Identification of three genes up-regulated in PU.1 rescued monocytic precursor cells
Gregory W. Henkel1,
Scott R. McKercher1 and
Richard A. Maki1
1 The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
Correspondence to: R. Maki; E-mail: maki{at}neurocrine.com
Transmitting editor: C. Paige
 |
Abstract
|
---|
The requirement of the transcription factor PU.1 for macrophage development has been well documented. However, the target genes regulated by PU.1 controlling macrophage maturation are not known. A granulocyte macrophage colony stimulating factor (GM-CSF)-dependent PU.1 null monocytic precursor cell was stably transduced with a PU.1-expressing retrovirus. The expression of PU.1 altered the surface expression of a few proteins expressed on monocytes; these cells, however, remained GM-CSF dependent and maintained an immature phenotype. In contrast to the PU.1 null cells, the cells expressing PU.1 responded to macrophage colony stimulating factor (M-CSF) with subsequent development into mature macrophages. Using suppressive subtractive hybridization between the PU.1 null and immature PU.1 rescued cells, three genes, MRP-14, Dap12 and CD53, were found expressed in the rescued cells, but not in the PU.1 null cells. In addition, these genes were modulated during M-CSF-induced maturation of the PU.1 rescued cells. The PU.1 null and rescued early monocytic cells provide a useful model to study the role of PU.1 in macrophage development.
Keywords: gene regulation, macrophages, transcription factor
 |
Introduction
|
---|
Transcription factors are integral for programming both cellular and tissue development. Hematopoiesis both in vitro and in vivo has been a useful model for studying the role of transcription factors in development (14). Knockout studies in mice have identified critical transcription factors, including Gata-1 (5), Gata-2 (6), myb (7), tal/Scl (8,9), AML1 (10), PU.1 (11,12), c-EBP factors (13,14), Ikaros (15), Pax5 (16) and E2A (17,18), necessary for both erythrocyte and leukocyte differentiation. However, little is known about the genetic targets regulated by these transcription factors. Recently, a partial explanation was provided for the incomplete granulopoiesis observed in C/EBP
null mice. Both the granulocyte-colony stimulating factor (G-CSF) and IL-6 receptors are dramatically reduced in this knockout animal (19,20). The low level of G-CSF receptor is consistent with a previous study demonstrating that C/EBP
regulates the G-CSF receptor promoter (21). Either the addition of soluble IL-6 receptor plus ligand or retrovirally transducing the G-CSF receptor into C/EBP
null progenitors restores granulocytic maturation (20). Therefore, one mechanism by which transcription factors can control hematopoietic lineage development is through regulating the expression of specific cytokine receptors.
In PU.1 null mice, there is commitment to the myeloid and B cell lineages, but no mature monocytes/macrophages or B cells are detectable (12,2225). Therefore, PU.1 functions in a lineage-restricted manner. It has been reported that adding back PU.1 to early PU.1 null progenitor cells from fetal liver restores both granulocyte and macrophage development (24,26,27). As in the case of the C/EBP
null mouse, the lack of complete myeloid development in the absence of PU.1 could be attributed to the absence or low expression of growth factor receptors, such as G-CSF, granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) receptors, reported to be regulated by PU.1 (21,2830). Although PU.1 null progenitors can commit to the neutrophil or monocytic lineage in the presence of G-CSF or GM-CSF respectively, total maturation of these two cell types does not occur (24,25). The restoration of the M-CSF receptor into PU.1 null progenitors through retroviral transduction allows the progenitors to proliferate in the presence of M-CSF, but not differentiate (24,27). Therefore, additional genes independent of these growth factor receptors are required for neutrophil and macrophage maturation.
Recently, we reported the generation of GM-CSF-dependent cell lines from PU.1 null fetal liver cultures (25). These cells demonstrate characteristics of early monocytes at approximately the monoblast stage of development. We have reintroduced PU.1 cDNA into the GM-CSF-dependent cells. Cells expressing PU.1 remain immature, but undergo maturation to macrophages in the presence of M-CSF. As a first step in understanding the genetic parameters required for macrophage maturation, suppressive subtractive hybridization between null and rescued cells was used to identify PU.1-regulated genes at this early stage of development. Three differentially expressed genes were found from the initial phase of this analysis.
 |
Methods
|
---|
Cell culture
The generation of GMpu4 (GM4) cells and culturing conditions was previously described (25); 5000 U/ml of rM-CSF (gift from Dr David Hume) and 0.1 ng/ml GM-CSF were used for macrophage maturation of PU.1 rescued clones. Complete maturation was seen after 57 days.
Retrovirus replacement of PU.1
The PU.1 cDNA was subcloned into the TET-ON retrovirus vector (Clontech, Palo Alto, CA). This vector carries both the modified chimeric VP16-Tet regulator gene and the tet response elements linked to a minimal CMV promoter. The retrovirus vector was initially cut with NotI and then filled in with Klenow DNA polymerase. Afterwards, the vector was cut with BamHI. The PU.1 cDNA with EcoRVBamHI ends was directionally cloned into the TET-ON retrovirus vector. The final construct was transfected into Phoenix-E packaging cell line (ATCC, Rockville, MD) using calcium phosphate. The cells were resuspended in 1 ml of viral supernatant and centrifuged at 2500 r.p.m. for 40 min to infect the GM4 cells. Cells were incubated at 37°C overnight and the next day they were fed fresh growth media. After another 24 h stably infected cells were selected in the presence of 1 µg/ml puromycin (Sigma, St Louis, MO). Cells that grew in the presence of puromycin were further characterized. Clones were generated by single-cell suspension. The intention of the original experiment was to control PU.1 expression using this tetracycline-inducible system. However, it was found during the course of the experiment that this particular Tet-inducible construct does not induce expression >2- to 3-fold above the uninduced expression level. There was, however, sufficient expression of PU.1 without induction to derive stable PU.1-expressing clones. The addition of doxycycline (a derivative of tetracycline) to the cells had no effect on PU.1 expression or maturation of the cells.
