasts into myotubes, thus establishing that this enzyme is critical to the development of skeletal muscle cells (14). These data establish an essential role for PI 3-kinase in the differentiation of both B lymphocytes and muscle cells, and they suggest a potential role of this signaling pathway in myeloid development.
IGF-I and its major inducer, growth hormone, augment a number of
functional activities of B cells, T cells, and macrophages (15, 16),
and both of these proteins are synthesized by leukocytes (17, 18, 19).
IGF-I also has been reported to promote the differentiation of
granulocytes (20, 21) and B lymphocytes (22, 23). For example, Landreth
et al. (1992) demonstrated that stromal cell-derived IGF-I increases
expression of the cytoplasmic µ heavy chain during pro-B cell
maturation (22) by promoting the expansion of IL-7-dependent pro-B
cells (23). Although IGF-I stimulates PI 3-kinase in murine promyeloid
cells (9), and the D3-phosphorylated phosphoinositides of this enzyme
activate PKC- (6), the potential role of IGF-I in macrophage
development is unknown. We recently reported that HL-60 cells express
abundant cell surface receptors for IGF-I that are biologically active,
as assessed by a 2.5-fold increase in proliferation following addition
of as little as 10 ng of IGF-I (24). In this study, we establish that
IGF-I also promotes the differentiation of these cells into macrophages
when cultured with vitamin D3. IGF-I, but not vitamin
D3, induces a substantial increase in the activity of
PKC-. More importantly, inhibition of the enzymatic activity of PI
3-kinase abrogates the enhancement of macrophage differentiation caused
by IGF-I and completely blocks the IGF-I-induced activation of PKC-.
These data show that IGF-I activates both PI 3-kinase and PKC- and
promotes the vitamin D3-induced differentiation of
promyeloid cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture medium was prepared from powdered RPMI 1640
(MediaTech, Herndon, VA) supplemented with 2 g/L sodium bicarbonate,
100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma Chemical Co.,
St. Louis, MO). FBS (HyClone Laboratories, Logan, UT), which was heat
inactivated for 30 min before use, as well as all other reagents
contained <25 pg endotoxin/ml (Limulus amebocyte lysate assay;
Associates of Cape Cod, Woods Hole, MA). The
1,25-(OH)2D3 (vitamin D3)
was kindly provided by Dr. Milan Uskokovic (Hoffmann-La Roche, Nutley,
NJ), and all-trans retinoic acid (RA) was purchased from
Sigma Chemical Co. Both vitamin D3 and RA were dissolved in
ethanol. Human rIGF-I was purchased from Intergen (Purchase, NY), and
nonglycosylated human rIGF-binding protein-3 (rIGFBP-3) was kindly
provided by Celtrix (Santa Clara, CA). The mouse anti-human IGF
type I receptor mAb (IR3, IgG1k, free of sodium azide)
was purchased from Oncogene Science (Uniondale, NY), the rat
anti-human CD11b mAb (Mac-1, IgG2bk) was from
BioSource International (Camarillo, CA), and the irrelevant
isotype-matched murine IgG1 and rat IgG2b control mAbs were purchased
from Sigma Chemical Co. The F(ab')2 fragment of
FITC-conjugated goat anti-rat Ab was obtained from Cappel (Durham,
NC). The mouse anti-phosphotyrosine (PY) Ab (4G10) and rabbit
anti-rat PKC- Abs were purchased from Upstate Biotechnology
(Lake Placid, NY).
Human HL-60 promyeloid cells were kindly supplied by American Type Culture Collection (Rockville, MD). Cells were grown in RPMI 1640 medium supplemented with 10% FBS at 37°C in 7% CO2 and 95% humidity. Cells in the exponential phase of the cell cycle were washed three times with RPMI and then deprived of residual serum growth factors by culturing for 24 h in serum-free medium supplemented with 12.5 µg/ml human transferrin (Sigma Chemical Co.) and 30 nM sodium selenite (Sigma Chemical Co.). In differentiation assays, cells were plated in this fresh serum-free RPMI medium at a density of 3 x 105 cells/ml. Cells were cultured with vitamin D3 (1 µM), RA (1 µM), or an equivalent concentration of ethanol (0.1%, v/v) for the indicated time period before analysis by flow cytometry.
