Biology Reference
In-Depth Information
were inconclusive with respect to the entire cAMP-signaling differentiation path-
way, it did establish ERK as an essential player in cAMP-mediated HL-60 cell
differentiation.
Beavo and colleagues performed a set of studies where chronic elevation of
cyclic nucleotides was mimicked by treating HL-60 cells with 8-Br-cAMP or 8-Br-
cGMP during the 3-day differentiation process in the presence of PMA (Bender and
Beavo
2006b
). Morphological differences were readily observed after differentia-
tion in the presence of the cAMP analog, but 8-Br-cGMP had a minimal effect on
morphology. Cell spreading in culture was inhibited in the presence of elevated
cyclic nucleotides and the cells form large clumps of round cells. These differences
were not attributed to any changes in macrophage function, but one could imagine
that differences in “cell spreading” could be the result of differences in migration
or secretion of chemotactic molecules or cytokines. Surface molecule expression
was also slightly changed with treatment but differentiation was not halted.
8-Br-cAMP significantly decreased the upregulation of CD11b, a monocyte/mac-
rophage marker, while it increased expression of the urokinase plasminogen acti-
vator (CD87); however, 8-Br-cGMP had no effect. These observed effects were
mimicked by the PKA-specific activator N
6
-Benzoyl-cAMP, and therefore likely
mediated through PKA.
HL-60 cells have also been used as a model system emulating human granulo-
cyte macrophage colony-stimulating factor (GM-CSF) differentiated monocytes
to determine the role of PDE1B in these cells. One variant of this isoform, the
cGMP-preferring enzyme PDE1B2 (Fidock et al.
2002
), is specifically upregulated
upon differentiation in both GM-CSF differentiated human monocytes and PMA
differentiated HL-60 cells (Bender et al.
2005
). PDE1B2 activity is regulated
by intracellular Ca
2+
and may provide a link between Ca
2+
and cAMP signaling
pathways. To determine the role of PDE1B2, stable knockdown of this isoform
by short hairpin RNAs was performed in differentiated HL-60 cells (Bender and
Beavo
2006b
). This specific isoform proved to be the major regulator of atrial
natriuretic factor-stimulated cGMP levels in HL-60 cells, and the authors subse-
quently searched for a functional role for this newly uncovered variant. It was
found that suppression of PDE1B2 expression alters some aspects of the macro-
phage-like phenotype, because cell spreading, phagocytic ability, and CD11b
expression were augmented. Unexpectedly, the cAMP analog 8-Br-cAMP, but
not 8-Br-cGMP, reversed the changes caused by PDE1B2 knockdown, indicating
a role for cAMP in control of these PDE1B2-dependent functions. Moreover,
despite the preference of PDE1B for cGMP as substrate, knockdown of PDE1B2
caused a decrease in basal cAMP levels, but showed no change in cGMP, and
a number of PKA phosphorylation consensus sites were altered. The cause of the
decrease in basal cAMP levels remains unclear. The expected increase in cGMP
with PDE1B2 knockdown may be contained within localized signaling microdo-
mains, and therefore undetectable by whole cell cGMP measurements. More likely,
there is some level of crosstalk between the cGMP and cAMP signaling path-
ways, possibly through inhibition of an adenylyl cyclase, or activation of another
cAMP-preferring PDE.
