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Molecular Endocrinology, doi:10.1210/me.2005-0530
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Molecular Endocrinology 21 (1): 259-273
Copyright © 2007 by The Endocrine Society

Activin A Decreases glucagon and arx Gene Expression in {alpha}-Cell Lines

Aline Mamin and Jacques Philippe

Diabetes Unit, Division of Endocrinology, Diabetes and Nutrition, University Hospital G, 1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Aline Mamin, Diabetes Unit, Division of Endocrinology, Diabetes and Nutrition, University Hospital Geneva, 24, rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: aline.mamin{at}medecine.unige.ch.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activin A is a potent growth and differentiation factor involved in development, differentiation, and physiological functions of the endocrine pancreas; it increases insulin and pax4 gene expression in ß-cells and can induce transdifferentiation of the exocrine acinar cell line AR42J into insulin-producing cells. We show here that Activin A decreases glucagon gene expression in the {alpha}-cell lines InR1G9 and {alpha}TC1 in a dose- and time-dependent manner and that the effect is blocked by Follistatin. This effect is also observed in adult human islets. Glucagon gene expression is inhibited at the transcriptional level by the Smad signaling pathway through the G3 DNA control element. Furthermore, Activin A decreases cell proliferation of InR1G9 and {alpha}TC1 cells as well as cyclin D2 and arx gene expression, whose protein product Arx has been shown to be critical for {alpha}-cell differentiation. Overexpression of Arx in Activin A-treated InR1G9 cells does not prevent the decrease in glucagon gene expression but corrects the inhibition of cell proliferation, indicating that Arx mediates the Activin A effects on the cell cycle. We conclude that Activin A has opposite effects on {alpha}-cells compared with ß-cells, a finding that may have relevance during pancreatic endocrine lineage specification and physiological function of the adult islets.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ACTIVINS ARE MEMBERS of the TGF-ß superfamily and are involved in the functional regulation of many organs, such as the pituitary, as well as in the growth and differentiation of many cell types and in determining embryonic axial patterning and function of foregut-derived organs (1, 2). Activin receptors are expressed in the primordia of foregut organs including the pancreas, stomach, intestine, and lung (3), and Activins are expressed in the pancreatic bud during development (4, 5). Activin A has been proposed to regulate pancreas development and endocrine determination (6, 7). In vitro, Activin A converts the exocrine AR42J cells into insulin-producing cells (8, 9) after inducing the expression of Neurogenin3 (10), a critical factor for endocrine differentiation, and Pax4, a paired homeobox transcription factor critical for ß-cell differentiation (10, 11). Activin A is also capable of inducing differentiation of human fetal pancreatic endocrine cells into ß-cells (12) and has been implicated in ß-cell neogenesis in vivo in the pancreatic duct during pancreatic regeneration (13, 14). Transgenic mice that express a dominant-negative mutant of the Activin type II receptors (15, 16) and mice deficient for Activin receptors (6) display reduced levels of differentiated islet cells. These results indicate that Activins play an important role in regulating differentiation of the endocrine pancreas.

In addition to their role during pancreas development and differentiation, Activins may also be important for glucose homeostasis during adulthood. Activin subunits are indeed present in pancreatic islets (17, 18, 19, 20), and Activin A has been shown to stimulate insulin secretion from both rat and human islets (21, 22) as well as inhibit glucagon secretion (23). In addition to these local effects, Activin A may increase glucose production from the liver (22).

In view of the well-defined actions of Activin A on ß-cell differentiation and function, we investigated its effects on {alpha}-cells using the hamster and mouse glucagon-producing cell lines InR1G9 (24) and {alpha}TC1 (25), respectively. We now report that Activin A markedly and rapidly decreases glucagon gene transcription through the G3 control element in a dose- and time-dependent manner with maximal effects observed at 24 h. This effect is mediated through the Smad signaling pathway. Furthermore, Activin A also profoundly decreases cell proliferation rate of InR1G9 and {alpha}TC1 cells, and this effect is accompanied by decreased expression of cyclin D2 and Arx, a homeobox-containing gene that is critical in promoting the acquisition of {alpha}-cell fate from endocrine progenitors (26, 27). Arx may mediate the Activin A effect on cell proliferation inasmuch as overexpression of Arx in Activin A-treated InR1G9 cells prevents this effect but not the inhibition of glucagon gene expression. We conclude that Activin A regulates glucagon gene expression at the transcriptional level and may be an important factor in the differentiation of endocrine cells by preventing {alpha}-cell proliferation and differentiation through inhibition of cyclin D2 and Arx and, as previously reported, by favoring ß-cell differentiation through stimulation of pax4 and insulin gene expression (10, 11).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Activins and Their Receptors in Islet Cells
The Activin ßA and ßB subunits as well as the type I, II, and IIB receptors have been reported to be expressed in human, mouse, and rat islets. In mouse islets, the ßA and ßB subunits are found in the {alpha}-cells both during development, from embryonic day 16.5, and in the adult (5, 28); in the rat, Activin ßA is essentially found in {alpha}-cells, but also in a few insulin-positive cells during development and in {delta}-cells in the adult (4, 19, 20). In the human pancreas, the ßA subunit is detected in {alpha}-cells and the ßB subunit in {delta}-cells (17). Overall, Activin ßA is found in {alpha}-cells during development and in the adult. To study the effects of Activin A on the glucagon-producing {alpha}-cells, we first investigated the expression of the Activin subunits and their receptor genes in insulin- and glucagon-producing cell lines as well as in mouse islets by RT-PCR, to characterize our experimental model (Fig. 1AGo). The Activin ßA subunit mRNA is present in all four cell lines and is more abundant in ß- compared with {alpha}-cell lines (ßTC vs. {alpha}TC1 and Hit-T15 vs. InR1G9). The Activin ßB subunit mRNA is detected at similar levels in InR1G9, {alpha}-, and ßTC cells but at much lower levels in Hit-T15. Activin receptors I, II, and IIB are expressed in all cell lines tested and in mouse islets at similar levels. We also analyzed the presence of both Activin ßA and ßB subunits by immunofluorescence and detected both of them in all four cell lines, except for the subunit ßB in Hit-T15 (Fig. 1BGo), a result in agreement with mRNA detection. We conclude that the Activin ßA or Activin ßB subunits and their receptors are expressed both in islets as previously reported (21, 22) and in {alpha}- and ß-cell lines.


