Originally published online as doi:10.1189/jlb.0503211 on September 2, 2003
Published online before print September 2, 2003
(Journal of Leukocyte Biology. 2003;74:1108-1116.)
© 2003
by Society for Leukocyte Biology
Insulin-dependent signaling regulates azurophil granule-selective macroautophagy in human myeloblastic cells
Kumiko Saeki*,
Zhang Hong*,
Masami Nakatsu*,
Tamotsu Yoshimori
,
Yukiko Kabeya
,
Akitsugu Yamamoto
,
Yasushi Kaburagi* and
Akira Yuo*,1
* Departments of Hematology and Metabolic Disorder, Research Institute, International Medical Center of Japan, Tokyo, Japan,
Department of Cell Genetics, National Institute of Genetics, Mishima, Japan,
Department of Cell Biology, National Institute for Basic Biology, Nagoya, Japan,
Department of Physiology, Kansai Medical University, Osaka, Japan
1Correspondence: Department of Hematology, Research Institute, International Medical Center of Japan, 1-21-1, Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: yuoakira{at}ri.imcj.go.jp
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ABSTRACT
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We show that insulin-dependent signals regulate azurophil granule-selective macroautophagy in human myeloid cells. Depletion of insulin from an insulin-transferrin-supplemented serum-free medium caused growth retardation of myeloblastic HL-60 cells, in which sequestration of electronic-dense cytoplasmic materials by autophagosomes was observed. Positive immunoreactivity with anti-CD68, anti-cathepsin D, and anti-myeloperoxidase antibodies indicated that the sequestrated materials were azurophil granules, the granulocyte/macrophage lineage-specific lysosome-like particles. By contrast, other organelles, including the mitochondria, endoplasmic reticulum, and Golgi apparatus remained intact, indicating that the macroautophagy selectively targeted azurophil granules. The addition of insulin induced rapid activations of p70S6K and Akt, and the cells were rescued from macroautophagy. Rapamycin, an inhibitor of mammalian target of rapamycin, did not block the insulin-mediated rescue from macroautophagy, although it nullified the activation of p70S6K and cell growth. Low doses of LY294002, a phosphatidyl-inositol-3-kinase inhibitor, which abolished cell growth and p70S6K activity but did not influence Akt activity, did not block the insulin-mediated rescue either. By contrast, low doses of Akt-specific inhibitors, which inhibited neither cell growth nor p70S6K activity, completely blocked the insulin-mediated rescue from macroautophagy. Thus, insulin-dependent signals are responsible for the control of azurophil granule-selective macroautophagy via Akt-dependent pathways, while p70S6K-dependent pathways promote cell growth.
Key Words: Azurophil granule-selective macroautophagy • CD68 • Cathepsin D • myeloperoxidase • LC3 • transferrin • IGF-1
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INTRODUCTION
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During differentiation, cytoplasmic organelles or particles are properly generated and degenerated. In myeloid cells, for example, the number of azurophil granules dynamically changes: They first appear at an early myeloblastic stage and, after a transient abundance at the subsequent promyelocyte stage, gradually disappear during later stages. However, the mechanism controlling the number of the particles has not been clarified.
Large cytoplasmic particles, as well as long-lived proteins, are often degraded by macroautophagy, a highly regulated nonselective and sometimes selective degradation process in eukaryotes (reviewed in [1
]). The process starts with the sequestration of target materials by isolation membranes. The isolation membranes extend and, fully sequestrating the targets, they form autophagosomes. Later, lysosomes combine with the autophagosomes to form autolysosomes, where target materials are enzymatically degraded.
In yeast, macroautophagy plays essential roles during sporulation and adaptation to nitrogen starvation [1
]. In mammalian cells, macroautophagy is observed in vivo and in vitro. The livers of starving animals [2
], as well as various cultured mammalian cells under nitrogen starvation [3
], undergo macroautophagy. Macroautophagy also plays roles during mammalian differentiation. For example, macroautophagy contributes to the elimination of whole organelles during erythrocyte maturation [4
, 5
], and specific glycoproteins are degraded by macroautophagy during colon cell differentiation [6
]. Furthermore, defects in macroautophagy are suggested to contribute to tumorigenesis [7
].
