Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells

Abstract

Monocytes and macrophages provide key targets for the action of novel anti-inflammatory therapeutics targeted at inhibition of PDE4 cAMP-specific phosphodiesterases. PDE4 enzymes provide the dominant cAMP phosphodiesterase activity in U937 human monocytic cells. Differentiation of U937 monocytic cells to a macrophage-like phenotype causes a marked reduction in total cellular PDE4 activity. Monocytic U937 cells express the long PDE4A4, PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 isoform. Differentiation of U937 cells to a macrophage-like phenotype causes a marked downregulation of PDE4D3 and PDE4D5, elicits a marked upregulation of PDE4B2 and induces the novel PDE4A10 long isoform. Comparable patterns are found in human peripheral blood monocytes and macrophages differentiated from them. Immunopurification of PDE4 subfamilies identifies long PDE4D isoforms as providing the major PDE4 activity in U937 monocytic cells. In U937 macrophage-like cells, the activity of the short PDE4B2 isoform predominates. No indication of either the expression or induction of PDE4C was evident. Activation of ERK exerts an inhibitory effect on total PDE4 activity in monocytic U937 cells, where the activity of long PDE4 isoforms predominates. The effect of ERK activation is switched to one of overall stimulation of total PDE4 activity in macrophage U937 cells, where the activity of the short PDE4B2 isoform predominates.10 The profound differentiation-induced changes in PDE4 isoform profile identified here suggests that the development of inhibitors specific for particular PDE4 isoforms may allow for selective effects on monocytes and macrophages to be achieved.

Figure 1

Differentiation of U937 monocytic cells. Shows Western blots comparing expression of (a) CD11b, (b) COX2 and (c) PKCβ doublet in nondifferentiated, monocyte-like (mono) and differentiated, macrophage-like (macro) U937 cells. In each case, the protein-loading amounts used reflected equal cell numbers. These data are typical of experiments done at least three times. In (d) is shown the protein content per cells for nondifferentiated, monocyte-like (mono) and differentiated, macrophage-like (macro) U937 cells.

Figure 2

Altered cAMP PDE activity upon differentiation of U937 monocytic cells. Shown for nondifferentiated, monocyte-like (mono) and differentiated, macrophage-like (macro) U937 cells is (a) the total cAMP PDE activity, the PDE4 rolipram-inhibited (10 μM) PDE activity and the PDE3 cilostamide-inhibited (1 μM) PDE activity, assayed at 1 μM cAMP substrate. The activity is given as pmol cAMP hydrolysed min⁻¹ 10⁶ cells⁻¹. (b) The % age of total PDE activity that is provided by PDE4 and by PDE3, through selective inhibition using rolipram (10 μM) and cilostamide (1 μM), respectively. (c) PDE1 activity towards cAMP is given here as that activity increased by the addition of Ca²⁺ (5 mM) and calmodulin (10 U) in the presence of EGTA (2 mM). We did note, however, that similar levels of activity were found when adding Ca²⁺ and calmodulin alone. These data are for experiments done three times with means±s.d., n=3.

Figure 3

Altered PDE4 isoform expression upon differentiation of U937 monocytic cells. Comparative analyses done for nondifferentiated, monocyte-like (mono) and differentiated, macrophage-like (macro) U937 cells. (a) Western blot using antisera specific for PDE4A isoforms; (b) Western blot using antisera specific for the PDE4A4 isoform; (c) Western blot using antisera specific for the PDE4A10 isoform; (d) RT–PCR for PDE4A10 transcripts; (e) Western blot using antisera specific for PDE4B isoforms and (e) Western blot using antisera specific for PDE4D isoforms. In all cases, comparisons were done using loadings that equated to equal numbers of cells. This, for western blots, was protein from 0.75 × 10⁶ cells per track. These data are typical of experiments done at least three times.

Figure 4

Altered PDE4 isoform expression upon monocyte-macrophage differentiation in U937 cells and human peripheral blood monocytes. Densitometric scanning of immunoblots similar to and including those shown in Figure 3 was carried out to compare the relative levels of expression of the indicated PDE4 isoforms. The isoforms detected and analysed were the long PDE4A4 (A4), PDE4A10 (A10), PDE4D3 (D3) and PDE4D% (D5) isoforms, as well as the short PDE4B2 (B2) isoform. Results are shown as ratios to indicate relative changes, and are done by comparing expression on a per cell basis, where equal expression is a value of 1 (unity). Data are shown for both the differentiation of U937 monocytic cells to macrophages and also primary human peripheral blood monocytes plated out and allowed to differentiate to macrophages, as in Methods. Data are shown as means±s.d. for n=3 separate experiments.

Figure 5

Altered PDE4 subfamily activity upon differentiation of U937 monocytic cells. (a) Shown for nondifferentiated, monocyte-like and differentiated, macrophage-like U937 cells is the relative contribution (%) of cAMP PDE activity supplied by each of the PDE4 subfamilies, as determined by selective immunopurification using specific antisera. In each case, a sufficient amount of antiserum was added to ensure complete immunopurification of that specific PDE4 subfamily, while at the same time not affecting any of the other PDE4 subfamilies, as determined by Western blotting of the remaining lysates. The total PDE4 activity immunoprecipitated using these antisera accounted for >90% of the total input lysate PDE4 activity, as determined by inhibition using 10 μM rolipram. No detectable activity was found using the PDE4C-specific antiserum (<2% total). (b) shows similar studies done on human peripheral blood monocytes and the macrophage form differentiating from them. These data are for experiments done three times with means±s.d., n=3.

Figure 6

ERK2 phosphorylation upon acute challenge with PMA. Shown are Western blots for both (a) nondifferentiated, monocyte-like and (b) differentiated, macrophage-like U937 cells. Identification was done using antisera specific for activated phospho ERK2 (P-ERK2) and also for ERK2 itself to indicate equal loading of lanes. These data are for a time course of cells treated acutely with PMA (50 nM) for the indicated time. Evaluation was done using loadings that equated to equal numbers of cells. In this instance, protein was from 0.75 × 10⁶ cells per track. These data are typical of experiments done at least three times.

Figure 7

Altered ERK regulation of PDE4 activity upon differentiation of U937 monocytic cells. Shown is the rolipram-inhibited cAMP PDE4 activity of lysates from both nondifferentiated, monocyte-like (a) and differentiated, macrophage-like (b, c) U937 cells challenged for the indicated time with PMA (50 nM). In some instances, cells were also treated with (a, b) the ERK inhibitor, UO126 (10 μM), (c) the PKA inhibitor H89 (1 μM) and (c) the COX inhibitor indomethacin (100 nM). In (d) is shown the ratio of PDE4/PDE3 activity in U937 monocytic and macrophage cells challenged acutely with PMA (50 nM) for 10 min prior to harvesting for PDE assay. PDE4 and PDE3 activities were determined here as those components inhibited by 10 μM rolipram and 1 μM cilostamide, respectively, added to assays done with 1 μM cAMP as substrate. All experiments were done three times with means±s.d., n=3. Activity data for PDE3 and PDE4 in monocyte-like and differentiated, macrophage-like U937 cells are as shown in Figure 2.