MOLECULAR AND CELLULAR BIOLOGY, Oct. 2008, p. 6373–6383 0270-7306/08/$08.00⫹0 doi:10.1128/MCB.00413-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 20
Histone Deacetylase Inhibitors Modify Pancreatic Cell Fate Determination and Amplify Endocrine Progenitors䌤† Ce´cile Haumaitre,* Olivia Lenoir, and Raphae¨l Scharfmann INSERM U845, Centre de Recherche Croissance et Signalisation, Universite´ Paris Descartes, Faculte´ de Me´decine, Ho ˆpital Necker, 75015 Paris, France Received 12 March 2008/Returned for modification 20 May 2008/Accepted 4 August 2008
During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of  cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes. opposite phenotype (11, 56). Furthermore, Arx and Pax4 display mutual transcriptional inhibition (8). Acetylation or deacetylation of histone terminal domains can regulate gene expression. Histone acetyltransferases and histone deacetylases (HDACs), respectively, loosen or compact chromatin structures and regulate cell proliferation/differentiation in various tissues (6, 7, 36, 44, 48, 59, 61). Based on sequence similarity, catalytic sites, and cofactor dependency, mammalian HDACs are grouped into the classical class I, II, and IV HDAC family (including HDAC1 to -3 and -8 in class I; HDAC4 to -7, -9, and -10 in class II; and HDAC11 in class IV) (12, 14) and the structurally unrelated sirtuin family (class III HDACs). Whereas class I HDACs are located in the nucleus and are ubiquitously expressed, class II HDACs can shuttle between the nucleus and the cytoplasm. Class II HDACs have a more restricted cell type pattern of expression (heart, brain, and skeletal muscle) and contain an N-terminal extension that links them to specific transcription factors and confers responsiveness to a variety of signal transduction pathways, thereby connecting the genome with the extracellular environment (14, 43). Small-molecule HDAC inhibitors (HDACi) are major tools for studying the connection between overall chromatin effects and cell lineage specification. Pharmacological inhibition of HDACs enables experimental manipulation and systematic analysis of chromatin remodeling (42). The effects of HDACi are selective (40, 60) and are thus often used to specifically inhibit HDACs (42, 46, 62). Valproic acid (VPA) and MS275 preferentially target class I HDACs (18, 27), whereas trichostatin A (TSA) and sodium butyrate (NaB) inhibit both class I and class II HDACs (13, 67). HDACi were successfully used to demonstrate the roles of HDACs in intestine (58), oligodendrocyte (41, 55), neuron (26), adipocyte (65), osteoblast (38), and T-cell (57) differentiation programs and are now being clinically evaluated as cancer drugs (46).
The mature pancreas contains exocrine tissue composed of acinar cells that secrete digestive enzymes via a branched network of ductal cells into the intestine and endocrine islets that produce hormones, such as insulin ( cells), glucagon (␣ cells), somatostatin (␦ cells), and pancreatic polypeptide (PP cells). The pancreas originates from the dorsal and ventral regions of the foregut endoderm directly behind the stomach. Signals derived from adjacent mesodermal structures, the notochord and dorsal aorta (33, 37), and the mesenchyme, which condenses around the underlying committed endoderm (4, 54), are involved in the control of pancreas development. Studies of genetically engineered mice have identified a hierarchy of transcription factors regulating pancreatic specification, growth, and differentiation (10, 31). The pancreas-committed endodermal region expresses the homeodomain factor PDX1 (30, 50). Next, the basic helix-loop-helix factor neurogenin 3 (NGN3) initiates the endocrine differentiation program in epithelial pancreatic progenitor cells. Indeed, Ngn3-deficient mice fail to generate any endocrine cells (19), and lineage-tracing experiments have also provided direct evidence that NGN3-expressing cells are islet progenitors (21). Subsequently, additional transcription factors determine the specific endocrine cell fate. Gain- and loss-of-function experiments are consistent with antagonistic roles for Pax4 and Arx in specifying endocrine subtypes (for /␦ or ␣/PP cells, respectively). Whereas Pax4-deficient mice display a selective loss of  and ␦ cells with a proportional increase in ␣ cells, Arx-deficient mice present the
* Corresponding author. Mailing address: INSERM U845, Centre de Recherche Croissance et Signalisation, Universite´ Paris Descartes, 156 rue de Vaugirard, Faculte´ de Me´decine, 75015 Paris, France. Phone: 33 140 615584. Fax: 33 143 060443. E-mail: cecile.haumaitre
[email protected]. † Supplemental material for this article may be found at http://mcb .asm.org/. 䌤 Published ahead of print on 18 August 2008. 6373
