Induction of Erythroid Differentiation by Altered Galpha 16 Activity as Detected by a Reporter Gene Assay in MB-02 Cells*

Sraboni Ghose, Hartmut Porzig, and Kurt BaltenspergerDagger

From the Institute of Pharmacology, University of Bern, Postfach 51, Friedbühlstrasse 49, CH-3010 Bern, Switzerland

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein alpha -subunit Galpha 16, which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of Galpha 16 inhibited cellular growth rates. A reporter gene system was established to assess the role of Galpha 16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the beta -globin promoter. Expression of Galpha 16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a Galpha 16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of Galpha 16, Galpha 16R186C, further stimulated differentiation to 195% of control values. Because the effect of Galpha 16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed Galpha 16 with G protein beta gamma -subunits can be excluded. The corresponding mutant of Galpha q (Galpha qR182C), the phylogenetically closest family member of Galpha 16, had no effect. The data define a specific role for Galpha 16-dependent signal transduction in cellular differentiation: deviations from optimal levels of Galpha 16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.

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INTRODUCTION
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Receptor-coupled heterotrimeric G proteins are known to mediate signals that modulate growth factor-dependent cellular proliferation (1). G protein-linked second messengers, such as Ca2+ or cAMP, as well as G protein beta gamma -subunits released upon G protein activation, may be involved in this modulation. Ultimately, these signaling elements are thought to act on mitogen-activated protein kinase or on c-Jun amino-terminal kinase modules, which serve as central regulators for cellular growth and differentiation (2, 3). In some endocrine tumors, mutations in clearly defined codons for conserved amino acids result in GTPase-deficient (i.e. GTP-bound, constitutively active) G protein alpha -subunits of Gs and Gi2, known as the products of the gsp and gip oncogenes (4, 5). In transfected cell lines, GTPase-deficient mutants of Galpha s and Galpha i2, as well as corresponding mutants of Galpha 12/13 and Galpha q, stimulate mitogenic responses. The mechanisms for mitogenic stimulation may involve elevated levels of cAMP (Galpha s) or activation of phospholipase Cbeta (Galpha q). However, in many cases, the signaling pathways involved are not clear.

Paradoxically, G protein-mediated signaling may also be associated with growth inhibition and cellular differentiation. Overexpression of GTPase-deficient Galpha q in rat pheochromocytoma cells induces neurite outgrowth (6). Constitutively active forms of Galpha 13 promote differentiation of P19 mouse embryonal carcinoma cells into an endodermal phenotype (7). Both appear to involve stimulation of the c-Jun amino-terminal kinase pathway. In mouse embryonic stem cells, expression of Galpha i2 or its GTPase-deficient mutant results in adipogenic differentiation (8). Furthermore, expression levels of members of all classes of G protein alpha -subunits are found to be regulated during cellular differentiation (9-14). These observations indicate a potentially widespread role of G protein alpha -subunits in differentiation programs. However, conclusive evidence that G protein regulation is the initiating event of the differentiation process is still lacking in most cases.

The molecular cloning of Galpha 16, a novel member of the Gq family of G proteins, and the analysis of its tissue distribution revealed that it is uniquely expressed in normal and in malignant hematopoietic cells. Its expression is confined to hematopoietic cell lines that were derived from early stages of differentiation and is absent or strongly down-regulated in differentiated normal cells or in leukemia cell lines after induction of differentiation (12, 13, 15-17). In peripheral blood T-lymphocytes, Galpha 16 expression is transiently up-regulated after lymphocyte activation, whereas the expression of Galpha i2 and of Galpha q remains unchanged (18). In order to determine whether Galpha 16 modulates T-cell activation, regulated expression of Galpha 16 was disrupted by stably overexpressing Galpha 16 or Galpha 16 antisense RNA in Jurkat T-cells, a human T lymphoma cell line. Activation of Galpha 16-deregulated Jurkat T-cells was inhibited as demonstrated by a reduced up-regulation of interleukin-2 and of the activation-specific surface antigen CD69 (18). These results suggest a critical role of tightly regulated Galpha 16 expression in lymphocyte activation.

Further information about potential roles of Galpha 16 accumulated mostly from experiments in nonhematopoietic cells by overexpression studies. In Swiss 3T3 fibroblasts, constitutive activation of Gq-dependent pathways by overexpression of GTPase-deficient mutants of Galpha q and Galpha 16 results in growth arrest or in reduced growth in response to platelet-derived growth factor or serum, respectively (19). In small cell lung carcinoma, overexpression of a GTPase-deficient mutant of Galpha 16 inhibits growth (20), and in rat pheochromocytoma cells, it induces differentiation (6). Surprisingly, Galpha 16 has also been reported to be involved in growth stimulatory events. Activation of the receptor for complement fragment C5a in human embryonic kidney cells leads to a pronounced activation of mitogen-activated protein kinase when coexpressed with Galpha 16 (21). Taken together, these findings imply that Galpha 16-dependent signaling may modulate cellular proliferation or differentiation, depending on the specific cellular environment.

