Contrasting patterns of expression of transcription factors in pancreatic ␣ and ␤ cells

Jie Wang*, Gene Webb†, Yun Cao‡, and Donald F. Steiner*†‡§

Departments of *Biochemistry and Molecular Biology and †Medicine and ‡The Howard Hughes Medical Institute, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637

Contributed by Donald F. Steiner, August 18, 2003 Pancreatic ␣ and ␤ cells are derived from the same progenitors but hybridization (10, 11), mRNA differential display (12), and play opposing roles in the control of glucose homeostasis. Distur- oligonucleotide microarrays (13–15). However, the underlying bances in their function are associated with diabetes mellitus. To molecules and mechanisms regulating the proliferation and identify many of the proteins that define their unique pathways of differentiation of the pancreatic endocrine cells (5) and the differentiation and functional features, we have analyzed patterns genetic factors responsible for the common forms of diabetes of gene expression in ␣TC1.6 vs. MIN6 cell lines by using oligonu- mellitus (16) largely remain elusive. Furthermore, our current cleotide microarrays. Approximately 9–10% of >11,000 transcripts knowledge of the proteome of the ␣ cell, the second major player examined showed significant differences between the two cell in the control of glucose homeostasis, is still very limited. Thus, types. Of >700 known transcripts enriched in either cell type, our knowledge remains incomplete regarding many key pro- transcription factors and their regulators (TFR) was one of the most cesses, including developmental origin, differentiation, metab- significantly different categories. Ninety-six members of the basic olism, regulation of secretory activity, and the physiological͞ zipper, basic helix–loop–helix, homeodomain, zinc finger, high pathological profiles of ␣ cells vs. the well studied ␤ cells. mobility group, and other transcription factor families were en- In attempts to identify, classify, and contrast factors important riched in ␣ cells; in contrast, homeodomain proteins accounted for for ␣ or ␤ cell function, we have applied Mu11K DNA microar- ␣ ␤ 51% of a total of 45 TFRs enriched in ␤ cells. Our analysis thus ray analysis to two well characterized and cell lines, clonal ␣ highlights fundamental differences in expression of TFR subtypes TC1.6 (17, 18), and MIN6 (19). These are well differentiated within these functionally distinct islet cell types. Interestingly, the cell lines that preserve similar characteristics (such as correct ␣ cells appear to express a large proportion of factors associated hormone processing and secretion of glucagon or insulin in with progenitor or stem-type cells, perhaps reflecting their earlier response to glucose stimulation), and that have arisen from appearance during pancreatic development. The implications of transformed mature islet cells having similar genetic back- these findings for a better understanding of ␣ and ␤ cell dysfunc- grounds (17–21). In the present study, attention has been tion in diabetes mellitus are also considered. concentrated on members of various functional categories that are enriched predominantly in the ␣ or ␤ cell phenotype with a ␣ ␤ special emphasis on the category of transcription factors and ancreatic islets consist of four endocrine cell types, , ,D, their regulators (TFR). Thus, activated transcription factor 3 Pand pancreatic peptide (PP). These cell types produce and (ATF3), hypoxia-inducible factor 1 ␣ (HIF1␣), four-and-a-half secrete the major islet hormones: glucagon, insulin, islet amyloid LIM domains 1 (FHL1), and other basic zipper (bZip), basic polypeptide (IAPP), somatostatin, and PP, respectively, which helix–loop–helix (bHLH), and zinc finger (ZF) proteins were ␣ regulate fuel and energy homeostasis (1). The cells secrete detected predominantly in pancreatic ␣ cells, whereas ␤ cells glucagon, which stimulates gluconeogenesis and glycogenolysis were enriched with members of the homeodomain (HD) group. ␤ to prevent hypoglycemia, whereas the cells increase insulin Our results indicate that various important families of transcrip- secretion in response to elevated blood glucose levels. Glucagon tion factors are quite differently represented in ␣ vs. ␤ cells and and insulin antagonistically regulate the balance of glucose suggest fundamental differences in their functional regulation. storage, production, and consumption to maintain physiological plasma glucose concentrations. Therefore, the ␣ and ␤ cells Materials and Methods together play a central role in glucose homeostasis. Cell Culture, Islet Isolation, and RNA Preparation. Cells (passages Excessive production and secretion of glucagon by the ␣ cells 23–25) of ␣TC1.6 (17, 18), MIN6 (19), and ␤TC3 (22) were is a common accompaniment to the two main types of diabetes. maintained as described previously. proprotein convertase (PC)2 Physiologically, glucagon secretion is suppressed by hyperglyce- null ␣ cells (an ␣ cell line established from these mice in the lab; mia. However, this normal homeostatic suppression is lost in G.W., unpublished data) were cultured under the same conditions diabetic states, which in turn perpetuates hyperglycemia by as the ␣TC1.6 cells. The medium was replaced 24 h before RNA stimulating hepatic glucose output (2). Another major typical extraction. Islets from adult PC2 null (23) and wild-type littermate manifestation of diabetes is an absolute or relative deficiency of mice were isolated as described (24). Total RNA from the cultured insulin from the ␤ cells, resulting in failure to adequately control cells and͞or islets was extracted with TRIzol Reagent (GIBCO), ϩ the blood glucose level (3). Disturbances of ␣ and͞or ␤ cell and poly(A ) RNA was then prepared by using the Oligotex function thus are central to the failure to maintain physiological mRNA Mini Kit (Qiagen, Valencia, CA). glucose levels and related metabolic concomitants of diabetes mellitus. Microarray Analysis. The biotinylated cRNA samples of ␣TC1.6 To understand the molecular basis for the development and and MIN6 cells were synthesized and hybridized in duplicates to specialized functions of ␣ and ␤ cells is an important goal for understanding and effectively treating diabetes. Although much effort has been expended on the biosynthesis of the islet Abbreviations: En, embryonic day n; ATF3, activated transcription factor 3; bHLH, basic helix–loop–helix; bZip, basic zipper; FHL1, four-and-a-half LIM domains 1; HD, homeodo- hormones (1) and on the pancreas and its development (4–7), main; HIF1␣, hypoxia-inducible factor 1 ␣; IAPP, islet amyloid polypeptide; PDX1, pancreatic much remains to be learned about the full complement of gene and duodenal homeobox gene 1; PC, proprotein convertase; TFR, transcription factors and products expressed in these cells. Examination of the ␤ cell their regulators; ZF, zinc finger. functional profile has recently been carried out by using various §To whom correspondence should be addressed. E-mail: [email protected]. techniques such as representational analysis (8, 9), subtractive © 2003 by The National Academy of Sciences of the USA