Cell isolation
GM4 cells stably transduced with the PU.1 retrovirus vector (GM4puR) were incubated with anti-CD11BFITC (PharMingen, San Diego, CA) followed by anti-FITCmicrobeads (Miltenyi Biotec, Auburn, CA). The CD11b+ cells were captured and isolated using a steel wool column and magnet (Miltenyi Biotec). The enrichment of CD11b+ cells was verified by flow cytometry.
Flow cytometry
The method and antibodies used for flow cytometry have been previously described (25). Additional antibodies used in this study included MHC class II I-Ab, CD80, CD86 (PharMingen) and scavenger receptor (Harlan Bioproducts, Indianapolis, IN).
Reverse transcription
Total RNA was isolated using Trizol (Life Technologies, Gaithersburg, MD). RNA (2 µg) was reverse transcribed and equivalent amounts of single-stranded cDNA from each cell line were used for PCR. Mouse PU.1 forward primer: 5'-GATGGAGAAGCTGATGGCTTGG-3'. Mouse PU.1 reverse primer: 5'-TTCTTCACCTCGCCTGTCTTGC-3'. Mouse ATP synthase F subunit forward primer: 5'-TCGTGCCGCTGAAG GAGAAGA-3'. Mouse ATP synthase F subunit reverse primer: 5'-GGAGTGGGGCGGGCAGTTAT-3'. The ATP synthase primers were a kind gift from Dr Melody Clark. The primers for c-fms have been previously reported (29). The cycling parameters for PU.1 and c-fms were 94°C 1 min 1 cycle 94°C 30 s, 62°C 30 s and 72°C 1 min for 32 and 36 cycles respectively followed by a 72°C extension for 5 min. The cycling parameters for ATP Synthase were 94°C 1 min 1 cycle 94°C 30 s, 58°C 30 s and 72°C 1 min for 32 cycles followed by a 72°C extension for 5 min.
Mobility shift
Nuclear extracts were prepared using a modified version of a technique described by Dignam et al. (32). Cells sit on ice in buffer A for 15 min and then are passed through a 25 gage needle 5 times. The free nuclei are pelleted and resuspended in buffer C and rocked for 30 min at 4°C. The debris was pelleted, and the supernatant was recovered and used in mobility shift assays. Equivalent amounts of nuclear extract were mixed with a 32P-labeled oligonucleotide containing a PU.1 consensus binding site (5'-AAGGGGAAGTGG-3') in a total reaction volume of 25 µl that consists of 20 mM HEPES/KOH, pH 7.9, 1 mM EDTA, 0.1% Triton X-100, 100 mM KCl, 100 µg/ml BSA, 700 ng poly(dIdC), 1 mM PMSF and 7% glycerol final concentration. The binding reaction was allowed to incubate for 30 min at room temperature. In the reactions containing antibody, the antibody was added 15 min prior to the addition of the labeled oligonucleotide. The N-PU.1 (1297) polyclonal antiserum was previously reported (33). The polyclonal antisera to whole PU.1 was made against a GST PU.1 fusion protein in our laboratory. The Ets-2 antiserum was a gift from Dr Craig Hauser. The reactions were run on a Trisglycine (25 mM Tris, 190 mM glycine and 1 mM EDTA, final concentration) 5% polyacrylamide gel.
Subtraction
mRNA was isolated from GM4 (driver) and PU.1 rescue clone GMpuR-3B6 (tester) using Invitrogen FastTrack kit 2.0 (Invitrogen, Carlsbad, CA). The suppressive hybridization method (34) was employed using the Clontech PCR-Select cDNA subtraction kit (Clontech, Palo Alto CA). The final PCR-derived subtracted material from the tester cDNA was blunted with T4 polymerase and phosphorylated with T4 kinase. The PCR products were then run on an agarose gel and various size fractions were collected for blunt-end cloning into Bluescript KS+ vector (Stratagene, La Jolla, CA). The vector was cut with EcoRV and treated with alkaline phosphatase. The ligation reaction was transformed into TOP10 One Shot Competent Cells (Invitrogen) and grown on plates with X-gal/IPTG for blue/white selection. Plasmids from white colonies were isolated and screened by restriction digest with PvuII. Plasmids with inserts were sequenced with the ABI Prism DNA sequencer (Perkin Elmer, Foster City, CA).
Northern
mRNA (13 µg) was run on a denaturing gel and transferred to a nylon membrane. Clonal fragments isolated from the subtraction were labeled using Random Primer II kit (Stratagene, La Jolla CA). Hybridization was done with Express Hyb hybridization solution using the manufacturers method (Clontech). A human ß-actin cDNA probe (Clontech) that cross-hybridizes with mouse was used as a control in these experiments.
 |
Results and discussion
|
---|
Restoration of PU.1 in PU.1 null early monocytic cells:
As previously reported, we have derived several GM-CSF- dependent clones from PU.1/ fetal liver cultures (25). These cells appear to be early committed monocytic precursors, but cannot develop further. In order to study the role of PU.1 in macrophage maturation we set out to restore PU.1 expression in these cells. The clone used for this experiment was GMpu4 (GM4). PU.1 cDNA was subcloned into a retroviral vector and transfected into a packaging cell line (see Methods). Virus-containing supernatant was used to infect GM4 cells. The retrovirus vector also contained a puromycin resistant gene. GM4 cells that successfully integrated the vector were selected in puromycin-containing media.