Although G16 may interact with a broad spectrum of receptors in some overexpressing systems (22), selective coupling of receptors to G16, but not to Gq, was observed for the C-X-C chemokine interleukin-8, as well as for complement fragment C5a, and for the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (23-25). In the human erythroleukemia (HEL)1 cell line, Galpha 16 specifically couples to the P2Y2 (P2U) purinoceptor (26), suggesting that Galpha 16 might assume specific roles in individual cells or cell lines. However, its role in hematopoietic cells is still poorly defined in view of its lineage-independent but differentiation stage-dependent expression.

In the present study, we examined the role of Galpha 16 in growth and differentiation of erythroleukemic cells. A reporter gene assay was established, to detect entry into erythroid differentiation in transiently transfected cells. The results indicate that changes in the expression level and functional activity of Galpha 16 lead to the induction of differentiation in the factor-dependent erythroleukemia cell line MB-02. The data suggest a new role of Galpha 16-dependent signaling in the decision between cellular proliferation and differentiation.

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Reagents-- Cell culture media and medium supplements were purchased from Life Technologies, Inc. Healthy donors who had received no recent medications were the source of human serum, which was heat-inactivated at 56 °C for 30 min and stored at -20 °C for up to 6 months. Granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (Epo), and stem cell factor (SCF) were generous gifts from Werthenstein-Chemie (Schachen, Switzerland), Cilag AG (Schaffhausen, Switzerland), and Dr. E. K. Thomas (Immunex Corp., Seattle, WA), respectively. Unless otherwise mentioned, all chemicals (analytical grade), were from Sigma Chemicals (Buchs, Switzerland) or from Merck AG (Dietikon, Switzerland).

Cell Culture and Induction of Differentiation-- MB-02 cells (27) were maintained in basal medium (RPMI 1640 medium, 10% human serum, 2 mM Glutamax I (Life Technologies, Inc.), 1 mM sodium pyruvate, 50 units/ml penicillin, 50 µg/ml streptomycin), supplemented with 5 ng/ml GM-CSF and kept at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. Cells were passaged every third day and replated at a density of 4 × 105/ml. The protocol for the induction of differentiation was adapted from Broudy et al. (28). Briefly, cells were washed once with phosphate-buffered saline and plated at a density of 106 cells/ml in fresh basal medium supplemented with 25 ng/ml SCF and 4 units/ml Epo and left undisturbed for a week prior to further routine passaging for another 7-10 days. Hemoglobin-producing cells were detected by benzidine staining of cellular suspensions (29).

The HEL cell lines, 3D4 and 1E3, with suppressed Galpha 16 expression were generated by stable transfection of parental HEL cells derived from clone 92.1 (American Type Culture Collection, Manassas, VA) with an antisense plasmid to Galpha 16 and have been described previously (26). G418 sulfate-resistant cells that expressed wild-type levels of Galpha 16 (9G10 and 7H6) were used as controls. All cell lines were maintained in the presence of G418 sulfate (400 µg/ml) in RPMI 1640 medium, which was supplemented with 2% fetal bovine serum, 2 mM Glutamax I, 1 mM sodium pyruvate, 50 units/ml penicillin, and 50 µg/ml streptomycin. For growth studies, cultures were counted every 48 h using a Coulter Counter (Coulter Electronics, Ltd.)

COS-1 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM Glutamax I, 1 mM sodium pyruvate, 50 units/ml penicillin, 50 µg/ml streptomycin (complete Dulbecco's modified Eagle's medium).

Plasmid Construction-- The reporter plasmid piGFP was assembled as follows: the full-length sequence encoding a highly fluorescent version of green fluorescent protein (GFP) was excised from phGFP-S65T (CLONTECH) using the SacI and XbaI sites and inserted into the cloning vector pUC19 (New England Biolabs). After EcoRI and SalI double digestion, the fragment including GFP was inserted into the similarly digested expression vector pEV3 (generously provided by Dr. H. Weir Zeneca Pharmaceuticals, Cheshire; United Kingdom). As a positive control, a plasmid constitutively expressing GFP (pcGFP) was generated by cloning the cytomegalovirus (CMV) promoter into piGFP upstream of GFP. The full-length sequence encoding CMV was cut out from the expression vector pcDNA3 (Invitrogen, Inc.) by digestion with EcoRI and MunI and then ligated into the unique EcoRI site of piGFP. As a nonfluorescent control, pclacZ was constructed replacing GFP in pcGFP with lacZ from the expression vector pCMV-lacZ (a gift from Dr. S. Rusconi, University of Fribourg, Switzerland).