In experiments in which a mAb directed against the IGF-I receptor
(IR3) was used, cells were incubated with IR3 (5 µg/ml) or an
irrelevant isotype-matched murine IgG1 Ab for 1 h at 37 C before
other treatments. Similarly, to study the effect of IGF-binding
proteins on macrophage differentiation, preincubation of IGFBP-3 (250
ng/ml) or the same amount of an irrelevant serum protein (BSA) was
conducted 1 h before other treatments.
Flow cytometry to detect cell surface CD11b
HL-60 cells undergo differentiation to a macrophage or a
granulocyte phenotype following addition of vitamin D3
and RA, respectively (25). Flow-cytometric methods were used to measure
initiation of macrophage differentiation by characterizing surface
expression of the -subunit of the ß2 integrin CD11b.
This cell membrane marker, which is absent on human immature myeloid
cells, has been widely used as an early differentiation marker in HL-60
cells (26, 27). Cells were washed once in PBS (1.5 M NaCl, 19 mM
NaH2PO4·H2O, and 8.4 mM
Na2HPO4) supplemented with 0.5% FBS and 0.25%
BSA, and were then incubated with an anti-CD11b (Mac-1) mAb or its
isotype-matched control mAb for 30 min at 4°C. After two washes with
dilution buffer, cells were then incubated with FITC-conjugated goat
anti-rat secondary Ab for another 30 min at 4°C. After two final
washes, cells were fixed in PBS containing 1% formaldehyde until
analysis by flow cytometry (EPICS V; Coulter Corp., Hialeah, FL).
Bitmaps were established to include at least 5000 cells of uniform size
using immunofluorescence intensity of control cells that were treated
identically and stained with an isotype-matched mAb.
Double staining with propidium iodide and Hoechst 33342 to detect apoptotic cells
The apoptotic population was quantitated by using a double-staining procedure employing both propidium iodide and Hoechst 33342 (28). The charged dye propidium iodide (Sigma Chemical Co.) is excluded by cells with intact membranes, whereas Hoechst 33342 (Sigma Chemical Co.) stains DNA in fixed cells. Those cells expressing low fluorescence intensity with both Hoechst 33342 and propidium iodide were used to define and quantitate the number of apoptotic cells. Serum-deprived HL-60 cells (1 x 106) were treated as indicated and washed once in PBS and incubated in 100 µl of propidium iodide (20 µg/ml) at 4 C for 30 min. Cells were washed three times and subsequently fixed in 1.9 ml 25% ethanol. Finally, 50 µl of Hoechst 33342 solution (60 µg/ml) was added, and cells were washed once more with PBS and then analyzed by flow cytometry.
Detection of PI 3-kinase activity
Anti-PY-associated PI 3-kinase activity was determined in cell
lysates, as described previously (9). Phosphatidylinositol lipid kinase
activity was measured in anti-PY-associated immunoprecipitates,
which is a more sensitive measure of PI 3-kinase activity than that
found in immunoprecipitates using an Ab against the p85 subunit of this
enzyme (29, 30). Serum-deprived cells (108) were
incubated with optimal concentrations of wortmannin (1 µM), LY294002
(25 µM), or DMSO solvent (0.1% v/v) for 1 h before other
treatments. The optimal dose of both inhibitors was selected based upon
our preliminary experiments to achieve a total blockage (>95% rather
than 50% inhibition) of enzymatic activity (data not shown) so that we
could better evaluate the role of PI 3-kinase in the activation of
PKC- and macrophage differentiation. Our working concentrations are
consistent with other reports in which wortmannin (1 µM) (9) and
LY294002 (50 µM) (31) effectively blocked PI 3-kinase activity in
cells of the hemopoietic lineage. Cells were then incubated with
vitamin D3 (1 µM) for 30 min before stimulation with
IGF-I (100 ng/ml) for 5 min. The cells were centrifuged at 400 x
g and homogenized at 4°C in cell lysis buffer containing
1% Nonidet P-40, 50 mM Tris · HCl, 100 mM NaCl, 50 mM NaF, 10 mM
sodium pyrophosphate, 2 mM orthovanadate, 2.5 mM benzamidine, 1 mM
PMSF, and 1 µM DTT. Cell lysates were then incubated with a mouse
anti-PY Ab 4G10 (2 µg; Upstate Biotechnology) at 4°C overnight
and then with protein A-agarose beads for 2 h. The
immunoprecipitated complex was washed twice with PBS/1% Nonidet P-40/1
mM DTT, twice with 0.5 M LiCl/1 mM DTT/100 mM Tris · HCl, and twice
with 10 mM NaCl/1 mM DTT/10 mM Tris · HCl. Lipid kinase activity
was measured directly by incubating the precipitated beads in a
reaction mixture containing L--phosphatidylinositol
(0.33 mg/ml), 20 mM Na HEPES, 0.4 mM EGTA, 0.4 mM NaPO4, 10
mM MgCl2, and 2 µCi/nmol [
-32P]ATP.