Figure 1
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  		Fig. 1. Activin Subunits and Activin Receptors Type I, II, and IIB Gene Expression in Islet Cell Lines and Pancreatic Islets

A, Semiquantitative RT-PCR were performed on RNA isolated from mouse islets and pancreatic endocrine cell lines using specific primers and run on a 2% agarose gel. One representative result is shown. The number of cycles corresponding to the illustrated band is indicated above each column (Nb cycles). B, Detection of Activin subunits ßA and ßB by immunofluorescence in InR1G9, Hit T15, and {alpha}- and ßTC cells.

 
Activin A Decreases glucagon mRNA Steady-State Level
Activin A is known to stimulate insulin gene expression (11, 29) and secretion (21, 22) in vitro and in vivo. Furthermore, during development, Activin A has been proposed to promote endocrine cell differentiation through the inhibition of Shh pathway and the formation and migration of pancreatic islets (30), as well as inducing formation and expansion of insulin cells in vitro (12) and in vivo (12, 13, 15, 16). Activin A may also inhibit glucagon secretion at low glucose (23). We thus studied the effects of Activin A on glucagon gene expression and {alpha}-cell proliferation. We first determined the effect of different concentrations of Activin A on glucagon mRNA steady-state levels in InR1G9 and {alpha}TC1 cells at different time points. We performed quantitative RT-PCR with specific primers for glucagon mRNA using TATA-binding protein (TBP) mRNA levels for normalization. Activin A significantly decreased glucagon mRNA levels in a dose-dependent [0.25 nM (by 38% for InR1G9 and 61% for {alpha}TC1), 0.5 nM (by 57% for InR1G9 and 69% for {alpha}TC1), and 2 nM (by 76% for InR1G9 and 74% for {alpha}TC1) (Fig. 2AGo)] and time-dependent manner: 12 h (0.5 nM, 34/65%, 2 nM, 30/51%), 24 h (0.5 nM, 60/73%; 2 nM, 67/75%), and 48 h (0.5 nM, 55/69%; 2 nM, 71/74%) (Fig. 2BGo). Of note, we observed a more rapid effect of Activin A in {alpha}TC1 compared with InR1G9 cells at the lowest concentrations; with the latter cells, we also observed a slight increase of glucagon mRNA levels, although not significant, at the lowest Activin A concentration at 48 h. We then tested the effect of Follistatin, known to specifically antagonize Activin A (5). Activin A (2 nM) and/or Follistatin (100 nM) (corresponding to the ED50 of Activin A effect) were added on both cell lines cells for 24 h. The inhibitory effect of Activin A on glucagon mRNA was abolished under these conditions, whereas Follistatin alone had no effect (Fig. 2CGo). We conclude that Activin A potently and rapidly inhibits glucagon gene expression with maximal effect as early as 24 h. This was not a general inhibitory effect of Activin A on RNA synthesis inasmuch as total RNA levels from control and Activin A-treated cells were not statistically different at 12 and 24 h, and specific mRNAs coding for transcription factors such as Pax6 or Foxa2 were not affected by Activin A treatment (data not shown).


Figure 2
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  		Fig. 2. Activin A Decreases glucagon mRNA Steady-State Levels

A and B, InR1G9 and {alpha}TC1 cells were treated with increasing concentrations of Activin A for 48 h (A) and with 0.5 and 2 nM at different time points (B). C, Both cell lines were treated with Activin A (2 nM), Follistatin (100 nM), or both. Gluc, glucagon. D, Human islets were treated with or without 2 nM Activin A or 100 nM Follistatin. Total RNA of three independent experiments was isolated from treated and control cells, and glucagon or insulin mRNA steady-state levels were measured by quantitative RT-PCR (three PCR for each reverse transcription) and corrected by the TBP mRNA level as a control. Results are mean values ± SEM. Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.02.