For cotransfection experiments, the different G protein genes were cloned into the expression vector pcDNA3. The plasmids pG16AS and pG16WT, harboring a full-length copy of the human Galpha 16 cDNA in antisense or sense direction, respectively, were described previously (26). In order to generate pG16RC, the GTPase-deficient, constitutively active mutant of Galpha 16, Galpha 16R186C, was recovered from pVL1393Galpha 16R186C (a gift from Drs. A. Dietrich and P. Gierschik, University of Ulm, Germany) and cloned into the unique XbaI site of pcDNA3. A plasmid expressing the corresponding GTPase-deficient mutant of Galpha q, pCISGalpha qR182C, was a gift of Dr. M. I. Simon (California Institute of Technology, Pasadena, CA). A plasmid encoding the GTPase-deficient mutant of Galpha i2, Galpha i2Q205L, was constructed by excising the coding region from pCW1Galpha i2Q205L (30) and inserting it into pcDNA3 at the HindIII site. pGi3AS was assembled using the BamHI fragment of pCISGalpha i3 (also from Dr. M. I. Simon), which was inserted in antisense orientation into pcDNA3.

Transient Transfection-- MB-02 cells were washed with phosphate-buffered saline and resuspended at a density of 2 × 106/ml in phosphate-buffered sucrose (sucrose, 272 mM; MgCl2, 1 mM; NaH2PO4, 7 mM; glucose, 20 mM; KCl, 1 mM; pH 7.4). In transfection experiments, 5 µg of piGFP in combination with 5 µg of one of the G protein-containing plasmids were used, or 5 µg of pcGFP and 5 µg of pclacZ (positive control), or 10 µg pclacZ (nonfluorescent control). 106 cells and the DNA were mixed in the electroporator cuvette (gap width, 4 mm) and equilibrated on ice for 5 min. Cells were then electroporated using a Gene Pulser unit (Bio-Rad) that was set to 350 V, 100 µF (exponential decay), and immediately plated into 1 ml of prewarmed (37 °C) complete RPMI medium with human serum and GM-CSF or Epo plus SCF (for noninduced or induced cells, respectively). For the transfection of COS-1 cells, 105 cells in complete Dulbecco's modified Eagle's medium were seeded into 35-mm dishes. After 24 h, the culture medium was replaced with Opti-MEM (Life Technologies, Inc.), and cells were transfected with a total of 1 µg of plasmid DNA per dish using the N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate (Boehringer Mannheim) reagent according to the manufacturer's instructions. Cells were harvested after 48 h, and the membrane fraction was analyzed by protein immunoblotting as described previously (26).

Flow Cytometry-- MB-02 cells were washed three times and resuspended in phosphate-buffered saline (1 ml) supplemented with 22 mM glucose. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Laser excitation was at 488 nm (argon laser) for GFP, and emission was measured at 515-545 nm. The single color analysis was gated on forward scatter and side scatter. This gate contained all viable cells and excluded cell debris and cellular aggregates. A threshold for intensity of fluorescence was set high enough to exclude autofluorescence and was determined by running samples of untransfected and lacZ-transfected cells prior to the analysis. A total of 10,000 events was counted and analyzed for each sample using the CellQuest software (Becton Dickinson). To correct for variability in transfection efficiencies from experiment to experiment, inducible GFP expression was normalized in each experiment to the average of fluorescent cells observed upon expression of GFP under the constitutively active CMV promoter, i.e. after transfection with pcGFP (three to four independent transfections per experiment).

Data Analysis-- Statistical analyses were performed using the software packages StatView, version 4.02 for Macintosh (Abacus Concepts, Inc., Berkeley, CA) or GraphPad Prism, version 2.0 (GraphPad Software, Inc., San Diego, CA).

