Biology Reference
In-Depth Information
2.2 U937
The U937 cell line is a human line established from a diffuse histiocytic lymphoma
and displays many monocytic characteristics (Harris and Ralph
1985
). The cGMP-
PDE profile of this cell line is very similar to that of a macrophage differentiated
from a human CD14+ monocyte in the presence of monocyte colony-stimulating
factor (M-CSF) in that it exhibits high PDE2 activity and some PDE1 activity
(Bender et al.
2004
). Changes in PDE4 activity have been observed in conjunction
with changes in cAMP levels. Characterization of PDE4 activity in monocytic
U937 cells showed that PDE4 activity can be augmented with a transient increase
in cAMP of 2-4 h in length. Also, the
V
max
of PDE4 was increased two-to threefold
in the presence of salbutamol, a
b
2 adrenergic agonist and the PDE4 inhibitor,
rolipram, but only if cAMP levels were elevated for more than 2 h. It was deter-
mined that PDE4 activity was increased through a PKA- and protein synthesis-
dependent mechanism (Torphy et al.
1992
). These data imply that cAMP signaling
feeds back to activate PDE4-mediated degradation of cAMP, thereby limiting the
duration and magnitude of signaling through that microdomain.
Houslay and colleagues examined cAMP-PDE activity levels and found that
PDE4 comprised the majority of PDE activity in U937 monocytic cells. However,
upon differentiation with PMA, PDE3 activity predominated and PDE4 activity
was decreased. Using antisera specific for PDE4 isoforms and RT-PCR, the inves-
tigators determined the splice variants of PDE4 that were changed during U937
differentiation to a macrophage-like cell. The N-terminal end of PDE4 isoforms
contain unique isoform-specific regions that can vary in length with long, short, and
supershort isoforms commonly observed. These isoforms contain variable trunca-
tions of the upstream conserved regions, or UCR domains. The long forms gener-
ally contain both UCR1 and UCR2 domains, while the short forms express only
UCR2 and supershort forms merely a portion of the UCR2 domain (Houslay et al.
2005
). A key distinguishing factor for these various isoforms is their susceptibility
to regulation by phosphorylation in their N-terminal UCR domains. Upon differen-
tiation to a macrophage, protein levels of the long isoform PDE4A10 and the short
isoform PDE4B2 were markedly increased, whereas long-form PDE4A4 remains
unchanged, and short forms PDE4D3 and PDE4D5 were downregulated (Shepherd
et al.
2004
). Additionally, it was found that phospho-ERK was increased with acute
PMA challenge, and this challenge gave a time-dependent inhibition of PDE4
activity in U937 monocytes and an increase in PDE4 activity in U937 macrophages,
both of which are dependent on ERK activation. The differential effects of ERK
activation can be directly attributed to the remodeling of the pattern of PDE4
isoform expression. The predominant PDE4 activity in monocytes is contributed
by long PDE4D isoforms, whereas in macrophages PDE4B short form activity is
the major source of PDE4 activity. This remodeling has functional consequences
since PDE4D long isoforms will become inhibited, PDE4B short forms will be
activated upon phosphorylation by ERK, and PDE4A isoforms are unaffected as