To initially screen the infected GM4 cells for the presence of PU.1, we analyzed the puromycin resistant population (GM4puR) by flow cytometry for expression of CD11b and F4/80. The promoter for CD11b has been shown to be regulated by PU.1 and, therefore, should be a good indicator of PU.1 expression (35). F4/80 was examined to see if the cells had changed their state of maturation. The parent cell, GM4, as reported was CD11b and F4/80 (Fig. 1A). The retrovirally transduced cells were also F4/80 and while the vast majority of the cells were CD11b, a small proportion of cells were CD11b+. The CD11b+ population was enriched using magnetic cell sorting and expanded (Fig. 1B). Both the CD11b+ and CD11b populations along with several clones isolated from both populations were characterized. Only the CD11b+ pool and cells isolated from this population expressed PU.1 and demonstrated a rescued phenotype described below. Data from this analysis will be presented in this report using representative clones from both the CD11b+ and CD11b population. Clone GMpuR-3B6 (3B6) was derived from the CD11b+ population and clone GMpuR-B2 (B2) was derived from the CD11b population.


View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1. Screening PU.1-transduced GM4 cells. (A) GM4 and the Gm4puR population were stained with either FITC-conjugated CD11b or F4/80 antibodies and analyzed by flow cytometry. The dotted tracing indicates background staining. The CD11b+ cells in the GM4puR population are indicated by an arrow. (B) The CD11b+ cells were isolated using magnetic cell sorting with an anti-FITC magnetic-labeled antibody. Comparison of the histoplots demonstrates the enrichment of the CD11b+ cells.
|
|
Expression of PU.1
To verify that CD11b expression was due to PU.1 expression, total RNA was isolated from GM4, 3B6 and B2, and used to look for PU.1 message by RT-PCR. As shown in Fig. 2(A), no PU.1 message was detected in GM4 or B2 cells, but a positive band was seen in 3B6. The RNA control for 3B6 was negative, demonstrating there was no contaminating DNA. As a control for cDNA integrity the expression of the mitochondrial gene, ATP synthase F subunit, was examined and was detected in all three samples.


View larger version (71K):
[in this window]
[in a new window]
|
Fig. 2. The expression of CD11b corresponds with PU.1 expression. (A) 3B6 is a clone derived from the CD11b+ population and B2 was derived from the CD11b population. The expression of PU.1 message in GM4, 3B6 and B2 was determined by RT-PCR. Total RNA (2 µg) was used for cDNA synthesis. Approximately 1% of the cDNA reaction was used for PCR of PU.1 and the positive control ATP synthase. An equivalent amount of total RNA from 3B6 was used in the PCR step to control for contaminating DNA. Control indicates a reagent blank. (B) The production of PU.1 protein was determined by mobility shift assay. Equivalent amounts of nuclear extract from GM4 and 3B6 were incubated with a 32P-labeled oligonucleotide containing a PU.1 consensus-binding site. The arrows show PU.1DNA complexes and the asterisks denote supershifted complexes with PU.1 antisera. N-PU.1 refers to antisera against an N-terminal peptide from PU.1.
|
|
The expression of PU.1 protein was examined by mobility shift assay. Equivalent amounts of nuclear extract from GM4 and 3B6 were incubated with an oligonucleotide containing a PU.1 consensus binding site. Nuclear extract from 3B6 displayed three proteinDNA complexes (Fig. 2B). These three bands represent degradation products of PU.1 bound to the DNA probe. A polyclonal antibody to the N-terminus of PU.1 was able to supershift the slower migrating complex while addition of a polyclonal antibody made to the whole PU.1 protein supershifts both the upper and middle complex. Addition of a polyclonal antibody to Ets-2 had no effect on the three complexes. No proteinDNA binding activity in GM4 extract equivalent to that of 3B6 was found. On the other hand, two larger complexes were detected that did not respond to the PU.1 antibodies, but were eliminated with the Ets-2 antibody. The identification of Ets-2-binding activity in the GM4 nuclear extract provides a positive control for extract integrity. As expected, no PU.1 protein was found in B2 nuclear extract (data not shown).
Consistent with the reported role of PU.1 regulating CD11b expression, the clones derived from the CD11b+ population were found to express both PU.1 message and protein while CD11b cells did not express PU.1.