    RESULTS AND DISCUSSION
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Down-regulation of Galpha 16 in HEL Cells Impairs Cell Growth-- We recently established several sublines of HEL cells that showed reduced expression of endogenous Galpha 16 protein after transfection with a plasmid harboring a full-length copy of Galpha 16 in antisense orientation (26). In these sublines, mobilization of intracellular Ca2+ through activation of the P2Y2 (P2U) purinoceptor by UTP is impaired, whereas Ca2+-mobilization via other receptors is not or is only partially affected, demonstrating a specific functional defect (26). Compared to controls with normal levels of Galpha 16, cellular growth was impaired in the Galpha 16-deficient sublines (Fig. 1). Population doubling times were significantly higher in the Galpha 16-suppressed cell lines 3D4 and 1E3 (41.4 ± 1.4 and 41.2 ± 1.6 h, respectively) than in the cell lines 9G10 and 7H6 expressing normal levels of Galpha 16 (34.8 ± 1.0, 33.2 ± 0.6 h, respectively) (mean ± S.E.). The results suggested that Galpha 16-mediated cellular signaling may be involved in the regulation of cellular proliferation. However, from these experiments, it could not be established whether Galpha 16-mediated inhibition of proliferation was associated with erythroid differentiation in hematopoietic cells, because the factor-independent HEL cells only partially differentiate in response to various inducers (31, 32). To address this question, a factor-dependent cell line, MB-02, was chosen that can be readily differentiated along the erythroid pathway (27, 28).


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Fig. 1.   Expression of antisense RNA to Galpha 16 increases population doubling times of HEL cells. Stably transfected HEL cells with reduced levels of Galpha 16 expression (Galpha 16-suppressed cell lines 3D4 and 1E3) or controls with unaltered Galpha 16 expression (Galpha 16-normal cell lines 7D6 and 9G10) were seeded at a density of 2 × 105 cells/ml. Forty-eight hours later, total numbers of viable cells were counted, and population doubling times were calculated. The box plot shows the summary of multiple measurements. The 10th, 25th, 50th (median), 75th, and 90th percentiles of the variables are indicated by vertical bars. One-way analysis of variance indicated significant differences between the means (po < 0.0001). Post hoc analysis by the method of Bonferroni-Dunn showed that differences between controls and Galpha 16-suppressed cell lines are highly significant (po <=  0.0014).

Differentiation of MB-02 Cells-- MB-02 cells maintained in the presence of GM-CSF differentiate along the erythroid pathway upon withdrawal of GM-CSF and subsequent treatment with SCF and Epo (28). Under our experimental conditions, the properties of the cell line corresponded to the ones described in the original literature: treatment for 10 days or longer with SCF/Epo resulted in a substantial proportion of cells that expressed hemoglobin, which was detected by benzidine staining (Fig. 2A). An average of 45 ± 5% of cells expressed hemoglobin upon induction, whereas noninduced cells expressed detectable levels only in 6 ± 2% of the population (Fig. 2B). MB-02 cells showed rapid growth in the presence of GM-CSF, whereas induction of differentiation with SCF/Epo led to a reduction in the population growth rate (Fig. 2C). Withdrawal of GM-CSF resulted in massive cell death within 24 h, confirming their factor dependence (not shown), whereas withdrawal of SCF during the induction process, i.e. continuation of induction in the presence of Epo alone, resulted in a population that was almost stationary in its number of viable cells (Fig. 2C). Under the latter condition, a higher proportion of cells expressed hemoglobin, but the overall viability of the population decreased sharply (not shown). Therefore, in our experiments, induction of cells was always performed in the presence of Epo and SCF.


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Fig. 2.   Hemoglobin expression and growth characteristics of MB-02 cells upon induction of differentiation by SCF/Epo. A, benzidine staining (dark gray) of hemoglobin in noninduced (maintained in GM-CSF) and induced (SCF/Epo for 14-16 days) MB-02 cells. B, cells treated as in A were counted under the microscope, and the percentage of benzidine positive cells from noninduced and induced cultures was calculated and plotted (mean ± S.E., n = 6). C, MB-02 cells were cultured in the presence of GM-CSF, SCF/Epo, or Epo alone during successive days and counted. The cumulative fold increase in cell numbers is plotted.

Assembly and Functional Evaluation of the Reporter System-- In order to study the effects of G protein overexpression in MB-02 cells, we used a transient transfection system to acutely manipulate G protein levels. As the transfection efficiency is low in hematopoietic cells, potential effects of such treatments could not be studied by biochemical or immunological methods. Thus, a reporter gene assay was established based on a plasmid that was co-transfected with the gene of interest. As a reporter for erythroid differentiation, the nucleotide sequence encoding GFP was placed under the control of the beta -globin promoter in the original plasmid pEV3 (33). In this reporter plasmid (piGFP), the beta -globin promoter is located downstream of locus control region sequences derived from the human beta -globin gene cluster (Fig. 3A). In constructs bearing the locus control region-beta -globin promoter arrangement, strong inducible expression from the beta -globin promoter has been shown upon induction of differentiation in erythroid cells (33). In addition, a plasmid (pcGFP) with constitutive expression of GFP under the control of the CMV promoter was constructed by splicing in CMV promoter sequences upstream of the GFP sequence (Fig. 3B).


