A lineage-specific protein kinase crucial for myeloid maturation

Abstract

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-beta (PKC-beta) and several stable PKC-beta transfectants, we found that PRKX gene expression is under control of PKC-beta; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.

Figure 1

Northern blot of PRKX mRNA levels. Induction of the PRKX gene expression during macrophage (A), granulocytic (B), and monocytic (C) maturation of HL-60 cells. Glyceraldehyde phosphate dehydrogenase probe was used to quantify the RNA loaded onto each lane. The X-CGD gene expression was used as a marker of myeloid maturation.

Figure 2

Expression of the PRKX gene in human granulocytes and lymphocytes analyzed by Northern blot analysis (A) and RT-PCR (B). Granulocytic HL-60 cells were used as a control. RT-PCR analysis did not reveal any PCR transcript in any of the tested human cell lines, including erythroid and megacaryocytic K-562 cells.

Figure 3

Light microscope images of HL-60 cells and human peripheral blood monocytes incubated with sense and antisense oligonucleotides to PRKX and induced to mature. (a) HL-60 cells; (b) HL-60 cells induced to mature with PMA; (c) HL-60 cells incubated with SENSE1 and induced with PMA; (d) HL-60 cells incubated with ANTISENSE1 and induced with PMA; (e) peripheral blood monocytes; (f) monocytes induced to mature with M-CSF; (g) monocytes incubated with SENSE1 and induced with M-CSF; (h) monocytes incubated with ANTISENSE1 and induced with M-CSF.

Figure 4

PRKX gene expression in untreated and in PMA-treated HL-60 cells incubated with either sense or antisense oligonucleotides. Expression of the X-CGD gene was used as a myeloid maturation marker.

Figure 5

Induction of the PRKX gene expression by PMA in HL-60 cells and in HL-525 stably transfected with an expression plasmid containing a full-length PKC-β cDNA (HL-525/3–2; HL-525/3–30) or a plasmid containing only the neomycin resistance gene (HL-525/NEO).