After a 15-min incubation at room temperature, and following
termination of the reaction by addition of 15 µl 4 N HCl, lipids were
extracted once in chloroform/methanol (1:1 (v/v)) and once in 0.15 N
HCl/methanol (1:1 (v/v)). Labeled phospholipid contained in the organic
phase was separated by TLC in chloroform/methanol/ammonium hydroxide
(75:58:17 (v/v)). Dried TLC plates were exposed to Kodak XRP5
autoradiographic film (Eastman Kodak, Rochester, NY) at -80°C for
24 h and subsequently exposed to PhosphorImager storage screens
(Molecular Dynamics, Sunnyvale, CA). Individual signal intensity was
measured on a series 400 PhosphorImager, using the ImageQuant 3.3
software (Molecular Dynamics) (9).
Measurement of PKC- activity
Cells (108) were preincubated with wortmannin
(1 µM), LY294002 (25 µM), or the DMSO diluent for 1 h. Cells
were then treated with vitamin D3 for 30 min before
incubation with IGF-I (100 ng/ml) for 5 min. Subsequently, cells were
extracted with cell lysis buffer containing 1% Nonidet P-40, 50 mM
Tris · HCl, 100 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 2
mM orthovanadate, 2.5 mM benzamidine, 1 mM PMSF, and 1 µM DTT. The
anti-PKC- rabbit antiserum (2 µg) or control rabbit serum was
added to equal amounts of cell lysates (200 µg), followed by
immunoprecipitation with protein A-agarose, as described above. The Ab
immunoprecipitated equal amounts of PKC- in each treatment group, as
confirmed by Western analysis using the same anti-PKC- Ab (data
not shown). The washed immununoprecipitates were subjected to the
kinase reaction for 30 min at 30°C in 50 µl of kinase buffer
containing 0.5 mM EGTA, 10 mM MgCl2, 20 mM Na HEPES (pH
7.4), 50 µM ATP, and 1 µCi [-32P]ATP with 2 µg
MBP as a substrate. The reaction was terminated by the addition of 5%
TCA, and 25 µl of reaction mixture was transferred to p81
phosphocellulose paper. After three rinses with 1% phosphoric acid,
radioactivity on the filter disc was determined in a Beckman LS6000IC
scintillation counter (Beckman Instruments, Fullerton, CA).
Statistical analysis
All experiments were repeated for at least three times. Data were analyzed using the Statistical Analysis System (32), with Student’s t test being used to detect differences between treatments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although generally recognized as a classical growth factor acting
at the G1 to S transition phase of the cell cycle
(33), IGF-I has been reported to promote differentiation of both
skeletal and neuronal tissues (34, 35). However, the potential role of
IGF-I in macrophage development is unknown. We recently reported that
HL-60 cells express easily detectable receptors for IGF-I (24), and
these cells are well known to differentiate along the macrophage
lineage in the presence of vitamin D3. As these progenitors
develop into more mature myeloid cells, they express the -subunit of
the ß2 integrin CD11b, and this surface marker is used
routinely to evaluate their maturation toward the macrophage phenotype
(26, 27). Indeed, induction of CD11b is very well correlated with the
expression of other myeloid differentiation markers, such as the CD14
LPS receptor (26) and CD67 (36). In these experiments, HL-60 cells were
cultured with vitamin D3 to induce their differentiation
along the macrophage lineage, and flow-cytometric histograms
depicting these results are shown in Figure 1
A. When HL-60 cells were
cultured in medium only, very few expressed the integrin adhesion
molecule, CD11b, amounting to only 6 ± 1% when replicated in
three independent experiments (Fig. 1B). In
serum-containing medium, vitamin D3 induced HL-60 cells to
differentiate along the macrophage lineage in a dose-dependent fashion
(data not shown). At an optimal dose of vitamin D3 (1
µM), expression of the CD11b differentiation marker increased from
6 ± 1% to 70 ± 5% (Fig. 1B; p
< 0.01; n = 3). HL-60 cells maintained in defined
serum-free medium were able to survive for at least 6 days, as
determined by >95% of these cells excluding trypan blue. Incubation
with vitamin D3 in this serum-free medium, however, induced
only a moderate increase in the proportion of cells expressing CD11b
(Fig. 1B; 5 ± 1% vs 25 ± 3%; p
< 0.01). Interestingly, addition of exogenous IGF-I (100 ng/ml) to
vitamin D3-treated cells resulted in a threefold increase
in the proportion of cells expressing CD11b (Fig. 1B;