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Fig. 3.   Schematic representations of expression plasmids piGFP and pcGFP. A, reporter plasmid piGFP, which expresses GFP upon activation of the beta -globin promoter. B, control plasmid pcGFP, which constitutively expresses GFP under the control of the CMV promoter. The construction of the plasmids is described in detail under "Experimental Procedures."

In order to test whether the reporter plasmid was indeed capable of detecting erythroid differentiation, cells treated with GM-CSF (noninducing conditions) or with SCF/Epo (inducing conditions) were transiently transfected with piGFP or with the control plasmid pcGFP. Expression of GFP was then detected by flow cytometry. As shown in Fig. 4A, the relative number of GFP-expressing cells was 2.7-fold higher in SCF/Epo-treated cells than in GM-CSF-treated controls when normalized to GFP expression under the constitutively active CMV promoter. The relative number of differentiating cells as measured by the reporter assay reached a maximum after 11-14 days of induction (Fig. 4B), consistent with previously published data on SCF/Epo-induced expression of globin proteins in this cell line (28). In order to quantify the extent of beta -globin gene induction in differentiating cells, their fluorescence intensities resulting from the expression of the reporter gene construct were compared with that of noninduced cells. The histogram in Fig. 4C shows an analysis in bins of 0.5 log units over the 4 log units of fluorescence intensities recorded by the FACScan. The first log unit of fluorescence (Fig. 4C, bins 1 and 2) represents autofluorescence as demonstrated by cells that were transfected with lacZ instead of the reporter gene construct. Overall, SCF/Epo-treated cells that were transfected with the reporter plasmid showed a 2.9-fold increase in their number of fluorescent cells contained in bins 3-8 when compared with noninduced cells (averages of 95.2 and 32.5 cells, respectively). The corresponding total fluorescence of induced cells was 4.3-fold higher than in noninduced cells (Fig. 4C). Thus, SCF/Epo treatment resulted in a substantial increase in the number of fluorescent cells in the gated window (encompassing bins 3-8) but only in a slight increase of average fluorescence per cell (+48%), which is reflected by higher ratios of fluorescent cells appearing in bins 5-8 than in bins 3 and 4 (3.7, 4.5, 6.5, and 3.3 versus 2.7 and 2.1, respectively). Apparently, cells that undergo spontaneous differentiation under noninducing conditions (see also Fig. 2) show beta -globin promoter activities almost equal to those of cells that were treated with SCF/Epo. Consequently, the number of fluorescent cells rather than gene expression levels on a per cell basis in the gated window was taken as a measure for erythroid induction.


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Fig. 4.   Detection of erythroid differentiation by the GFP-reporter assay in induced MB-02 cells. A, induced and noninduced MB-02 cells were transfected with either pcGFP or piGFP and analyzed by flow cytometry after 24 h. Numbers of fluorescent cells observed in piGFP transfectants were normalized to the numbers of fluorescent cells in pcGFP transfectants to account for different transfection efficiencies of noninduced and induced (14 days) cells (4.54 and 1.64% of total numbers of viable cells, respectively). Induced cells (n = 12) showed a 2.7-fold increase in normalized numbers of fluorescent cells as compared with noninduced cells (n = 11). The difference between noninduced and induced cells is highly significant (po < 0.0002, unpaired t test). B, time course of the appearance of differentiated MB-02 cells during SCF/Epo treatment. Cells were transfected with 5 µg of either pcGFP or piGFP and analyzed by flow cytometry 24 h later at the different time points indicated in the figure. Cells that were transfected with pclacZ (5 µg) were used as a control to size the acquisition window. Inducible GFP fluorescence from the reporter plasmid was normalized to constitutive GFP fluorescence (resulting from the expression of pcGFP), and the results were plotted. The means (± S.E.) of four different transfections are shown. C, frequency histogram of fluorescence intensities of control (pclacZ)- and piGFP-transfected MB-02 cells (data from B). Fluorescence intensities (logarithmic scale) were collected in 1024 channels. For the frequency histograms, the entire range of fluorescence was divided into eight equal gates. Thus, data from 128 channels each (representing 0.5 log units of fluorescence) were binned and plotted (bins 1-8, bin 1 representing the lowest and 8 the highest fluorescence intensities). The mean of four transfections for noninduced (day 0) and six transfections for induced (day 14) cells are shown (±S.E.). A total of 10,000 cells were counted, and the fluorescence intensities were analyzed. In this experiment, transfection efficiencies in noninduced and induced cells matched closely (1.15 and 1.09%, respectively), allowing for direct comparison of numbers of fluorescent cells (for details, see under "Experimental Procedures").