 
To investigate the effects of Activin A in adult islets, we isolated human islets that express Activin I, II, and IIB receptors as well as the Activin ßA and ßB subunits (17), treated them with 2 nM for 48 h, and measured glucagon mRNA levels (Fig. 2DGo). Glucagon was slightly but significantly diminished by Activin A treatment by 12%. As a control, insulin mRNA levels, which are known to be increased by Activin A treatment, were measured and found to be increased by 35%. The quantitatively mild effects observed in islets compared with cultured cells may be due to the relatively high abundance of endogenous Activin A. We thus treated the islets for 48 h with 100 nM Follistatin, to antagonize a putative effect of endogenous Activin A. Glucagon mRNA levels were slightly increased but not significantly, suggesting alternative explanations for the clear difference of the Activin effects between cell lines and islets such as the opposing influence of other peptides or effects related to cell proliferation observed in cell lines.

Activin A Decreases glucagon Gene Expression through the G3 Control Element
The first –2.5 kb of the glucagon gene promoter are sufficient for cell-specific and maximal expression of the glucagon gene (31). To investigate the mechanisms by which Activin A decreases glucagon gene expression, we first assessed whether Activin decreases transcription. We performed transient transfections with the wild-type 5'-deleted rat glucagon gene promoter or with the promoter containing various deletions or point mutations in InR1G9 and {alpha}TC1 cells treated or not with 1 nM Activin A for 48 h. Chloramphenicol acetyltransferase (CAT) assays were then performed. In InR1G9 cells, Activin A markedly decreased promoter activity up to 89% with –2.5GluCAT and 63% with –292GluCAT (Fig. 3AGo). This effect disappeared when the promoter was truncated to –200 bp upstream the TATA-box, indicating that one or more Activin A-responsive elements lie upstream of –200 bp. Similar results were observed in {alpha}TC1 cells (decreased promoter activity up to 35% with –2.5GluCAT and 61% with –292GluCAT). No significant decrease was observed with –175 and –31GluCAT promoters (Fig. 3BGo). However, due to much lower transfection efficiency in {alpha}TC1 cells compared with InR1G9 cells, the inhibitory effects of Activin A on short promoters or promoters containing point mutations could not be reliably analyzed in {alpha}TC1 cells. Because the major drop in the effect of Activin A occurred between –292 and –200 bp, we focused more detailed analyses primarily on this promoter region in InR1G9 cells. Between –292 and –200 bp, G3 is a well-defined box, where Pax6, Pbx1, and Prep1 have been shown to bind and regulate glucagon gene expression (32, 33). To study whether G3 is involved in the Activin A response, we transfected InR1G9 cells with the G3 and G2 boxes or the G3 box alone linked to –138 bp of the glucagon gene promoter. We observed a 76% decrease of the transcriptional activity with G3–138 and a 58% decrease with G3G2–138, whereas no effect was seen with the G2 box linked to –138CAT or –138CAT alone (Fig. 3CGo). To more precisely define the responding site within the G3 control element, we performed transfection experiments with DNA constructs carrying point mutations in the G3 box, one in the distal part where Pax6 binds (M6), and the other on the proximal part where Pbx1/Prep1 interact (M3) (33, 34). Mutation M6, but not M3, completely abolished the Activin A effect on glucagon transcription (Fig. 3DGo) either when using G3M6–138 or -292 containing the same mutation (–292G3M6), indicating that the distal part of G3 is involved in the effects of Activin A on glucagon gene expression.


Figure 3
Figure 3
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  		Fig. 3. Activin A Effect Is Mediated by the G3 Control Element of the glucagon Gene Promoter

A schematic representation of the rat glucagon gene promoter (with its previously defined DNA control elements represented by boxes) and of the glucagon reporter gene constructs are shown above each panel. Cells were cotransfected with 3 µg of the indicated reporter plasmids and 1 nM Activin A was added for 48 h. A, 5'-Deleted constructs of the glucagon gene promoter were used to precisely define the smallest promoter sequence mediating the Activin A effect in both InR1G9 (A) and {alpha}TC1 cells (B). Cytomegalovirus (CMV)-CAT represents the positive control and is not affected by Activin A. Experiments were performed on three independent times. C, Specific DNA control elements of the promoter were cloned 5' of the –138-bp sequence to localize sequences mediating Activin A effects. Experiments were performed in InR1G9 cells on four independent times. D, Mutational analyses of the G3 control element were performed to precisely define the DNA sequences involved in the Activin A effect. Experiments were performed in InR1G9 cells on four independent times. Results are expressed as relative CAT activities corrected by transfection efficiency (placental alkaline phosphatase activity) (mean ± SEM). Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.02.

 
Pax6 Protein Level and Binding Activity Are Not Affected in Cells Treated with Activin A
Pax6 is known to bind the distal part of the G3 element of the glucagon gene promoter and to strongly activate gene expression (33). The M6 mutation that abolishes the Activin A effect on glucagon gene transcription prevents Pax6 binding (33). We therefore tested whether Pax6 protein levels or binding activity were affected in InR1G9 cells treated with Activin A. We thus first performed Western blot analyses using 10 µg of nuclear extracts, a Pax6 antibody recognizing the p46 and p48 forms and a TFIIE-{alpha} antibody for control (Fig. 4AGo). There was no significant decrease of Pax6-p48 or -p46 upon treatment with Activin A. The same results were obtained with whole-cell extracts (data not shown). These results were strengthened by the finding that Pax6 mRNA was also unchanged between Activin A-treated and untreated cells (data not shown). We then studied the binding capacity of Pax6 on G3 by EMSA analyses using 10 µg of nuclear extracts (Fig. 4BGo). No difference was observed in the intensity of the Pax6 DNA complex between Activin A-treated and untreated cells.