78 ± 5%; p < 0.01) in serum-free medium,
suggesting that IGF-I can totally replace the serum requirement for
macrophage differentiation. IGF-I or FBS alone, in the absence of
vitamin D3, failed to promote the differentiation of HL-60
cells (6 ± 1% and 7 ± 1%, respectively). Similarly, we
observed that a small percentage of cells treated with vitamin
D3 alone became adherent, irregularly shaped cells with
mononuclear, but not polymorphonuclear, nuclei. Cells treated with
vitamin D3 alone also expressed detectable amounts of CD14
and the nonspecific esterase, -naphthyl acetate esterase, both of
which are expressed predominantly on cells of the macrophage/monocytic
series (26, 37, 38). However, addition of IGF-I (100 ng/ml) in
conjunction with vitamin D3 markedly increased the
proportion of adherent mononuclear cells that expressed these
markers (data not shown). These are the first data to show that
exogenous IGF-I, a classical growth factor, promotes cellular
differentiation along the macrophage lineage.
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CSFs such as IL-3 promote differentiation of promyeloid cells by
preventing their apoptotic cell death (39). We and others have
demonstrated that IGF-I inhibits apoptosis of murine promyeloid cells
(9), fibroblasts (40), and PC12 pheochromocytoma cells (8). We
therefore asked whether vitamin D3 induces HL-60 cells
to undergo apoptosis in serum-free medium, and whether IGF-I prevents
this cell death, thus allowing the completion of vitamin
D3-initiated macrophage differentiation processes. As
summarized in Table I, vitamin
D3 alone induced macrophage differentiation in a small
proportion of HL-60 cells when compared with those in serum-free medium
only (6 ± 2% vs 24 ± 3% CD11b-positive cells;
p < 0.01; n = 3). However, vitamin
D3 did not induce apoptotic cell death (6 ± 2%
apoptotic cells) compared with that of cells incubated in serum-free
medium (6 ± 3% apoptotic cells). Furthermore, IGF-I tripled
expression of the CD11b differentiation marker (24 ± 3% vs
73 ± 4%; p < 0.01) in vitamin
D3-treated cells cultured in serum-free medium compared
with those incubated with vitamin D3 alone. IGF-I did not
affect the proportion of apoptotic cells in either the absence (5
± 2%) or presence (5 ± 2%) of vitamin D3 in
serum-free medium, and similar results were observed in the presence of
FBS. In contrast, IGF-I promoted expression of the CD11b Ag in
RA-treated HL-60 cells cultured in serum-free medium compared with
those incubated with RA alone (Table I; 22 ± 2% vs 50 ±
4%; p < 0.01) while rescuing these cells
differentiating along the granulocytic pathway from apoptosis (31
± 3% vs 6 ± 3% apoptotic cells; p < 0.01).
Similar to results presented in Figure 1
, cells incubated with either
differentiating agent in the presence of either IGF-I in serum-free
medium or 10% FBS expressed equivalent amounts of CD11b, again
suggesting that IGF-I can replace the serum requirement for
differentiation along the myeloid lineage. These data establish that
IGF-I promotes development of HL-60 cells along both the granulocytic
and monocytic lineages, but that IGF-I does not act as a survival
factor to promote macrophage differentiation.
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IGF-I binds to the IGF-I receptor as well as the insulin receptor,
although with a 50- to 100-fold reduction in affinity (41). A murine
mAb against the IGF-I receptor, IR3, specifically recognizes the
extracellular -subunit of the IGF-I receptor (42) and inhibits a
number of receptor-mediated activities (24). We therefore asked whether
IR3 would inhibit IGF-I-enhanced macrophage differentiation of
vitamin D3-induced HL-60 cells. Cells were
preincubated with IR3 (5 µg/ml) for 30 min before addition of
IGF-I (100 ng/ml). As expected, IGF-I significantly increased the
proportion of CD11b-expressing cells treated with vitamin
D3 from 25 ± 4% to 76 ± 4% (Fig. 2; p < 0.01;
n = 3). Neutralization of the IGF-I receptor with
IR3 led to a complete inhibition of this IGF-I-promoted cell
differentiation in the presence of vitamin D3 (Fig. 2;
76 ± 4% vs 26 ± 3%; p < 0.01). However,
at the same concentration, an irrelevant isotype-matched control Ab
(IgG1) had no effect (Fig. 2; 73 ± 2%). These results clearly
demonstrate that the IGF-I-promoted enhancement of differentiation is
mediated exclusively by the IGF-I receptor.