Flow cytometry analysis
To determine if other phenotypic changes occurred in 3B6 relative to GM4 and B2 we used a variety of antibodies to look at early and late surface markers of macrophage development. Both ER-MP12 and ER-MP20 mAb have been used to characterize early monocytic precursors (36). The antigens recognized by these antibodies are CD31 (PECAM-1) and Ly-6C respectively (37,38). Both of these markers are down-regulated in mature macrophages (39,40). Upon examination by flow cytometry it was revealed that GM4, 3B6 and B2 expressed equivalent levels of CD31 and Ly-6C (Fig. 3). In addition, none of the clones expressed late markers such as scavenger receptor, CD80 and CD86. All three clones still had the appearance of early precursor cells as determined by WrightGiemsa stain (data not shown). Therefore, based on surface marker expression and the morphology of the cells, 3B6 was still arrested at an early stage of development. However, there were some changes in surface marker expression on 3B6 compared to GM4 and B2. The Moma-2 mAb recognizes both mature macrophages and monocytic precursors (41). Although all three clones expressed Moma-2, the expression of Moma-2 on 3B6 relative to GM4 and B2 was
10-fold higher. In addition, expression of CD18 and Gr-1 is higher on 3B6. Finally, low levels of MHC class II and F4/80 surface antigens were found on 3B6, but were not detected on either GM4 or B2 cells. It is unknown at this time whether PU.1 directly or indirectly modulates the expression of these surface markers. It has been demonstrated that the promoters for the CD18 (42) and F4/80 (S. Gordon, pers. commun.) genes contain a functional PU.1-binding site (42). Likewise, a PU.1-binding site in the MHC class II I-Aß Y box has been identified (43). Therefore, the increased expression of CD18, F4/80 and class II in 3B6 could be due to direct interaction of PU.1. It has been reported that PU.1 may repress I-Aß expression (44), but because the context of the I-Aß regulatory sites in the reporter construct was different than the in vivo conformation it is difficult to rule out a positive role for PU.1 in regulating class II expression.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3. Surface marker comparison of GM4, 3B6 and B2 cells. The cells were incubated with either unconjugated [ER-MP12 (CD31), CD80 or scavenger receptor], FITC-conjugated [Ly-6C (ER-MP20), Moma-2, CD11b, Gr-1, MHC class II, F4/80 or CD86] or phycoerythrin-conjugated (CD18) antibodies. FITC-conjugated secondary antibodies were used to detect CD31, CD80 and scavenger receptor. The light tracing shows background staining, the dark tracing refers to GM4 cells, the dash tracing to 3B6 and the dotted tracing to B2.
|
|
Differentiation of 3B6
The M-CSF receptor gene c-fms has been reported to be regulated in part by PU.1 (28,29). Therefore, we tested to see if c-fms message could be detected in 3B6. As shown in Fig. 4, no c-fms message could be detected in GM4 or B2 cells, but c-fms message was found in 3B6.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4. Expression of PU.1 induces c-fms expression. The expression of the M-CSF receptor gene c-fms in clones GM4, 3B6 and B2 was determined by RT-PCR. The conditions for PCR are described in Methods. The same cDNA preparation used to look for PU.1 expression was also used to look for c-fms. Therefore, the controls can be seen in Fig. 2(A).
|
|
M-CSF is important for macrophage growth, differentiation and survival (4,46). Because the M-CSF receptor message was expressed in 3B6 cells we asked whether or not culturing the cells in M-CSF might induce maturation. To test this possibility the GM-CSF concentration was reduced by 10-fold and M-CSF was added to the media. By 57 days the plates were completely covered in adherent macrophages (Fig. 5A). Cells were isolated and examined histologically by WrightGiemsa (Fig. 5B). 3B6 maintained in GM-CSF have the appearance of immature monocytic cells, but after several days in media with M-CSF the cells look like mature macrophages with large highly vacuolated cytoplasm and small eccentric nucleus. M-CSF was required since reducing the level of GM-CSF alone did not induce maturation. The PU.1 null GM4 (Fig. 5A) or B2 cells (data not shown) grown under the same conditions did not mature.


View larger version (172K):
[in this window]
[in a new window]
|
Fig. 5. M-CSF induces macrophage maturation of 3B6, but not GM4. Cultures of GM4 and 3B6 cells were either incubated in 1 ng/ml GM-CSF or 0.1 ng/ml GM-CSF plus 5000 U/ml rM-CSF for up to 7 days. Changes in morphology were visualized by (A) phase contrast and (B) WrightGiemsa.
|
|
The surface marker phenotype of the M-CSF-derived macrophages was indicative of mature cells. The expression of early markers such as CD31, Ly-6C and Gr-1 was reduced, while late markers such as the scavenger receptor, CD80 and CD86 were now detected (Fig. 6). Also, levels of MHC class II increased. No apparent increase in F4/80 expression was observed but when the mature macrophages were stimulated with lipopolysaccharide (LPS), the level of F4/80 expression increased 4- to 5-fold (data not shown). As another measure of maturation, the 3B6 M-CSF-derived macrophages responded to IFN-
activation indicated by a 5-fold increase in class II expression (data not shown). Class II expression did not change in IFN-
-treated immature 3B6 cells.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6. Surface marker changes occur as 3B6 cells undergo M-CSF-induced maturation. Both early and late macrophage markers are examined by flow cytometry on 7-day-old M-CSF treated and untreated 3B6 cells. The light tracing shows background staining, the dark tracing shows untreated 3B6 cells and the dotted tracing shows M-CSF-treated 3B6 cells after 7 days in culture.
|
|
Subtraction
It could be argued that the role of PU.1 in macrophage development is to induce the expression of the M-CSF receptor. Clearly, the expression of the M-CSF receptor is a necessary component in the maturation of the PU.1 rescued cells. However, as mentioned in the Introduction, exogenous expression of c-fms in PU.1 null progenitors does not restore macrophage differentiation in the presence of M-CSF (24,27). Furthermore, macrophage development can take place in the absence of M-CSF as demonstrated in the op/op mouse (47). Therefore, PU.1 must be regulating other genes during macrophage development in addition to the M-CSF receptor.