Figure 4
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  		Fig. 4. Pax6 Protein Level and Binding Activity Are Not Affected in Cells Treated with Activin

A, Western analyses of the p48 and p46 forms of Pax6 from nuclear extracts (NE) from InR1G9 cells treated or not with Activin A (1 and 2 nM) for 48 h. Results were corrected by the internal control TFIIE-{alpha}. One representative experiment of five is represented. B, EMSA experiment with NE from InR1G9 cells treated or not with Activin A (1 and 2 nM) for 48 h. The G3 box of the glucagon gene promoter, known to bind Pax6 and Pbx1/Prep1, was used as a probe. One representative experiment of seven is represented.

 
The Smad pathway is the most common and well-defined pathway between the binding of Activin A to its receptors and the resulting effects on gene expression (35). To test the involvement of Smad proteins in the regulation of glucagon gene transcription, we transfected InR1G9 cells with small interfering RNA (siRNA) directed against hamster Smad4, the common Smad partner for Smad2 or Smad3 in the canonical Smad pathway. Three different siRNA were transfected together, twice during an interval of 24 h, at a final concentration of 300 nM (100 nM each). Activin A was added after the second transfection for a further period of 24 h. Endogenous Smad4 expression was decreased of 86.4% in the absence or 93.7% in the presence of 2 nM Activin (Fig. 5AGo). Transfection of scramble siRNA in the same conditions did not affect Smad4 level. TFIIE-{alpha} was used for quantification. We also quantified Smad2 protein levels by Western blot analyses, which was not affected by treatment with siRNA against Smad4, indicating specificity of siRNA (data not shown). Glucagon mRNA level was then measured and found to be decreased to 50% after treatment with Activin A when cells were not transfected or transfected with scramble siRNA. By contrast, when endogenous Smad4 was decreased by siRNA, the effect of Activin A was abolished. We conclude that Activin A acts through the Smad pathway to affect glucagon gene expression.


Figure 5
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  		Fig. 5. Smad Proteins Mediate the Effect of Activin A on glucagon Gene Expression

InR1G9 cells were transiently transfected with siRNA directed against hamster-Smad4 mRNA (or with scramble siRNA) in the presence or absence of 2 nM Activin A for 24 h. A, Nuclear extracts were isolated and analyzed for endogenous Smad4 levels by Western blots (a representative experiment is shown). Results were corrected by TFIIE-{alpha} as a control. The mean of three experiments ± SEM is shown. B, Total RNA was isolated from InR1G9 cells treated with either scramble siRNA or siRNA directed against Smad4, and glucagon (Gluc) mRNA steady-state level was measured by quantitative RT-PCR and corrected by the TBP expression as a control (mean of three experiments ± SEM). Asterisks indicate statistical significance as follows: **, P < 0.02.

 
Apart from the Smad pathway, TGF-ß/Activins can signal through non-Smad pathways, such as ERK, p38 MAPK and p42/44 MAPK, Ras GTPase, Rho-like GTPase, and protein phosphatase 2A (35). To test a potential involvement of p38 or p42/44 MAPK in the Activin A effect, we measured phosphorylation of the latter two MAPK in InR1G9 after treatment with 2 nM Activin A for 5, 10, 30, 60, and 120 min; MAPK are known to be stimulated rapidly and transiently. p42/44 and p38 were not stimulated by Activin A and inhibitors of these kinases did not prevent Activin A effects (data not shown). Thus, these alternative pathways are not involved in the Activin A effect on glucagon mRNA levels.

Activin A Affects Cell Proliferation But Not Survival
To investigate whether Activin A affects cell death and proliferation, we first performed FACS assay using 7-amino-actinomycin D (7-AAD) to determine cell apoptosis. Treatment of InR1G9 or {alpha}TC1 cells with up to 2 nM Activin A for 24 and 48 h did not affect the rate of cell apoptosis, whereas treatment with H2O2, a known apoptotic agent, led to a 9- and 2-fold increase in InR1G9 and {alpha}TC1 cell apoptosis, respectively (Fig. 6AGo). To assess cell proliferation, we performed 5-bromo-2'-deoxyuridine (BrdU) immunostaining at 48 h with 2 nM Activin A treatment. We observed a 36% decrease of BrdU immunostaining for {alpha}TC1 cells and 44% for InR1G9 cells (Fig. 6BGo). Furthermore, we performed total cell count of InR1G9 or {alpha}TC1 cells at 48 h with and without 2 nM of Activin A and observed a marked decrease in cell number at 48 h after treatment with Activin A. We extrapolated the doubling time using cell count after 48 h. Normal doubling time of untreated InR1G9 and {alpha}TC1 cells was 44 and 63 h, respectively, whereas in the presence of Activin A, it was increased to 76 and 85 h (data not shown). We conclude that Activin A affects cell proliferation but not cell death. To more precisely define the cell cycle phase affected by Activin A, we performed FACS analysis using BrdU as an S phase marker and propidium iodide as a G phase marker. In both InR1G9 and {alpha}TC1 cells, we observed an accumulation in the G1 phase (+8.3% for {alpha}TC1, +3.5% for InR1G9), with a concomitant decrease in the S and G2 phases (Fig. 6CGo). Although weak, these changes are significant and indicate that Activin A provokes a cell cycle arrest in the G1 phase. Because cyclin D2 plays an essential and specific role in the G1 phase and in the replication of terminally differentiated {alpha}- and ß-cells in vivo (36), we quantified cyclin D2 mRNA in Activin A-treated and untreated InR1G9 or {alpha}TC1 cells. In the presence of 2 nM Activin A, cyclin D2 mRNA decreased by 62% at 48 h for InR1G9 cells and 34% for {alpha}TC1 cells (Fig. 6DGo). We also measured mRNA levels of other proteins that may affect the cell cycle, such as c-Myc, CDC25A, and p27kip, but none of them was found to be changed by Activin A treatment (data not shown).