Compared with the relatively clear understanding of the membrane dynamics of macroautophagy and its molecular basis [reviewed in 8
], the signal transduction that controls macroautophagy seems rather unclear. It has been reported that class I phosphatidyl-inositol-3-kinase (PI3K) products reduce macroautophagy in human colon cells, whereas class III PI3K products enhance it [9
]. In yeast, inhibition of a phosphatidylinositol kinase homologue Tor by rapamycin induces macroautophagy even under nutrient-rich conditions [10
]. Insulin signalings, which activate mammalian target of rapamycin (mTOR) [11
], suppress starvation-induced macroautophagy in rat livers [2
]. Indeed, an involvement of mTOR, although only partial, was shown in the control of macroautophagy in rat livers [12
]. However, rapamycin-independent pathways regulate the leucine limitation-induced macroautophagy in murine muscle cells [13
]. Thus, diverse systems are involved in the regulation of macroautophagy, although the precise mechanisms involved remain unknown.
Insulin-dependent signals play important roles not only in liver cells, but also in myeloid cells. For example, insulin-dependent signals promote the growth of myelo-progenitor cells via insulin-receptor substrate (IRS)-dependent pathways [14
]. We hypothesized, by an analogy to macroautophagy in liver cells, that insulin-dependent signals might play a role in suppressing myeloid macroautophagy. To assess this issue, we investigated the effects of insulin, which is commonly used for the maintenance of myeloid cells under serum-free conditions, on the macroautophagy of human myeloblastic cells.
We show that insulin-dependent signals block the induction of azurophil granule-selective macroautophagy. The downstream signaling events and the physiological relevance of this phenomenon are discussed.
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MATERIALS AND METHODS
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Cells and reagents
HL-60 cells were maintained in RPMI 1640 medium (Life Technologies Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS) (JRH Bioscience, Lenexa, KS). For some experiments, cells were cultured in RPMI 1640 medium supplemented with 5µg/ml of insulin (Sigma Chemical Co., St. Louis, MO) and 5µg/ml of human holo-transferrin (Sigma) with and without 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) (Calbiochem Co., La Jolla, CA, USA) and rapamycin (Calbiochem). Akt-specific inhibitors of SH-5 and SH-6 [15
] were purchased from Alexis Corporation (Montreal, Canada).
Western blotting and immunostaining
The 5 x 105 cells were lysed in 100 µl of 1 x Laemmli’s sample buffer and boiled. A 10-µl aliquot of this lysate was subjected to SDS-PAGE on a 12.5% gel (5x104 cells/lane) and transferred to nitrocellulose membranes. Western blotting was performed using anti-Akt, antiphosphorylated Akt, anti-Stat5 and antiphosphorylated Stat 5, antiextracellular signal-regulated kinase (ERK) and phosphorylated. ERK antibody, anti-p38 and anti-phosphorylated p38, anti-c-Jun N-terminal kinase (JNK) and antiphosphorylated JNK, anti-p70S6K and phosphorylated p70S6K (Thr389), anti-FKBP and antiphosphorylated FKBP, anti-GSK-3ß and antiphosphorylated GSK-3ß, anti-PAK and antiphosphorylated PAK antibodies (Cell Signaling Technology, Inc., Beverly, MA), anti-insulin receptor substrate (IRS)-1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-IRS-2 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and PY20 (Wako Pure Chemical Industries, Osaka, Japan) in the first antibody reactions. The second antibody reaction and the final detection procedure were performed as described previously [16
]. For immunostaining, the cells were fixed on slide glasses with a cytospin apparatus (Cytospin2, SHANDON, Pittsburgh, PA) with further fixation with acetone/methanol solution (1:3). The immunostaining procedure was performed as described elsewhere using anti-LC3 [3
], anti-CD68, antimyeloperoxidase (Caltag Laboratories, An-Der-Grub, Austria) and anticathepsin D antibody (Calbiochem Co., La Jolla, CA).
