Induction Of PU Research Articles

Transcriptional control of granulocyte and monocyte development

PU.1 directs the hematopoietic stem cell to the lymphoid-myeloid progenitor (LMP) and interacts with GATA-binding protein 1 to inhibit commitment to the megakaryocyte-erythroid progenitor. The CCAAT/enhancer-binding protein (C/EBP)alpha then directs the LMP to the granulocyte-monocyte progenitor (GMP) stage, while inhibiting lymphoid development via cross-inhibition of Pax5 and potentially other regulators. Increased PU.1 activity favors monocytic commitment of the GMP. Induction of PU.1 by C/EBPalpha and interaction of PU.1 with c-Jun elevates PU.1 activity. Zippering of C/EBPalpha with c-Jun or c-Fos also contributes to monocyte lineage specification. An additional factor, potentially an Id1-regulated basic helix-loop-helix protein, may be required for the GMP to commit to the granulocyte lineage. Egr-1, Egr-2, Vitamin D Receptor, MafB/c: Fos and PU.1:interferon regulatory factor 8 complexes direct further monocytic maturation, while retinoic acid receptor (RAR) and C/EBPepsilon direct granulopoiesis. Both C/EBPalpha and RARs induce C/EBPepsilon, and PU.1 is also required, albeit at lower levels, for granulocytic maturation. HoxA10 and CAAT displacement protein act as transcriptional repressors to delay expression of terminal differentiation. Gfi-1 and Egr-1,2/Nab2 complexes repress each other to maintain myeloid lineage fidelity. NF-kappaB directly binds and cooperates with C/EBPbeta to induce the inflammatory response in mature myeloid cells and potentially also cooperates with C/EBPalpha to regulate early myelopoiesis.

Transcription Factor PU.1 Controls Transcription Start Site Positioning and Alternative TLR4 Promoter Usage

Human and mouse show markedly different sensitivities toward bacterial endotoxins, and recent evidence suggests that a species-specific regulation of the lipopolysaccharide receptor Toll-like receptor 4 (Tlr4) may contribute to this phenomenon. To gain further insight into mechanisms of Tlr4 regulation, we conducted a detailed in vivo and in vitro study of the murine Tlr4 gene, including analysis of transcription start site location, transcription factor occupancy, and activities of its proximal regulatory sequences. Our analyses identified a PU.1-dependent myeloid promoter, which is conserved between humans and mouse. We also identified an additional, distal promoter, located approximately 200 bp upstream of the myeloid-specific promoter, which is a functional target of E-box binding factors. In contrast to humans, where non-myeloid cells utilize both promoters, the distal Tlr4 promoter initiates all Tlr4 transcripts in murine non-myeloid cells, indicating that species-specific differences in TLR4 mRNA regulation may primarily exist in non-myeloid cell types. Interestingly, PU.1 null murine myeloid progenitor cells predominantly use the distal promoter, and the conditional induction of PU.1 expression in these cells leads to the rapid switch of transcription initiation to the proximal myeloid promoter. This indicates a direct role for PU.1 in determining the transcriptional start site and in recruiting the basal transcription machinery to myeloid promoters.

C/EBPα induces PU.1 and interacts with AP-1 and NF-κB to regulate myeloid development

C/EBPα and PU.1 are key regulators of early myeloid development. Mice lacking C/EBPα or PU.1 have reduced granulocytes and monocytes. Consistent with a model in which induction of PU.1 by C/EBPα contributes to monocyte lineage specification, mice with reduced PU.1 have diminished monocytes but retain granulocytes, C/EBPα directly activates PU.1 gene transcription, and exogenous C/EBPα increases monocytic lineage commitment from bipotential myeloid progenitors. In addition to C/EBPα, AP-1 proteins also have the capacity to induce monocytic maturation. C/EBPα:c-Jun or C/EBPα:c-Fos leucine zipper heterodimers induce monopoiesis more potently than C/EBPα or c-Jun homodimers or c-Fos:c-Jun heterodimers. C/EBPs and NF-κB cooperatively regulate numerous genes during the inflammatory response. The C/EBPα basic region interacts with NF-κB p50, but not p65, to induce bcl-2, and this interaction may be relevant to myeloid cell survival and development.