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Normal human serum contains abundant amounts of IGF-I (
200
ng/ml) and IGF-II (700 ng/ml) (43), and similar amounts of IGF-I are
contained in FBS (44). IGF-I was identified recently as the relevant
serum factor responsible for maintaining the survival of human vascular
smooth muscle cells (45). IGF-II also binds and activates the IGF-I
receptor in HL-60 cells (24). Since HL-60 cells are conventionally
differentiated in FBS-containing medium (as shown in Table I), we
tested whether IGF-I in serum is also responsible for promoting
macrophage differentiation induced by vitamin D3. To
test this hypothesis, cells were preincubated with IR3 (5 µg/ml)
for 30 min before culturing with vitamin D3 in 10%
FBS-containing medium (Fig. 3). The
proportion of CD11b-positive macrophages in vitamin
D3-induced HL-60 cells increased from 24 ± 5% to
70 ± 3% (p < 0.01; n =
3) by addition of 10% FBS. Preincubation with IR3 in cells cultured
with vitamin D3 in the presence of FBS reduced CD11b
expression to 35 ± 3% (p < 0.01), which
was statistically similar to that of cells treated with vitamin
D3 alone. At the same concentration, the irrelevant
isotype-matched control Ab had no effect. Human rIGFBP-3 also has a
high affinity for IGF-I and has been shown to inhibit a number of
activities of IGF-I (24, 46). We therefore tested the possibility that
IGFBP-3 would inhibit vitamin D3-induced macrophage
differentiation in serum-containing medium. Similar to the results
obtained with IR3, addition of IGFBP-3 (250 ng/ml) reduced CD11b
expression to 34 ± 4% (p < 0.01) in
vitamin D3-stimulated HL-60 cells (Fig. 3), amounting to a
78% inhibition. At the same concentration (250 ng/ml), an irrelevant
serum protein (BSA) had no effect, excluding the possibility of a
nonspecific protein effect of IGFBP-3. We interpret these data to
indicate that both an Ab to the receptor and a protein that
specifically binds the ligand are capable of inhibiting the unbound
form of IGF-I in serum, and therefore suppress the development of HL-60
cells into macrophages.
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PI 3-kinase has been demonstrated to play a key role in normal B
lymphocyte differentiation (13). Indeed, inhibition of this lipid
kinase completely blocks the CD40 ligand-induced Ig production. We
therefore asked whether PI 3-kinase is activated during IGF-I-promoted
macrophage differentiation in promyeloid cells induced by vitamin
D3. We found that vitamin D3 alone was
unable to activate anti-PY-immunoprecipitable PI 3-kinase activity,
whereas addition of IGF-I resulted in a sevenfold increase in kinase
activity (Fig. 4B;
p < 0.01; n = 3). Indeed, the
IGF-I-induced increase in PI 3-kinase activity was nearly the same in
either the presence or the absence of vitamin D3 (7-
± 1- vs 8- ± 2-fold, respectively; p >
0.10).
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B; p < 0.01;
n = 3) the IGF-I-induced enhancement of
phosphatidylinositol phosphorylation in cells treated with vitamin
D3.
