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Prolactin Induction of Insulin Gene Transcription: Roles of Glucose and Signal Transducer and Activator of Transcription 5¹

Departments of Pediatrics and Cell Biology, Division of Pediatric Endocrinology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Donald E. Fleenor, Box 3080, Duke University Medical Center, Durham, North Carolina 27710. E-mail: fleen001{at}mc.duke.edu

	Abstract

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GH and PRL stimulate insulin production in pancreatic ß-cells through induction of insulin gene transcription. The transcriptional effects of GH are mediated through the binding of signal transducer and activator of transcription-5 (STAT5) to a consensus recognition sequence (TTCnnnGAA) in the rat insulin-1 promoter. In this study we demonstrate that PRL also induces the binding of STAT5 proteins to the rat insulin-1 STAT5 motif. However, the magnitude of binding of STAT5 nuclear proteins, as assessed by electrophoretic mobility shift assays, was only 1/30th that of the binding of the same STAT5 proteins to the ß-casein STAT5 site. The differences in the affinities of the rat insulin-1 and ß-casein STAT5 motifs are explained in part by differences in promoter sequences flanking the STAT5 sites. To assess the importance of the STAT motif in PRL induction of insulin gene transcription, we deleted the STAT5 consensus sequence in the rat insulin 1 promoter, cloned the truncated promoter upstream of the luciferase reporter gene, and transfected the construct into rat insulinoma (INS-1) cells. The transcriptional activity of this construct was compared with that of the wild-type promoter. Although deletion of the STAT5 site in the promoter reduced the basal luciferase activity, the response to PRL was unaffected. PRL also induced transcription of constructs containing the wild-type human insulin promoter or the rat insulin-2 promoter, which contain no classic STAT5 sequences. The transcriptional effect of PRL was manifest even when cells were incubated in glucose-free medium, indicating that the action of the hormone is not mediated solely through changes in glucose uptake or glucose metabolism. To identify PRL-responsive regions of the rat and human insulin promoters, we constructed a series of promoter truncations and assessed their responsiveness to PRL. A PRL-responsive region of the rat insulin-1 promoter was localized between nucleotides -165 and -109. A PRL-responsive region of the human insulin promoter was localized between nucleotides -346 and -250. Additional regions of the human and rat insulin-1 promoters were required for PRL induction of a heterologous, minimal thymidine kinase promoter, suggesting that there are multiple PRL-responsive elements in the insulin genes. These observations suggest a glucose- and STAT5-independent pathway by which PRL may induce insulin gene transcription.

	Introduction

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GH AND THE lactogenic hormones PRL and placental lactogen (PL) stimulate ß-cell proliferation, insulin production, and glucose-dependent insulin secretion in pancreatic islets (1, 2, 3, 4, 5). The effects of the lactogenic and somatogenic hormones on insulin production are mediated through induction of insulin gene transcription. Galsgaard et al. presented evidence that GH stimulation of rat insulin-1 transcription is mediated through the Jak/STAT pathway using a STAT5 DNA-binding motif centered at -327 (6). Like GH, PRL stimulates nuclear translocation of STAT5 in rat insulinoma (INS-1) cells (7); however, the importance of the STAT5 DNA-binding motif in PRL action is unclear. In this series of studies we used electrophoretic mobility shift assays and deletion analysis to assess the importance of the STAT5 motif in PRL induction of rat insulin-1 gene expression. We also examined the effect of PRL on transcription of constructs containing the human insulin promoter or the rat insulin-2 promoter, which contain no consensus STAT5 recognition sequences. To identify PRL-responsive regions in the human and rat insulin-1 promoters, we constructed a series of promoter truncations and assessed their responsiveness to PRL and glucose.

	Materials and Methods

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Cell culture
Rat insulinoma (INS-1) cells were provided by M. Asfari (INSERM, Paris, France). INS-1 cells express both the rat Ins-1 and Ins-2 genes and synthesize and release insulin in response to glucose (8). The cells also express receptors for PRL and GH, which stimulate cellular proliferation, insulin production, glucose uptake, and an increase in glucose transporter 2 messenger RNA levels (9). The cells were used at passages 50–100; there was no change in the behavior of the cells or their responses to PRL or glucose throughout the course of the study.

