Introduction

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IL-3 is a multilineage hematopoietic cytokine that can support the survival and proliferation of immature multipotential progenitors and cells committed to a number of myeloid lineages (Ihle, 1992). IL-3 also supports the proliferation of factor-dependent cell lines such as FDCP-1 (Dexter et al., 1980) and B6SUtA1 (Sorensen et al., 1989). The receptor for human IL-3 consists of an -subunit and a -subunit, but both lack detectable catalytic domains (Ihle, 1992). Recent studies on postreceptor cytokine signaling have revealed that the binding of IL-3 to its receptor molecules results in their dimerization, thus activating two signaling pathways: a ras-mediated (ras-raf-1-MAP kinase) pathway (Satoh et al., 1992; Vojtek et al., 1993; Kyriakis et al., 1992) and a ras-independent JAK (Janus kinase)-STAT (signal transducer and activator of transcription) system (Silvennoinen et al., 1993; Ihle et al., 1994).

PKC (Nishizuka, 1984; 1988; 1992) has been reported to play an important role in the proliferation of IL-3-dependent cell lines. IL-3 stimulation of FDCP-1 cells has been reported to induce the translocation of PKC from cytosole to membrane (Farrar et al., 1985). Although this observation has been confirmed in some studies (Whetton et al., 1986; Pelech et al., 1990), it has not been confirmed in others (Ihle, 1992). Recently, it has been shown that PKC activates Raf-1 by direct phosphorylation in NIH 3T3 fibroblasts (Kolch et al., 1993, Burgering and Bos, 1995). These findings are very important in demonstrating the cross-talk of PKC activation and ras-mediated IL-3 signal transduction pathways. However, it is not certain that these observations are applicable to normal hematopoiesis.

In the presence of IL-3, certain cytokines, such as IL-6 (Ikebuchi et al., 1987), granulocyte colony-stimulating factor (G-CSF; Ikebuchi et al., 1988), IL-11 (Musashi et al., 1991a), c-kit ligand (Tsuji et al., 1991) and IL-12 (Jacobson et al., 1993; Hirayama et al., 1994), have been observed to be capable of exerting synergistic effects on the proliferation of primitive hematopoietic progenitors. These particular cytokines are termed "synergistic factors" (Ogawa, 1993). On the basis of these studies, it has been suggested that IL-3 can support the proliferation of progenitors that have left the dormant state of the cell cycle (G0) but that it is unable to trigger the recruitment of primitive progenitors into the cell cycle (Suda et al., 1985; Leary et al., 1989). On the other hand, these synergistic factors augment IL-3-dependent colony growth by shortening the G0 period (Ikebuchi et al., 1987; 1988; Musashi et al., 1991a; Tsuji et al., 1991; Hirayama et al., 1994). Interestingly, in addition to its synergistic effects on IL-3-dependent colony formation, c-kit ligand stimulates the proliferation of progenitors that have left the G0 state, so it is able to exert synergistic effects on other "synergistic factors" (Tsuji et al., 1991). Thus the synergistic interaction of these cytokines is somewhat complicated, and it is important that their signal transduction pathways, along which cellular responses will be made, be clarified.

As a first step, we studied the PKC activator TPA (Niedell et al., 1983) in order to determine whether it could enhance the IL-3-dependent proliferation of primitive hematopoietic progenitors in mice, in an effort to clarify the significance of PKC in the proliferation of primitive hematopoietic progenitors.