PKC- is activated by IGF-I, but not vitamin
D3, during macrophage differentiation
Members of PKC family, in particular aPKC-, have recently been
demonstrated to mediate signals from growth factors during cell growth
and differentiation, and have also been suggested to be one of the
downstream signaling targets of PI 3-kinase (6). Of particular
relevance is the finding that overexpression of PKC- in human
promonocytic U937 cells induces their differentiation along the
monocyte pathway (3). We therefore tested the possible involvement of
endogenous PKC- in IGF-I signaling during macrophage
differentiation. HL-60 cells were incubated in serum-free medium with
vitamin D3 (1 µM) in the presence or absence of
IGF-I (100 ng/ml) for 5 min. Cell lysates were then immunoprecipitated
with an anti-PKC- Ab, and the resulting kinase activity was
measured in the immunocomplex. While vitamin D3 alone
failed to activate PKC-, addition of IGF-I to vitamin
D3-treated cells induced a sixfold increase in protein
phosphorylation in the anti-PKC--precipitated complex (Fig. 5; p < 0.05;
n = 3). Indeed, the IGF-I-elicited increase in the
PKC- activity was not statistically different in either the presence
(6 ± 1) or absence (10 ± 3) of vitamin D3. More
importantly, inhibition of PI 3-kinase was directly related to the
diminished PKC- activity (Fig. 5) because treatment with either
wortmannin (1 µM) or LY294002 (25 µM) led to a 95 ± 3% and
82 ± 2% inhibition, respectively, of PKC- activity
(p < 0.05). These results established that
PKC- is activated during the development of HL-60 cells into
macrophages, and that IGF-I rather than vitamin D3 is
responsible for induction of this enzyme. The IGF-I-induced activation
of PKC- is likely to be a downstream target of PI 3-kinase.
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Since IGF-I induces significant PKC- activity, and this
activation is blocked by two different inhibitors of PI 3-kinase, we
tested the important possibility that these PI 3-kinase inhibitors
might also suppress IGF-I-enhanced macrophage differentiation. As
expected, vitamin D3 induced CD11b expression in only
a small proportion of cells (Fig. 6;
26 ± 4%; n = 3), and addition of IGF-I led to a
threefold increase in expression of this leukocyte differentiation
marker (79 ± 4%; p < 0.01). Both PI 3-kinase
inhibitors, LY294002 (25 µM) and wortmannin (1 µM), suppressed
IGF-I-enhanced CD11b expression (Fig. 6; 26 ± 2% and 26 ±
3%, respectively; p < 0.01) to levels equivalent to
cells treated with only vitamin D3. This is consistent with
the finding that vitamin D3 alone induces the activity of
neither PI 3-kinase (Fig. 4) nor PKC- (Fig. 5). In the absence of
IGF-I, LY294002 and wortmannin did not affect CD11b expression in cells
treated with vitamin D3 alone (Fig. 6; 22 ± 2% and
21 ± 2%, respectively), indicating that the two PI 3-kinase
inhibitors were specific for IGF-I. These results, together with the
earlier experiments measuring PKC- and PI 3-kinase activation,
strongly suggest that IGF-I-enhanced macrophage differentiation occurs
concomitantly with activation of PI 3-kinase and its putative
downstream PKC- pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by a mechanism that involves PI 3-kinase, and that IGF-I plays a
critical role in the development of macrophages from their progenitor
cells. Immature myeloid progenitors express very little CD11b, which is
a component of the heterodimeric CR3 complement receptor, and in this
work we demonstrate that addition of IGF-I to vitamin
D3-treated progenitors increases the expression of
CD11b by threefold (Fig. 1). Indeed, this enhancement of
differentiation by exogenous IGF-I is mediated via its own receptor
rather than the potential involvement of the insulin receptor (Fig. 2)
since a mAb directed against the IGF-I receptor completely abrogates
IGF-I-potentiated macrophage differentiation. These data further
suggest that IGF-I is a critical peptide in serum that promotes
macrophage maturation because both IGFBP-3 and a mAb against the IGF-I
receptor profoundly inhibit vitamin D3-induced CD11b
expression in serum-containing conditions (Fig. 3). More importantly,
IGF-I-enhanced macrophage differentiation occurs concomitantly with the
activation of PI 3-kinase and its putative downstream target PKC-
(Figs. 4 and 5). Inhibition of the activity of PI 3-kinase blocks the
activation of both PKC- (Fig. 5) and the IGF-I-promoted enhancement
of macrophage differentiation (Fig. 6). Taken together, the present
results argue for a novel role for IGF-I during the development of
macrophages by a mechanism that induces the activation of both PI
3-kinase and PKC-.