The results shown in Fig. 4A indicate a ratio of differentiated cells of 46 ± 6% in SCF/Epo-treated cells. The reporter gene assay closely reflects the relative number of differentiated MB-02 cells, as detected by benzidine staining of cells after treatment with SCF/Epo (Fig. 2B), which resulted in induction rates of 45 ± 5% of the population. However, it appears that the reporter assay is more sensitive than benzidine staining at lower levels of beta -globin promoter activity: in noninduced cultures, 6 ± 2% of the cells were benzidine-positive, whereas 17 ± 1% of the cells showed increased fluorescence in the reporter assay (compare Figs. 2B and 4A). In COS-1 cells, transient transfection of pcGFP resulted in strong fluorescence, whereas no fluorescence was observed in cells transfected with piGFP (not shown). These results rule out the possibility that low levels of the reporter gene might have been expressed in the absence of activators of the beta -globin promoter. Thus, the reporter system provides a valid and sensitive assay for monitoring erythroid differentiation in MB-02 cells.

Induction of Differentiation in MB-02 Cells upon Changes in Galpha 16 Expression-- Using the reporter assay, we then examined the effect of G protein expression on beta -globin promoter activation in nondifferentiated cells. Cells were co-transfected with the inducible reporter plasmid together with a plasmid encoding either wild-type Galpha 16 or the GTPase-deficient mutant of Galpha 16 (Galpha 16R186C). Cultures transfected with wild-type Galpha 16 showed a significant increase of GFP-expressing cells to 154% of levels observed in control cells transfected with pclacZ (Fig. 5A). Expression of the GTPase-deficient mutant of Galpha 16 led to an even higher proportion (195% of control) of transfected cells that showed beta -globin promoter activity, which was also significantly higher than for the wild-type alpha -subunit. In order to test whether this effect was specific for Galpha 16 or resulted from the mere overexpression of G protein alpha -subunits, a GTPase-deficient mutant of Galpha q, Galpha qR182C, was also expressed. Galpha q represents the phylogenetically closest relative of Galpha 16, which also belongs to subfamily I of Galpha q proteins (34). In contrast to Galpha 16R186C, no induction was observed when Galpha qR182C was expressed (Fig. 5B). Expression of a GTPase-deficient member of the more distant Galpha i family, Galpha i2Q205L, resulted in a slight increase to 127 ± 19% (mean ± S.E.) of control values, which, however, was not significantly different from the control (not shown). Although from the same subfamily of G proteins, Galpha q apparently is not able to functionally substitute for Galpha 16 in inducing differentiation of MB-02 cells. We previously observed that in HEL cells the P2U purinoceptor specifically couples to Galpha 16, leading to similarly exclusive Galpha 16-dependent signaling (26). These experiments demonstrate that the specificity of Galpha 16-dependent signal transduction is not limited to specificity in the receptor-G protein coupling but may also result from the interaction of Galpha 16 with downstream effector systems. The inducing effect of Galpha 16 appears to depend on functional activity of Galpha 16, because the GTPase-deficient mutant showed a significantly stronger induction than the wild-type form. The inability of Galpha i2 and of Galpha q to effectively induce beta -globin promoter activity indicates that differentiation strictly depends on Galpha 16 rather than being a general effect of G protein alpha -subunit overexpression.


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Fig. 5.   Alterations of Galpha 16 activity trigger entry into erythroid differentiation in MB-02 cells. Noninduced MB-02 cells that were maintained in GM-CSF were transiently transfected (electroporation) with piGFP and various G protein constructs or lacZ as indicated. After 72 h, cells were analyzed by flow cytometry for induced fluorescence. Inducible GFP expression as detected in piGFP transfected cells was then normalized to the total number of transfected cells as measured in parallel for each experiment by transfection with pcGFP. The percentages of fluorescent cells 72 h after transfection of cultures with pcGFP were in the range of 0.41-1.95% of gated, viable cells. A, the ratio of cells showing inducible fluorescence as a percentage of transfected cells is shown; the figure represents 16 (lacZ, control), 21 (G16RC), 16 (G16AS), and 15 (G16WT) independent transfections and flow cytometric analyses, which were performed in 5-8 different experiments. One-way analysis of variance of the experimental data indicated that the observed means are not equal (po < 0.0015). Post hoc analysis by the method of Dunnett (comparing the control value against all other values) indicated significant differences of the means against the control at the level of po < 0.01 (*) or po < 0.05 (#). ¥ indicates significant differences between G16RC and G16WT and between G16RC and G16AS when tested by paired t test for each individual experiment (po < 0.05). B, MB-02 cells were transfected with G16RC or a GTPase-deficient, constitutively active form of Galpha q, Galpha qR182C (GqRC). Each column represents the mean ratio (± S.E.) of fluorescent cells observed from eight transfections. One way analysis of variances indicated that the observed means were not equal (p < 0.0001), and post hoc analysis by Dunnett's method indicated a statistically significant difference between the control and G16RC (p < 0.01 (§)), but no difference between GqRC and control (p > 0.05 (¶)).