	Materials and Methods

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Cell preparation. Ten to 15-week-old male BDF₁ (C57 B1/6 × DBA/2 F1 hybrids) mice were obtained from Charles River Japan (Atsugi, Japan). A single-cell suspension was prepared from the pooled femurs of the mice, which had been injected with 150 mg/kg b. w. of 5-FU (Kyowa Hakko Kogyo Co., Tokyo, Japan) i.v. through their tail veins 2 days before examination (Day-2 post 5-FU marrow cells) in order to enrich the noncycling hematopoietic primitive progenitors (Hodgson and Bradley, 1979; Suda et al., 1983). Lineage-negative (Lin) Day-2 post 5-FU marrow cells were isolated as described by Shih et al. (1992) with some minor modifications. Briefly, light-density cells were separated by density centrifugation above Ficoll-Conray (specific gravity, 1077) from the Day-2 post 5-FU marrow cells. They were then incubated at 4°C for 45 min in a cocktail of antibodies: anti-Thy 1.2 (Pharmingen, San Diego, CA), B220 (CD45R, Pharmingen), Gr-1 (Pharmingen), and Mac-1 (CD11b, Boehringer Mannheim Biochemica, Germany). After washing twice, sheep anti-rat IgG (Fc)-conjugated immunomagnetic beads (Dinabeads, Dynal A.S., Oslo, Norway) were added to the cell suspension and incubated at 4°C for 45 min. Lineage-specific-antigen-positive (Lin+) cells were removed by a magnetic particle concentrator (Dynal), and Lin cells were recovered from the supernatant. The cell/bead ratio applied was 1:30.

Factors and agents. The source of recombinant murine IL-3 was medium conditioned by Chinese hamster ovary (CHO) cells that had been genetically engineered to produce murine IL-3 to high titer (approximately 70,000 U/ml). Human recombinant Ep was a generous gift from Kirin Brewers Co. (Tokyo Japan). TPA and OAG were purchased from Sigma Chemical Co. (St. Louis, MO). Calphostin C, a specific inhibitor of PKC, was purchased from Kyowa Medics Co. (Tokyo, Japan). The IC₅₀ values of calphostin C against PKC, cyclic AMP-dependent protein kinase (A-kinase) and tyrosine kinase (p60^v-src) were reported to be 0.05 µM, >50 µM and >50 µM, respectively (Kobayashi et al., 1989 a, b).

Clonal cell culture. Methylcellulose cell culture was performed in 35-mm Lux suspension culture dishes (No. 5221R, Nunc Inc., Naperville, IL) as described previously (Musashi et al., 1991a, b). One milliliter of culture contained 5 × 10⁴ Day-2 post 5-FU marrow cells or 2000 lineage-negative cells, alpha-medium (Flow Laboratories Inc., Rockville, MD), 1.2% methylcellulose (Wako Junyaku Co., Tokyo, Japan), 30% fetal calf serum (Hyclone Laboratories, Logan, UT), 1% fraction V bovine serum albumin (Sigma), 2 U/ml of recombinant human Ep, 100 µM 2-mercaptoethanol (2-ME, Eastman Kodak, Rochester, NY), hematopoietic factors and agents. The dishes were incubated in a humidified atmosphere flushed with 5% CO₂ at 37°C. Colonies consisting of 50 or more cells were counted on indicated days under an inverted microscope according to colony types (Nakahata et al., 1982) as indicated in the tables. Briefly, GM colonies consisted of large, round macrophages and polygonal neutrophils. GEMM colonies were recognized by the red or dark brown color of hemoglobin in aggregated erythrocytes and by huge megakaryocytes adding to granulocytes and macrophages. The blast cell colonies consisted of a homogeneous population of up to 1000 loosely arranged, frequently clumped, round cells with no signs of terminal differentiation such as granulocytes, macrophages, erythrocytes, and megakaryocytes. The blast cell colonies would later develop into GEMM colonies or large GM colonies.

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Effects of long-term preincubation of cells with TPA on colony growth. Long-term incubation of cells with TPA resulted in a down-regulation of PKC (Kitajima et al., 1988; Goodnight et al., 1994). To examine whether TPA exerts its effects through the activation of PKC, one million Day-2 post 5-FU marrow cells were incubated in alpha-medium with 2 × 10⁸ M TPA or vehicle for 48 hr. Neither the FCS nor the cytokines were supplemented in the cell suspension. After washing, the cells were incubated in a methylcellulose culture containing IL-3 either alone or in combination with TPA.

