Biology Reference
In-Depth Information
they do not contain ERK phosphorylation sites (Baillie et al.
2000
). This presents an
attractive therapeutic possibility if PDE4 isoform-selective inhibitors can be devel-
oped, allowing the differential regulation of monocytes and macrophages.
U-937 differentiation can be induced by increasing cAMP levels, either using
PGE
2
, the adenylyl cyclase agonist forskolin, or a nonhydrolyzable cAMP analog,
db-cAMP (Brodsky et al.
1998
; Shayo et al.
1997
). Surprisingly, activation of the
G
a
s-coupled histamine receptor is not able to induce U937 differentiation, despite
its ability to activate adenylyl cyclase and cause a transient increase in intracellular
cAMP (Shayo et al.
1997
). Upon further investigation, it was discovered that the
histamine receptor rapidly becomes desensitized through a GRK2-dependent mech-
anism and that a sustained elevation of cAMP is required for induction of differen-
tiation (Fernandez et al.
2002
; Legnazzi et al.
2000
; Shayo et al.
2001
). The stable
overexpression of the histamine H2 receptor (H2R) alone also induced differentia-
tion (Monczor et al.
2006
). The increased expression of H2R leads to an increase
in basal cAMP levels and a leftward shift of the dose-response curve. There was
a maximal response to agonist similar to those cells expressing normal amounts of
H2R, indicating that overexpression of H2Rs results in increased numbers of
functional spare receptors in this system. In addition to the generation of spare
receptors, there was a compensatory increase in GRK2 expression and PDE activity
stimulation. Despite the onset of these regulatory mechanisms, an H2 agonist and
the PDE4 inhibitor, rolipram, were still able to induce differentiation in these cells,
contrary to the previous observations in na¨ve cells (Monczor et al.
2006
).
The time course of cAMP elevation was determined to be critical for the
induction of U937 differentiation. Using the cAMP-elevating agonists amthamine
(a histamine H2 receptor agonist), PGE
2
(an EP receptor agonist), and forskolin (an
adenylyl cyclase agonist), Shayo and colleagues were able to demonstrate a time
dependence to cAMP-induced differentiation in U937 cells (Shayo et al.
2004
).
Amthamine caused a transient increase in cAMP returning to baseline in about 3 h,
whereas forskolin and PGE
2
created a more prolonged elevation of cAMP, which
was still sustained at the final measurement of 4 h. This prolonged cAMP eleva-
tion was necessary for differentiation of U937 cells, as indicated through the
observed change in differentiation markers such as an increase in C5aR expression,
arrested proliferation, and a decrease in c-myc protein levels. The mechanism of
this discrepancy seems to be H2R dependent occurring through GRK2-mediated
desensitization of the receptor. PGE
2
and forskolin bypass H2R and therefore are
able to sustain cAMP elevation. Homologous desensitization of the EP receptor
by PGE
2
was evident immediately upon stimulation and persisted for 40 min.
However, a sustained cAMP elevation via either PGE
2
or forskolin can also induce
heterologous desensitization of the H2 receptor in a PKA/PKC-dependent manner.
The data also showed a transient upregulation of c-Fos with amthamine and
sustained c-Fos expression with PGE
2
and forskolin. This correlated well with the
fact that sustained c-fos expression is known to be essential for U937 differentia-
tion. Overall, these studies indicate that cAMP levels must be sustained long term to
induce U937 differentiation.