The cells were maintained at 37 C in 5% CO₂ in RPMI 1640 medium (11.1 mM glucose; Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 1 mM pyruvate, 10 mM HEPES (pH 7), and a 1% antibiotic-antimycotic solution. Cells transfected by electroporation were allowed to recover for 24 h in complete medium and then treated with 1 µg/ml ovine PRL or diluent in basal medium containing DMEM (5.5 mM glucose) supplemented with 0.1% human serum albumin, 10 µg/ml transferrin, 0.1 nM T₃, 50 µM ethanolamine, 50 µM phosphoethanolamine, and 1% antibiotic-antimycotic solution (all from Sigma, St. Louis, MO). Selected experiments were performed in cells incubated for 20 h in glucose-free basal medium supplemented with 3 mM sodium pyruvate. During the incubation no glucose could be detected in this medium using a colorimetric assay (Sigma).

Electrophoretic mobility shift assays
INS-1 cells in basal medium were treated with 1 µg/ml ovine PRL or diluent for 30 min, washed with cold PBS, and harvested by scraping. An extract of nuclear proteins was then prepared by a modification of the method of Ganguly et al. (10). The cells were centrifuged at 200 x g for 5 min, washed with PBS, and resuspended in 10 ml ice-cold buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 1 mM NaF, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, 5 µg/ml aprotinin, and 2 µg/ml leupeptin. After a 10-min incubation on ice, the cells were centrifuged at 10,000 x g for 10 sec. The pelleted nuclei were resuspended in 1 ml buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, 0.5 mM DTT, 0.2 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, 5 µg/ml aprotinin, and 2 µg/ml leupeptin. Nuclear proteins were liberated during a 20-min incubation on ice. The supernatant was clarified by a 2-min centrifugation, and the extract was aliquoted and stored at -80 C until used. Protein concentrations were determined using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Probes were prepared by labeling double-stranded oligonucleotides with [-³²P]ATP and T4 polynucleotide kinase. Binding was performed in a 30-µl volume with 200 pg radiolabeled probe, 10 µg protein, 5 mM Tris (pH 7.9), 15 mM HEPES (pH 7.9), 80 mM KCl, 3.5 mM MgCl₂, 5 mM EDTA, 0.1% Tween 20, 5 mM DTT, 10% glycerol, and 100 µg/ml poly(dI-dC). After incubation at room temperature for 30 min, the products were electrophoresed on 4% polyacrylamide gels containing 2.5% glycerol and 0.25 x TBE (22 mM Tris, 22 mM boric acid, and 0.5 mM EDTA). A 50-fold excess of unlabeled double stranded oligonucleotides was used as cold competitor. For supershift studies, the nuclear proteins were incubated for 30 min at room temperature with polyclonal antisera (200 ng) raised against STAT5a and STAT5b (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) before addition of the probe.

Construction and transient expression of luciferase reporter plasmids
The rat Ins-1 promoter (-382/+172, gift from G. Bell, University of Chicago, Chicago, IL), the rat Ins-2 promoter (-375/+175, generated by PCR from genomic DNA), the human Ins promoter (-502/+242, gift from G. Bell), and a minimal thymidine kinase promoter (-121/+31, generated by PCR from pRL-TK, Promega Corp., Madison, WI) were cloned into the promoterless luciferase reporter plasmid, pGL3 (Promega Corp.). Promoter truncations were generated using convenient restriction sites or by PCR. Each plasmid was verified by sequencing. The electroporation of INS-1 cells and luciferase assays were performed as described previously (11). A Rous sarcoma virus-ß-galactosidase (RSV-ßgal) expression plasmid was included as a transfection control.

Statistical analysis
All experiments were performed in triplicate or quadruplicate and were repeated at least three times. Statistical differences among sample means were tested by ANOVA, followed by the Newman-Keuls test; P < 0.05 was considered significant.

	Results

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Previous studies showed that GH stimulates transcription of the rat insulin 1 gene through induction of binding of STAT5 to its consensus sequence located between nucleotides -331 and -323 of the rat insulin-1 promoter. To determine whether PRL induces insulin gene expression through a similar mechanism, we first examined by electrophoretic mobility shift assays the binding of nuclear extracts from PRL-treated and control INS-1 cells to a radiolabeled, double stranded oligonucleotide that contained the rat insulin-1 STAT5 binding sequence (actgcaactTTCTGGGAAatgaggtgg). As a control for STAT5 binding to the insulin 1 promoter we included a probe that contains the STAT5 sequence (agatTTCTAGGAAttcaatcc) of the bovine ß-casein promoter.

