Constitutive somatostatin receptor subtype 2 activity attenuates GH synthesis

Abstract

Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2(WT) cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2(DRY) stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR(2DRY) did not exhibit an enhanced octreotide response as did GpSSTR2(WT) cells; however, both SSTR2(WT)-enhanced yellow fluorescent protein (eYFP) and SSTR2(DRY)-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2(WT) cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2(DRY) cells. In vivo SAHA also increased GH release from GpSSTR2(WT) but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2(WT) compared with that of either GpSSTR(2DRY) or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Figure 1.

Cell characterization. A and B, Comparative characterization of GH, PRL, SSTR1, and SSTR2 gene expression in untreated WT GC and GH₃ cells grown in DMEM enriched with 10% FBS. RNA was collected, and TaqMan qRT PCR was performed. C, Rat GH gene expression in WT GC cells treated with octreotide or vehicle (100 nM; daily for 3 consecutive days), after which RNA was collected and analyzed by TaqMan qRT PCR. D, Rat GH peptide levels measured by RIA in WT GC medium with or without octreotide (100 nM). ***, P < .001; ****, P < .0001. NT, not treated.

Figure 2.

Characterization of stable GC transfectants for ligand-dependent and -independent SSTR2 function. A, SSTR2 mRNA expression in GpCon and GpSSTR2_WT cells and pooled male rat pituitary glands (n = 3). Cells were plated for 48 hours after which RNA collected, and rat pituitary glands were harvested immediately after sacrifice and preserved in RNAlater. Samples were analyzed simultaneously using SSTR2 TaqMan qRT PCR assays. A human SSTR2 TaqMan assay that recognizes both rodent and human SSTR2 was used. B, SRIF inhibition of intracellular cAMP levels in GpCon and GpSSTR2_WT cells cotreated with IBMX and forskolin (1 μM) in DMEM supplemented with 0.3% BSA for 30 min. C, Baseline intracellular cAMP in untreated and IBMX (1 mM)–treated in GpCon and GpSSTR2_WT cells. Cells were then lysed for intracellular cAMP measurements with the LANCE cAMP assay kit. NT, not treated.

Figure 3.

Effects of SSTR2 overexpression on baseline GH expression. A, GH mRNA levels 48 hours after plating. B, GH levels measured in the medium of GpCon and GpSSTR2_WT cells at 24 and 48 hours after plating. C, Mouse serum IGF-I (m-IGF-1) levels in SCID mice before (n = 30) and 30 days after inoculation of GpCon tumors (n = 10) or GpSSTR2_WT tumors (n = 10). A total of 200 000 cells were suspended in 100 μL of PBS and Matrigel and injected sc. Mice were killed 30 days later, tumors and blood were harvested, and serum mouse IGF-I was measured using an ELISA kit. **, P < .01; ****, P < .0001. NT, not treated.

Figure 4.

Effect of DRY motif mutation (R140A) on constitutive SSTR2 activity. A, Baseline medium GH levels 48 and 72 hours after plating. ****, P < .0001 compared with GpCon cells; ##, P < .01 and ####, P < .0001 compared with GpSSTR2_WT cells. B, Baseline GH mRNA levels measured 48 hours after plating by a TaqMan gene expression assay. C, Luciferase activity measured 48 hours after transient transfection of rat GH promoter (−4192/+167) luciferase. D and E, Dose dependence of intracellular cAMP levels with increasing doses of octreotide in the 3 cell lines: octreotide potency (D) and octreotide efficacy (E). F, SSTR2_WT-eYFP and SSTR2_DRY-eYFP internalization. Cells were plated on coverslips 24 hours before transient transfection with either plasmid for 48 hours and then were treated with increasing doses of octreotide for 1 hour or not treated (NT). Cells were then washed, fixed, and visualized with a fluorescent confocal microscope. Green, eYFP; blue, DNA. ****, P < .0001 compared with GpCon cells.