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Past research on pancreatic development mainly dealt with the regulatory roles of specific transcription factors, with little focus on the roles of coregulators, such as HDACs. Since the acetylation state of nucleosomal histone modulates chromatin structure and epigenetically regulates gene expression, we hypothesized that this mechanism might control the timing of pancreatic differentiation and embryonic pancreas cell fate decisions. Here, we used an in vitro model in which endocrine and exocrine cells develop from E13.5 rat pancreases in a way that replicates in vivo pancreas development perfectly (2, 22) and explored the role of HDACs in pancreatic development by treating embryonic explants with HDACi. This treatment did not affect cell proliferation but did have profound effects on exocrine tissue cell fate decisions by suppressing acinar differentiation and promoting ductal differentiation. Importantly, we found that HDACi treatment enhanced the development of NGN3-positive (NGN3⫹) endocrine progenitors and modified the endocrine subtype lineages choices. Specifically, TSA and NaB treatments increased the pool of endocrine precursor cells that subsequently gave rise to a larger pool of insulin⫹ cells. Our data demonstrate that the maintenance of acetylation (i.e., HDAC inhibition) has a specific, dominant function in pancreatic lineage development and highlight the HDACi’s ability to modulate pancreatic cell determination and amplify specific cellular subtypes. This approach may be useful for developing novel cell replacement therapies in diabetes. MATERIALS AND METHODS Animals and dissection of dorsal pancreatic rudiments. Pregnant Wistar rats were purchased from the CERJ (Le Genest, France). The first day postcoitum was taken as embryonic day 0.5 (E0.5). Pregnant female rats at 13.5 days of gestation were killed by CO2 asphyxiation, according to the French Animal Care Committee’s guidelines. Dorsal pancreatic buds from E13.5 rat embryos were dissected as described previously (2). Organ culture, HDACi treatments, and BrdU incorporation. Pancreases were laid on 0.45-m filters (Millipore) at the air-medium interface in petri dishes containing RPMI 1640 (Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 g/ml), HEPES (10 mmol/liter), L-glutamine (2 mmol/liter), nonessential amino acids (1⫻; Invitrogen), and 10% heat-inactivated calf serum (HyClone). The cultures were maintained at 37°C in humidified 95% air-5% CO2. The medium was changed every other day. Explants were cultured in the presence of VPA or TSA (Sigma). We determined the optimal concentrations of both inhibitors by testing increasing doses of VPA (from 0.75 mM to 3 mM) and TSA (from 50 nM to 200 nM). Because VPA concentrations above 1.5 mM and TSA concentrations above 125 nM resulted in increased cell death (data not shown), we used concentrations that led to phenotypic effects without toxicity: 1 mM VPA and 100 nM TSA. MS275 and NaB (Sigma) were used at 1 M and 125 M, respectively. For cell proliferation assays, 10 M bromodeoxyuridine (BrdU) (Sigma) was added to the medium during the last hour of culture. Immunohistochemistry and quantification. Tissues were fixed in 10% formalin, preembedded in low-gelling agarose, and embedded in paraffin. All sections (4 m thick) of each pancreatic explant were collected and processed for immunohistochemistry, as described previously (15, 47). Antibodies were used at the following dilutions: mouse anti-insulin (Sigma), 1/2,000; guinea pig antiinsulin (Dako), 1/200; mouse antiglucagon (Sigma), 1/2,000; rabbit antiamylase (Sigma), 1/300; goat anti-osteopontin/SPP1 (R&D Systems), 1/200; rabbit antiPDX1 (15), 1/1,000; mouse anti-BrdU (Amersham), 1/2; and rabbit anti-Ngn3 (22), 1/1,000. The fluorescent secondary antibodies were fluorescein anti-rabbit and anti-goat antibodies (Jackson Immunoresearch; 1/200), Texas red antimouse antibody (Jackson Immunoresearch; 1/200), Alexa Fluor 488 anti-rabbit antibody (Biogenex; 1/400), and AMCA anti-guinea pig antibody (Jackson Immunoresearch; 1/200). Nuclei were stained blue with Hoechst 33342 (0.3 g/ml; Invitrogen). Ngn3 detection was performed as previously described (22) using the Vectastain elite ABC kit (Vector Laboratories). Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica) and digitized using cooled
MOL. CELL. BIOL. three-charge-coupled-device cameras (C5810 or C7780; Hamamatsu). The surface area of each staining was quantified with IPLab (Scanalytics). The surface areas per section were summed to obtain the total surface area per explant in mm2. At least three explants were analyzed per condition, and the results are expressed as means plus standard errors of the mean (SEM). Statistical significance was determined using Student’s t test. TUNEL labeling. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) experiments were performed using an in situ cell death detection kit (Roche) and followed by insulin and amylase immunostainings. In situ hybridization. In situ hybridization was performed as previously described (16), and colorimetric development was performed with 5-bromo-4chloro-3-indolyl phosphate (Promega) and nitroblue tetrazolium (Roche). No signal was obtained when a sense riboprobe was used. RNA extraction and real-time PCR. Total RNA was extracted from pools of at least three pancreases using an RNeasy Microkit (Qiagen) and reverse transcribed using Superscript reagents (Invitrogen). Real-time PCR was performed with the 7300 Fast real-time PCR system; each reaction consisted of a mixture of Taqman universal PCR master mix with a specific labeled probe (Applied Biosystems) (22). The comparative method of relative quantification (2⫺⌬⌬CT) (39) was used to calculate the expression levels of each target gene, normalized to peptidylpropyl isomerase A/cyclophilin A. The data are presented as changes in gene expression. At least three pools of explants were analyzed per condition, and the results are expressed as means plus SEM. Statistical significance was determined using