As HEL cells with suppressed Galpha 16 showed reduced growth rates (Fig. 1) but were not well suited to examine Galpha 16-dependent effects on cellular differentiation, MB-02 cells were also transfected with a Galpha 16 antisense-plasmid to down-regulate endogenous Galpha 16. Interestingly, transcription of antisense-RNA also significantly increased the number of GFP-positive cells to 156% of control values (Fig. 5A). Since differentiation is associated with reduced rates of proliferation, this inducing effect of Galpha 16-down-regulation in MB-02 cells is consistent with its effect on proliferation that was detected in HEL cells (Fig. 1). However, the observation that either reducing or up-regulating the levels of Galpha 16 protein expression triggers differentiation may not easily be reconciled. Although this phenomenon has also been observed for acquiring functional competence of Jurkat T cells (18), the underlying mechanism cannot be deduced from the available data and warrants further investigation. A cell-specific, optimal level of Galpha 16 activity seems to be required for proliferation, and any deviation from it will decrease growth rates and result in increased ratios of differentiating cells.

Expression of Antisense RNA Inhibits Galpha 16 Expression in Transiently Transfected COS-1 Cells-- Due to low transfection efficiencies, expression of Galpha 16 protein or antisense-dependent down-regulation of endogenous Galpha 16 could not be verified directly in MB-02 cells. Therefore, expression of Galpha 16 protein and its down-regulation through antisense RNA was verified by protein immunoblotting of solubilized membranes of transiently transfected COS-1 cells. Transfection with Galpha 16R186C or with wild-type Galpha 16 resulted in a single band with a relative mobility identical to the single immunoreactive band observed in membranes of MB-02 cells (Fig. 6). As expected, in COS-1 cells that were transfected with the control plasmid pclacZ, no immunoreactivity was detected, as these cells do not express endogenous Galpha 16. Transfection of smaller amounts of the Galpha 16 plasmid resulted in lower expression levels, indicating that under these conditions, plasmid-dependent protein expression was in a dynamic range, thus allowing for the detection of copy number-dependent alterations of expression. As shown in Fig. 6, co-transfection of Galpha 16 with the Galpha 16 antisense construct resulted in a marked down-regulation of Galpha 16 expression when compared with controls that were transfected with Galpha 16 alone. A plasmid leading to a Galpha i3 antisense RNA transcript was not capable of reducing the expression of Galpha 16, indicating specific interaction of the Galpha 16-transcript with Galpha 16 antisense sequences (Fig. 6, compare lanes G16WT+lacZ and G16WT+Gi3AS).


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Fig. 6.   Galpha 16 antisense expression down-regulates Galpha 16 expression in COS-1 cells. COS-1 cells were transfected with plasmids encoding Galpha 16R186C (G16RC), lacZ (lacZ), or wild-type Galpha 16 (G16WT) or with Galpha 16 (G16AS) or Galpha i3 (Gi3AS) antisense plasmids as indicated in the figure. A total of 1 µg of DNA was used in each transfection. Co-transfections were done with pG16WT (0.15 µg) plus either pclacZ (0.85 µg), pG16AS (0.85 µg), or pGi3AS (0.85 µg). Forty-eight hours after transfection, membrane fractions were prepared, and the proteins (15 µg of total protein per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted for Galpha 16 using the antiserum AS 339 (35) as described earlier (26). The membrane fraction (15 µg of protein) of MB-02 cells was also analyzed, showing expression of endogenous Galpha 16 in this cell line. The figure shows the immunoblot, which was developed using the enhanced chemiluminescence protocol (Amersham Pharmacia Biotech).

