Section, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 .... point lithium chloride (final concentration, 10 mM) was added.
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3284-3288, May 1988 Biochemistry
Interleukin 3 stimulates proliferation via protein kinase C activation without increasing inositol lipid turnover A. D. WHETTON*, P. N. MONK*, S. D. CONSALVEY*, S. J. HUANG*, T. M. DEXTERt, AND C. P. DOWNESt *Department of Biochemistry and Applied Molecular Biology, UMIST, Sackville Street, Manchester M60 1QD, United Kingdom; tExperimental Haematology Section, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, United Kingdom; and TSmith Kline and French Research Ltd., The Frythe, Welwyn, Herts., AL9 6AR, United Kingdom
Communicated by Eugene P. Cronkite, January 4, 1988
ABSTRACT Interleukin 3 (IL-3) is required for the survival and proliferation of the FDCP-Mix 1 multipotent stem cell line. IL-3 or phorbol esters can rapidly translocate protein kinase C from a cytosolic to a membrane-bound form in these cells. Phorbol esters were able to partially replace the requirement of FDCP-Mix 1 cells for IL-3. Down-modulation of protein kinase C levels by chronic treatment with phorbol ester markedly reduced the ability of the cells to proliferate in response to either IL-3 or phorbol esters. These data indicate that IL-3 can activate protein kinase C, leading to the survival and proliferation of stem cells. Protein kinase C is activated conventionally by complexing with diacylglycerol which accumulates in the cell membrane after agonist-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2]. However, there was no detectable breakdown of Ptdlns(4,5)P2 when IL-3 was added to FDCP-Mix 1 cells, nor was there detectable accumulation of inositol phosphates in response to IL-3. In contrast, rapid hydrolysis of PtdIns(4,5)P2 and accumulation of inositol 1,4,5-trisphosphate was elicited by readdition of horse serum to serum-starved cells, thus indicating that these cells possess the necessary machinery to undergo agonist-mediated inositol phospholipid breakdown. We conclude that the mechanism whereby IL-3 can activate protein kinase C leading to proliferation is not associated with inositol phospholipid hydrolysis.
(8). The possible mechanism whereby IL-3 can evoke this response has also been investigated. IL-3 has been shown to translocate and activate a Ca2+-sensitive, phospholipiddependent protein kinase (protein kinase C) (9). This enzyme is the major receptor for the tumor-promoting phorbol esters, which can act as mitogens in many different cells because of their ability to activate protein kinase C (10-12). The phorbol esters can also translocate protein kinase C in IL-3-dependent cell lines (9, 13), and this translocation is associated with increased survival and proliferation of the cells in the absence of IL-3 (13). This infers that protein kinase C may have an essential function leading to survival and proliferation of hemopoietic progenitor cells. Activation of protein kinase C in response to growth factors is believed to be achieved by the generation of 1,2diacylglycerol after receptor-stimulated breakdown of phosphatidylinositol (Ptdlns) 4,5-bisphosphate [Ptdlns(4,5)P2]. The other product of this reaction is inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], a second messenger that increases intracellular (cytosolic) Ca2+ levels [Ca2+]i (14, 15). We have assessed the effect of IL-3 on inositol phospholipid metabolism and protein kinase C activation in IL-3dependent stem cells. Although protein kinase C activation appears to be an element in IL-3-mediated proliferation, this does not appear to be achieved by hydrolysis of inositol phospholipids.