Student’s t test. Protein extracts and Western blot analysis. Tissue lysates were prepared from pools of at least five pancreases using a Complete Lysis-M kit (Roche). Equal amounts of proteins were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for separation and transferred onto 0.45-m nitrocellulose membranes. After being blocked with milk, the membranes were probed with different antibodies: rabbit anti-HDAC1, rabbit anti-HDAC2, rabbit antiHDAC3, rabbit anti-HDAC4, rabbit anti-HDAC6, rabbit anti-HDAC7 (Abcam), and rabbit anti-HDAC5 (Sigma); rabbit anti-histone acetyl-H3 (Lys9) and rabbit anti-histone acetyl-H4 (Lys12) (Upstate); and mouse antiactin antibody (Sigma). The results were visualized with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) and enhanced chemiluminescence (LumiGLO; Cell Signaling). HDAC enzymatic-activity assay. Total cellular HDAC enzymatic activity was measured using an HDAC assay kit (Millipore). Briefly, 6 g of control or VPAor TSA-treated cell extracts (prepared as described above) were incubated in a 96-well plate with a fluorometric substrate in HDAC assay buffer for 45 min at 37°C. An activator solution was then added to release the fluorophore from the deacetylated substrates, and the fluorescence was measured in a plate-reading fluorimeter (excitation, 390 nm; emission detection, 460 nm).
RESULTS HDACs are downregulated during pancreas development. We first analyzed the expression of different HDACs during rat pancreas development. Using Western blotting, we found that both class I (HDAC1 to -3) and class II (HDAC4 to -7) HDACs were expressed at E13.5 and E17.5 and in the adult pancreas. The expression levels of most HDACs (with the exception of HDAC3) decreased during development (Fig. 1A). We next measured total HDAC activity from E13.5 to the adult pancreas and observed an 86.1% ⫾ 6.5% decrease between the two stages (Fig. 1B). To determine whether decreased HDAC activity was correlated with increased histone acetylation, we performed Western blot analysis with antibodies directed against histone acetyl-H3 and acetyl-H4 residues. We found that the overall degree of histone acetylation increased from E13.5 to the adult (Fig. 1C). These results show that HDACs are expressed and then developmentally regulated in the pancreas. Decreased HDAC expression is associated with decreased HDAC activity and increased histone acetylation throughout pancreas development. To study the role of HDACs in pancreas development, we treated rat pancreatic explants with VPA and TSA. We cultured E13.5 rat pancreases on floating filters at the air-medium
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FIG. 1. HDAC expression and activity during pancreas development in vivo. (A) Western blot analysis of the expression of HDAC proteins (HDAC1 to -3, class I; HDAC4 to -7, class II) in vivo in E13.5, E17.5, and adult rat pancreatic extracts. (B) Total HDAC enzymatic activity from E13.5 (set to 100%) to adult pancreatic extracts. (C) Western blot analysis of histone acetylation with antibodies against histone acetyl-H3 and acetyl-H4 residues in E13.5, E17.5, and adult rat pancreatic extracts. The values are means plus SEM of three independent experiments. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001.
interface for up to 14 days. Under such conditions, acinar and endocrine cells develop in a way that replicates pancreas development in vivo (2, 22). We found that the in vitro HDAC expression pattern replicates the one found in vivo, with decreased expression of most HDACs (Fig. 2A). We next measured total HDAC activity from day 0 to day 14 and found a 65.4% ⫾ 1.6% decrease between the two stages. We verified that VPA and TSA were acting as HDACi, and found average decreases of 93.2% ⫾ 2.3% and 99.2% ⫾ 0.9% with VPA and TSA, respectively (Fig. 2B). The decreased HDAC activity was correlated with an increase in histone acetylation observed by Western blotting with histone acetyl-H3 and -H4 antibodies (Fig. 2C, compare control lanes after 1, 3, 5, and 7 days of
culture). Moreover, we found that both HDACi induced histone hyperacetylation (Fig. 2C). Altogether, these results show that HDAC expression and activity are regulated during pancreas development in vivo and in vitro. Our data validate the use of VPA and TSA for potently modulating HDAC activity in pancreatic explants. HDACi treatment suppresses acinar differentiation and promotes ductal differentiation. We first compared the morphologies of pancreases cultured in the presence or absence of HDACi. At E13.5, the pancreas is composed of an epithelium surrounded by mesenchymal tissue. Under control conditions, the epithelium grew and spread into the mesenchyme (Fig. 3A to D). With VPA or TSA treatment, epithelial growth occurred
FIG. 2. HDAC expression and activity in pancreases cultured in vitro with and without HDACi. (A) Western blot analysis of the expression of different HDAC proteins in pancreatic extracts after 1, 3, 5, and 7 days in culture (D1, D3, D5, and D7, respectively). (B) Total HDAC enzymatic activity in control and VPA- or TSA-treated cell extracts from day 0 (E13.5; D0; set to 100%) to day 14 (D14). (C) Western blot analysis of histone acetylation after 1, 3, 5, or 7 days in culture in control and VPA- or TSA-treated pancreases. The values are means plus SEM of three independent experiments. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001.