Figure 6
Figure 6
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  		Fig. 6. Activin A Does Not Affect the Apoptotic Rate but Significantly Decreases InR1G9 and {alpha}TC Cell Proliferation

A, FACS analyses using the apoptotic dye 7-AAD. InR1G9 and {alpha}TC1 cells were treated with Activin A for 24 or 48 h, with 0.5 or 2 nM Activin A. Results are expressed as a percentage of apoptotic and necrotic cells to total cell number (10,000 cells; three independent experiments). Treatment with H2O2 was used as positive control. Results are expressed as mean values ± SEM. B, BrdU immunostaining (green) was performed after 48 h of 2 nM Activin A treatment on InR1G9 or {alpha}TC1 cells. Propidium iodide (PI; red) was used for total cell staining. Proliferating cells are visualized in yellow (one representative picture; left panel). A minimum of 200 cells were counted in three different experiments, and means were used for graphic representation (right panel). Results are represented as the percentage of BrdU-positive cells to total cell number (mean values ± SEM). C, FACS analyses after BrdU and PI staining. S phase is visualized by BrdU staining, G1 and G2 phases can be separated by PI staining and DNA content. Three independent experiments were performed. Means ± SEM are represented. D, Semiquantitative RT-PCR (35 cycles for cyclin D2 and 25 cycles for actin) were carried out on RNA isolated from either InR1G9 or {alpha}TC1 cells treated (+) or not (–) with Activin A (2 nM) for 48 h and subjected to 2% agarose gel analysis. Left panel shows one individual experiment. Quantification was performed using the ImageQuant application (Molecular Dynamics) and expressed as relative cyclin D2 mRNA abundance (mean ± SEM). Asterisks indicate statistical significance as follows: **, P < 0.02.

 
Arx mRNA Levels Decrease in Response to Activin A
Arx, a homeobox-containing gene, has been recently shown to be critical for {alpha}-cell development (27). Indeed, in mutant mice with no Arx proteins, {alpha}-cells are absent, suggesting that Arx may be involved in {alpha}-cell differentiation, {alpha}-cell proliferation, and/or glucagon gene expression. Because Activin A affects glucagon gene expression and glucagon-producing cell proliferation, we investigated the effects of Activin A on arx mRNA levels. We performed semiquantitative RT-PCR on InR1G9 or {alpha}TC1 cells treated or not with Activin A. Activin A decreased arx mRNA at both 24 and 48 h by 66 and 41%, respectively, for InR1G9, and 40 and 21% for {alpha}TC1 cells (Fig. 7AGo). To examine the relationship between the decrease in arx and glucagon gene expression as well as cell proliferation, we overexpressed Arx in InR1G9 cells and measured glucagon mRNA levels and cell proliferation in the presence or absence of Activin A. In the absence of Arx antibodies, we quantified cell transfection efficiency with a green fluorescent protein reporter plasmid; 60–70% of InR1G9 cells were transfected in these experiments. Transfection of the arx cDNA resulted in a 20-fold increase in arx mRNA compared with mock-transfected cells (Fig. 7BGo). However, Activin A effects on glucagon mRNA levels were unaffected, suggesting that Arx does not mediate Activin effects on glucagon gene expression (Fig. 7CGo). We then measured cell proliferation by BrdU staining in arx cDNA- and mock-transfected cells (Fig. 7DGo). Activin A decreased the number of proliferating cells from 45–32% as well as Arx mRNA levels in control cells (Fig. 7AGo), whereas it had no effect when Arx was overexpressed in control cells. We conclude that Arx is a mediator of the effect of Activin A on cell proliferation.


Figure 7
Figure 7
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  		Fig. 7. Activin A Diminishes arx mRNA Levels, and Overexpression of Arx in Activin A-Treated Cells Abolishes Activin A Effect on Proliferation But Not on Glucagon mRNA Levels

A, Semiquantitative RT-PCR (35 cycles for Arx and 25 cycles for Actin) were carried out on RNA isolated from either InR1G9 or {alpha}TC1 cells treated or not with 2 nM Activin A for 24 or 48 h and subjected to agarose gel analyses. Left panel shows one representative experiment. Quantification was performed using the ImageQuant application (Molecular Dynamics) and expressed as relative arx mRNA abundance (right panel; mean ± SEM). B–D, InR1G9 cells transfected with the mouse arx cDNA or the empty vector and treated or not with 2 nM Activin A for 48 h were analyzed for overexpression of Arx [semiquantitative RT-PCR (32 cycles)] (B), relative glucagon (Gluc) mRNA levels (quantitative PCR) (C), and cell proliferation rate (BrdU staining) (D), as described previously. Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.02.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Activins belong to the TGF-ß superfamily of growth and differentiation factors and are critical for the regulation of many systems including reproduction, wound repair, liver regeneration, tubular morphogenesis, neuronal development, and pancreatic endocrine and exocrine differentiation (2, 37).