Interleukin 3 (IL-3) is a multilineage hemopoietic growth factor that can promote the survival, proliferation, and development of multipotent stem cells and myeloid committed progenitor cells from the granulocyte/macrophage, erythroid, eosinophil, megakaryocytic, mast cell, and basophilic lineages (1, 2). The wide range of biological activity of IL-3 suggests that it is an important regulatory molecule in murine and human hemopoiesis (3). Furthermore, the IL-3 dependence of hemopoietic progenitor cells may be overcome during leukemic transformation (4-6). In view of these observations, elucidation of the mechanism whereby IL-3 exerts its effects on stem and progenitor cells may be an essential element in understanding the regulation of hemopoiesis and also the possible causes of IL-3 independence and leukemic transformation. When hemopoietic stem cells are cultured in the presence of IL-3 they develop into clones containing multiple lineages: in the absence of IL-3 they die (2, 7). We have investigated the nature of this phenomenon using cell lines that are absolutely dependent on the continuous presence of IL-3 for their survival and growth in vitro. IL-3 has been shown to stimulate the primary metabolism of such factor-dependent cells leading to the maintenance of ATP levels within the cells; removal of IL-3 leads to afall in the primary metabolism of the cells culminating in reduced ATP levels and cell death
METHODS Cell Culture. FDCP-Mix 1 cells were routinely maintained in Fischer's medium containing 20% (vol/vol) horse serum. WEHI-3B cell-conditioned medium [10%o (vol/vol)] was added as a source of IL-3, which is essential for the survival and proliferation of FDCP-Mix 1 cells. The cells were routinely subcultured twice weekly to give between 5 x 104 and 2 x 105 cells per ml (16). Cellular Viability and DNA Synthesis. Measurements of cellular viability and thymidine incorporation (to assess DNA synthesis) were performed as described by Whetton et al. (13). Measurement of Ca2 /Phospholipid-Dependent Protein Kinase Activity. Exponentially growing FDCP-Mix 1 cells were centrifuged at 800 x g for 10 min, the supernatant was removed, and the cell pellet was resuspended in fresh Fischer's medium. The cells were then centrifuged again, the pellet resuspended in Fischer's medium, and the suspension centrifuged once more. The resulting pellet was then resuspended to a cell density of 5 x 105 per ml in Fischer's medium and incubated for 2 hr in a gassed CO2 incubator at 37°C. After this time the cells were centrifuged, and the pellet was Abbreviations: IL-3, interleukin 3; Ptdlns, phosphatidylinositol; PtdInsP, Ptdlns monophosphate; PtdIns(4,5)P2, Ptdlns 4,5-bisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; [Ca2+] , intracellular (cytosolic) Ca2+ concentrations; TPA, phorbol 12-tetradecanoate 13-acetate; InsP, inositol monophosphate(s); InsP2, inositol bisphosphate(s); InsP3, inositol trisphosphate(s).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3284
Biochemistry: Whetton et al. resuspended to a cell density of 1 x 107 per ml in Fischer's medium. After incubation of the cells with the appropriate additives, membrane and cytosol fractions were prepared as described by Farrar et al. (9), and the cytosolic and membrane-associated Ca2+/phospholipid-dependent protein kinase C activity was determined (9). Labeling of FDCP-Mix 1 Cells with [3H]Inositol. Cultures of FDCP-Mix 1 cells were centrifuged as described above and resuspended in 5% (vol/vol) horse serum/5% (vol/vol) WEHI-3B-conditioned medium supplemented with 5 p.Ci of [3H]inositol per ml (1 Ci = 37 GBq). The FDCP-Mix 1 cells were then incubated for 18 hr at 37TC. This procedure did not result in any loss of cellular viability. FDCP-Mix 1 cells were then centrifuged, washed as detailed above, and resuspended in Fischer's medium without additives to a density of 5 x i05 per ml and incubated for 2 hr in a gassed CO2 incubator set at 370C. After this the cells were centrifuged and resuspended to 0.25-1.0 x 107 cells per ml in Fischer's medium. At this point lithium chloride (final concentration, 10 mM) was added to inhibit breakdown of inositol phosphates to free inositol. Analysis of [3H11]Inositol Phosphates. After addition of lithium, aliquots of FDCP-Mix 1 cell suspension (250 ,ul) were incubated with the appropriate additive at 370C. The reaction was terminated by the addition of 250 1.l of 7% (wt/vol) perchloric acid. The solution was centrifuged to remove the precipitate, and 400 1.l of supernatant was removed. To this was added 300 ,ul of 1,1,2-trichlorotrifluoroethane/tri-noctylamine, 1:1 (vol/vol) to neutralize excess perchloric acid (17). The resultant solution was centrifuged, and 350 ,lI of the aqueous upper phase was removed. This was made up to 5.0 ml with water, and EDTA was also added to a final concentration of 1 mM. The solution was taken for anion-exchange chromatography (18). The following eluants were used: water (10 ml, to elute free inositol); 60 mM ammonium formate/5 mM disodium tetraborate (10 ml, to elute any deacylated inositol phospholipids); 200mM ammonium formate/100 mM formic acid [10 ml, to elute inositol monophosphates (InsP)]; 400 mM ammonium formate/100 mM formic acid [10 ml, to elute inositol bisphosphates (InsP2], and 