C/EBPα Binds and Activates the Distal PU.1 Enhancer.

C/EBPα and PU.1 are both key regulators of myeloid development. C/EBPα (−/−) mice have markedly reduced granulocyte-monocyte progenitors, and PU.1 (−/−) mice lack B cells and monocytes and have greatly reduced neutrophil numbers. Genetic evidence indicates that higher levels of PU.1 are required for monocyte as compared to granulocyte development. Exogenous PU.1 or C/EBPα converts B-lineage to monocytic cells and increases monocyte as compared to granulocyte production from transduced myeloid progenitors cultured in IL3/IL6/SCF or in GM-CSF. We previously demonstrated that activation of C/EBPα-ER with estradiol induces PU.1 mRNA within 4 hr even in the presence of cycloheximide and that C/EBPα binds and activates the PU.1 promoter via a site conserved in the murine and human genes. However, binding to the promoter site in a gel shift assay was weak compared to the strong binding site in the neutrophil elastase promoter. To obtain further evidence for regulation of the PU.1 gene by C/EBPα, we have now investigated regulation of the 240 bp proximal segment of the PU.1 distal enhancer located at −14 kb. We focused on the proximal segment of the enhancer due to is strong transcriptional activating activity when linked to the PU.1 promoter, compared to the repressive effect of the distal enhancer segment. Five sites within the proximal enhancer (C1–C5) fit the C/EBP consensus site T(T/G)NNGNAA(T/G) or differed from this consensus by 1 bp. Each was evaluated for binding to C/EBPα expressed transiently in 293T cells. Site C1 bound C/EBPα weakly, and C2 and C5 each bound C/EBPα strongly. Binding of C/EBPα to the endogenous proximal enhancer segment was also demonstrated in the HF1 myeloid cell line using the chromosomal immunoprecipitation (ChIP) assay. HF1 cells were employed for this assay due to their high-level expression of C/EBPα. Sites C1, C2, and C5 were mutated individually within the enhancer, which was then linked to the PU.1 promoter and the luciferase cDNA. Mutation of C1 minimally reduced the activity of this reporter, mutation of C2 reduced activity 2-fold, and mutation of C5 reduced activity 5-fold, on average, upon transient transfection into the more readily tranfectable 32DPKCδ cell line. These data provide further support for the conclusion that C/EBPα directly activates the PU.1 gene and that induction of PU.1 by C/EBPα, potentially in cooperation with additional factors such as c-Jun, helps direct bipotent myeloid progenitors to commit to the monocytic lineage.

C/EBPα directs monocytic commitment of primary myeloid progenitors

AbstractC/EBPα is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBPα in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBPα-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBPα-ER to near the physiologic range. C/EBPα-ER increased Mac1+/Gr1–/MPO–/low monocytes 1.9-fold while reducing Mac1+/Gr1+/MPOhi granulocytes 2.5-fold at 48 hours, even in 0.01 μM estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 μM estradiol. C/EBPα-ER induced PU.1 mRNA, and PU.1-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBPα contributes to monopoiesis. A C/EBPα variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-κB p50 stimulated granulopoiesis, suggesting their cooperation with C/EBPα during monocytic commitment.

Downregulation of PU.1 Is Required for Subtype of Multiple Myeloma.