In order to identify other genes potentially regulated by PU.1 that play a role in macrophage maturation, we employed the suppressive subtractive hybridization method to identify differentially expressed genes. To reduce the complexity and find genes directly regulated by PU.1, we used RNA from the PU.1 null GM4 clone and from the uninduced PU.1 rescued 3B6 clone. In this case the GM4 cDNA served as the driver and the 3B6 cDNA as the tester. The direction of this subtraction favors the identification of genes expressed in 3B6 cells not found in GM4. Several gene sequences were found from this subtraction, but only three were differentially expressed between 3B6 and GM4 as determined by Northern blot analysis (Fig. 7A). These three cDNA fragments were sequenced and found to be known genes. MRP14 is a calcium-binding protein (48), Dap12 is a transmembrane protein with a single ITAM domain (49,50) and CD53 is a surface protein belonging to the tetraspanin family of proteins (51). All three genes have been shown to be expressed in cells of the monocytic lineage.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 7. Three genes involved in signaling are differentially expressed between 3B6 and GM4 cells. (A) RNA (2 µg) from GM4, 3B6 and B2 were analyzed by Northern using three 32P-labeled cDNA probes obtained from a subtracted 3B6 library. The subtraction was between GM4 and 3B6. A ß-actin probe was used to control for loading and integrity of RNA among the samples analyzed. (B) MRP-8 message was examined since this protein is known to dimerize with MRP-14. The equivalent expression of MRP-8 among the three clones further substantiates the subtraction results.
|
|
As seen in the Northern blot, the GM4 and control B2 cells do express low levels of MRP14, but when PU.1 is expressed, a 10-fold increase in MRP-14 was observed. Normally, MRP14 is expressed in early monocytic cells and in infiltrating monocytes during inflammation, but not in resting macrophages (48). MRP14 forms an active complex by dimerizing with its partner MRP8 in a calcium-dependent manner (52). This active complex translocates to the plasma membrane and the cytoskeleton (53). Not much is known about the function of this hetero-complex. We examined GM4, B2 and 3B6 for the expression of MRP8, and found nearly equivalent levels of this message in all cell lines (Fig. 7B). It is unclear if PU.1 directly regulates MRP-14 expression, although recently a 1200-bp promoter/enhancer region from the MRP14 gene was shown to contain several PU.1-binding sites (54).
In contrast to MRP14, there was no detectable expression of either Dap12 or CD53 in PU.1 null GM4 and B2 clones. Dap12 was originally identified in NK cells, and recently shown to mediate activation signaling from Ly-49D and Ly-49H receptors (55,56). Dap12 contains a single ITAM motif that recruits protein tyrosine kinases Zap70 and syk, which leads to activation of ERK and calcium mobilization (57). Dap12 knockout mice or mice carrying a non-functional mutation in the Dap12 ITAM region demonstrate normal production of monocytes and macrophages. However, there is an accumulation of dendritic cells in the skin of these mice (58,59). The function of the Dap12-deficient and dominant-negative mutant macrophages, however, appears to be compromised as evident by resistance to experimental autoimmune encephalomyelitis and hapten-specific contact sensitivity respectively. It is suggested that Dap12 may play a role in regulating migration and the antigen-presenting function of macrophages/dendritic cells. Several Dap12-associated receptors have been identified in myeloid cells, including myeloid Dap12-associating lectin-1 (MDL-1), signal-regulatory protein ß (SIRP-ß), and triggering receptor expressed on myeloid cells-1 and -2 (TREM-1 and -2) (6063). The TREM-2/Dap12 pathway has been shown to be important for regulating dendritic cell maturation (63). Another published report demonstrated that cross-linking a FLAG-tagged version of Dap12 in M1 monoblast cells was sufficient to induce macrophage maturation (64). Therefore, the accumulated data on Dap12 suggests it has a direct role in macrophage development. Whether or not PU.1 directly regulates expression of Dap12 has yet to be determined. Further work studying the promoter region of the Dap12 gene is needed.
Not much is known about the function of CD53. CD53 belongs to the tetraspanin family of transmembrane proteins, and members of this family have been implicated in cell activation, proliferation, adhesion, motility, differentiation and cancer (65). In monocytes the cross-linking of CD53 induces monocyte oxidative burst and NO production as well as calcium mobilization (66,67). Tetraspanin proteins form complexes with adhesion receptors of the integrin family and likely play a role in integrin signaling, such as the recruitment of kinases (68). Recently, an analysis of the promoter of the human CD53 gene has revealed several ets-1/PU.1-binding sites and the mutation of some of these sites resulted in the loss of expression of a reporter gene to which the promoter had been linked (69). Based on this recently published work and the work presented here, PU.1 appears to be an important regulator of CD53 expression.
Further characterization of these three proteins in macrophage development is required. The use of antisense technology and/or dominant-negative mutants to these proteins in the PU.1 rescued cell line may help elucidate their role in macrophage maturation and function.
To find out whether or not the expression of these genes is modulated during maturation, mRNA was collected from several time points during the maturation of 3B6 in M-CSF-containing media. Cells were monitored for stages of maturation by flow cytometry and changes in morphology (data not shown). By day 5 the majority of cells were mature macrophages. A small increase in CD53 message could be detected as the cells matured (Fig. 8). The message for Dap12, on the other hand, increased dramatically during M-CSF induced maturation of 3B6 (64). This is consistent with a recently published report showing increased Dap12 expression during macrophage differentiation of mouse bone marrow-derived cells. In contrast to CD53 and Dap12, MRP-14 mRNA decreased as the cells mature. This is consistent with previously published data showing high levels of MRP-14 expression in early monocytic cells and no expression in resting tissue macrophages (70,71).