Morphological examination
The cells were washed with phosphate-buffered saline (PBS), fixed on slide glasses using a cytospin apparatus (Cytospin2, SHANDON), stained with Wight and Giemsa solution (Muto Pure Chemical Co., Ltd, Tokyo, Japan) and observed by fluorescent microscopy (Olympus). For immunologically stained cells, fluorescent microscopic observation was performed along with Normarsky differentiated interference contrast microscopy (Olympus). For electronmicroscopic study, the cells were directly fixed with 2% glutaraldehyde and postfixed with 1% osmium tetroxide. The cell pellets were embedded in epon resin and cut with an ultramicrotome to 70 nm thick. The sections were stained with uranyl acetate and lead citrate and photographed by Bio Medical Laboratories Co. Ltd. (Tokyo, Japan). The quantification of azurophil granule-selective macroautophagy was performed by counting the number of the cells bearing large cytoplasmic vacuoles that contained myeloperoxidase-positive azurophilic particles. At least 100 cells were analyzed under microscopy in each experiment.
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RESULTS
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Insulin depletion induces azurophil granule-selective macroautophagy
Human myeloid cells can be maintained in an insulin-transferrin-supplemented serum-free medium for at least one month. To assess the requirement of insulin in this state, human myeloblastic HL-60 cells were cultured with or without insulin in serum-free medium. The cells remained viable, exporting trypan blue particles (data not shown), and the DNA content analysis showed no increment in the sub-G1 dead population (Fig. 1A
). Indeed, the number of viable cells gradually increased with time (Fig. 1B)
. Re-addition of insulin after two-day-depletion sufficiently rescued cell growth (Fig. 1C) . By contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF), which reportedly shares a downstream signaling with insulin, failed to rescue the growth (Fig. 1C)
. Light microscopic observation demonstrated the existence of multiple cytoplasmic vacuoles filled with azurophilic materials in the insulin-depleted cells as in the case of macroautophagy (Fig. 2A
). Again readdition of insulin rescued the cell morphology (Fig. 2B)
. To confirm the occurrence of macroautophagy in insulin-depleted cells, the expression of LC3 was examined. As shown in Fig. 2C , the vacuoles were brightly stained by anti-LC3 antibody, indicating that the vacuoles were indeed autophagosomes. Electron microscopic study further illustrated sequestrated materials appearing like azurophil granules (Fig. 2D
, closed arrowhead), some of which were fused to become a giant particle (Fig. 2D
, an open arrowhead). Interestingly, all the other cytoplasmic organelles including the Golgi apparatus, endoplasmic reticulum, and mitochondria remained intact (Fig. 2D
, arrows). To determine whether the sequestrated materials actually originated from the azurophil granules, the existence of various azurophil granule proteins was examined. The sequestrated materials were positively stained by anti-cathepsin D antibody (Fig. 3A
). The vacuoles in the insulin-depleted cells were further stained by anti-CD68 antibody (Fig. 3B)
. Moreover, double immunostaining using anti-myeloperoxidase and anti-LC3 antibodies indicated that myeloperoxidase-positive materials, which identified the azurophil granules, were surrounded by LC3-positive autophagosomes (Fig. 3C)
.
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Figure 1. The effects of insulin on survival and growth of myeloid cells. (A) HL-60 cells were cultured with 10% FCS (upper), 5 µg/ml of transferrin (middle), and 5 µg/ml of transferrin and 5 µg/ml of insulin (lower). After 2 days, cells were fixed and DNA content analysis was performed. Representative data from two independent experiments were shown. (B) Cells were cultured with FCS, transferrin only, transferrin plus insulin, and transferrin plus GM-CSF as indicated, and the viable cell number was counted by a time course study. The data are expressed as mean ± SD of three independent experiments. (C) Cells, which had been cultured in transferrin-supplemented serum-free medium for 2 days were further incubated under various conditions as indicated and the viable cell number was counted by a time course study. The data are expressed as mean ± SD of three independent experiments.
