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Regulation of protein kinase C (PKC) expression by iron: effect of
different iron compounds on PKC-beta and PKC-alpha gene expression and role
of the 5'-flanking region of the PKC-beta gene in the response to ferric
transferrin
O Alcantara, L Obeid, Y Hannun, P Ponka and DH Boldt
Audie L. Murphy VA Hospital, Department of Medicine, San Antonio, TX.
We have studied effects of ferric transferrin (FeTF), ferric lactoferrin
(FeLF), ferric complexes of pyridoxal- or salicylaldehyde- isonicotinoyl
hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on
expression of protein kinase C (PKC) mRNA transcripts in a variety of
cultured cell lines. FeTF supported an increase of PKC-beta mRNA
transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B- lymphoblastoid
(Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte
(U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an
increase of PKC-beta mRNA transcripts in any of these cell lines.
Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in
HL-60, K562, and U937 cells only. Preincubation of cells with
desferrioxamine (DF), a cell-permeable iron chelator, abolished the
increments of PKC-beta mRNA observed in response to FeTF or FAC. In
contrast to results with PKC-beta, neither FeTF nor FAC caused an increase
of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA
regulatory elements of the PKC-beta gene, we prepared genetic constructs
containing various portions of the human PKC-beta 5'-flanking DNA linked to
the firefly luciferase gene. Constructs were cotransfected with the
neomycin resistance plasmid, Pwl- neo, into HRE H9 cells, and stable
transfectants were selected in G418. Treatment with FeTF of transfectants
bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking
region increased luciferase activity and mRNA transcripts 2.5-fold. This
increase was blocked by DF. Neither luciferase activity nor mRNA increased
with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp
of the PKC-beta 5'-flanking region. These data provide direct confirmation
that iron is involved in regulation of PKC-beta but not PKC-alpha gene
expression in many cell lines. The form in which iron is presented to these
cell lines appears to affect its availability for this function, and cells
vary in their capabilities to use nontransferrin iron to support PKC- beta
gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF
is mediated by DNA sequences located between -2200 bp and -587 bp in the
5'-flanking region of the human PKC-beta gene.
Volume 84,
Issue 10,
pp. 3510-3517,
11/15/1994
Copyright © 1994 by The American Society of Hematology
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