FIG. 1.

HDAC inhibitors act synergistically with cAMP agonist to promote somatostatin mRNA accumulation. (A) Lanes 1 to 10, Northern blot assays of total RNA from D5 cells expressing chromosomal copies of the rat somatostatin gene. Cells were treated with control vehicle (C), 10 μM forskolin (F), 15 mM sodium butyrate (But), 100 ng of TSA per ml, 100 nM trapoxin (TPX), or combinations of these reagents, as indicated, for 4 h. RNA was hybridized with a ³²P-labeled somatostatin riboprobe (som) or α-tubulin cDNA probe (tub), as indicated. Lanes 11 to 22, time course analysis of somatostatin mRNA levels in D5 cells treated with forskolin (F), TSA (T), or both agents (FT) for times listed above the lanes. (B) CREB is required for synergistic effects of cAMP agonist and HDAC inhibitors on somatostatin gene expression, as determined by Northern blot assay of total RNA from D5 cells that were transfected with dominant negative A-CREB CMV expression vector (Acidic CREB) or CMV expression vector without insert (Empty Vector) plus RSV-GFP marker. After FACS sorting for transfected cells, GFP-positive cells were treated either without (C) or with 10 μM forskolin (F) and/or TSA (T). (C and D) HDAC inhibitors do not modulate cellular PKA activity or catalytic subunit expression levels. (C) Western blot assay of PKA catalytic subunit levels in D5 cells following treatment with forskolin or forskolin plus TSA for times listed above the lanes; (D) cellular PKA activation assay performed on extracts from D5 cells treated either with forskolin (Forsk) or forskolin plus TSA for various times. The line graph shows percentage of total cellular PKA activated by each treatment as measured by phosphorylation of synthetic Kemptide fragment. Total cellular PKA activity in each sample was estimated by incubating samples with 20 μM dibutyryl-cAMP to dissociate residual PKA holoenzyme.