Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells

Abstract

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI(14-22), and was not affected by 8-pCPT-2'-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI(14-22). Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.

Figure 1.

PDE4 activity in B16-F10 cells. Homogenates were prepared and assayed for PDE activity with or without 10 μM rolipram as described in the materials and methods. The error bars represent the mean ± SD of three different experiments.

Figure 2.

mRNA expression of PDE4 isoforms in B16-F10 cells as determined by RT-PCR. (A) Expression of PDE4 mRNAs in B16-F10 cells. Molecular markers (M) and B16-F10 cell PDE4s (B16-F10): PDE4A (4A), PDE4B (4B), PDE4C (4C) and PDE4D (4D). Positive controls (P.C.) were also used: mouse heart PDE4A (4A) and mouse kidney PDE4C (4C). (B and C) Expression of PDE4B2 and PDE4D5 splice variant mRNAs in B16-F10 cells. Molecular markers (M), B16-F10 cell PDE4B2 (4B2) and PDE4D5 (4D5) are indicated.

Figure 3.

PDE4 inhibitors do not affect B16-F10 cell growth. Cells were plated in 96-well plates and cultured with different concentrations as indicated of the following reagents: (A) 8-Br-cAMP, (B) rolipram and (C) denbufylline. The error bars represent the mean ± SD of three different experiments. The treatments that differed significantly from the control are noted (^*P<0.05). Effects of (D) rolipram and (E) denbufylline on intracellular cAMP concentration. cAMP concentration was measured as described in Materials and methods. Values represent the mean ± one-half of the range of two independent experiments.

Figure 4.

Role of PDE4 in B16-F10 cell migration. B16-F10 cells were transferred to inserts for migration assays as indicated in Materials and methods. Inserts were incubated with different concentrations of the reagents: (A) 8-Br-cAMP, (B) rolipram and (C) denbufylline. The cells were counted as described in Materials and methods. The error bars represent the mean ± SD of three different experiments. The treatments that differed significantly from the control are noted (^*P<0.05).

Figure 5.

Downstream PKA signaling mediates the effects of PDE4 on B16-F10 cell migration. The cells were transferred to inserts for migration assays with indicated concentrations of the following reagents: (A) PKI_14–22, (B) 8-pCPT-2′-O-Me-cAMP and (C) rolipram with or without PKI_14–22. The cells were counted as described in Materials and methods. The error bars represent the mean ± SD of three different experiments. (A and B) The treatments that differed significantly from the control are noted (^*P<0.05). (C) Rolipram (50 μM) plus PKI_14–22 (20 μM) is compared with rolipram (50 μM) without PKI_14–22 (*P<0.05).