Preincubation of cells. To infer the signal transduction of TPA by using the protein kinase inhibitors, Day-2 post 5-FU bone marrow cells were preincubated for 12 hr with TPA either alone or in combination with the inhibitors. After washing twice, the cells were cultured in methylcellulose containing IL-3.

Statistical analysis. Student's t test was used for the statistical analysis. For analysis of variance (ANOVA), an F test was performed on the data before Student's t test.

	Results

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Colony formation derived from Day-2 post 5-FU marrow cells. We first analyzed the colony formation derived from Day-2 post 5-FU marrow cells supported by IL-3 in combination with TPA. Whereas TPA alone did not stimulate colony growth, it did augment colony formation when in combination with IL-3, maximally at 10⁷ M. This particular concentration of TPA, in combination with IL-3, increased colony growth by 175% ± 16% (mean ± S.D.) in nine of the experiments compared with the IL-3 plus vehicle control. Table 1 shows a representative result. A higher concentration of TPA (10⁶ M) seemed to suppress colony growth. The final concentration of DMSO that contained 10⁷ M TPA was 0.01%. Because concentrations of DMSO less than 0.1% did not affect IL-3-dependent colony formation (data not shown), this suppressive effect of TPA is not due to DMSO. Another PKC activator, OAG, at a concentration of 10⁶ M and 10⁷ M also, significantly enhanced IL-3-dependent colony formation. However, the degree of increase was less than that for those augmented with TPA. The addition of supplement OAG (final concentration, 10⁷ M) on the second day to those cultures containing 10⁷ M OAG resulted in an increase in colony numbers (19 ± 2) compared with those supplemented with vehicle (16 ± 2) and those that had not been supplemented (15 ± 2). These data suggest that OAG may be inactivated rapidly in the culture dishes.

Serial observation of blast cell development in Day-2 post 5-FU marrow cells. To examine the time course of colony development, we observed the culture dishes every third day for a serial observation of blast cell development. As shown in figure 1, although TPA augmented IL-3-dependent colony growth, unlike IL-6, it did not hasten the appearance of colonies supported by IL-3. The addition of TPA did not affect the synergistic effects of IL-3 + IL-6.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Time course of colony formation. Day-2 post 5-FU marrow cells were cultured with 2 U/ml of Ep and designated agents. Colonies were counted on every third day. Colony numbers show mean number for four dishes, each containing 5 × 104 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Growth rate of individual blast cell colonies. Solid lines shows blast cell colonies that later developed into GM colonies; dashed lines show those that later revealed GEMM expression. The data are from colonies identified in each of the four dishes seeded with 5 × 104 day-2 post 5-FU bone marrow cells in the presence of IL-3 either alone or in combination with TPA.

Colony formation in pooled blast cells. It is not clear whether TPA acted on primitive progenitors. To clarify this, we used pooled blast cells instead of Day-2 post 5-FU marrow cells. Because the pooled blast cells that constituted the blast cell colony did not contain stromal or mature cells and contained a relatively homogeneous population, as well as having a high plating efficiency (Nakahata et al., 1982; Musashi et al., 1991a), we extracted and pooled those blast cell colonies found to be smaller than 150 cells per colony as stimulated by IL-3 on day 9 of the culture. After washing, the pooled blast cells were reincubated with IL-3 or IL-3 + TPA in cultures containing 30% fetal calf serum, 1% bovine serum albumin, and 2 U/ml of Ep. Both IL-3 + TPA and IL-3 + vehicle stimulated secondary colony formation in the pooled blast cells, and the plating efficiencies were 15% to 20% and 8% to 12%, respectively (table 3). These data suggested that TPA had, to some extent at least, acted on the early progenitors directly.

Colony formation in lineage-negative bone marrow cells. To analyze further the direct effects of TPA on primitive progenitors, we purified progenitor cells partially by density cut and immunomagnetic bead selection. TPA increased IL-3-dependent colony formation derived from Lin cells about 6-fold, whereas the mean increment by TPA of IL-3-dependent colonies derived from Day-2 post 5-FU marrow cells was 175%. Thus TPA-enhanced IL-3-dependent colony formation was more sensitive to TPA in Lin bone marrow cells than in Day-2 post 5-FU marrow cells (table 4). Again, TPA increased not only the GM colonies but also the GEMM colonies. Lin+ cells did not give rise to colonies in response to IL-3 either alone or in combination with TPA.