Figure 5.

SAHA effects on somatotroph GH synthesis and secretion. A, Medium mouse GH (mGH) in primary mouse pituitary cultures treated with SAHA (100 nM) for 48 hours. B, Rat GH promoter luciferase activity with increasing SAHA concentration treatment for 48 hours in WT GC cells. ***, P < .001; ****, P < .0001 compared with not treated (NT).

Figure 6.

SAHA effects on GH synthesis and secretion in GpCon and GpSSTR2_WT cells. A, SAHA (5–500 nM, for 48 hours) effects on GH levels in GpCon and GpSSTR2_WT cell medium corrected to WST-1 measurement. B, Western blot analysis for GH and Pou1f1 (Pit-1) compared with actin in GpCon and GpSSTR2_WT cells, treated with SAHA (100 nM) for 48 hours and compared with vehicle-treated cells (not treated [NT]). C, GpCon, GpSSTR2_WT, and GpSSTR2_DRY cells were treated with either SAHA (100 nM) or vehicle for 48 and 72 hours, medium GH levels were measured by RIA, and results are presented as fold change from GH levels in matched vehicle-treated cells (NT). D, Rat GH mRNA levels in GpCon, GpSSTR2_WT, and GpSSTR2_DRY cells, 48 hours after SAHA (100 nM) or vehicle treatment. Cells were lysed, RNA was extracted, and GH mRNA levels were measured by TaqMan gene expression assays. Results are presented as percentage of change from vehicle-treated cells (NT). ****, P < .0001 compared with GpCon cells.

Figure 7.

In vivo GpCon and GpSSTR2_WT tumor-harboring SCID mouse responses to SAHA. A, Tumor-derived rGH level corrected to tumor weight (nanograms per milliliter per milligram; n = 10 per group). B, Serum rPRL levels (nanograms per milliliter; n = 10 per group). C, Pituitary weight (milligrams; n = 5 pre group). D, Mouse pituitary GH (mGH) mRNA corrected for actin mRNA (n = 5 per group). E, Mouse serum IGF-I (mIGF1) levels (nanograms per milliliter; n = 10 per group). F, Mouse liver IGF-I mRNA corrected for actin mRNA (n = 5 per group). Two-month-old male SCID mice were assigned to 2 groups of 20 mice per group: mice harboring sc GpCon and those with GpSSTR2_WT tumors. Seven days after tumor cell inoculation, mice in each group were further divided to 2 subgroups of mice treated either with SAHA (100 mg/kg) or vehicle (Con; DMSO 5%) twice weekly for 2 weeks. Blood, liver, pituitary, and tumor tissue were collected for analysis. Rat GH and PRL levels were measured by RIA, and mouse IGF-I measured by ELISA and qRT PCR by TaqMan assays. *, P < .05; **, P < .01; ****, P < .0001.

Figure 8.

ChIP analysis of endogenous rat GH promoter H3K9 acetylation. A, Rat GH promoter qRT PCR primer location and sequence. B, H3K9 acetylation fold enrichment with respective primer sets in untreated (NT) and SAHA (100 nM)–treated GpCon and GpSSTR2_WT cells. Results depicted are derived from four pooled experiments. C, H3K9 acetylation fold enrichment with each respective primer set in untreated and octreotide (100 nM)–treated GpCon and GpSSTR2_WT cells. Results are derived from 2 pooled experiments. D, H3K9 acetylation fold enrichment with respective primer sets in untreated (NT) and SAHA (100 nM)–treated GpCon, GpSSTR2_WT, and GpSSTR2_DRY cells. Cells were treated every 24 hours with SAHA or octreotide (1 μM) for 48 hours and fixed, and H3K9 acetylation enrichment was analyzed using ChIP-IT Express Enzymatic Magnetic Chromatin Immunoprecipitation and Enzymatic Shearing Kits, following the manufacturer's instructions. *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001.