The PKC- isoform has been demonstrated recently to mediate cellular
differentiation in adipocytes (4) and neuronal cells (50). Although
overexpression of PKC- stimulates the phenotypic expression of
monocytic maturation markers (3), the possibility that this endogenous
Ser/Thr kinase is activated in clonal progenitor cells induced by
vitamin D3 has not been addressed. Our experiments
demonstrate that IGF-I not only promotes macrophage differentiation,
but also stimulates PKC-, and that both of these events are
dependent upon the activation of a wortmannin- and LY294002-sensitive
pathway, putatively PI 3-kinase. These findings are of importance
because the stimulation of PI 3-kinase by IGF-I is required to prevent
apoptosis of promyeloid progenitor cells (9) and PC 12 pheochromocytoma
cells (8). Recent evidence has demonstrated that protein kinase B
(c-Akt) is a downstream signaling target of PI 3-kinase since
suppression of PI 3-kinase activity leads to inhibition of this enzyme
in vivo (12, 51). Although certain members of both nPKC and aPKC, such
as PKC-, are likely to be direct downstream targets of PI
3-kinase-triggered signals (6), the possibility of a potential linkage
between PI 3-kinase and PKC- in IGF-I-promoted macrophage
differentiation has not yet been tested. Our experiments demonstrate
that IGF-I enhances macrophage differentiation as well as activation of
both PI 3-kinase and PKC-, and that inhibition of PI 3-kinase
results in suppression of both PKC- and expression of the
differentiation marker CD11b. Interestingly, the
jun/fos transcriptional factor acts
synergistically with the vitamin D receptor to bind AP-1 DNA binding
sites. Overexpression of PKC- leads to the induction of this
transcription factor (3), suggesting a direct interaction between this
Ser/Thr kinase and the AP-1 heterodimer. Although members of the
mitogen-activated protein (MAP) kinase family have been reported to
transduce extracellular signals during differentiation in other cell
types, including immature thymocytes (52), adipocytes (53), and
myoblasts (54), preliminary data do not appear to support a major role
for Erk1 and Erk2 in IGF-I-enhanced macrophage differentiation (data
not shown). This conclusion is supported by a recent report that
identified PKC- as a novel cell growth suppressor that acts in
v-raf-transformed NIH-3T3 cells via a Raf/MEK/ MAP
kinase-independent mechanism (55).
Although these data clearly establish that IGF-I activates both PI
3-kinase and PKC-, our experiments do not exclude the possibility
that other PI 3-kinase family members may be involved in the signaling
events that lead to enhanced macrophage differentiation. It is now
recognized that there is a growing family of PI 3-kinase-related
proteins, currently consisting of at least nine members (reviewed in
56 . All of the carboxyl-terminal regions of these proteins share
homology with those found in the catalytic domain of the classical PI
3-kinases. The enzymatic activity of two mammalian homologues of this
PI 3-kinase family, mTOR (57) and DNA-PKcs (58), is inhibited in vitro
by both wortmannin and LY294002 at concentrations similar to those used
in our in vivo experiments. However, neither mTOR nor DNA-PKcs are
currently known to phosphorylate phosphatidylinositol, whereas IGF-I
clearly induces the activity of PI 3-kinase. At the concentrations used
in our experiments, neither wortmannin nor LY294002 directly inhibits
the enzymatic activity of other non-PI 3-kinase-related signaling
molecules that are ubiquitously expressed, such as Raf, MEK, PKC,
protein kinase A, or Src kinase (49, 59). Although myosin light chain
kinase can be inhibited by LY294002 at concentrations higher than 100
µM (40% inhibition), it is not blocked at the concentration (25
µM) used in our study (60). These data are consistent with the idea
that one or more members of the PI 3-kinase family are responsible for
the activation of PKC- in IGF-I-treated myeloid progenitor
cells.
Although IGF-I has been viewed as a progression factor that is required for promoting cells to advance through the cell cycle (33, 61), our findings that this peptide also enhances vitamin D3-induced development of macrophages are consistent with those of IGF-I augmenting the differentiation of fat (62), muscle (34), and nerve cells (35). By using a defined serum-free system, we demonstrate that IGF-I increases the expression of a surface differentiation marker, CD11b, as promyeloid progenitors develop into mature macrophages in the presence of vitamin D3. IGF-I acts to promote rather than to initiate this process because it is ineffective in the absence of vitamin D3. These results support accumulating evidence that this classical growth factor significantly promotes cell differentiation. For example, both IGF-I and IGF-II, acting through the IGF-I receptor, stimulate myogenesis in the absence of other inducers of differentiation (63, 64). Indeed, autocrine secretion of IGF-II causes spontaneous differentiation (65), whereas expression of antisense IGF RNA blocks skeletal muscle differentiation in vitro (66). Recent data show that IGF-I also promotes initial progression through the cell cycle and subsequently induces differentiation in myoblast cells (34), suggesting a biphasic effect of IGF-I in promoting clonal expansion, followed by cellular differentiation.