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FIG. 3. Morphological effect of HDACi treatment on the embryonic rat pancreas. E13.5 rat pancreases were cultured at the air-medium interface for different periods (day 1 to day 7) without or with HDACi. VPA and TSA were used at 1 mM and 100 nM, respectively. Representative images after 1 (A, F, and K), 3 (B, G, and L), 5 (C, H, and M), and 7 (D, I, and N) days in culture are shown. In panels A to C, F to H, and K to M, the epithelium is circled in red. A branched epithelium (red arrowheads) was observed with HDACi treatment after 3 days in culture (panels G and L versus B and H, and M versus C). Cystic structures (red asterisks) were observed with HDACi treatment, and especially with TSA, after 7 days in culture (panels I and N versus D). (E, J, and O) Hematoxylin/eosin staining after 7 days of culture. Cystic structures were seen (black asterisks). Fewer acinar structures were observed with VPA and TSA than in the control (black arrows). Scale bar, 50 m.
and branching increased (Fig. 3G, H, L, and M), and after 7 days of culture, the tips of the branched epithelium formed cystic structures (Fig. 3I and N). Hematoxylin/eosin staining revealed cystic structures and less-developed acinar structures with VPA and TSA than with controls (compare Fig. 3J and O with E). We found similar results with MS275 and NaB (see Fig. S1 in the supplemental material). To precisely define the molecular events resulting from HDAC inhibition, we analyzed markers for the different pancreatic lineages. We found no effect of HDACi on the ratio of epithelial to mesenchymal tissue (by analyzing E-cadherin and vimentin expression, respectively) (data not shown). We next analyzed the effects of HDACi on cell differentiation, focusing first on the exocrine lineage. We used real-time PCR to monitor the expression of the P48/Ptf1a (34) and Mist1 (53) transcription factors, involved in acinar cell differentiation, and amylase, a marker of differentiated acinar cells. Under control conditions, the expression of P48 and Mist1 increased over the first 5 days of culture, followed by increased amylase expression (Fig. 4A). The increase in levels of P48, Mist1, and amylase was strongly reduced by VPA or TSA (Fig. 4A). Quantification of the surface area occupied by amylase⫹ cells indicated that acinar cell differentiation was much less in pancreases cultured with VPA or TSA (Fig. 4A). For ductal cell differentiation, we used real-time PCR to evidence a major increase in SPP1 (32) expression, especially after 5 and 7 days of culture with VPA or TSA (Fig. 4B). Immunohistological analysis showed that SPP1 staining was greater in HDACitreated pancreases and was mainly found around cystic structures. Quantification of the surface area occupied by SPP1⫹ cells showed a fourfold increase on HDACi treatment, indicating enhanced ductal cell development (Fig. 4B). Similar results with exocrine differentiation were found with MS275 and NaB (see Fig. S2 in the supplemental material).
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Overall, our results show that HDACi treatment leads to a dramatic decrease in acinar lineage differentiation, whereas ductal lineage differentiation is strongly enhanced. HDACi treatment strongly promotes the Ngn3 proendocrine lineage. To define whether HDAC activity was involved in the regulation of the endocrine lineage, we focused on the expression of Ngn3, a specific pancreatic endocrine progenitor marker (19). In control pancreatic explants, Ngn3 mRNA levels measured by real-time PCR increased after 1 day of culture, peaked at day 3, and decreased thereafter. With VPA or TSA, we observed a dramatic increase in Ngn3 expression from day 1 and a peak at day 7 (14-fold higher than in controls) (Fig. 5A). Hence, the Ngn3 expression profile was extended and amplified with HDACi treatment. We found similar results with MS275 and NaB (see Fig. S3 in the supplemental material). At day5, we visualized Ngn3 mRNA expression by in situ hybridization and observed enhanced Ngn3 expression, with positive cells throughout the epithelium in VPA- and TSA-treated explants (Fig. 5B). Finally, immunohistochemical analysis at day 7 (Fig. 5C) showed that NGN3⫹ cells were extremely scarce in control pancreases, while many were present in VPA- and TSAtreated explants. Thus, our results show that the