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Figure 2. The effects of insulin on survival and growth of myeloid cells. (A) HL-60 cells were cultured with 10% FCS (left), 5 µg/ml of transferrin and 5 µg/ml of insulin (middle), and 5 µg/ml of transferrin (right). After 2 days, cells were fixed and stained with Wright-Giemsa solution. Representative data from five independent experiments were shown. Almost all of the cells showed prominent cytoplasmic vacuole formation, and about one third of the cells showed definite azurophlic particle-containing large vacuoles. (B) Cells that had been cultured in transferrin-supplemented serum-free medium for 2 days were further incubated in the presence of insulin, and cell morphology was examined by a time course study by Wright-Giemsa staining. Representative data from three independent experiments were shown. (C) The cells were cultured with 10% FCS (upper), 5 µg/ml of transferrin and 5 µg/ml of insulin (middle), and 5 µg/ml of transferrin (right). After 2 days, cells were fixed and immunostained using anti-LC3 antibody. Cells were examined by light microscopy with Normarsky differentiated interference contrast (DIC, left) or by fluorescent microscopy (right). Almost all the granules in vacuoles were positively stained by anti-LC3 antibody. Representative data from three independent experiments were shown. (D) The cells were cultured with 10% FCS (upper left), 5 µg/ml of transferrin and 5 µg/ml of insulin (upper right), and 5 µg/ml of transferrin (lower) for 2 days and electron microscopic observation was performed. Closed arrows indicate the intact cytoplasmic organelles (Mt: mitochondria, sER: smooth endoplasmic reticulum, rER: rough endoplasmic reticulum, G: Golgi apparatus), an open arrow indicates isolation membrane, arrowheads indicate the sequestrated azurophil granules by autophagosomes. Representative data from two independent experiments were shown.
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Figure 3. The expression of azurophil granule proteins is shown. (A) Cells were cultured with 10% FCS (upper), 5 µg/ml of transferrin and 5 µg/ml of insulin (middle), and 5 µg/ml of transferrin (lower) for 2 days, fixed and immunologically stained using anticathepsin D antibody (right panels). DIC indicates the photograph taken by the Normarsky differentiated interference contrast apparatus (left). Almost all of the granules in vacuoles were positively stained by cathepsin D antibody. Representative data from three independent experiments were shown. (B) and (C) The cells which had been cultured in transferrin-supplemented serum-free medium for 2 days were fixed and stained using (B) anti-CD68 antibody or antimyeloperoxidase and (C) anti-LC3 antibodies. Representative data from more than three independent experiments were shown.
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Thus, myeloblastic cells reversibly undergo azurophil granule-selective macroautophagy in addition to growth retardation after insulin depletion.
Analysis of the downstream signals after insulin stimulation
We next examined the downstream signaling events following insulin stimulation. The cells that had undergone macroautophagy after two-day depletion of insulin were restimulated with insulin, and the serine/threonine phosphorylation states of various proteins were examined by Western blotting. As shown in Fig. 4A
, insulin specifically activated Akt and p70S6K, while GM-CSF, which failed to rescue macroautophagy, did not at all activate Akt and only faintly activated p70S6K. Although ERK is reportedly activated by insulin in certain cases, insulin-dependent activation of ERK, as well as p38 and JNK, was not detected in our system. To further clarify the proximal signaling events, Western blotting using anti-phosphotyrosine antibody was performed. As shown in Fig. 4B
, 160 kD and 190 kD proteins were tyrosine-phosphorylated after insulin stimulation. The insulin-dependent phosphorylation of the 190 kD protein was even detected following immunoprecipitation using anti-IRS-2 antibody (Fig. 4C)
, indicating that the 190 kD protein was IRS-2. On the other hand, the insulin-dependent phosphorylation of the 160 kD protein was detected after immunoprecipitation using anti-IRS-1 antibody, although we failed to detect IRS-1 per se by Western blotting after immunoprecipitation (data not shown).