View larger version (83K):
[in this window]
[in a new window]
|
Fig. 8. Dap12 and MRP-14 are reciprocally modulated as 3B6 cells mature in the presence of M-CSF. mRNA was isolated from 3B6 on different days of culture in the presence of M-CSF and analyzed by Northern using 32P-labeled Dap12, MRP-14, CD53 or ß-actin probes.
|
|
In conclusion, a PU.1 null monocytic precursor and a PU.1-rescued counterpart that can be induced to mature in the presence of M-CSF provide a unique model not only to study the role of PU.1 in macrophage development, but also to discover the genetic requirements necessary for macrophage maturation.
 |
Acknowledgements
|
---|
This work was supported by National Institutes of Health grant nos AI30656 (R. A. M.) and AI09307 (G. W. H.). The authors thank Dr Bruce Torbett for helpful discussions and critically reading the manuscript.
 |
Abbreviations
|
---|
G-CSFgranulocyte colony stimulating factor
GM-CSFgranulocyte macrophage colony stimulating factor
M-CSFmacrophage colony stimulating factor
TREMtriggering receptor expressed on myeloid cells
 |
References
|
---|
- Shivdasani, R., Orkin, S. 1996. The transcriptional control of hematopoiesis. Blood, 87: 4025.[Free Full Text]
- Tenen, D., Hromas, R., Licht, J., Zhang, D. 1997. Transcription factors, normal myeloid development, and leukemia. Blood, 90: 489.[Free Full Text]
- Valledor, A., Borras, F., Cullell-Young, M., Celada, A. 1998. Transcription factors that regulate monocyte/macrophage differentiation. J. Leukoc. Biol., 53: 405.
- Glimcher, L., Singh, H. 1999. Transcription factors in lymphocyte developmentT and B cells get together. Cell, 96: 13.[ISI][Medline]
- Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., DAgati, V., Orkin, S. H., Costantini, F. 1991. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature, 349: 257.[ISI][Medline]
- Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W., Orkin, S. H. 1994. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature, 371: 221.[ISI][Medline]
- Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schreiner, C. M., Miller, T. A., Pietryga, D. W., Scott, W. J., Jr., Potter, S. S. 1991. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell, 65: 677.[ISI][Medline]
- Shivdasani, R., Mayer, E., Orkin, S. 1995. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature, 373: 432.[ISI][Medline]
- Robb, L., Elwood, N., Elefanty, A., Kontgen, F., Li, R., Barnett, L., Begley, C. 1996. The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J., 15: 4123.[Abstract]
- Okuda, T., vanDeursen, J., Hiebert, S. W., Grosveld, G., Downing, J. 1996. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell, 84: 321.[ISI][Medline]
- Scott, E. W., Simon, M. C., Anastasi, J., Singh, H. 1994. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science, 265: 1573.[ISI][Medline]
- McKercher, S. R., Torbett, B. E., Anderson, K. L., Henkel, G. W.,Vestal, D. J., Baribault, H., Klemsz, M., Feeney, A. J., Wu, G. E., Paige, C. J., Maki, R. A. 1996. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J., 15: 5647.[Abstract]
- Lekstrom-Himes, J., Xanthopoulos, K. G. 1998. Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J. Biol. Chem., 273: 28545.[Abstract/Free Full Text]
- Yamanaka, R., Lekstrom-Himes, J., Barlow, C., Wynshaw-Boris, A., Xanthopoulos, K. G. 1998. CCAAT/enhancer binding proteins are critical components of the transcriptional regulation of hematopoiesis. Int. J. Mol. Med., 1: 213.[ISI][Medline]
- Wang, J. H., Nichogiannopoulou, A., Wu, L., Sun, L., Sharpe, A. H., Bigby, M., Georgopoulos, K. 1996. Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity, 5: 537.[ISI][Medline]
- Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F., Busslinger, M. 1994. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell, 79: 901.[ISI][Medline]
- Zhuang, Y., Soriano, P., Weintraub, H. 1994. The helix-loop-helix gene E2A is required for B cell formation. Cell, 79: 875.[ISI][Medline]
- Bain, G., Maandag, E. C., Izon, D. J., Amsen, D., Kruisbeek, A. M., Weintraub, B. C., Krop, I., Schlissel, M. S., Feeney, A. J., van Roon, M., van der Valk, M., te Riele, H. P. J., Berns, A., Murre, C. 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell, 79: 885.[ISI][Medline]
- Zhang, D. E., Zhang, P., Wang, N. D., Hetherington, C. J., Darlington, G. J., Tenen, D. G. 1997. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc. Natl Acad. Sci. USA, 94: 569.[Abstract/Free Full Text]
- Zhang, P., Iwama, A., Datta, M. W., Darlington, G. J., Link, D. C., Tenen, D. G. 1998. Upregulation of interleukin 6 and granulocyte colony-stimulating factor receptors by transcription factor CCAAT enhancer binding protein alpha (C/EBP alpha) is critical for granulopoiesis. J. Exp. Med., 188: 1173.[Abstract/Free Full Text]