expression profile of the proendocrine transcription factor NGN3 was strongly enhanced and maintained with HDACi treatment, leading to an increased pool of endocrine progenitor cells. HDACi treatment enhances the endocrine ␣/PP lineage. We next analyzed the effect of HDACi on each of the pancreatic endocrine cell types. We first focused on the expression of Arx, known to support an ␣ and PP cell fate (8, 9, 11). We observed a major increase in Arx expression with VPA or TSA treatment at all stages of culture. At day 7, Arx expression was enhanced around 3-fold with VPA and 11-fold with TSA (Fig. 6A). We next used real-time PCR to monitor the expression of glucagon and PP synthesized by ␣ and PP cells, respectively. Both VPA and TSA treatment produced a major increase in glucagon and PP expression at all stages of culture. At day 7, we observed a 5-fold and a 25-fold increase in glucagon expression and a 4-fold and a 6-fold increase in PP expression with VPA and TSA, respectively (Fig. 6A). Immunohistological analysis revealed a fourfold increase in the number of glucagon-expressing cells upon VPA or TSA treatment (Fig. 6B). Similar results were found with MS275 and NaB (see Fig. S4 in the supplemental material). Together, these results show that HDACi treatment promotes ␣ and PP lineage differentiation. Opposing effects of VPA and TSA on the endocrine /␦ lineage. We used real-time PCR to monitor the expression of Pax4, known to support /␦ cell fate (56), over 7 days of culture. VPA produced a dramatic decrease in Pax4 expression as early as day 1, associated with a dramatic decrease in insulin expression and almost full abolition of somatostatin expression (Fig. 7A). Moreover, the expression of NeuroD1, another Ngn3 target subsequently expressed in  cells (28, 49), was significantly lower in VPA-treated pancreases. These results were confirmed immunohistologically, with a major decrease in the number of insulin⫹ cells at days 5 and 7 (Fig. 7B). Even in tissues cultured for 14 days with VPA, insulin expression remained dramatically low (Fig. 7D). Few insulin⫹ cells were observed immunohistochemically (Fig. 7E).
FIG. 4. Effect of HDACi treatment on exocrine differentiation. (A) Real-time PCR quantification of P48/Ptf1a, Mist1, and amylase mRNAs after 0, 1, 3, 5, and 7 days in culture (D0, D1, D3, D5, and D7, respectively) with and without VPA or TSA treatment. Immunohistological analyses of pancreases after 5 and 7 days in culture with and without VPA or TSA treatment were performed. Acinar differentiation was evaluated by amylase staining (green). Nuclei were stained in blue with Hoechst stain. The absolute surface areas occupied by amylase⫹ cells that developed after 5 and 7 days of culture with and without VPA or TSA treatment were quantified. The values are means plus SEM of at least three independent experiments. NS, no significant difference; ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001. Scale bar, 100 m. (B) Real-time PCR quantification of SPP1 mRNA after 0, 1, 3, 5, and 7 days of culture with and without VPA or TSA treatment. Immunohistological analysis of pancreases after 7 days in culture with and without VPA or TSA treatment was performed. Ductal differentiation was evaluated using anti-SPP1 staining (green). Nuclei were stained in blue with Hoechst stain. The absolute surface areas occupied by SPP1⫹ cells that developed after 7 days of culture with and without VPA or TSA treatment were quantified. The values are means plus SEM of at least three independent experiments. NS, no significant difference; ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001. Scale bar, 100 m. 6377
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FIG. 5. HDACi treatment enhances the pool of Ngn3 endocrine progenitor cells. (A) Real-time PCR quantification of Ngn3 mRNA after 0, 1, 3, 5, 7, 9, 11, and 14 days in culture (D0, D1, D3, D5, D7, D9, and D11 respectively), with and without VPA or TSA treatment. The values are means plus SEM of at least three independent experiments. ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001. (B) (Top) Detection of Ngn3 transcripts by in situ hybridization in pancreases cultured for 5 days with and without VPA or TSA treatment. (Bottom) Detection of Ngn3 protein by immunohistochemistry in pancreases cultured for 7 days with and without VPA or TSA treatment. Scale bar, 100 m.
