PDE1B2 regulates cGMP and a subset of the phenotypic characteristics acquired upon macrophage differentiation from a monocyte

Abstract

Monocyte-to-macrophage differentiation with the cytokine granulocyte-macrophage colony-stimulating factor induces expression of the cyclic nucleotide phosphodiesterase PDE1B2. However, what role PDE1B2 plays in macrophage biology has not been elucidated. We have addressed this question by inhibiting PDE1B2 induction by using RNA interference. Using a retrovirus-based system, we created HL-60 stable cell lines that express a short-hairpin RNA targeting PDE1B2. HL-60 cells treated with phorbol-12-myristate-13-acetate differentiate to a macrophage-like phenotype and up-regulate PDE1B2. However, expression of PDE1B2 short hairpin RNA effectively suppresses PDE1B2 mRNA, protein, and activity up-regulation. Using the HL-60 PDE1B2 knockdown cells and agonists for either adenylyl or guanylyl cyclase, it was found that PDE1B2 predominantly regulates cGMP and plays a lesser role in cAMP regulation in response to cyclase agonists. Furthermore, in intact HL-60 cells, PDE1B2 activity can be regulated by changes in Ca+2 levels. Inhibiting PDE1B2 up-regulation does not prevent HL-60 cell differentiation, because several markers of macrophage differentiation are unaffected. However, suppression of PDE1B2 expression alters some aspects of the macrophage-like phenotype, because cell spreading, phagocytic ability, and CD11b expression are augmented. The cAMP analog 8-Bromo-cAMP reverses the changes caused by PDE1B2 knockdown. Also, PDE1B2 knockdown cells have lower basal levels of cAMP and alterations in the phosphorylation state of several probable PKA substrate proteins. Thus, the effects of PDE1B2 on differentiation may ultimately be mediated through decreased cAMP. In conclusion, PDE1B2 regulates a subset of phenotypic changes that occur upon phorbol-12-myristate-13-acetate-induced differentiation and likely also plays a role in differentiated macrophages by regulating agonist-stimulated cGMP levels.

Fig. 1.

RNAi suppresses PDE1B2 induction. HL-60 cells were infected with a retrovirus encoding expression of a PDE1B2-targeting shRNA (RNAi) or a shRNA with a scrambled sequence (Scr), and stable cell lines were generated by puromycin selection. (A) PDE1 activity in untreated cells (Ctl) and cells differentiated for 3 days with PMA (+PMA) was determined. (B) The expression of PDE1B2 protein in PMA differentiated cells was detected by immunoprecipitation with the ACC-1 antibody and Western blotting with an α-PDE1B antibody. Activity values are means ± SEM of four separate differentiations, and the Western blot shown is representative of three separate differentiations.

Fig. 2.

PDE1B knockdown alters PMA differentiated HL-60 cell phenotype. HL-60 cells expressing either a scrambled shRNA (Scr) or a PDE1B-targeting shRNA (RNAi) were differentiated for 3 days with PMA and cell phenotype was analyzed. (A) Cell morphology was examined by using phase-contrast microscopy. PDE1B2 shRNA cells display increased spreading in comparison with cells infected with the scrambled shRNA. (B) Phagocytosis of 2-μm fluorescently labeled microspheres by differentiated HL-60 cells was determined after 4 h by FACS for the scrambled and PDE1B2 shRNA cells. Values shown are means ± SEM of four separate experiments. *, Significantly different from scrambled cells (P < 0.05).

Fig. 3.

PDE1B2 regulation of cAMP and cGMP in response to agonists. HL-60 cells expressing either scrambled or PDE1B2 shRNA were differentiated for 3 days with PMA. (A) Cells were treated with forskolin for 15 min. Cyclic nucleotides were then extracted by incubating the cells in ice-cold EtOH/HCl for 10 min. cAMP levels were determined by EIA. Cells were also treated with various concentrations PGE₂ for 15 min after a 10-min pretreatment with 200 μM 3-isobutyl-1-methylxanthine. Values shown are means ± SEM from four separate determinations. (B) HL-60 cells were treated with 100 nM ANF, and the cGMP content of cells was determined at various time points by EIA. (C) Cells were treated with 100 nM ANF for 10 min after 10 min of pretreatment with varying concentrations of the calcium ionophore ionomycin. cGMP values for the cells expressing the scrambled shRNA are plotted on the left y axis, and the values from the cells expressing the PDE1B2 shRNA are plotted on the right y axis. All cGMP values are means ± SEM from three separate experiments.

Fig. 4.

8-Br-cAMP prevents HL-60 cell spreading, decreases CD11b expression, and decreases phagocytic ability. HL-60 PDE1B2 shRNA (RNAi) and scrambled shRNA (Scr) cells were differentiated for 3 days with 100 nM PMA in the presence of 500 μM 8-Br-cAMP or 8-Br-cGMP. After 3 days, cellular morphology was assessed by using phase-contrast microscopy (A). (B) CD11b and CD87 expression was measured by FACS, and phagocytosis of fluorescent microspheres was determined for cells differentiated in the presence or absence of 500 μM 8-Br-cNTs. Numbers reported are mean fluorescence values. Pictures shown are representative of at least three separate differentiations under each condition, whereas FACS and phagocytosis values are means ± SEM from four separate experiments. *, Significantly different from scrambled shRNA cells (P < 0.05).

Fig. 5.

PDE1B2 knockdown lowers basal cAMP and alters PKA substrate phosphorylation. HL-60 PDE1B2 shRNA (RNAi) and scrambled shRNA cells (Scr) were differentiated for 3 days with 100 nM PMA. Basal cAMP and cGMP levels were determined by EIA. Values shown are means ± SEM from eight separate differentiations. *, Significantly different from scrambled shRNA cells (P < 0.05). (B) Lysates from cells treated for 3 days with 100 nM PMA were Western-blotted by using an antibody that recognizes a phospho-Ser/Thr PKA substrate motif. Untreated cells (Ctl), PMA treated cells (+PMA), and PMA-treated cells with 8-Br-cNT added (+PMA/8-Br-cAMP) are compared. Protein bands that were consistently different between the two cell populations are boxed in and described in the text. The gel shown is representative of three experiments, and quantitation is graphed in Fig. 10.