There are several recent reports that certain mutations in critical transcription factors could be associated with leukemogenesis; however, it remains unclear whether such mutations could be linked to the pathogenesis of multiple myeloma (MM). In the present study, we attempted to determine whether mutations in transcription factors could be found in the patients with MM, utilizing MM cell lines and freshly isolated MM cells, purified with CD138 antibody and magnetic beads. When we looked at the expression of Blimp-1, XBP-1, NF-kB (p65 and p50), and PU.1 using semi-quantitative RT-PCR, all except PU.1 were found expressed in all MM cell lines and freshly isolated MM cells examined. All the PCR products were sequenced, but no significant mutations were identified. As examined with RT-PCR, PU.1 was found completely downregulated in 6 of 8 MM cell lines, while only faint to no expression of PU.1 was seen in 12 of 41 freshly isolated MM cells. As PU.1 message was sequenced, no mutations were found in 29 PU.1-positive MM cells or 2 MM cell lines that had relatively high PU.1 expression. Considering that PU.1 is essential for myeloid and lymphoid differentiation and that mice with null mutation in the PU.1 gene have no granulocytes, monocytes/macrophages or B lymphocytes, together with a recent report by others that AML developed in mice with PU.1 downregulation by 20%, we asked whether PU.1 downregulation might be associated with the oncogenesis or maintenance of MM. To examine the effects of PU.1 expression on MM cells, we generated tet-off conditional PU.1 expression model of PU.1-negative U266 MM cell line. Surprisingly the induction of PU.1 expression resulted in cell cycle arrest and apoptosis of those cells. Cell cycle analysis using flow cytometry revealed that PU.1-expressing U266 cells were in the G1 arrest. May-Giemsa staining revealed that such cells were swollen, enlarged, and vacuolous. These changes were completely reverted when the expression of PU.1 was silenced by tetracycline addition. There was no change in expression levels of cell cycle-related genes (p27, cyclin D1, cyclin E) and apoptosis-related genes (Bcl-2, Bcl-XL, and A1), while caspase3 cleavage was observed by 72 hr following PU.1 induction. We are currently examining the mechanism(s) of PU.1 downregulation in MM cells and a downstream cascade of PU.1 expression during cell death.

Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia

We hypothesized that PU.1 and PU.1 interacting proteins (PIP) binding to the Toll-like receptor 4 (TLR4) promoter is involved in endotoxin-induced upregulation of TLR4 gene expression. Our results employing chromatin immunoprecipitation assays indicate that PU.1 binds to the murine TLR4 promoter both in macrophage cells and, most importantly, in whole lung tissue. Treatment of RAW 264.7 cells with endotoxin induced the association of PU.1 and the TLR4 promoter in a time-dependent manner, and this was closely tied to interactions between the TLR4 promoter and the PIP interferon regulatory factors (IRF)4 and IRF8. PU.1 binding was related to increases in steady-state TLR4 mRNA and total TLR4 protein in RAW cells. Endotoxemia in animals caused the similar inducible interaction between PU.1 and IRF4 and the TLR4 promoter in lung tissue of mice that was treated with a single intraperitoneal injection of endotoxin. PU.1 binding to the TLR4 promoter was not enhanced in the lung tissue of endotoxin-resistant C3H/HeJ mice in response to endotoxemia. Transient transfection studies in RAW cells indicate that inducible binding of PU.1 to the TLR4 promoter is abrogated by a Ser148 to Ala mutation in PU.1. These data suggest that induction of PU.1/PIP binding to the TLR4 promoter is involved in endotoxin response in vivo and may mediate transcriptional changes in TLR4 gene expression.

Exogenous C/EBPα Increases the Monocytic Maturation of Normal Murine Myeloid Progenitors.