In contrast, TSA strongly activated Pax4 and NeuroD1 expression. However, no differences in insulin and somatostatin expression were found after 7 days in culture either by realtime PCR (Fig. 7A) or by immunohistochemistry (Fig. 7B and data not shown). Hence, we extended the culture period. After 11 days, TSA-treated pancreases developed translucent buds at the periphery of the pancreas, corresponding to an outer mass of insulin⫹ cells (Fig. 7C and E). Quantification of insulin staining showed that the -cell mass had increased almost twofold under such conditions (Fig. 7E). These results were confirmed using real-time PCR: insulin expression was increased twofold at day 9, threefold at day 11, and fivefold at day 14 compared with controls (Fig. 7D). Somatostatin expression was also increased after 14 days in culture (data not shown), demonstrating that the /␦ lineage was promoted by TSA. Interestingly, MS275 showed results similar to those with VPA, whereas NaB showed results similar to those with TSA (see Fig. S5 in the supplemental material). Taken together, these results indicate that VPA suppresses the /␦ lineage, whereas TSA enhances it. The effects of HDACi treatment on pancreatic differentiation are independent of proliferation and apoptosis. The observed effects of HDACi on pancreatic development could be
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attributed to a direct effect of HDAC inhibition on the differentiation program itself or to an indirect effect on proliferation/apoptosis. First, we asked whether the massive increase in the number of NGN3⫹ cells was accompanied by an increased proliferation of early PDX1⫹ pancreatic precursor cells that would give rise to more NGN3⫹ endocrine progenitor cells (21). We cultured pancreases for 1 day with or without VPA and TSA and added BrdU to the medium during the last hour of culture. No difference in BrdU incorporation was seen (Fig. 8A to C). The percentages of PDX1⫹/BrdU⫹ cells were 30.4% ⫾ 1.4% under control conditions, 29.3% ⫾ 2.3% with VPA, and 31.9% ⫾ 1.4% in TSA-treated pancreases. We next determined whether the greater number of NGN3⫹ cells was associated with increased proliferation of such cells. After 3 days of culture, very few NGN3⫹/BrdU⫹ cells could be found under control conditions, and neither VPA nor TSA increased the proliferation of NGN3⫹ cells (Fig. 8D to F). Given the observed increase in glucagon-expressing cells with HDACi treatment, we also tested for increased proliferation of such cells. As for NGN3⫹ cells, we observed a low proliferative rate for glucagon⫹ cells with no difference between controls and pancreases treated for 5 days with VPA or TSA (Fig. 8G to I). Finally, using the TUNEL method, we found that neither VPA nor TSA treatment modified the number of apoptotic cells expressing amylase or insulin, indicating that the lower numbers of amylase and insulin cells observed with VPA treatment were not due to apoptosis (Fig. 8J to L). These results show that HDACi treatment does not modify the proliferation/apoptosis balance and further suggest that the effects of HDACi treatment on pancreas differentiation are direct. DISCUSSION Recent work has identified transcription factors that regulate pancreatic cell lineages (10, 31). However, the mechanisms regulating cell fate choices during pancreatic development are still unclear. Histone modifications play crucial roles in the transcriptional regulation of most eukaryotic genes and have been linked to cell differentiation control (in muscle and cardiac cells, for example) (3, 44). Here, we investigated the connection between overall histone acetylation and pancreatic cell fate determination and established that regulation of HDAC enzymatic activity is crucial. We highlighted the use of HDACi as major tools for regulating pancreatic cell fate decisions and amplifying specific cell subtypes. Recent invalidation studies with mice revealed that the various HDACs are not functionally redundant. Hdac1-deficient mice died at E10.5 due to proliferation defects and developmental delay (36). HDAC1 and -2 were shown to regulate cardiac development (59). HDAC5 and HDAC9 are involved in the heart’s response to stress signals (6), HDAC4 in chondrocyte hypertrophy (61), and HDAC7 in the maintenance of vascular integrity (7). To date, pancreatic phenotypes have not been reported in specific HDAC-deficient mice. Here, we found that class I (HDAC1 to -3) and class II (HDAC4 to -7) proteins were expressed in the embryonic pancreas. Based on such results, it will be interesting to further analyze pancreatic phenotype in HDAC-deficient mice and/or to knock down selective HDACs via short hairpin RNA using a procedure that
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FIG. 6. HDACi treatment increases endocrine ␣/PP lineage differentiation. (A) Real-time PCR quantification of Arx, glucagon, and PP mRNAs after 0, 1, 3, 5, and 7 days in culture (D0, D1, D3, D5, and D7, respectively) with and without VPA or TSA treatment. (B) Immunohistological analyses of pancreases after 5 and 7 days in culture with and without VPA or TSA treatment. ␣-cell development was evaluated using antiglucagon staining (red). Nuclei were stained in blue with Hoechst stain. The absolute surface areas occupied by glucagon⫹ cells that developed after 5 and 7 days in culture with and without VPA or TSA treatment were quantified. The values are means plus SEM of at least three independent experiments. NS, no significant difference; ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001. Scale bar, 100 m.