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Figure 4. Protein phosphorylation after insulin stimulation. HL-60 cells which had been cultured in transferrin-supplemented, serum-free medium for 2 days were stimulated by insulin for 5 min. (A) The cell lysates were prepared and Western blotting was performed using the indicated antibodies (the left panel indicates the result using phosphorylation-specific antibodies and the right panel indicates that using total protein-recognizing antibodies. First lane; before stimulation, second lane; five-minute incubation without stimulation, third lane; insulin stimulation, fourth lane; GM-CSF stimulation). Representative data from two independent experiments were shown. (B) The cell lysates were immunoprecipitated using the indicated antibodies and immunoblotting using antiphosphotyrosine antibody was performed. Representative data from four independent experiments were shown. (C) The cell lysates were immunoprecipitated using anti-IRS-2 antibody, and immunoblotting using antiphosphotyrosine antibody or anti-IRS-2 antibody was performed. Representative data from two independent experiments were shown.
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Thus, insulin activates Akt and p70S6K signaling via IRS-2-dependent, and possibly, via IRS-1-dependent pathways.
Effects of kinase inhibitors on insulin-mediated rescue from macroautophagy
To further determine the involvement of Akt and p70S6K in macroautophagy regulation, the effects of various kinase inhibitors were tested. First, the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), was examined. After two-day depletion of insulin, macroautophagic cells were cultured in the presence of insulin with or without rapamycin, and the cell growth and morphology were examined. In our serum-free culture system, as low as 1 nM of rapamaycin sufficiently blocked the insulin-mediated p70S6K phosphorylation (Fig. 5B
) and insulin-dependent cell growth (Fig. 5A)
, whereas Akt activity remained intact (Fig. 5B) . Morphological analysis showed that the insulin-mediated rescue from macroautophagy was not prevented by rapamycin (Fig. 5C and 5D)
, indicating that mTOR/p70S6K activity is not essential for the rescue from macroatuophagy. Next, we examined the effects of LY294002, an inhibitor of phosphatidyl-inositol-3-kinase (PI3K). As shown in Fig. 6B
, 5 µM of LY294002 sufficiently blocked the insulin-stimulated p70S6K activity and insulin-promoted cell growth, whereas Akt activity remained intact (the fold increment in phospho-Akt band densities after insulin treatment is 37.1 ± 3.7 and 36.6 ± 2.4 (means±SD in three independent experiments) in DMSO-pretreated and 5 µM LY294002-retreated samples, respectively). Again, rescue from macroautophagy was not prevented. In contrast, as high as 20 µM of LY294002 prevented Akt activity (the phospho-Akt band density was reduced to 5.6±1.1) and nullified the insulin-mediated rescue from macroautophagy (Fig. 6C)
. Because cell growth was severely damaged by this high dose of LY294002 (Fig. 6A)
, the loss of recovery from macroautophagy might possibly be a secondary consequence of the cytotoxic effects of LY294002. To exclude this possibility, we tested the effects of newly synthesized Akt-specific inhibitors of SH-5 and SH6 [15
]. Although they reportedly inhibit cell growth and decrease viability when used at 10 µM, they showed no apparent cytotoxicity at much lower doses, although the Akt activities were partially suppressed and phosphorylation of FKHR, one of the downstream targets of Akt, was diminished (Fig. 7A
). Interestingly, insulin-mediated rescue from macroautophagy was clearly blocked in these states (Fig. 7C)
, indicating that even partial inhibition of Akt had critical effects on macroautophagy regulation. Concerning GSK-3ß, another downstream target of Akt, neither insulin-dependent up-regulation nor Akt-inhibitor-mediated suppression of phosphorylation was observed in our system, indicating that GSK-3ß is not involved in macroautophagy regulation.
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Figure 5. The effects of rapamycin. (A) The cells which had been cultured in transferrin-supplemented serum-free medium for 2 days were cultured in the presence of insulin along with the indicated doses of rapamycin. The number of viable cells was counted by a time course study. The results of three independent experiments are shown. (B) The insulin-depleted cells were stimulated by insulin along with the indicated doses of rapamycin. After 5 min, cell lysate was prepared and phosphorylation states of p70S6K and Akt were examined by Western blotting. Representative data from three independent experiments were shown. (C) and (D) The cells in (A) at day 1.5 were fixed and stained with Wright-Giemsa solution (C), and the number of azurophil granule-containing autophagosome-positive cells was counted (D). Representative data from three independent experiments were shown in (C) and statistically analyzed in (D).