Within hematopoiesis C/EBPα is expressed specifically in both the granulocytic and monocytic myeloid lineages. Mice lacking C/EBPα have reduced neutrophil and monocyte production, due in part to diminished expression of the G-CSF, M-CSF, and GM-CSF receptor mRNAs. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells, and induction of late markers of granulopoiesis by C/EBPα-ER in 32Dcl3 cells was dependent upon its ability to slow G1 to S progression. We have now assessed the effect of C/EBPα-ER on the myeloid maturation of murine marrow progenitors. Regulation of the transgene using estradiol avoids biasing cell populations during transduction. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPαER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient, subjected to lineage-depletion, which removed 98% of Mac1+ and Gr1+ cells, and placed in liquid or methylcellulose culture with IL3/IL6/SCF +/− estradiol. By 48 hrs, 80% of the cells had acquired myeloid surface markers. Mac1+/Gr1+ cells were granulocytes and Mac1+/Gr1− cells were monocytes, as confirmed by Wright’s-Giemsa staining of cytospins. Estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold in 5 independent assessments. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68/macrosialin RNA, a 2-fold increase in M-CSF receptor and PU.1 RNA, and a 4-fold decrease in NE RNA by 48 hrs. In contrast, estradiol had little effect on the development of monocytes or granulocytes from vector, pBabePuro-transduced cells. Consistent with these findings, in an initial experiment we observed increased granulocytes when progenitors from floxxed C/EBPα mice (kindly provided by P. Johnson) were transduced with pBabeNeo-CRE, compare with pBabeNeo, subjected to lineage-depletion, and cultured in IL3/IL6/SCF. Loss of C/EBPα was demonstrated by PCR and Western blotting. During the first 48 hrs after transfer of transduced wild-type cells to estradiol, C/EBPα-ER slowed the proliferation of myeloid progenitors 2.6-fold, increased their G1/S ratio 2.8-fold, and reduced myeloid CFUs 5.1-fold. CFU-M, CFU-G, and CFU-GM were affected equally. Transduction with C/EBPαL12V-ER, harboring a defective leucine zipper, or with C/EBPαBR3-ER, mutant in 4 basic region residues, slowed proliferation in liquid culture 2-fold, reduced myeloid CFUs 1.5-fold, and had little effect on the G1/S ratio, perhaps indicating a role for DNA-binding in the regulation of the cell cycle by C/EBPα in normal myeloid progenitors (CFUs) and precursors. The L12V variant did not affect lineage output, whereas C/EBPαBR3 reduced monocytic maturation 1.4-fold and increased granulocyte formation 1.4-fold, in 4 experiments. C/EBPαp30, expressed in a subset of AML cases and lacking an N-terminal transactivation domain, increased the G1/S ratio and reduced myeloid CFUs 1.6-fold and had no effect on myeloid maturation. In summary, exogenous C/EBPα increases the monocytic and reduces the granulocytic maturation of transduced myeloid progenitors cultured in cytokines whose receptors are not regulated by C/EBPα. Cell cycle inhibition may play a role in this lineage bias, as the the L12V and p30 variants had both a reduced ability to slow G1 progression and to induce monocytes. Induction of PU.1 may also favor monocytic maturation. Finally, results with BR3 compared with L12V, neither of which bind DNA, suggest that alteration of a protein interaction with the non-DNA contact surface of the C/EBPα basic region favors granulocyte formation.

Opposing Roles for Class IA PI-3Kinase and Src Family Kinase in Regulating Myeloid Cell Growth and Lineage Commitment.

In adults, hematopoietic stem/progenitor (SC/P) cell expansion and lineage commitment is regulated by coordinated signals from cytokine receptors and transcription factors (TFs). Studies performed in cell lines using cytokine receptors lacking the binding sites for Src and PI-3Kinase family members have suggested a role for these signaling molecules in regulating growth and survival. However, the physiologic role(s) for these signaling molecules in primary SC/P cell growth and lineage commitment are poorly understood. Furthermore, the mechanism(s) via which these kinases regulate the expression of TFs in primary SC/Ps are not known. We demonstrate that Lyn Src family kinase and p85α subunit of class IA PI-3Kinase play opposing roles in regulating growth and lineage commitment in primary SC/Ps, in part by regulating the threshold of PI-3Kinase signaling, including the levels of phosphatidylinositol-3,4,5,-trisphosphate (PIP3) and Akt. Loss of Lyn in SC/P cells results in elevated PIP3 levels and hyperactivation of Akt. In contrast, loss of p85α subunit of class IA PI-3Kinase results in reduced levels of PIP3 and Akt activation. Functionally, Lyn−/− or Lyn+/− SC/P cells show enhanced growth and commitment towards myeloid lineage, including granulocytes, monocytes and mast cells, which is associated with enhanced and accelerated induction of myeloid lineage markers including c-Kit and the high affinity receptor for IgE. The enhanced myeloid lineage commitment in Lyn−/− SC/P cells is also associated with hyper induction of PU.1, CEB/Pα as well as members of the GATA family. In spite of high degree of homology between various Src family kinases, reconstitution of Lyn−/− SC/Ps with Lyn, but not with other related family members such as Hck or Fgr resulted in the rescue of Lyn−/− SC/P cell phenotype, suggesting that Lyn functions with specificity in regulating SC/P cell growth and survival, including lineage commitment. Enhanced lineage commitment, growth and survival as well as PIP3 levels seen in Lyn−/− SC/Ps were in part due to impaired activation of SHIP in these cells. Remarkably, in spite of continued reduction in the activation of SHIP and hyperactivation of Stat5 and Erk MAP kinase in Lyn−/− SC/P cells, haplo-insufficiency or complete deficiency of p85α subunit of class IA PI-3Kinase in the setting of Lyn deficiency or Lyn-haplo-insufficiency not only corrected the elevated PIP3 levels, cytokine induced hypersensitivity and lineage commitment to near wildtype levels, but in some cases made it worst than that seen in wildtype controls. Taken together, utilizing a genetic and a biochemical approach, our results illustrate a complex network of interactions between cytokine receptors and TFs involving Lyn, p85α and SHIP in positive and negative regulation of growth and lineage commitment in primary SC/P cells. Furthermore, our results demonstrate that the level of PIP3 in SC/P cells plays a critical role in determining the fate and lineage commitment in these cells. In vivo consequences on the long-term repopulating ability of stem cells lacking both Lyn and p85α subunit of class IA PI-3Kinase will be discussed.