needs to be adapted in our experimental system. Moreover, HDAC expression was regulated, with significantly decreased expression levels of class I and class II HDACs upon differentiation. This was correlated with decreased HDAC enzymatic activity. This pattern has been observed during osteogenesis and adipogenesis (38, 65). Histone acetylation generally contributes to the formation of a transcriptionally competent environment by relaxing the chromatin structure, allowing transcription factors to access the target DNA. In contrast, histone deacetylation compacts chromatin and leads to transcriptional repression (20, 35). Therefore, increased histone acetylation during pancreas development could lead to enhanced gene expression. This suggests that downregulation of HDACs could be a prerequisite for cell differentiation into specific subtypes. To define the role of histone acetylation in embryonic pancreatic development, we used an in vitro culture model. We had previously established that embryonic pancreases cultured in vitro at the air-medium interface developed as in vivo, with exocrine and endocrine differentiation (2, 22). Since most HDACs were expressed during pancreatic development, we chose an overall HDAC inhibition strategy rather than specifically but arbitrarily inactivating each HDAC. We used different HDACi—VPA (18, 51), TSA (17, 66, 67), MS275 (27), and NaB (13)—that have been used in several cell lines (42, 46), and validated their use in pancreatic explants, an approach similar to that used with intestinal explants (58). We found that HDACi treatment strongly affected exocrine differentiation. Both VPA and TSA increased duct cell differentiation (forming cystic structures) and decreased acinar dif-
ferentiation. This phenotype might be due to metaplasia, a process in which one tissue is replaced by another. In the pancreas, it has been suggested that ductal metaplasia occurs via transdifferentiation of acinar cells into ductal cells (45). This requires loss of acinar cell marker expression, concomitant with the initiation of duct cell differentiation. In such a case, intermediate cells (coexpressing acinar and ductal markers) should be found. However, in the present study, we did not observe dual-positive cells (data not shown). Alternatively, the observed phenotype might be a consequence of ductal cell expansion with parallel acinar cell loss. We did not observe increased acinar cell apoptosis or ductal cell proliferation on HDACi treatment. This phenotype can be compared to that of Hnf6 and Kif3a mutant mice, which both display ductal cell expansion and cyst formation without any change in ductal cell proliferation (5, 52). We thus propose that the observed HDACi-induced ductal expansion is due to increased duct cell differentiation during pancreas development. Importantly, we found that HDACi treatment enhanced the development of NGN3⫹ endocrine progenitors. During development, PDX1⫹ precursors in the pancreatic epithelial tree give rise to various pancreatic cell types, including NGN3⫹ cells (21). It has been shown that increasing the proliferation of PDX1⫹ precursors, with either mesenchyme or FGF10, enhances the pool of NGN3⫹ endocrine progenitors (2). However, we found that HDACi treatment did not increase the proliferation of PDX1⫹ precursors, suggesting that FGF10 and HDACi increase the pool of NGN3⫹ endocrine progenitors in different ways. Moreover, HDACi treatment did not increase proliferation of poorly proliferative NGN3⫹ cells themselves
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FIG. 8. HDACi treatment does not modify proliferation or apoptosis. Pancreases were pulsed with BrdU during the last hour of culture. BrdU staining was analyzed immunohistochemically (red) with Pdx1 staining (green) at day 1 (D1) (A, B, and C), with Ngn3 staining (green) at day 3 (D, E, and F), and with glucagon staining (green) at day 5 (G, H, and I). (G, H, and I) Proliferative PDX1 cells at day 1 are yellow. Nuclei were stained in blue with Hoechst stain at day 5. (J, K, and L) TUNEL staining was visualized (green) at day 5 with insulin staining (blue) and amylase staining (red). Scale bar ⫽ 50 m.
(2, 25). Taken as a whole, our data indicate that the HDACiinduced increase in Ngn3 expression was independent of cell proliferation. Data from mice deficient in Gdf11 or Smad2 (factors involved in TGF- signaling) and those with a perturbed Notch pathway show the regulation of NGN3⫹ cells by both pathways (1, 23, 24). Furthermore, it has been reported that HDAC1 regulates retinal neurogenesis by suppressing Wnt and Notch signaling pathways (63). Hence, it will be interesting to investigate connections between HDACs and the Notch, Wnt, and transforming growth factor  signaling pathways in pancreatic differentiation. Another important possibility is that HDACs act directly on the Ngn3 promoter at the histone level. In such a case, the HDACi’s effect on Ngn3 would constitute a previously uncharacterized and potentially therapeutically relevant direct mechanism of action, which we are currently investigating. In recent years, it has been shown that HDACi also have other nonhistone protein substrates