Effects of long-term preincubation of cells with TPA on colony growth. Next we examined the involvement of PKC in the proliferation of primitive progenitors stimulated by IL-3 alone or in combination with TPA. When the cells were preincubated with a vehicle, a difference in colony numbers was observed between those supported by IL-3 and those supported by IL-3 + TPA. However, IL-3 and IL-3 + TPA gave rise to almost the same number of colonies (13 vs. 11) after preincubation with TPA (table 5). These results suggested that 48 hr of incubation with TPA may have down-regulated the PKC in the progenitor cells, resulting in the loss of the enhancing effect of TPA.

Effects of preincubation with TPA alone and in combination with protein kinase inhibitors on IL-3-dependent colony formation. To infer a signal transduction pathway of TPA, we preincubated Day-2 post 5-FU marrow cells for 12 hr with TPA alone or in combination with the inhibitors. After washing twice, the cells were cultured with IL-3. The specific PKC inhibitor calphostin C abrogated the enhancing effects of TPA at a concentration of 200 nM; this concentration was 4 times higher than the IC₅₀ value against PKC and much lower than the IC₅₀ value against PTK and A-kinase. Genistein and herbimycin A also abrogated the effects of TPA at 10 µg/ml and 200 ng/ml, respectively. This concentration of genistein was almost the same as that of the IC₅₀ against tyrosine kinase, and the concentration of herbimycin A was markedly lower than the concentration of IC₅₀ against tyrosine kinase (table 6). The incubation of Day-2-post 5-FU marrow cells for 12 hr with DMSO, calphostin C, genistein or herbimycin A had no effect on the viability of these cells. The viabilities of the Day-2 post 5-FU marrow cells incubated with DMSO (0.1%-0.001%), calphostin C (50-200 nM), genistein (1-100 µg/ml) and herbimycin A (50-200 ng/ml) were 97.9% to 98.4%, 98.9% to 99.0%, 96.9% to 99.2% and 98.1% to 98.7%, respectively. Although these results suggest that TPA augmented IL-3-dependent colony formation, probably through activation not only of PKC but also of certain tyrosine kinases, direct evidence of this activation of PKC and tyrosine kinase is needed for confirmation.

	Discussion

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In this study we showed that TPA directly augmented the IL-3-dependent colony formation of primitive hematopoietic progenitors through the activation of PKC and certain tyrosine kinases. TPA did not augment colony formation at all the doses evaluated; at 10⁶ M, there was a reduced number of colonies. Although the suppressive effect of TPA at high concentration has already been reported (Kanakura et al., 1991), its mechanism is as yet unclear. The weaker action of OAG might be due to its rapid metabolism. Repeated supplement of OAG is required to produce a cellular response similar to that triggered by a single dose of TPA (Nishizuka, 1992). Our data from the addition of supplement OAG also appear to support this observation.

TPA is well known to induce the monocytic differentiation of myeloid leukemia cells, such as HL-60 (Rovera et al., 1979; Huberman and Callaham, 1979) and KG-1 (Ferrero et al., 1983). TPA, alone (Stuart and Hamilton, 1980; Burgess and Nicola, 1983) or in combination with IL-6 (Heyworth et al., 1993; Whetton et al., 1994), can also stimulate the proliferation of GM-CFC. But the extent of these stimulative effects of TPA is not limited to GM lineage: as we have shown, TPA in combination with IL-3 increased not only GM colonies but also multilineage GEMM colonies (table 2).