800 mM ammonium formate/100 mM formic acid [10 ml, to elute inositol trisphosphates (InsP3)]. High-performance liquid chromatography was used to confirm the identity of labeled components of the acid-soluble fractions from FDCP-Mix 1 cells by the method previously described (19). Analysis of [3H]Inositol-Labeled Phospholipids. Inositol phospholipids were extracted from perchloric acid-precipitated material, deacylated, and analyzed by anion-exchange chromatography as described by Downes and Wusteman (20). Phorbol Dibutyrate Binding Studies. FDCP-Mix cells were incubated for 16 hr in the appropriate medium and then washed three times to remove serum, IL-3, and any other additives. The cells were resuspended to a density of about 1 x 106 per ml in binding medium (140 mM NaCl/5 mM KCI/1.8 mM CaCI2/1 mM MgCl2/25 mM Hepes, pH 7.0) at 4°C (21). To assess the specific [3H]phorbol dibutyrate binding, cells were incubated with 50 nM [3H]phorbol dibutyrate for 90 min. To determine the amount of nonspecific binding, excess unlabeled phorbol dibutyrate (25 ,uM) was added to control incubations. After incubation the cells were harvested on glass fiber filters and washed. FDCP-Mix cells from control cultures specifically bound about 0.3 pmol of [3H]phorbol dibutyrate per 1 x 106 cells. Measurement of [Ca2"Ji. FDCP-Mix 1 cells (-1 x 107 per ml) were loaded with the fluorescent Ca2 + indicator quin 2 by incubation with the acetoxymethyl ester derivative of quin-2 (10 ,LM) (22) at 37°C in Fischer's medium for 20-30 min. Cells were then washed twice and resuspended in 145 mM NaCI/5 mM KCI/1 mM Na2HPO4/1 mM CaC12/0.5 mM MgCl2/10 mM glucose/10 mM Hepes, pH 7.4. Cells (1-2 x 106 per ml; 2 ml) were transferred to a spectrofluorimeter cuvette, and
Proc. Natl. Acad. Sci. USA 85
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fluorescence measurements were made with a Perkin-Elmer LS5 fluorescence spectrometer fitted with a thermostatted cuvette holder and magnetic stirring facility. The measurements were taken at 370C. Maximum fluorescence (Fmax) was determined by adding Triton X-100 (final concentration, 0.1%) or digitonin (final concentration, 50 AuM). The minimum fluorescence (low free-Ca2+ concentrations) was determined by adding EGTA (final concentration, 2 mM) and Tris-HCl (final concentration, 20 mM), giving a pH of 8.3. These values were used to determine [Ca2+]i (22). An alternative method of determining [Ca2+]i using the cation ionophore ionomycin and Mn2 + also was used as described (23). None of these additions to the cells produced significant changes in Fmax or autofluorescence. IL-3. The IL-3 used in these experiments was a highly purified preparation (a single band on silver-stained sodium dodecyl sulfate/polyacrylamide gel electrophoresis gels). IL-3 activity was determined as described by Bazill et al. (24) using the FDC-P2 cell line.
RESULTS The Effect of Phorbol Esters on FDCP-Mix 1 Cell Proliferation. FDCP-Mix 1 cells represent a continuously growing population of multipotent hemopoietic stem cells that require IL-3 for survival and proliferation (16): in its absence they die within 16-48 hr. No other growth factor tested can replace this requirement for IL-3, including granulocyte/macrophage colony-stimulating factor, macrophage colony-stimulating factor, and also those growth factors present in horse serum or fetal calf serum (16). Indeed, that IL-3 is the sole requirement for facilitating survival and proliferation is shown by the finding that in serum-free conditions, IL-3 was the only agent required to stimulate growth and DNA synthesis over a 2- to 3-day period. The phorbol esters phorbol 12-tetradecanoate 13-acetate (TPA) and phorbol dibutyrate could, at least in part, replace the requirement for IL-3 (Fig. 1 and ref. 13). Phorbol, an analogue of TPA that cannot activate protein kinase C, was unable to increase proliferation in the absence of IL-3. IL-3-Mediated Translocation of Protein Kinase C. Addition of IL-3 to FDCP-Mix 1 cells led to a rapid translocation of protein kinase C from a soluble to a particulate form (Table 1 and ref. 13). This translocation is also stimulated by the phorbol ester TPA (13). These data suggest that one of the primary effects of IL-3 on stem cells may be the translocation of protein kinase C, an effect normally considered to indicate enhanced cellular activity of the protein kinase. Down-Modulation of Phorbol Dibutyrate Binding Sites. To assess whether protein kinase C plays an essential role in IL-3-stimulated proliferation, we investigated the effect of reducing the number of phorbol dibutyrate binding sites present in FDCP-Mix 1 cells. Chronic treatment with phorbol esters has been shown to decrease the number of cellular phorbol dibutyrate binding sites (25) (the majority of which are believed to be protein kinase C) (10-12). This downmodulation of binding sites could also be observed in FDCPMix 1 cells treated with phorbol dibutyrate or TPA. There was a 50% reduction in the amount of specific [3H]phorbol dibutyrate binding within 6 hr of the addition of 100 ng of either phorbol dibutyrate or TPA per ml. After 10 min of incubation with these agents (followed by the washing procedure described in Methods), the amount of [3H]phorbol dibutyrate bound was reduced by