- Smith, L. T., Hohaus, S., Gonzalez, D. A., Dziennis, S. E., Tenen, D. G. 1996. PU.1 (Spi-1) and C/EBP alpha regulate the granu locyte colony-stimulating factor receptor promoter in myeloid cells. Blood, 88: 1234.[Abstract/Free Full Text]
- Anderson, K. L., Smith, K. A., Conners, K., McKercher, S. R., Maki, R. A., Torbett, B. E. 1998. Myeloid development is selectively disrupted in PU.1 null mice. Blood, 91: 3702.[Abstract/Free Full Text]
- Anderson, K., Smith, K., Pio, F., Torbett, B., Maki, R. 1998. Neutrophils deficient in PU.1 do not terminally differentiate or become functionally competent. Blood, 92: 1576.[Abstract/Free Full Text]
- DeKoter, R. P., Walsh, J. C., Singh, H. 1998. PU.1 regulates both cytokine-dependent proliferation and differentiation of granulo cyte/macrophage progenitors. EMBO J., 17: 4456.[Abstract/Free Full Text]
- Henkel, G. W., McKercher, S. R., Leenen, P. J., Maki, R. A. 1999. Commitment to the monocytic lineage occurs in the absence of the transcription factor PU.1. Blood, 93: 2849.[Abstract/Free Full Text]
- McKercher, S. R., Henkel, G. W., Maki, R. A. 1999. The transcription factor PU.1 does not regulate lineage commitment but has lineage-specific effects. J. Leukoc. Biol. 66: 727.[Abstract]
- Anderson, K. L., Smith, K. A., Perkin, H., Hermanson, G., Anderson, C-G., Jolly, D. J., Maki, R. A., Torbett, B. E. 1999. PU.1 and the granulocyte- and macrophage colony-stimulating factor receptors play distinct roles in late-stage myeloid cell differentiation. Blood 94: 2310.[Abstract/Free Full Text]
- Reddy, M. A., Yang, B. S., Yue, X., Barnett, C. J. K., Ross, I. L., Sweet, M. J., Hume, D. A., Ostrowski, M. C. 1994. Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes. J. Exp. Med., 180: 2309.[Abstract]
- Zhang, D. E., Hetherington, C. J., Chen, H. M., Tenen, D. G. 1994. The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol. Cell. Biol., 14: 373.[Abstract]
- Hohaus, S., Petrovick, M. S., Voso, M. T., Sun, Z., Zhang, D. E., Tenen, D. G. 1995. PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol. Cell. Biol., 15: 5830.[Abstract]
- Schmitt, R. M., Bruyns, E., Snodgrass, H. R. 1991. Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes & Development, 5: 728.[Abstract]
- Dignam, J. D., Lebovitz, R. M., Roeder, R. G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res., 11: 1475.[Abstract]
- Pongubala, J. M. R., Magulapalli, S., Klemsz, M. J., McKercher, S. R., Maki, R. A., Atchison, M. L. 1992. PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity. Mol. Cell. Biol. 12: 368.[Abstract]
- Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., Siebert, P. D. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl Acad. Sci. USA, 93: 6025.[Abstract/Free Full Text]
- Pahl, H. L., Scheibe, R. J., Zhang, D. E., Chen, H. M., Galson, D. L., Maki, R. A., Tenen, D. G. 1993. The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. J. Biol. Chem., 268: 5014.[Abstract/Free Full Text]
- de Bruijn, M. F., Slieker, W. A., van der Loo, J. C., Voerman, J. S., van Ewijk, W., Leenen, P. J. 1994. Distinct mouse bone marrow macrophage precursors identified by differential expression of ER-MP12 and ER-MP20 antigens. Eur. J. Immunol., 24: 2279.[ISI][Medline]
- Ling, V., Luxenberg, D., Wang, J., Nickbarg, E., Leenen, P. J., Neben, S., Kobayashi, M. 1997. Structural identification of the hematopoietic progenitor antigen ER-MP12 as the vascular endothelial adhesion molecule PECAM-1 (CD31). Eur. J. Immunol., 27: 509.[ISI][Medline]
- McCormack, J. M., Leenen, P. J., Walker, W. S. 1993. Macrophage progenitors from mouse bone marrow and spleen differ in their expression of the Ly-6C differentiation antigen. J. Immunol., 151: 6389.[Abstract/Free Full Text]
- Leenen, P. J., Melis, M., Slieker, W. A., Van Ewijk, W. 1990. Murine macrophage precursor characterization. II. Monoclonal anti bodies against macrophage precursor antigens. Eur. J. Immunol., 20: 27.[ISI][Medline]
- Leenen, P. J., de Bruijn, M. F., Voerman, J. S., Campbell, P. A., van Ewijk, W. 1994. Markers of mouse macrophage development detected by monoclonal antibodies. J. Immunol. Methods, 174: 5.[ISI][Medline]
- Kraal, G., Rep, M., Janse, M. 1987. Macrophages in T and B cell compartments and other tissue macrophages recognized by monoclonal antibody MOMA-2. An immunohistochemical study. Scand. J. Immunol., 26: 653.[ISI][Medline]
- Rosmarin, A. G., Caprio, D., Levy, R., Simkevich, C. 1995. CD18 (beta 2 leukocyte integrin) promoter requires PU.1 transcription factor for myeloid activity. Proc. Natl Acad. Sci. USA, 92: 801.[Abstract]
- Shackelford, R., Adams, D. O., Johnson, S. P. 1995. IFN-gamma and lipopolysaccharide induce DNA binding of transcription factor PU.1 in murine tissue macrophages. J. Immunol., 154: 1374.[Abstract/Free Full Text]
- Borras, F. E., LLoberas, J., Maki, R. A., Celada, A. 1995. Repression of I-A beta gene expression by the transcription factor PU.1. J. Biol. Chem., 270: 24385.[Abstract/Free Full Text]