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Figure 6. The effects of LY294002. (A) Cells were cultured with 5 µg/ml of transferrin or 5 µg/ml of insulin and transferrin along with the indicated doses of LY294002, and the number of viable cells was counted by a time course study. The results of three independent experiments are shown. (B) The cells that had been cultured in transferrin-supplemented serum-free medium for 2 days were stimulated by insulin along with the indicated doses of LY294002. After 5 min, cell lysate was prepared and Thr389-phosphorylation of p70S6K and Ser473-phosphorylation of Akt were examined by Western blotting. Because p70S6K has other phosphorylation sites than Thr389 such as Thr229, Ser411 Thr421, and Ser424, some of the mobility-shifted p70S6K bands were not detected by this phosphorylation-specific antibody. The representative data from three independent experiments were shown. Representative data from three independent experiments were shown. (C) and (D) Cells in (A) at day 3.5 were fixed and stained with Wright-Giemsa solution (C) or the number of azurophil granule-containing autophagosome-positive cells was counted (D). Representative data from three independent experiments were shown in (C) and statistically analyzed in (D).
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Figure 7. The effects of Akt-specific inhibitors of SH-5 and SH-6 (A) Cells were cultured with 5 µg/ml of transferrin (lane 1) or 5 µg/ml of transferrin and 5 µg/ml of insulin (lane 2) along with 0.03 µM (lane 5) and 0.1 µM (lane 6) of SH-5 or 0.1µM (lane 3) and 0.3 µM (lane 4) of SH-6. After a 3-day incubation, viable cell number was counted. The results of three independent experiments are shown. (B) The cells that had been cultured in transferrin-supplemented serum-free medium for 2 days were stimulated by insulin along with 0.03 µM (lane 5) and 0.1 µM (lane 6) of SH-5 or 0.1 µM (lane 3) and 0.3 µM (lane 4) of SH-6. After 5 min, cell lysate was prepared and phosphorylation states of p70S6K and Akt were examined by Western blotting (lane 1; no stimulation, lane 2; insulin stimulation without Akt inhibitors). Representative data from three independent experiments were shown. (C) The cells in (A) at day 1.5 were fixed and stained with Wright-Giemsa solution. Representative data from three independent experiments were shown. (D) The number of azurophil granule-containing autophagosome-positive cells in (A) at day 1.5 was counted and statistically analyzed.
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These findings together suggest that insulin regulates azurophil granule-selective macroautophagy via IRS/PI3K/Akt-dependent pathways, whereas IRS/PI3K/mTOR/ p70S6K-dependent pathways regulate cell growth.
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DISCUSSION
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In this paper, we showed a novel role of insulin-dependent signaling in human myeloid cells, namely that insulin inhibits the induction of azurophil granule-selective macroautophagy. Our study is the first report to show the involvement of macroautophagy in the selective degradation of organelles other than peroxisomes, which can be selectively catalyzed by macroautophagy during methanol adaptation in yeast [17
].
Myeloid cells are very unique in that they lack the typical structures of lysosomes, that is, the dense bodies or matured lysosomes [18
]. Azurophil granules were once thought to be myeloid lysosomes because of their abundance in lysosomal enzymes such as cathepsins. However, the lysosome-associated membrane proteins (LAMP) were barely expressed in azurophil granules, although they contained mannose 6-phosphate-containing glycoproteins (Man 6-P GP) [18
]. Indeed, LAMP was absent in all the identified granule populations in myeloid cells, but was found in multivesicular bodies (MVB) and the multilaminar compartment (MLC). Thus, it is now believed that azurophil granules are the storage sites of lysosomal enzymes while MVB and MLC are the genuine lysosomes. Whether the lysosomal enzymes are actually transferred from azurophil granules to MVB and MLC and whether any populations of MVB and MLC contain lysosomal enzymes still remain unknown. At least, enzyme-rich matured lysosomes have not been detected by electron microscopy [18
]. Thus, the process of lysosome-mediated proteolysis, including macroautophagy, seems equivocal in myeloid cells, and one might even suspect the occurrence of macroautophagy per se in them. However, we previously showed that human myeloblastic cells actually underwent macroautophagy when bcl-2 expression was down-regulated [19
]. This observation at the same time assures the existence of maturated lysosomes. Azurophil granule-selective macroautophagy might be one possible way to transfer lysosomal enzymes to MVB and MLC. To reveal the physiological relevance of azurophil granule-selective macroautophagy in myeloid cells with an appropriate proteolytic process is a matter of great interest.
