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To determine which PDEs were controlling the pools of cAMP regulating
surface marker and chemokine expression, selective inhibitors of PDE2, 3, and 4
were used in combination with a low dose of forskolin. The low dose of forskolin
showed a minimal change in mRNA and protein levels of these genes (Hertz et al.
2009a ). However, when that same dose of forskolin was administered with a PDE
inhibitor it caused a large response. This is exactly what would be predicted for
a synergistic effect of PDE inhibition coupled with adenylyl cyclase activation.
Using this technique, it was determined that PDE4 primarily controls the signaling
microdomain regulating expression of surface markers and chemokines.
The intracellular signaling pathways involved in regulation of these chemokines
seem to be largely downstream of Epac, not PKA as seen for cAMP's effect on most
of the proinflammatory cytokines (Hertz et al. 2009a ). Cyclic nucleotide analogs
which specifically activate Epac induce chemokine expression at both the mRNA
and the protein levels, while a PKA-specific activator had a minimal effect on
chemokine levels. Downstream signaling pathways leading to changes in macro-
phage inflammatory gene expression are generally regulated through the canonical
NF- k B pathway. Chemokine production was attenuated with a general inhibitor of
the NF- k B signaling pathway, the IKK1/2 inhibitor BMS-345541, but surprisingly
remained elevated after treatment with a peptide inhibitor of the canonical p50/p65
NF- k B heterodimer. While inflammatory cytokine production is normally regulated
through the p50/65 NF- k B pathway in macrophages, it seems that production
of these chemokines via elevated cAMP may occur through the noncanonical
p50/p105 NF- k B pathway. Also affecting this pathway was a negative regulator
of NF- k B, the transcription factor ATF3, which exhibits both reduced mRNA levels
and attenuated binding to the CXCL7 promoter when forskolin is present during
differentiation (Hertz et al. 2009a ). The relief of inhibition of ATF3 on promoter
regions containing NF- k B would promote expression of many inflammatory genes
(Gilchrist et al. 2006 ).
The signaling pathways downstream of cAMP can also be changed during
differentiation. Human monocytes stimulated with LPS produce large amounts of
TNF- a , IL-6, and IL-8. The release of these chemokines can be reduced with a p38
kinase inhibitor. However, in macrophages differentiated in GM-CSF for 12 days,
p38 inhibition has a lesser effect on production of these cytokines while TNF- a
production is effectively blocked with an ERK inhibitor (Tudhope et al. 2008 ). This
suggests that kinase usage can also change as a monocyte differentiates to a
macrophage, with monocytes favoring p38 kinase and macrophages preferring
ERK to control cytokine production.
3 Dendritic Cell Differentiation
Monocytes differentiated in the presence of GM-CSF + IL-4 gain a dendritic cell
phenotype (Geissmann et al. 2010 ). The PDE profile of these dendritic cells (DCs)
showed lower PDE4 levels than monocytes, increased PDE3, and a moderate
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