The results suggest a causative role for a Galpha 16-dependent signaling pathway in the induction of differentiation in MB-02 cells. The identification of constitutively active, GTP-bound Galpha 16 as the active molecular entity suggests that in a natural environment, activation of Galpha 16 in response to receptor activation may indeed induce differentiation. This conclusion is supported by the results from the experimental up- or down-regulation of wild-type Galpha 16, which is expected to translate into changes in basal levels of GTP-bound Galpha 16, and consequently into changes of Galpha 16-dependent signaling activity. It is not known which receptors may engage Galpha 16 in MB-02 cells. Therefore, an assessment of the role of agonists that activate Galpha 16-coupled receptors is not yet possible. The observation that the GTPase-deficient Galpha 16 shows a higher efficacy than the wild-type form also excludes the possibility that Galpha 16 may act solely as a receptor-independent regulator of G protein signaling, e.g. by capturing beta gamma -subunits that might have been liberated by activation of other G proteins.

Transfection of MB-02 cells with the Galpha 16R186C mutant resulted in a 1.9-fold increase of induced cells as compared with the number observed in control-transfected cells (Fig. 5A). This increase is substantial in view of the 2.7-fold increase observed when cells were induced to differentiate by SCF/Epo (Fig. 4A). Importantly, cells induced with SCF/Epo received the differentiating stimulus for 14 days, whereas transfected cells had to be scored after 3 days due to the transient nature of transfection. It was not possible to subject cells to transfection after 3 days of differentiation by SCF/Epo due to the fragile nature of the cultures at this time point. However, as demonstrated in Fig. 4B, SCF/Epo treatment required several days to fully differentiate MB-02 cells. Thus, Galpha 16R186C---and deregulation of Galpha 16 activity in general---may be considered as potent inducers of differentiation.

In this context, it should be noted that our attempts to generate stable HEL cell lines transfected with Galpha 16R186C were not successful. It appeared that expression of constitutively active Galpha 16 inhibits proliferation in these cells to an extent that precluded the isolation of cell clones. Expression of the functionally similar GTPase-deficient mutant Galpha 16Q212L in nonhematopoietic cells apparently led to growth retardation, but stable cell lines could still be established (19). Galpha 16 may thus act as a much stronger (negative) regulator of proliferation in hematopoietic cells than in other cell lines. Whether this could be caused by a unique coupling of Galpha 16-dependent signaling to downstream effector systems or by stronger coupling to effector systems that are used by other members of the Galpha q-family is not known.

In conclusion, in hematopoietic cells an increase of Galpha 16 function or its down-regulation have profound effects on proliferation and may cause erythroid differentiation in specific cell lines. It remains to be established whether differentiation along the erythroid pathway is specifically determined by Galpha 16 or whether Galpha 16 preconditions hematopoietic cells for differentiation independently of lineage determination. Deviations from optimal levels of Galpha 16 activity seem to be associated with acquiring functional competence by inducing either differentiation or activation of cellular proliferation, as seen in T-lymphocytes (18). One might speculate that agonists employing Galpha 16-coupled receptors directly regulate the proportion of functionally competent cells. With the reporter assay based on transient transfection of cells and subsequent analysis by flow cytometry, it should now be possible to identify the Galpha 16-dependent signaling pathways involved in these processes. Furthermore, these studies could be extended to primary hematopoietic progenitor cells, in which G protein expression cannot be easily manipulated by other techniques.

    ACKNOWLEDGEMENTS

We are indebted to Dr. D. Morgan for generously providing us with the cell line MB-02. We thank Drs. P. Gierschik and A. Dietrich (University of Ulm, Germany) for providing us with the plasmids encoding Galpha 16R186C; Dr. M. I. Simon (California Institute of Technology, Pasadena, CA) for plasmids encoding Galpha 16, Galpha qR182C, and Galpha i3; Dr. S. Rusconi (University of Fribourg, Switzerland) for a plasmid encoding lacZ; and Drs. C. C. Malbon and C. Moxham (State University of New York, Stony Brook, NY) for providing us with a plasmid encoding Galpha i2Q205L. We also thank Drs. Claudio Vallan and Steven Merlin for expert technical support using the FACS facilities at the Department of Clinical Research, University of Bern, and Drs. K. Spicher and G. Schultz (Free University of Berlin, Germany) for antiserum (AS 339) raised against Galpha 16.

    FOOTNOTES

* Funded by Grant KFS 183-9-1995 from Cancer Research Switzerland and Grant 31-39678.93 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 41-31-632-3290; Fax: 41-31-632-4992; E-mail: kurt.baltensperger{at}pki.unibe.ch.

    ABBREVIATIONS

The abbreviations used are: HEL, human erythroleukemia; GM-CSF, granulocyte-macrophage colony-stimulating factor; Epo, erythropoietin; SCF, stem cell factor; GFP, green fluorescent protein; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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