Similar to the development of adipocytes and neuroblastoma cells, both
lymphocytes and myeloid cells require two signals for differentiation.
For example, IGF-I alone does not induce the differentiation of human
SH-SY5Y neuroblastoma cells, but it potently promotes their
differentiation in the presence of PMA as an induction factor (35).
More significantly, IGF-I alone is able to replace the serum
requirement for differentiation of 3T3-L1 preadipocytes induced by
dexamethasone and 1-methyl-3-isobutylxanthine (62), suggesting IGF-I
acts as a critical factor to support fat cell development. In the
absence of these inducers of differentiation, however, IGF-I has no
effect. During hemopoiesis, stromal cell-derived IGF-I induces the
formation of pre-B cells from murine bone marrow progenitor cells, as
assessed by expression of cytoplasmic µ heavy chains (22). This B
cell differentiation process is abrogated by either an anti-IGF-I
Ab or pretreatment of stromal cells with an antisense oligonucleotide
to IGF-I mRNA. However, IGF-I does not induce pre-B cell development in
the absence of IL-7 (23), which is similar to our findings that IGF-I
does not induce macrophage differentiation in the absence of vitamin
D3. Instead, IGF-I, as well as c-kit ligand,
potentiates IL-7-induced B cell maturation. Similarly, during myeloid
cell development from primary human bone marrow cells, IGF-I also
promotes the formation of granulocytic colonies induced by granulocyte
CSF, granulocyte-macrophage CSF, or IL-3, but as in our experiments,
has little effect in the absence of these initiation signals (21).
Therefore, it appears that at least two signals are needed for the
optimal development of myeloid progenitor cells into macrophages: 1) a
specific inducer such as vitamin D3 is required for
initiation of the differentiation process, and 2) IGF-I activates both
PI 3-kinase and PKC- and enhances, but does not induce, macrophage
differentiation. This is similar to the classical requirements for
macrophage activation in which both priming (e.g., IFN-) and
inducing (e.g., LPS) signals are clearly defined (67).
The mechanism by which IGF-I synergizes with vitamin D3 to
promote the differentiation of hemopoietic cells remains unknown.
Indeed, since vitamin D3 has only been used to induce
hemopoietic precursor cells to differentiate toward the
monocyte/macrophage lineage in serum-containing medium (26, 27), it has
been difficult to separate the role of serum from that of vitamin
D3 in this process. Our data clearly show that the use of a
defined serum-free system for myeloid cell differentiation is necessary
to separate the signals derived from vitamin D3 and IGF-I.
During macrophage differentiation in vitamin D3-stimulated
HL-60 cells in serum, expression of both c-jun and
c-fos has been identified as a lineage-specific marker of
this process (68). The steroid/nuclear vitamin D receptor acts
synergistically with a number of nonreceptor transcriptional factors,
such as jun/fos, at the level of cooperative DNA
binding at the AP-1 site (69). Since IGF-I activates the AP-1
transcriptional factor (70), which is also regulated by PKC- (3, 55), perhaps a mechanism by which IGF-I synergizes with vitamin
D3 to promote macrophage differentiation is at the level of
activation of the AP-1 transcriptional factor.
In conclusion, we have established a new and important role for IGF-I
in promoting vitamin D3-induced macrophage differentiation.
By using a defined serum-free differentiation system, we demonstrate
that although vitamin D3 initiates the differentiation
program, IGF-I, acting through its own receptor, dramatically promotes
this process, as indicated by a threefold increase in expression of the
CD11b mature leukocyte Ag. More importantly, IGF-I enhances macrophage
differentiation via a pathway that includes the concomitant activation
of both PI 3-kinase and PKC-. Inhibition of IGF-I-inducible PI
3-kinase abrogates PKC- activity and suppresses vitamin
D3-induced macrophage differentiation. Taken together,
these results suggest a novel role for the classical growth factor
IGF-I in promoting macrophage differentiation and activating PKC-,
both of which occur concomitantly with the activation of PI
3-kinase.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Keith W. Kelley, University of Illinois, Laboratory of Immunophysiology, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; aPKC, atypical protein kinase C; IGF, insulin-like growth factor; IGFBP-3, insulin-like growth factor-binding protein-3; MBP, myelin basic protein; nPKC, novel protein kinase C; PI 3-kinase, phosphatidylinositol 3'-kinase; PY, phosphotyrosine; RA, retinoic acid; MAP, mitogen-activated protein.
Received for publication April 24, 1997. Accepted for publication October 16, 1997.
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