Phenothiazine neuroleptics signal to the human insulin promoter as revealed by a novel high-throughput screen

Abstract

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic beta-cell. A cell line from human islets in which the expression of insulin and other beta-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of beta-cell differentiated function.

FIG. 1

E47 expression. (A) E47 mRNA level was measured by quantitative RT-PCR in 3 independent preparations of T6PNE cells and human islets from 2 different donors. (B) T6PNE and parental uninfected cells (T6PN) were assayed by immunohistochemistry for E47 (red) in the presence and absence of 4 μM tamoxifen. In the absence of tamoxifen, most cells did not have detectable E47 either in the cytoplasm or the nucleus. Some cells did have relatively weak nuclear E47, presumably due to leakiness of the modified estrogen receptor. With tamoxifen addition, nuclear E47 was greatly increased. DAPI (blue). (C) Insulin expression was responsive to the dose of tamoxifen, reaching a maximum level at 4 μM. Error bars are standard error of the mean (SEM) of 3 replicates. (D) E47 induction by tamoxifen resulted in upregulation of glucokinase, SUR-1, and MafA. Assay was done twice, and GAPDH control was equivalent between samples.

FIG. 2

Activation of E47 induces growth arrest in T6PNE cells. (A) Cells cultured in the absence or presence of tamoxifen (4 μM) were counted on the indicated days, demonstrating substantial growth arrest with the addition of tamoxifen *p < 0.05, **p < 0.001. (B) BrdU (green) and DAPI (blue) staining of T6PNE cells, demonstrating the absence of BrdU incorporation in the presence of tamoxifen (4 μM). (C) Microarray analysis was used to determine genes in T6PNE cells that were affected by E47, with particular attention to CDKIs. T6PNE were treated with tamoxifen (4 μM) and assayed for the expression of 24,000 human genes using Illumina BeadArray microarrays. Each dot on the scatter plot (power function) represents a single gene. The values on the x- and y-axes represent the level of hybridization to the oligonucleotide on the array, which is an indirect measure of the level of mRNA in the sample. p57^Kip2 was undetectable in the absence of tamoxifen and was expressed at an approximately 2000-fold higher level in cells in which E47 was induced by tamoxifen. p21^Cip1 was induced 2-fold.

FIG. 3

Kip2 controls proliferation in T6PNE cells. A retroviral vector expressing green fluorescent protein (GFP) alone (A) or expressing GFP and Kip2 (B) was used to infect T6PNE cells in the absence of tamoxifen. Cell cycle status of GFP-positive cells was assessed by BrdU staining (red). No GFP-BrdU double-positive cells were found in wells infected with the bicistronic GFP-Kip2 vector (B), whereas many cells infected with the GFP control vector did take up BrdU (arrows in A). DAPI is shown in blue. To inhibit Kip2 expression, T6PNE cells were transfected with a plasmid encoding an shKip2 (D) or scrambled (C) RNA along with GFP. The efficacy of the Kip2 shRNA construct was assessed by Western blot analysis (E). In T6PNE cells in which growth was inhibited by tamoxifen (4 μM)–mediated induction of E47, transfection of the shKip2 but not control shRNA (visualized by co-coexpression of GFP) caused cell cycle reentry as determined by colocalization of BrdU (red) and GFP (these are green cells with yellow nuclei; 6 such cells are marked by white arrows in D).

FIG. 4

High-throughput screening for insulin promoter modulators. (A) T6PNE cells infected with a lentiviral vector expressing an insulin promoter-eGFP cassette exhibited a tamoxifen dose-dependent increase in green fluorescence similar to that seen with endogenous insulin mRNA levels (Fig. 1C). (B) Scatter plot of a high-throughput screen with T6PNE expressing the insulin promoter–GFP cassette. In total, 1040 known drugs from the NIH/JDRF Custom Collection were screened in duplicate. Each set of replicates is depicted as either red squares or blue triangles. The duplicates for each compound were assayed on separate 384-well plates, but the values for each compound are shown consecutively for ease of visualization. Duplicate values for the 3 compounds that passed the primary confirmatory and secondary assays (berbamine, chlorpromazine, and ethopropazine) are circled.

FIG. 5

Effect of phenothiazines on insulin promoter activity. (A) Quantitative RT-PCR of green fluorescent protein (GFP) mRNA and endogenous insulin mRNA. Ethopropazine and chlorpromazine increased endogenous insulin mRNA and eGFP mRNA relative to untreated cells. Insulin mRNA levels were normalized to GAPDH. Error bars are SEM of 3 independent experiments. (B) Retesting of all phenothiazines in the NIH/JDRF library for effect on the insulin promoter. Values are expressed as fold change in GFP-positive cells relative to vehicle-treated cells. Each compound is shown at its maximal tolerated dose, which was 20 μM for all compounds except fluphenazine, perphenazine, and trifluoperazine, which were used at 10 μM because of toxicity at higher doses (n = 6; experiment was performed 3 times; error bars are standard error; *p < 0.05, **p < 0.01). (C) Effect of chronic administration of ethopropazine on insulin promoter activity. Ethopropazine (15 μM) was administered to T6PNE cells for 12 days. Media were changed every 3 days. A lower concentration of drug was used than in acute treatment to minimize toxic effects on the cells. Values are expressed as fold change in GFP-positive cells relative to vehicle-treated cells (n = 3; error bars are SEM; *p < 0.05).