- Stanley, E. R., Chen, D. M., Lin, H. S. 1978. Induction of macrophage production and proliferation by a purified colony stimulating factor. Nature, 274: 168.[ISI][Medline]
- Tushinski, R. J., Oliver, I. T., Guilbert, L. J., Tynan, P. W., Warner, J. R., Stanley, E. R. 1982. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy. Cell, 28: 71.[ISI][Medline]
- Begg, S. K., Radley, J. M., Pollard, J. W., Chisholm, O. T., Stanley, E. R., Bertoncello, I. 1993. Delayed hematopoietic development in osteopetrotic (op/op) mice. J. Exp. Med., 177: 237.[Abstract]
- Kerkhoff, C., Klempt, M., Sorg, C. 1998. Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9). Biochim. Biophys. Acta, 1448: 200.[ISI][Medline]
- Lanier, L. L., Corliss, B. C., Wu, J., Leong, C., Phillips, J. H. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature, 391: 703.[ISI][Medline]
- Tomasello, E., Olcese, L., Vely, F., Geourgeon, C., Blery, M., Moqrich, A., Gautheret, D., Djabali, M., Mattei, M. G., Vivier, E. 1998. Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12. J. Biol. Chem., 273: 34115.[Abstract/Free Full Text]
- Maecker, H., Todd, S., Levy, S. 1997. The tetraspanin superfamily: molecular facilitators. FASEB, 11: 428.[Abstract/Free Full Text]
- Teigelkamp, S., Bhardwaj, R. S., Roth, J., Meinardus- Hager, G., Karas, M., Sorg, C. 1991. Calcium-dependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14. J. Biol. Chem., 266: 13462.[Abstract/Free Full Text]
- Roth, J., Burwinkel, F., van den Bos, C., Goebeler, M., Vollmer, E., Sorg, C. 1993. MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate filaments in a calcium-dependent manner. Blood, 82: 1875.[Abstract]
- Nacken, W., Lekstrom-Himes, J. A., Sorg, C., and Manitz, M. P. 2001. Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils. J. Cell. Biochem., 80: 606.[ISI][Medline]
- Lanier, L. L., Corliss, B. C., Wu, J., Leong, C. and Phillips, J. H. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature, 391: 703.[ISI][Medline]
- Smith, K. M., Wu, J., Bakker, A. B., Phillips, J. H., Lanier, L. L. 1998. Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J. Immunol., 161: 7.[Abstract/Free Full Text]
- Lanier, L. L. and Bakker, A. B. 2000. The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol. Today, 21: 611.
- Bakker, A. B., Hoek, R. M., Cerwenka, A., Blom, B., Lucian, L., McNeil, T., Murray, R., Phillips, J. H., Sedgwick, J. D. and Lanier, L. L. 2000. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity, 13: 345.[ISI][Medline]
- Tomasello, E., Desmoulins, P. O., Chemin, K., Guia, S., Cremer, H., Ortaldo, J., Love, P., Kaiserlian, D. and Vivier, E. 2000. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity, 13: 355.[ISI][Medline]
- Bakker, A. B., Baker, E., Sutherland, G. R., Phillips, J. H., Lanier, L. L. 1999. Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells. Proc. Natl Acad. Sci. USA, 96: 9792.[Abstract/Free Full Text]
- Dietrich, J. M., Seiffert, C. M., Buhring, H. J. and Colonna, M. 2000. Cutting edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells. J. Immunol., 164: 9.[Abstract/Free Full Text]
- Bouchon, A., Dietrich, J. and Colonna, M. 2000. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J. Immunol., 164: 4991.[Abstract/Free Full Text]
- Bouchon, A., Hernandez-Munain, C., Cella, M. and Colonna, M. 2001. A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells. J. Exp. Med., 194: 1111.[Abstract/Free Full Text]
- Aoki, N., Kumura, S., Takiyama, Y., Atsuta, Y., Abe, A., Sato, K. and Katagiri, M. 2000. The role of the DAP12 signal in mouse myeloid differentiation. J. Immunol., 165: 3790.[Abstract/Free Full Text]
- Maecker, H. T., Todd, S. C., and Levy, S. 1997. The tetraspanin superfamily: molecular facilitators. FASEB J., 11: 428.[Abstract/Free Full Text]
- Olweus, J., Johansen-Lund, F., Horejsi, V. 1993. CD53, a protein with four membrane-spanning domains, mediates signal transduction in human monocytes and B cells. J. Immunol., 151: 707.[Abstract/Free Full Text]
- Bosca, L., Lazo, P. 1994. Induction of nitric oxide release by MRC OX-44 (anti-CD53) through a protein kinase C-dependent pathway in rat macrophages. J. Exp. Med, 179: 1119.[Abstract]
- Zhang, X. A., Bontrager, A. L. and Hemler M. E. 2001. Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins. J. Biol. Chem., 276: 25005.[Abstract/Free Full Text]
- Hernandez-Torres, J., Yunta, M., and Lazo P. A. 2001. Differential cooperation between regulatory sequences required for human CD53 gene expression. J. Biol. Chem., 276: 35405.[Abstract/Free Full Text]
- Lagasse, E., Weissman, I. L. 1992. Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage. Blood, 79: 1907.[Abstract]
- Goebeler, M., Roth, J., Henseleit, U., Sunderkotter, C., Sorg, C. 1993. Expression and complex assembly of calcium-binding proteins MRP8 and MRP14 during differentiation of murine myelomonocytic cells. J. Leukoc. Biol, 53: 11.[Abstract]