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involved in regulation of gene expression and cell proliferation/ death (64). HDAC inhibition may cause the accumulation of acetylated forms of these proteins, thereby modifying their functions. Further investigations will be performed to identify such targets in the pancreas and, particularly, to evaluate their potential contributions to the regulation of Ngn3 expression. NGN3⫹ progenitor cells can differentiate into ␣, , ␦, and PP cells (19). Differentiation into the ␣/PP and /␦ lineages is mediated by Arx and Pax4, two targets of Ngn3 (10). We found that all the HDACi tested increased Arx expression and promoted the ␣/PP lineage. In contrast, VPA/MS275 and TSA/ NaB had opposing effects on the /␦ lineage. VPA or MS275 treatment induced almost total abolition of insulin- and somatostatin-expressing cells, together with decreased Pax4 expression. Due to the previously observed reversible nature of histone modification (41), we anticipated that the VPA-specific blockade on the /␦ lineage would be overcome on withdrawal of the treatment. We tested this hypothesis by removing VPA after 7 days and culturing the cells for up to 14 days; we then observed the recovery of insulin- and somatostatin-expressing cells (data not shown). These data support the hypothesis that HDACi have direct effects on endocrine cell fate regulation. In contrast, we found that TSA or NaB had a positive effect on the /␦ lineage, with increased Pax4 expression followed by an increase in insulin- and somatostatin-expressing cells. Importantly, these results show that the increased pool of NGN3 endocrine progenitor cells produced by TSA or NaB treatment give rise to a larger pool of endocrine cells. We propose a working model for unraveling the role of HDACs in the development of pancreatic cells from endodermal progenitors (see Fig. S6 in the supplemental material). During this development window, HDACi have minor effects on cell proliferation but control cell differentiation tightly. Given the specificities of VPA and MS275 (preferential inhibition of class I HDACs) (18, 27) and of TSA and NaB (inhibition of both class I and II HDACs) (13, 17, 66, 67), we propose that HDACs have specific roles in pancreatic differentiation. All tested HDACi precluded the establishment of the acinar lineage and favored alternative lineages, the ductal and proendocrine NGN3 lineages, suggesting the involvement of class I HDACs in this process. It will now be particularly interesting to identify the class I HDACs and transcription factor complexes involved in these controls. Downstream of Ngn3, all HDACi promoted the ␣/PP lineage, but only TSA and NaB promoted the /␦ lineage. Temporal control of Ngn3 activity was shown to be associated with
FIG. 7. Opposite effects of VPA and TSA treatments on endocrine /␦ lineage differentiation. (A) Real-time PCR quantification of Pax4, NeuroD1, insulin, and somatostatin mRNAs after 0, 1, 3, 5, and 7 days in culture (D0, D1, D3, D5, and D7, respectively) with and without VPA or TSA treatment. (B) Immunohistological analyses of pancreases after 5 and 7 days in culture with and without VPA or TSA treatment. -Cell development was evaluated using anti-insulin staining (red). Nuclei were stained in blue with Hoechst stain. The absolute surface areas occupied by insulin⫹ cells that developed after 5 and 7 days in culture with and without VPA or TSA were quantified. (C) Representative images of pancreases cultured for 9, 11, and 14 days with and without VPA or TSA treatment. Note that with TSA treatment, at days 11 and 14, translucent buds could be seen (black arrows). (D) Real-time PCR quantification of insulin mRNA after 7, 9, 11, and 14 days of culture with and without VPA or TSA treatment. (E) Immunohistological analyses of pancreases after 14 days of culture with and without VPA or TSA treatment. -Cell development was evaluated using anti-insulin staining (red). Nuclei were stained in blue with Hoechst stain. Note that in TSA-treated explants, insulin staining (white arrow) corresponds to the translucent bud seen in panel C (black arrow). The absolute surface areas occupied by insulin⫹ cells that developed after 14 days of culture without or with VPA or TSA treatment were quantified. The values are means plus SEM of at least three independent experiments. NS, no significant difference; ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.005; ⴱⴱⴱ, P ⬍ 0.001. Scale bar, 100 m.
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the generation of different endocrine cell types (29). In our study, both VPA and TSA increased Ngn3 expression, with a longer and more intense expression profile, but had different effects on the /␦ lineage. We thus favor the hypothesis that the effects observed on endocrine cellular subtypes are not due to temporal modification of Ngn3 activity but to HDACi specificity, and thus to different functions for class I and II HDACs in the regulation of endocrine-specific cell subtypes. Class I HDACs may inhibit Arx expression, as all HDACi increased Arx expression. Conversely, the decreased expression of Pax4 with VPA/MS275, but not TSA/NaB, suggests that class II HDACs inhibit Pax4 expression. Thus, class I and class II HDACs could be involved in the regulation of Arx and Pax4, which exert mutual transcriptional inhibition (8). Unfortunately, no class II-specific HDACi are available to easily test this hypothesis, as we did for class I HDACs. Future studies, including chromatin immunoprecipitation experiments, will be necessary to highlight this regulatory mechanism and to specify which HDACs and transcription factor complexes are involved. In the present study, we sought to establish whether overall epigenetic mechanisms control pancreatic differentiation. We showed that HDACs are crucial regulators of pancreatic cell fate determination and that class I and class II HDACs have different roles. These results demonstrate that HDACi are potent tools for modifying pancreatic lineages and highlight pancreatic plasticity. Specifically, we discovered that TSA and NaB treatments lead to an increased pool of endocrine progenitors and thus an increased pool of endocrine cells. Our work provides new insights into the control of pancreatic differentiation and may provide new therapeutic options involving small molecules for targeting pancreatic cells in cell replacement strategies in diabetes.
MOL. CELL. BIOL.
9.
10. 11. 12. 13. 14. 15. 16. 17.
18.
19. 20. 21. 22. 23.
ACKNOWLEDGMENTS We are grateful to Isabelle Lenin-Werhle, Annie Basmaciogullari, and Virginie Aiello for technical assistance. O.L. has a fellowship from the Ministe`re de la Recherche et de la Technologie. This work was supported by the 6th European Union Framework Program (Beta-Cell Therapy Integrated Project), the Association pour la Recherche sur le Diabe`te, and the Association Fran¸caise des Diabetiques.
24.
25. 26.
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