We show here that Activin A markedly inhibits glucagon gene expression in a dose- and time-dependent manner reaching maximal effects at 2 nM and 24 h, respectively, in two different glucagon-producing cell lines, InRIG9 and {alpha}TC1. This inhibition is completely abolished by Follistatin, which binds Activin A with high affinity and neutralizes its interaction with type II receptors, emphasizing the specificity of the effect. In adult islets, Activin A also decreases glucagon mRNA levels while increasing insulin mRNA levels. We found that the regulation of the glucagon gene by Activin A is mediated, at least partly, by a transcriptional effect through the G3 DNA element, which interacts with Pax6 on its distal part and Pbx1/Prep1 on its proximal part (32, 34). It is possible, however, that Activin A represses glucagon gene expression through additional DNA control elements inasmuch as deletional and mutational analyses of the glucagon gene promoter suggested that the full effect of Activin A observed with –2.5 kb of the promoter was somewhat decreased with shorter constructs or with the mutated G3 constructs; more detailed promoter analyses between –292 and –2500 bp will have to be performed. Of interest, Activin has been previously shown to negatively regulate pax6 gene expression in the neural plate (38) and to activate the FSH ß-subunit gene through the interactions of Smad 2 and 3 with Pbx1 and Prep1 (39). Mutational analyses of G3 revealed that mutation of the Pax6 binding site, which prevents Pax6 binding (34), completely abolished the Activin A effect, whereas mutation of the proximal Pbx1/Prep1 binding site was silent, clearly indicating that the Pax6 binding site is involved in mediating the Activin A inhibition.

Rather than affecting Pax6 protein abundance or binding activity, we find that Activin A inhibits glucagon gene expression by the activation of the Smad-signaling pathway. Although Pax6 has been reported to bind both the G3 and G1 elements of the glucagon gene promoter (33, 34), we have not observed any change in transcription of –138GluCAT, which contains G1; basal expression of –138GluCAT being low, low-level inhibition by Activin A might not have been detected. However, G2–138GluCAT activity was not regulated by Activin A either, suggesting that G1, which binds several transcription factors in addition to Pax6, is not responsive to Activin A.

An additional effect of Activin A that we observed on glucagon-producing cells is a decrease in cell proliferation. This decrease was rather marked inasmuch as after 48 h in culture, the number of InR1G9 cells in the presence of 1 nM Activin A increased only by 25%, whereas it more than doubled in the controls compared with time 0. Activin A nearly doubled the doubling time of InR1G9 cells at 2 nM. Cell number at 24 h, however, was not significantly different from the control in the presence of Activin A, and thus effects on cell proliferation could not account for the marked decrease in glucagon gene expression. In {alpha}TC1 cells, the doubling time was also prolonged after Activin A treatment, although slightly less compared with InR1G9 cells. Although the mechanism by which Activin A decreases cell proliferation rate remains unknown, this effect was accompanied by the repression of cyclin D2, a key component of the cell cycle machinery that associates with partner cyclin-dependent kinase CDK4 and CDK6 to drive cells into S phase (40), and is involved in the replication of terminally differentiated {alpha}- and ß-cells in vivo (36). Furthermore, Activin A also repressed arx gene expression. Arx, a homeobox-containing gene localized on the X chromosome, was demonstrated to be critical in the {alpha}-cell specification process. Mice deficient for Arx lack mature {alpha}-cells, whereas the numbers of ß- and {delta}-cells are increased (26). In these mice, the development of the early glucagon-expressing cells remains unaffected, but mature {alpha}-cells fail to appear at later stages. These data indicate that the loss of Arx leads to the replacement at least partially of mature {alpha}-cells by ß- and {delta}-cells, suggesting that Arx promotes the acquisition of the {alpha}-cell fate by endocrine progenitors and does so by antagonizing ß- and {delta}-cell commitment. We thus hypothesize that the inhibition of arx gene expression by Activin A may underlie the repression of {alpha}-cell differentiation. Our results indicate that the decrease in cell proliferation but not in glucagon gene expression observed with Activin A is mediated, at least in part, by Arx; the mechanisms by which Arx affects cell proliferation will be critical to better understand {alpha}-cell physiology.

Activins, like TGF-ß, can inhibit or stimulate cell growth (36); for instance, treatment of AR42J exocrine cells with Activin A results in growth arrest (8, 9, 10), whereas it can stimulate ß-cell neogenesis in vitro and in vivo, along with Betacelluline (12, 13, 15, 16). Furthermore, Activin A appears to favor ß-cell proliferation and differentiation at the expense of the {alpha}-cells (41). Activin A indeed induces differentiation of AR42J cells into insulin-producing cells and activates Pax4 gene expression both in these cells and in insulin-producing cells (8, 9, 10, 11). In addition, in the developing chick dorsal pancreatic bud, Activin treatment resulted in a relative increase of insulin cells by 60–90%, depending on the concentration, at the expense of glucagon cells (42).