In terms of the synergistic effects of synergistic factors on IL-3-dependent colony formation, the synergistic factors did not influence the cell doubling time of blast cells but shortened the G0 period of blast cell CFC. Because TPA, unlike other synergistic factors, did not hasten the appearance of the blast cell colonies or shorten the G0 period of the blast cell CFC (figs. 1 and 2), it seems possible that TPA acts through some other mechanism than the shortening of the G0 period of progenitor cells, such as suppression of the programmed cell death of progenitor cells. It has been reported that not only IL-3 (Williams et al., 1990; Collins et al., 1992) but also TPA can rescue bone marrow cells from apoptosis through activation of PKC (Lotem et al., 1991). Further studies are needed to confirm the relationship between the rescue of progenitor cells by TPA from apoptosis and the enhancing effect of TPA on IL-3-dependent colony formation in primitive progenitor cells.

Next we examined whether TPA acted on primitive progenitors directly or indirectly through accessory cells contained in the cultures. To do this, we used blast cells obtained from the blast cell colonies that had developed in the presence of IL-3. These pooled blast cells are not primitive progenitor cells in themselves, however, they can give rise to secondary colonies, including GEMM colonies (Nakahata and Ogawa, 1982; Musashi et al., 1991a). The lower plating efficiencies than those found in previous experiments might be attributable to different culture conditions, such as the quality of fetal calf serum. TPA also enhanced the IL-3-dependent colony formation derived from lineage-negative Day-2 post 5-FU marrow cells. The multiple increase of colonies by TPA from lineage-negative cells was higher than that from Day-2 post 5-FU marrow cells (6-fold vs. 1.8-fold).

Then we focused on the signal transduction of TPA in IL-3-dependent colony formation derived from Day-2 post 5-FU marrow cells. First, we examined the effects on colony formation of prolonged pretreatment of the progenitors with TPA. Although we did not measure PKC activity in the bone marrow cells before and after the prolonged incubation with TPA, it has been reported that incubation with 2 × 10⁸ M TPA for 48 hr resulted in a down-regulation of PKC (Nishizuka, 1984; Goodnight et al., 1994). TPA did not enhance IL-3-dependent colony growth further after 48 hr of incubation, which suggests the involvement of PKC in the enhancing effect of TPA on IL-3-dependent colony formation. Second, we used protein kinase inhibitors against PKC and against tyrosine kinase. In these studies, the specificity of the inhibitors is critical. Calphostin C is a recently developed PKC inhibitor. It works by binding to the regulatory domain of PKC (Nishizuka, 1988; 1992) and does not share a common homology with other protein kinases, so it gives specific inhibitory effects. Calphostin C has a 1000 times lower IC₅₀ value against PKC than against tyrosine kinase or A-kinase. Because high concentrations of calphostin C have been reported to be somewhat cytotoxic to human tumor cells (Bruns et al., 1991), we examined the cytotoxic effects of calphostin C on Day-2 post 5-FU marrow cells. Twelve hours of incubation with calphostin C at a concentration of 50 to 200 nM did not influence cell viability. The fact that calphostin C abrogated the augmentation of IL-3-dependent colony formation by TPA suggests that PKC might be involved in the enhancing effects of TPA. We cannot, however, exclude the possibility that calphostin C blocks the signaling not only of TPA but also of IL-3, because calphostin C decreased colony numbers to less than that achieved with IL-3 stimulation only.

Genistein and herbimycin A, specific tyrosine kinase inhibitors, also inhibited the enhancing effects of TPA. One tyrosine kinase that may be activated by TPA and blocked by these inhibitors is MAP kinase (Gilmore and Martin, 1983; Vila and Weber, 1988), which is activated by both phosphorylation of threonine and tyrosine residues. Because genistein and herbimycin A, as well as calphostin C, completely abrogated IL-3-dependent colony formation, we could not ignore the possibility that these tyrosine kinase inhibitors might block the signaling of both TPA and IL-3, of which the intracellular signal is transduced via tyrosine kinase JAK 2 (Silvennoinen et al., 1993).

Further studies aimed at providing direct evidence of the involvement of PKC in the enhancing effects of TPA on the IL-3-dependent colony formation of primitive hematopoietic progenitors are underway.