Figure 1 shows that PRL induces binding of INS-1 cell nuclear extracts to both the rat insulin-1 and the ß-casein promoter probes (complex 1); the oligonucleotide probes also form a second complex (2) with a protein that is expressed constitutively. Complex 1 is supershifted with polyclonal antisera to STAT5a and -5b (Fig. 2), indicating that the complex contains STAT5 protein(s). The polyclonal antiserum to STAT5a interacts only with STAT5a (12) (information provided by the manufacturer); thus, the supershifted complex contains STAT5a. On the other hand, the STAT5b antiserum reacts with both STAT5a and -5b; thus, we cannot assess reliably the relative amounts of STAT5a and -5b contained in complex 1.

View larger version (80K): [in this window] [in a new window]   Figure 1. Electrophoretic mobility shift assay comparing binding of INS-1 cell nuclear proteins to the STAT5 sites within the rat insulin-1 and bovine ß-casein promoters. The rat insulin-1 probe contained the sequence 5'-actgcaactTTCTGGGAAatgaggtgg-3' and the ß-casein probe contained the sequence 5'-agatTTCTAGGAAttcaatcc-3'. Nuclear extracts were incubated with equivalent amounts of double stranded probes that were labeled to equivalent specific activities. Similar results were obtained in four separate experiments.

View larger version (73K): [in this window] [in a new window]   Figure 2. Electrophoretic mobility shift assay using antisera to STAT5a and STAT5b. The STAT5a antibody reacts with STAT5a, whereas the STAT5b antibody reacts with both STAT5a and -5b. Complex 1 is supershifted with antisera to STAT5a and –5b, demonstrating that this complex contains STAT5 proteins.

View larger version (14K): [in this window] [in a new window]   Figure 3. Electrophoretic mobility shift assay comparing the binding of INS-1 cell nuclear proteins to STAT5 sequences in the rat insulin-1 promoter (5'-actgcaactTTCTGGGAAatgaggtgg-3'), the bovine ß-casein promoter (5'-agatTTCTAGGAAttcaatcc-3'), and a hybrid motif containing the ß-casein promoter core STAT5 sequence within the rat insulin-1 promoter flanking sequences (5'-actgcaactTTCTAGGAAatgaggtgg-3'). Similar results were obtained in three separate experiments.

To identify PRL-responsive regions in the insulin genes we generated a series of truncations of the human and rat insulin-1 promoters and examined their responses to PRL. Deletion analysis of the human insulin promoter identified a PRL-responsive region between -346 and -250 (Fig. 4). Similarly, a PRL-responsive region in the rat insulin-1 promoter was identified between -165 and -109 (Fig. 5). We noted an increase in PRL response when the promoter was truncated from -306 to -165. It is unclear at this time whether this reflects the presence of a transcriptional repressor. Expression plasmids containing less than 250 bp of the human insulin promoter or less than 109 bp of the rat insulin-1 promoter failed to respond to PRL. A small (30–50%) increase in luciferase activity above baseline noted with these minimal truncated promoters was also observed using the RSV-ßgal expression plasmid, suggesting that this represents a nonspecific effect of PRL. There was no reduction in the basal (uninduced) expression levels of the rat insulin-1 or human insulin promoters after deletion of the PRL-responsive regions (data not shown); thus, the loss of PRL responsiveness did not reflect a nonspecific loss of promoter activity.

View larger version (11K): [in this window] [in a new window]   Figure 4. PRL induction of human insulin promoter truncations driving a luciferase reporter gene. The INS-1 cells were incubated in medium containing 5.5 mM glucose. The effect of PRL on luciferase activity encoded by plasmids H(-501/+242) and H(-346/+242) was statistically significant (P < 0.01). Note, however, the decline in PRL induction of luciferase activity as the promoter is truncated from -346 to -250. The small response to PRL (1.4- to 1.5-fold increase in luciferase activity) observed in experiments using the shorter promoters (-250/+242 or -171/+242) was also observed in experiments using the RSV-ßgal or minimal thymidine kinase expression plasmids. Thus, the 40–50% increase in luciferase activity is probably due to a nonspecific effect of PRL. Note that deletion of the first intron and the majority of the first exon has no effect on PRL responsiveness (4.1-fold increase in luciferase activity using the -501/+14 promoter). Similar results were obtained in three separate experiments.

View larger version (13K): [in this window] [in a new window]   Figure 5. PRL induction of rat insulin-1 promoter truncations driving a luciferase reporter gene. The INS-1 cells were incubated in medium containing 5.5 mM glucose. The effect of PRL on luciferase activity encoded by plasmids R(-382/+172), R(-306/+172), and R(-165/+172) was statistically significant (P < 0.01). Note the decline in PRL induction of luciferase activity as the promoter is truncated from -165 to -109. As noted in Fig. 4, the 60–70% increase in luciferase activity observed in experiments using the shorter promoters (-109/+172 or -82/+172) probably represents a nonspecific effect of PRL. Note again that deletion of the first intron and the majority of the first exon has no effect on PRL responsiveness (4.7-fold increase in luciferase activity using the -170/+12 promoter). Similar results were obtained in six separate experiments.