Combination of retinoic acid and tumor necrosis factor overcomes the maturation block in a variety of retinoic acid-resistant acute promyelocytic leukemia cells

AbstractRetinoic acid (RA) overcomes the maturation block in t(15:17) acute promyelocytic leukemia (APL), leading to granulocytic differentiation. Patients receiving RA alone invariably develop RA resistance. RA-resistant cells can serve as useful models for the development of treatments for both APL and other leukemias. Previously, we showed that RA and tumor necrosis factor (TNF) promote monocytic differentiation of the APL cell line NB4 and U937 monoblastic cells. Here, we report that combining TNF with RA leads to maturation of several RA-resistant APL cells along a monocytic pathway, whereas UF-1, a patient-derived RA-resistant cell line, showed characteristics of granulocytic differentiation. We found distinct differences in gene regulation between UF-1 cells and cells showing monocytic differentiation. Although IRF-7 was up-regulated by TNF and RA in all cells tested, expression of c-jun and PU.1 correlated with monocytic differentiation. Furthermore, synergistic induction of PU.1 DNA binding and macrophage colony-stimulating factor receptor (m-CSF-1R) mRNA was observed only in cells differentiating into monocytes. Using neutralizing antibodies against m-CSF-1R or its ligand, we found that inhibiting this pathway strongly reduced CD14 expression in response to RA and TNF, suggesting that this pathway is essential for their synergy in RA-resistant leukemia cells. (Blood. 2004;104:3335-3342)

Regulation of granulocyte and monocyte differentiation by CCAAT/enhancer binding protein α

CCAAT/enhancer binding protein α (C/EBPα)-ER induces 32Dcl3 neutrophilic differentiation and inhibits 32DPKCδ maturation to macrophages in response to phorbol ester. In 32Dcl3 cells, C/EBPα-ER rapidly induces the PU.1 and C/EBPα RNAs even in the presence of cycloheximide, suggesting that these are direct C/EBPα genetic targets. C/EBPα strongly binds and modestly activates the murine PU.1 promoter via an evolutionarily conserved binding site. C/EBPα-ER variants incapable of binding DNA still slow G1 progression but do not induce differentiation. N-terminally truncated C/EBPα variants, including the p30 isoform expressed in a subset of AMLs, also retain the ability to slow 32D cl3 proliferation, whereas the C/EBPα(BRM2)-ER variant does not slow G1 progression, has a reduced capacity to induce early granulocytic markers, and does not induce terminal maturation. In 32DPKCδ cells, C/EBPα-ER strongly inhibits endogenous or exogenous JunB induction, dependent upon the outer surface of the C/EBPα basic region, but does not inhibit c-Jun, PU.1, or C/EBPβ expression. Exogenous JunB restores AP-1 DNA binding but does not overcome inhibition of monopoiesis by C/EBPα-ER. In summary, we propose that while C/EBPα is required for development of immature granulocyte–monocyte progenitors, C/EBPα subsequently inhibits monopoiesis, via inhibition of JunB express and via additional activities, and induces granulopoiesis, via induction of PU.1, C/EBPε, and cell cycle arrest.