We observed an interesting effect of the PI3K inhibitor of LY294002: low doses of LY294002, below 10 µM, inhibited only p70S6K signaling, whereas as high as 20 µM of LY294002 inhibited both p70S6K and Akt signaling, indicating that p70S6K signaling requires a high activation of PI3K, while Akt signaling requires only a low activation. This may be due to the differences in their signal transduction complexity: Akt is directly activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1), a PI3K substrate, while p70S6K can be indirectly activated [20
]. Moreover, p70S6K activity per se is required for its maximal activation by multiple PI3K-dependent inputs [21
]. Thus, p70S6K may be more susceptible to kinase inhibitors.
In addition to PI3K, mTOR is also reportedly required for activation of p70S6K [22
]. This was indeed the case in our system (Fig. 5) . However, the effects of rapamycin on macroautophagy regulation do not seem simple. Rapamycin-dependent pathways reportedly contribute to the induction of macroautophagy in yeast [10
], and similar results were obtained in mammalian cells. However, rapamycin-independent pathways actually exist and regulate mammalian macroautophagy [13
]. Thus, the signaling involved in the regulation of macroautophagy is rather complex. Our results, which demonstrate that Akt-dependent pathways inhibit azurophil granule-selective macroautophagy in myeloid cells, might provide an answer concerning the signaling for rapamycin-independent macroautophagy.
The dose of insulin we used here was 5 µg/ml, which is a commonly used dose for the maintenance of myeloid cells in serum-free culture. Because the half-maximal stimulatory concentration of insulin was 0.5 nM (ca. 2.5 µg/ml) [23
], the dose of insulin we used here may have fully activated the insulin receptor. However, the insulin-like growth factor (IGF) receptor may also have been activated, although only partially. Because the IGF receptor, as well as the insulin receptor, was expressed in human myeloid cells (data not shown), and because IRSs can also be activated through the IGF receptor, we cannot exclude the possibility that IGF-dependent signals contributed to the regulation of azurophil granule-selective macroautophagy. Further investigations are required.
Because the membrane trafficking processes are important during macroautophagy, the expression of membrane proteins may be affected by insulin depletion-induced azurophil granule-selective macroautophagy. We observed that the surface expression of CD11b, a differentiation-associated adhesion molecule, was slightly up-regulated in insulin-depleted macroautophagic cells (unpublished observation by K.S.). Although other differentiation markers, such as oxidative burst activity, were not altered by the induction of macroautophagy, there may exist a possible link between differentiation and macroautophagy.
The in vivo relevance of insulin signaling in regulating azurophil granule-selective macroautophagy is not known. Azurophil granules function not only as the storage sites of lysosomal enzymes, but also as major storage sites of nitrogen per se. In the case of starvation, the serum insulin levels greatly fall and azurophil granule-selective macroautophagy may be induced to supply amino acids by degrading inactive lysosomal enzymes. If severe starvation persists, lysosomal enzymes would become exhausted and the antibacterial functions of phagocytes may be greatly damaged. The relationship between nutritive conditions and the susceptibility to bacterial infection in the terms of the amounts of lysosome enzymes in phagocytes is a subject for further study.
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ACKNOWLEDGEMENTS
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This work was supported by a Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan.
Received May 11, 2003;
revised July 8, 2003;
accepted July 15, 2003.
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