cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-signaling control cGMP and Ca(2+) homeostasis in melanoma cells

Abstract

Malignant melanoma is one of the most aggressive human neoplasms which develop from the malignant transformation of normal epithelial melanocytes and share the lineage with retinal cells. cGMP-phosphodiesterase 6 (PDE6) is one of the cancer-retina antigens newly identified in melanoma cells. Normally, PDE6 hydrolyzes the photoreceptor second messenger cGMP allowing the visual signal transduction in photoreceptor cells. cGMP also play an important signaling role in stimulating melanogenesis in human melanocytes. Here, we present evidence that PDE6 is a key enzyme regulating the cGMP metabolism in melanoma cells. Decrease in intracellular cGMP leads to calcium accumulation in melanoma cells. In these cells, cGMP-phosphodiesterase 6 can be activated by another cancer-retina antigen, transducin, through Wnt5a-Frizzled-2 cascade, which leads to a lowering of cGMP and an increase in intracellular calcium mobilization. Thus, the aberrant expression of PDE6 may control cGMP metabolism and calcium homeostasis in melanoma cells.

Fig. 1

Protein expression of PDE6 and PDE5. a Expression of PDE6 subunits in NEM (1), HaCaT (2) and melanoma cell lines Ma-Mel-04 (3), Ma-Mel-12 (4), UKRV-Mel-31 (5), Ma-Mel-11 (6), Ma-Mel-21 (7), and MeWo (8), as well as Y79 retinoblastoma cell line as a positive control (9); b expression of PDE6 subunits in untransfected controls (1) or in cell lines transfected with shDNA plasmids (2) as indicated on the right side; c expression of PDE5 in heart as a positive control (1, 9), NEM (2), HaCaT (3), and melanoma cell lines Ma-Mel-04 (4), Ma-Mel-12 (5), UKRV-Mel-31 (6), Ma-Mel-11 (7), Ma-Mel-21 (8), MeWo (10), UKRV-Mel-06 (11), UKRV-Mel-14a (12), UKRV-Mel-21 (13), SK-Mel23 (14), Ma-Mel-05 (15), and Ma-Mel-21 (16)

Fig. 2

PDE activity in melanoma cell lines is attributed to PDE6. a Exemplary determination of PDE activity in melanoma cell line Ma-Mel-05. Luminescence signal (y axis) in relative luminescence unit (RLU) which is proportional to the cGMP concentration was measured at different concentrations of cGMP (x axis) without (broken line) or with (solid line) whole protein extract of the cell line (0.35 mg/ml). For details, see "Materials and methods". b Dispersion of PDE activity in melanoma cell lines, HaCat and NEM, depends on the pattern of PDE6 expression. c PDE activity was measured in melanoma cell lines transfected with shDNA plasmids. Statistically significant differences (P < 0.05) are marked with an asterisk

Fig. 3

cGMP accumulation and [Ca²⁺]_i mobilization depend on PDE6. Melanoma cell lines with different patterns of PDE6 expression and the HaCaT cell line were cultivated for 24 h with 300 nM ZAP (a) or melanoma cell lines were transfected with shRNA coding plasmids (b) and then the accumulation of cGMP in the cells was measured. c Melanoma cell lines and the HaCaT cell line were cultivated with 300 nM ZAP or transfected with shRNA coding plasmids, and then the [Ca²⁺]_i was measured. Statistically significant differences (P < 0.05) are marked with an asterisk

Fig. 4

Protein expression of transducin, Wnt5a, and FZ2. Protein expression in Y79 (1), NHEM (2), HaCat (3), UKRV-Mel-06 (4), -14a (5), -21 (6), -31 (7), SK-Mel-23 (8), Ma-Mel-04 (9), -05 (10), -11 (11), -12 (12), -17 (13), -21 (14), -37 (15), -52 (16), and MeWo (17)

Fig. 5

Wnt5a activates PDE6 through the FZ2 and transducin cascade. a PDE6 activity was measured in melanoma cell lines with different pattern of PDE6 expression and in HaCaT cell line incubated with or without 20 ng/ml Wnt5a for 1 h. b PDE6 activity after stimulation with Wnt5a was measured in Ma-Mel-04 and Ma-Mel-12 melanoma cells pretreated with 50 ng/μl antibody against FZ2 for 30 min. c PDE6 activity after stimulation with Wnt5a was measured in melanoma cells cultivated with 50 ng/ml of pertussis toxin for 24 h. d PDE6 activity in untrasfected and transfected with shRNA coding plasmids for PDE6-γ, Ma-Mel-04 with and without additional stimulation/inhibition with Wnt5a or PT. Statistically significant differences (P < 0.05) are marked with an asterisk

Fig. 6

Wnt5a stimulates the increase of the [Ca²⁺]_i mobilization in Ma-Mel-04 and Ma-Mel-12 melanoma cells, but not in HaCaT cell line. Untreated and ZAP- or pertussis toxin-treated cells were loaded with fura-2/AM and then 20 ng/ml of Wnt5a was added to start the reaction. Measurement of fluorescence by 340 and 380 nm excitation was performed each 60 s during 15 min, and the ratio 340/380 was calculated. For details, see “Materials and methods”

Fig. 7

A schematic presentation of three types of melanoma cells in regard to PDE6. See text for details