Cross-talk between regulators of myeloid development: C/EBPalpha binds and activates the promoter of the PU.1 gene.

CCAAT/enhancer-binding protein (C/EBP)alpha and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBPalpha expressed in 293T cells bound the site centered at -68 most potently. C/EBPalpha from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPbeta did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The -68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the -68 C/EBP-binding site in a -85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPalpha and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPalpha-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPalpha DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPalpha and C/EBPalpha-ER with the PU.1 promoter. Finally, PU.1 RNA is reduced several-fold in immortalized C/EBPalpha (-/-) compared with (+/-) cells. Together, these findings indicate that C/EBPalpha binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPalpha may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

AbstractPotential redundancy among members of the CCAAT/enhancer-binding protein (C/EBP) family in myeloid cells is indicated by the ability of C/EBPβ to replace C/EBPα in vivo, by the expression of granulocyte colony-stimulating factor receptor (G-CSFR) on C/EBPα−/− cell lines, and by our finding that as with C/EBPα–estrogen receptor (C/EBPα-ER), either C/EBPβ-ER or C/EBPδ-ER can induce terminal granulopoiesis in 32D cl3 cells. To assess the consequences of globally inhibiting C/EBPs, we employed KαER, containing a Kruppel-associated box (KRAB) transrepression domain, the C/EBPα DNA-binding domain, and an ER ligand-binding domain. C/EBPs have a common DNA-binding consensus, and activation of KαER repressed transactivation by endogenous C/EBPs 50-fold and reduced endogenous G-CSFR expression. In 32D cl3 cells coexpressing exogenous G-CSFR, activation of KαER prevented and even reversed myeloperoxidase, lysozyme, lactoferrin, and C/EBPε RNA induction by G-CSF. In contrast, induction of PU.1 and CD11b, a gene regulated by PU.1 but not by C/EBPs, was unaffected. A KαER variant incapable of binding DNA owing to an altered leucine zipper did not affect 32D cl3 differentiation. Transduction of KαER into murine hematopoietic progenitor cells suppressed the formation of granulocyte colony-forming units, even in cytokines that enable C/EBPα−/−progenitors to differentiate into neutrophils. The formation of macrophage and of granulocyte-macrophage colony-forming units were also inhibited, but erythroid burst-forming units grew normally. Thus, in 32D cl3 cells and perhaps normal progenitors, C/EBPs are required for granulopoiesis beyond their ability to induce receptors for G-CSF and other cytokines. One requisite activity may be activation of the C/EBPε gene by C/EBPα, as either C/EBPα-ER or C/EBPβ-ER rapidly elevated C/EBPε RNA in 32D cl3 cells in the presence of cycloheximide but not actinomycin D.

Protein Kinase C Activation Increases Binding of Transcription Factor PU.1 in Murine Tissue Macrophages

PU.1 is a transcription factor found in macrophages, B cells, neutrophils, and hemopoietic stem cells. In macrophages PU.1 regulates a number of genes, includingc-fms,CD11b, CD18, and FcγR1b. Previously, in primary macrophages PU.1 binding to the sequence GAGGAA was found to be induced by treatment with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Here we investigated the role of protein kinase C (pKC) in the induction of PU.1 binding in macrophages. We report that pharmacological activation of pKC increases PU.1 binding, while inactivation of pKC inhibits the increases in PU.1 binding by agents which activate pKC in macrophages (LPS and tumor necrosis factor-α), but not by an agent which does not activate pKC (IFN-γ). pKC activation may therefore be one pathway by which PU.1 binding may be increased in primary macrophages.