C/EBP epsilon mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut).

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBP epsilon) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBP epsilon activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBP epsilon(-/-) mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBP epsilon and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBP epsilon in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBP epsilon mRNA. We therefore hypothesize that C/EBP epsilon positively regulates SGP gene expression, and that C/EBP epsilon is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBP epsilon promoter is regulated by CDP/cut during myeloid differentiation.

Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation.

In this study, we sought to identify factors responsible for the positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are capable of directing myeloid-specific LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possible role of these sites in LF expression. Cotransfection studies of LF-89luc plasmid with increasing concentrations of a C/EBPalpha expression vector in myeloid cells resulted in a linear transactivation of luciferase reporter activity. Electrophoretic mobility shift assays found that the C/EBP site is recognized by C/EBPalpha and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells, and neither Sp1 mutant plasmid was transactivated by a C/EBPalpha expression plasmid to the same extent as wild-type LF-89luc. We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous Sp1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBPalpha expression plasmid. Furthermore, cotransfection of LF-89luc plasmid simultaneously with C/EBPalpha and Sp1 expression plasmids resulted in an increase in luciferase activity greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in mediating LF expression. (Blood. 2000;95:3734-3741)

Modulation of a calcium/calmodulin-dependent protein kinase cascade by retinoic acid during neutrophil maturation.

Retinoic acid is a lipophilic derivative of vitamin A that can cause differentiation in a variety of cell types. A large body of evidence has shown that normal retinoid signaling is required for proper neutrophil maturation in vitro and in vivo. In this study, we have found that calcium/calmodulin dependent (CaM) protein kinase kinase alpha (CaMKKalpha) is upregulated in an immediate early fashion during retinoic acid induced neutrophil maturation. Furthermore, we describe the expression and modulation of various components of the CaM kinase cascade during neutrophil maturation. We have confirmed upregulation of CaMKKalpha protein by Western analysis and further show that CaMKKbeta is expressed, although its protein levels are constant throughout induction. We also find that neutrophil progenitor cells express both CaMKI and CaMKIV transcripts. RNase protection and Western analysis show that CaMKIV is downregulated during neutrophil maturation. In contrast, CaMKI transcript and protein is expressed in uninduced cells and is induced by all-trans retinoic acid. These data represent the first report of a CaM kinase cascade in myeloid cells and suggests that this cascade may mediate some of the well-characterized effects of calcium on neutrophil function. These observations also support the idea that the retinoic acid receptors play a major role in mediating neutrophil specific gene expression and differentiation.

CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter.

Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non-LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing base-pair (bp) -387 to bp -726 of the LF promoter, but not in a -916-bp plasmid. Transfection of a -916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the -916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non-LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non-LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor-induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF-induced maturation of the murine pluripotent myeloid cell line 32Dcl3.

Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL-3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival.

NB4 cells show bilineage potential and an aberrant pattern of neutrophil secondary granule protein gene expression.

NB4 is an acute promyelocytic leukemia cell line that has been shown to be inducible to terminal neutrophil maturation with all-trans retinoic acid (ATRA). HL60 cells are differentially inducible with 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide (DMSO) to monocytes or granulocytes, respectively. HL60 cells induced with DMSO undergo defective neutrophil maturation, manifested by a coordinate failure of secondary granule protein gene expression. We observed a similar defect in granulocytic maturation in ATRA-induced NB4 cells. In addition, because normal promyelocytes are known to have bilineage potential, we have investigated differentiation along monocytoid lines induced with TPA. We observed a striking phenotypic change along monocytoid/macrophage lines with TPA induction. Flow cytometry showed a TPA-induced increase in HLA-DR expression, and Northern blot analysis showed induction of expression of CD18, c-fos, and human neutrophil gelatinase (HNG). HNG is unique among the neutrophil secondary granule protein genes in that it is expressed in both the neutrophil and monocyte lineages. This again parallels our findings in TPA-induced HL60 cells, which retain the ability to express HNG. These findings confirm bilineage potential in NB4 cells. They also support the hypothesis of coordinate neutrophil secondary granule protein gene expression and a defect in this control as part of the leukemic phenotype.

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.

Bovine papillomavirus type I induces resistance to Ca+(+)-induced terminal differentiation in murine keratinocytes.

Bovine papillomavirus type 1 (BPV-1) was expressed in established mouse BALB/MK epidermal keratinocytes. In each of the transfected cell lines, DNA synthesis was stimulated by epidermal growth factor (EGF) and inhibited by type beta transforming growth factor to an extent similar to that in parental cells. In contrast, the BPV-1-transfectants were resistant to the induction of terminal differentiation by extracellular Ca(+)+. First, BPV transfectants continued to respond to EGF in the presence of Ca(+)+, whereas the growth of BALB/MK cells was arrested. The characteristics of EGF-binding were identical in the two cell lines. Second, the Ca+(+)-switch failed to induce the activation of epidermal transglutaminase in BPV-1-transfectants. Thus, BPV-1 caused mouse keratinocytes to become resistant to Ca+(+)-induced differentiation without otherwise affecting the control of cell growth.

Ability of herpes simplex virus (HSV) types 1 and 2 to induce clinical disease and establish latency following previous genital infection with the heterologous HSV type.

Guinea pigs were infected intravaginally with either herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2). One month postinoculation, animals were inoculated with the heterologous HSV type and observed for clinical disease and shedding of virus. One month after superinfection, animals were killed, and tissues were cocultivated to detect latent virus. Although the severity of clinical disease and the degree of shedding of virus were greatly reduced by prior infection with the heterologous virus type, superinfection did occur in 82%-90% of animals. Nervous system latency with the superinfecting virus was established in 20% of animals superinfected with HSV-2 and 55% superinfected with HSV-1. Of 21 animals tested, 5 had latent infection with both viruses, 6 with the superinfecting virus only, and 6 with the initial virus only. Protection from nervous system latency with the superinfecting virus correlated best with levels of serum neutralizing HSV antibody before superinfection.