It has been proposed that, during early pancreas morphogenesis, precursor cells express both Arx and Pax4 transcription factors (26). The selective activation of Arx or Pax4 would then promote an {alpha}-cell specification or a ß/{delta}-cell fate, respectively. The {alpha}-cell or ß/{delta}-cell fate will be insured by the repressive effect of Arx on Pax4 gene transcription through the interaction with its promoter, whereas Pax4 antagonizes Arx gene transcription by binding to a selective 3' enhancer (26). The mechanisms by which the selective activation of the arx or pax4 genes occurs are not known but are likely to result from one or more soluble factors. Activin A might play such a role by favoring the ß/{delta}-cell fate at the expense of the {alpha}-cell. Another member of the TGF-ß signaling pathway, growth differentiation factor 11, has recently been shown to negatively regulate the production of Ngn3-positive pancreatic islet precursor cells and to be required for ß-cell maturation and ß-cell mass (43). Indeed, mice lacking growth differentiation factor 11 have a 50% reduction in ß-cell numbers, evidence of arrested ß-cell development and a 3-fold increase in {alpha}-cell mass. Whether different TGF-ß like factors act at different times during development or whether some of these factors are redundant awaits further study. Differentiation of liver cells during development has been proposed to be regulated by similar mechanisms; indeed, a gradient of Activins/TGF-ß signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages (44).

Interestingly, Activins have been reported to stimulate insulin and inhibit glucagon secretion in islets (21, 22, 23), suggesting that the Activin signal is not only crucial to the developing pancreas but might be also involved in the maintenance of the normal function of {alpha}- and ß-cells. The Activin ßA subunit is produced by the {alpha}-cell, whereas the ßB subunit is found in both {alpha}- and ß-cells (6, 14, 17, 18, 19, 20); the Activin receptors are also present in the islets as well as Follistatin (5, 19). A regulatory network between Activins and Follistatin similar to that identified in the pituitary to regulate reproduction may thus be involved in glucose homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
Recombinant human Activin A (338-AC) and human Follistatin (669-FO) were purchased in R&D Systems Europe (Abingdon, UK); kinase inhibitor SB203580 was purchased from Calbiochem-Merck (Darmstadt, Germany) and U0126 from BioMol (Plymouth Meeting, PA).

Cell Culture and DNA Transfection
The glucagon-producing hamster InR1G9 (24) and mouse {alpha}TC1 (25) were grown in RPMI 1640 medium (R-6504; Sigma, Basel, Switzerland) containing 11 mM glucose and supplemented with 2 g/liter NaHCO3, 5% fetal calf serum, 5% newborn calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Mouse islets were isolated from exocrine pancreas by collagenase digestion and purification on an histopaque gradient. Human islets were kindly provided by Dr. D. Bosco (Division of Visceral and Transplant Surgery, University Hospital, Geneva, Switzerland) and isolated as described previously (45, 46, 47); they were cultured in RPMI 1640 medium (R-1383; Sigma, Basel, Switzerland) supplemented with 2 mM glucose, 2 g/liter NaHCO3, 5% fetal calf serum, 5% newborn calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfections of InR1G9 and {alpha}TC1 cells for CAT assays were done using the diethylaminoethyl-dextran method as described previously (48) using 3 µg of reporter plasmid and 0.15 µg of RSV-Luc, a plasmid containing the luciferase gene driven by simian virus 40 promoter, added to monitor transfection efficiency. Reporter plasmids consisted in the CAT reporter gene driven by different fragments of the wild-type rat glucagon gene promoter [–2.5GluCAT, –292GluCAT, –200GluCAT, –175GluCAT, –138GluCAT, G3–138GluCAT, G2–138GluCAT, G3G2–138GluCAT, –31GluCAT (49)], or mutated in the Pax6 or Pbx1/Prep1 binding sites on G3 (–138G3M6GluCAT or –292G3M6GluCAT and –138G3M3GluCAT, respectively) (34) as well as the cytomegalovirus promoter as negative control. Cells extracts were prepared 48 h after transfection and analyzed for CAT and luciferase activities (50). Quantification of acetylated and nonacetylated forms was done with a Typhoon 9400 (Molecular Dynamics, Sunnyvale, CA). Raw data are presented as CAT activity normalized by luciferase activity and are the mean ± SEM of at least three experiments carried out in duplicate. Transfection of the mouse arx cDNA [kind gift of Dr. K. Kitamura (Mitsubishi Kasei Institute of Life Science, Tokyo, Japan); subcloned in the pSG5 vector (Stratagene, La Jolla, CA)] in InR1G9 cells, was done using Transfectin (Bio-Rad Laboratories, Reinach, Switzerland) with 4 µg cDNA and 5 µl Tranfectin in six-well plates.

siRNA Assays
Three different Stealth siRNA were designed by Invitrogen (Carlsbad, CA) against homologous sequences between mouse and hamster Smad4. Appropriate scrambles were obtained at the same time (percentage of GC content identical with Smad4 siRNA). Transfections were performed twice sequentially using Lipofectamine 2000 (Invitrogen) as recommended by supplier.

Total Cell Count, BrdU, and Apoptosis Measurement
To quantify the rate of apoptosis, unfixed cells were stained with 7-AAD (A9400; Sigma, Basel, Switzerland) as described (51) and passed through FACS analyses (FACScan). Total cell count was performed using a Neubauer modified cell. BrdU immunostaining (BD Biosciences, San Jose, CA) was done as described by supplier after 48 h of 2 nM Activin treatment. Propidium iodide (1845; Sigma) was used for nuclear staining of total cell number.

