Figure 5

α-MSH-induced CREB/CRE activation requires EPAC-1/2 and ERK-1/2 activity.

(A) mHypoA-2/10-CRE cells stimulated with 1 μM of α-MSH for various periods of time were subjected to western-blot analysis using either a phospho-specific antibody against p-ERK-1/2 or against the total tubulin protein as a loading control. Data were quantified by densitometry and ratios between p-ERK-1/2 and tubulin signals calculated (N = 13). Asteriks indicate a significant difference using one-way ANOVA followed by Dunnett’s analysis. (B) Cells pretreated with ESI-09 (20 μM) or with the carrier DMSO (0.2) for 30 min and then stimulated with 1 μM α-MSH for 2.5 and 5 min were subjected to western-blot analysis using either a phospho-specific antibody against p-ERK-1/2 or against the total tubulin protein as a loading control. Data of 8 (2.5 min) or 10 (5 min) independent experiments were quantified by densitometry, ratios between p-ERK-1/2 and tubulin signals calculated, α-MSH-induced ERK-1/2 phosphorylation normalized to not stimulated cells. Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test. Asterisks indicate a significant difference using the two-sample Student’s t-test. (C) Cells pretreated with PD-184352 (10 μM) or with the carrier DMSO (0.1%) for 30 min and then stimulated with 1 μM α-MSH for 5 and 10 min were subjected to western-blot analysis using either a phospho-specific antibody against p-CREB (Ser-133) or against the total tubulin protein as a loading control. Data were quantified by densitometry, ratios between p-CREB and tubulin signals calculated, α-MSH-induced ERK-1/2 phosphorylation normalized to not stimulated cells (N = 6). Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test. Asterisks indicate a significant difference using the two-sample Student’s t-test. In (D), mHypoA-2/10-CRE cells or HEK-293-MC4R cells transfected with the CRE reporter plasmid were preincubated with PD-184352 (10 μM) or with the carrier DMSO (0.1%) for 30 min and then stimulated with 1 μM α-MSH for 4 h. Data of 10 (mHypoA-2/10-CRE cells) or 5 (HEK-293-MC4R cells) independent experiments performed in quadruplicates were compiled and shown as inhibition of α-MSH-induced CRE reporter activation in %. Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test.