View larger version (11K): [in this window] [in a new window]   Figure 6. PRL induction of rat insulin-1 promoter truncations in INS-1 cells incubated for 20 h in glucose-free medium. The effect of PRL on luciferase activity encoded by plasmids R(-382/+172), R(-306/+172), and R(-165/+172) was statistically significant (P < 0.01). Similar results were obtained in three separate experiments.

View larger version (25K): [in this window] [in a new window]   Figure 7. PRL induction of various insulin promoter fragments cloned upstream of a minimal thymidine kinase promoter and expressed in INS-1 cells. For each group, n = 4. Note the lack of PRL response using the -501/-171 human insulin promoter fragment and the restoration of responsivity with the -501/-40 fragment. The rat insulin-1 promoter fragment -170/-80 failed to respond specifically to PRL (when corrected for RSV-ßgal activity). However, PRL responsiveness increased with the inclusion of bases -80 to -43.

	Discussion

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A role for STAT5 in PRL action in the pancreatic ß-cell is suggested by several lines of evidence. PRL induces nuclear migration of STAT5a and -5b in rat INS-1 cells and induces binding of INS-1 nuclear proteins to STAT5 sequences in the rat insulin-1 promoter. Nevertheless, the results of our study suggest that the STAT5 motif may not be essential for PRL induction of insulin gene transcription. First, the STAT5 motif in the rat insulin-1 promoter appears to have a lower affinity for STAT5 than the STAT5 motif in the ß-casein promoter. The lower affinity of the insulin-1 STAT5 motif is related in part to differences in the sequences flanking the STAT sites in the insulin-1 and ß-casein genes. Second, deletion of the STAT5 motif at -331/-323 in the rat insulin-1 promoter has no effect on PRL induction of insulin gene transcription. Finally, PRL induces transcription of constructs containing the wild-type human and rat insulin-2 promoters, which have no classic STAT5-binding sequences.

It should be noted that deletion of the rat insulin-1 promoter region containing the STAT5 (and other) binding sequences reduced markedly the basal transcription activity. This finding suggests that this region is essential for full transcription of the insulin gene. We cannot exclude the possibility that ß-cell STAT proteins induced by PRL bind to variant STAT sequences in the insulin promoters or that STAT proteins act as cofactors for other transcription factors. In addition, we cannot exclude the possibility that PRL induction of STAT5 expression activates transcription of other trans-acting factors that regulate insulin gene transcription.

Nevertheless, our findings suggest that PRL induction of insulin gene transcription is mediated through the activation of promoter sequences distinct from or in addition to STAT5. In the human insulin gene, we have identified a PRL- responsive region between nucleotides -346 and -250, and an additional region between -170 and -40 appears to be necessary for full PRL action. In the rat insulin-1 promoter, a PRL-responsive region is localized between nucleotides -165 and -109, whereas a second region between bases -80 and -40 appears necessary for full PRL activity. Thus, there appear to be multiple PRL-responsive elements in the insulin promoter. The PRL-responsive regions in the human and rat insulin promoters share a number of consensus binding sites, including A boxes (ATTA) and E boxes (CAnnTG). However, the promoter elements critical for PRL action have not yet been identified. It is therefore unclear how these sites are activated by PRL binding to pancreatic ß-cells. It is clear that PRL induction of insulin gene transcription is not mediated solely by induction of glucose transport or glucose metabolism, because PRL action is retained in cells incubated in glucose-free medium. Additional studies will be required to identify signaling pathways and promoter elements that are critical for PRL induction of insulin gene transcription. It will be necessary to demonstrate that such pathways operate in primary pancreatic islets, which might behave differently from rat insulinoma cells.

Through induction of pancreatic insulin production and ß-cell proliferation, the lactogens play important roles in carbohydrate metabolism during pregnancy and postnatal life. The induction of rat insulin-2 transcription by PRL is of additional interest because the rat insulin-2 gene is expressed in the yolk sac and fetal liver (31, 32). The yolk sac and fetal liver also express PRL receptors (33, 34, 35), but additional investigations will be necessary to determine whether PRL or PL regulates insulin production in these tissues.

	Footnotes

 
¹ This work was supported by grants from the NICHHD (HD-24192) and Eli Lilly & Co. (both to M.F.).

Received December 15, 2000.

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