RT-PCR Analyses
Total RNA was isolated from adult mouse and human islets, ßTC [mouse insulin-producing (52)], {alpha}TC1 [mouse glucagon-producing (25)], Hit-T15 [hamster insulin-producing (52)], and InR1G9 [hamster glucagon-producing (25)] cell lines using either Trizol reagent (Invitrogen) for cell lines according to the supplier or an RNA isolation kit (RNeasy kit; Qiagen, Basel, Switzerland) for islets. First-strand cDNA synthesis was performed with 10 ng/µl random hexamer primers (Promega, Madison, WI) and SuperScript II Reverse Transcriptase (Invitrogen). For semiquantitative PCR, one tenth of the resulting cDNA was used with the Goldstar DNA Polymerase (Eurogenetec, Seraing, Belgium). PCR results were quantified in the linear part of the amplification curve, as determined by sampling every two cycles. Actin was used as an internal PCR efficiency control because it was not influenced by cell treatments. For quantitative PCR, one hundredth of the resulting cDNA was used with Quantitect SYBR Green PCR kit (Qiagen), in a Light-Cycler (Roche Diagnostics, Rotkreuz, Switzerland). TBP was used as an internal PCR efficiency control, because it was not influenced by cell treatments. Primers are listed in Table 1Go.


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  		Table 1. List of PCR Primers, with Sequence, Annealing Temperature, and Elongation Time

 
Imunocytochemistry
Cell lines were cultured on glass slides overnight and immunocytochemistry was performed for Activin ßA and ßB subunits with primary antibodies from Abcam (Cambridge, MA) [mouse monoclonal antibody to inhibin ßA (no. ab27945) and inhibin ßB (no. ab27946), respectively] as recommended by the supplier, and with Alexa 568 goat antimouse IgG (Invitrogen) as secondary fluorescent antibody.

EMSAs
Whole-cell or nuclear extracts from InR1G9 cells treated or not with Activin A were prepared according to Kumar and Chambon (53) and Schreiber et al. (54). EMSAs were performed as described previously (55) using 10–20 µg of nuclear extracts and oligonucleotide containing the site G3 (5'-GCTGAAGTAGTTTTTCACGCCTGACTGAGATTGAAGGGTGTATTTC) of the rat glucagon gene, labeled with [{gamma}-32P]dATP and polynucleotide kinase (NEB, Frankfurt, Germany).

Kinase Assay
Cells were scraped into 500 µl of lysis buffer containing 50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 40 mM ß-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 µM Na3VO4, 0.3 mM leupeptin, 1 µM pepstatin A, 1 mM phenylmethylsulfonylfluoride, and 100 nM okadaic acid (pH 7.4). After three freezing/thawing cycles, the homogenates were centrifuged for 10 min at 14,000 rpm at 4 C, and supernatants were collected. Whole-cell extracts were spotted on a Western blot and p38 or p42/44 MAPK phosphorylated or not were revealed using the following primary antibodies: phosphorylated p38 MAPK and phosphorylated p42/44 (Cell Signaling Technology, Denvers, MA), total p42/44 and total p38 MAPK (Santa Cruz Biotechnology, LabForce, Nunningen, Switzerland). The secondary antibody was a horseradish peroxidase-labeled goat antirabbit antibodies (CovalAb, Oullins, France), used in the conditions described by the supplier.

Western Blot Analyses
Whole-cell or nuclear extracts were isolated as described (53, 54) from InR1G9 cells, treated or not with Activin A. Ten to 20 µg of each protein extract were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred electrophoretically to polyvinylidene difluoride membranes. Immunoblotting was performed with polyclonal antibodies to rabbit Pax6 diluted 1/500 [kind gift from S. Saule (Centre National de la Recherche Scientifique, Unité Mixte de Recherche 146, Institut Curie, Orsay, France)] or Smad4 monoclonal mouse antibody (sc-7966; Santa Cruz Biotechnology, Santa Cruz, CA) and IgG antisera conjugated with horseradish peroxidase diluted 1/2000 (antirabbit; Covalab, Lyon, France; antimouse: Santa Cruz Biotechnology, sc-2005). The signal was detected with Super Signal West Pico Trial kit (Pierce, Rockford, IL). Protein loading was normalized by immunodetection of rabbit TF2IIE-{alpha} diluted 1/500 (Santa Cruz Biotechnology). At least three independent experiments were performed with nuclear/whole-cell extracts.

Data Analyses
Data are presented as mean ± SEM, and statistical significance was tested by Student’s t test on raw data for mRNA analyses and on logarithm of raw data for transfection analyses. Asterisks indicate statistical significance as follows: *, P < 0.05; **, P < 0.02.


   ACKNOWLEDGMENTS
 
We thank S. Saule for generously providing Pax6 antibodies and Dr. K. Kitamura for providing the mouse arx cDNA.


   FOOTNOTES
 
This work was supported by Swiss National Fund 3200-065162.

A.M. and J.P. have nothing to declare.

First Published Online September 20, 2006

Abbreviations: 7-AAD, 7-Amino-actinomycin D; BrdU, 5-bromo-2'-deoxyuridine; CAT, chloramphenicol acetyltransferase; siRNA, small interfering RNA; TBP, TATA-binding protein.

Received for publication December 23, 2005. Accepted for publication September 11, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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