12660–12665 ͉ PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1735286100 Downloaded by guest on September 30, 2021 Fig. 2. Relative sizes of various subgroups of transcription factors in the TFR group of ␣TC1.6 or MIN6 cells (see text for details).

cells vs. 7% in ␤ cells) (Fig. 5, which is published as supporting information on the PNAS web site). This finding might be consistent with the hypothesis that because ␣ cells differentiate early in pancreatic development, this lineage may preserve multipotential characteristics of its less well differentiated or Fig. 1. Summary of enriched molecules predominantly in ␣TC1.6 or MIN6 undifferentiated progenitors, as suggested also by the high cells. Of Ͼ11,000 transcripts, the percentage of those molecules in ␣TC1.6 or percentage of TFR among early expressed molecules [peaking at MIN6 cells with expression equal to or greater than the averaged value of the embryonic day (E)14.5] in embryonic pancreatic cells (25). indicated fold change in duplicates between the two cell lines is shown above TFR members described to play a role in regulating gene each bar. expression and͞or organogenesis in pancreas (Table 3, which is published as supporting information on the PNAS web site) helped validate the microarray data. Thus, brain-4 and c-Maf Mu11K oligonucleotide array (Affymetrix, Santa Clara, CA) as appeared as predominant factors in the ␣ cell line, whereas the described (13). Analysis of the data was performed by GENECHIP ␤ cell marker pancreatic and duodenal homeobox gene 1 SUITE (Ver. 4.0.1, Affymetrix). The threshold for determining (PDX1) and several other molecules, including IAPP, Ins1, Ins2, significant differences of expression level between the two cell Glut-2, PC1͞PC3, and glucokinase (data not shown), were types was set by using a provided algorithm. The molecules enriched in the MIN6 cells. Other reported ␤ cell-enriched having an average fold change of 3 or greater in either cell type factors such as Nkx6.1, Lmx1.1, HB9, and MafA were absent are shown in the following tables. from the microarray. Factors such as Pax-6 and Pbx1, which are expressed in differentiated ␣ and ␤ cells, as well as other islet cell RT-PCR. cDNA was synthesized by reverse transcription from types (for reviews, see refs. 5 and 7), did not differ significantly total RNA (1 ␮g) as described (24). The amount of cDNA used (␣͞␤Ͻ3), consistent with previous observations. Among other for PCR was normalized by the amplified levels of labeled TFRs, CRE-binding protein, which has been described as a BIOCHEMISTRY ␤-actin with [␥-32P]ATP (Amersham Pharmacia Biotech) by regulator of transcription of the glucagon, insulin, and soma- using semiquantitative PCR. The sequences of primers and the tostatin genes (7, 26, 27), also did not show significant differ- length of their respective cDNA fragments are shown in Table ences. Clearly, its generally ubiquitous expression extends to the 2, which is published as supporting information on the PNAS pancreatic islets. These findings indicate that the microarray web site, www.pnas.org. data are reliable. Moreover, the very great enrichment of both IAPP and PC1͞PC3 (412.7- and 29.7-fold) in the ␤ cells provides Immunohistochemical Studies. Rabbit antiserum to ATF3, goat further evidence that both the MIN6 and ␣TC1.6 cells are well antisera to HIF1␣, and FHL1 (Santa Cruz Biotechnology), as differentiated, because both IAPP and PC1͞PC3 have been well as guinea pig antiglucagon (Linco Research Immunoassay, reported to be present in fetal, but not in mature, ␣ cells (28). St. Charles, MO), were purchased commercially. The 5-␮m-thick sections of pancreas were used for immunofluorescent double The TFR Group. The TFR proteins were divided into several staining at room temperature. After blocking with 10% normal subgroups (Fig. 2) on the basis of their conserved structural serum in 0.05% Tween 20͞PBS buffer for 2 h, the sections were features. The members of this category, shown in Table 1, totaled incubated with both antiglucagon antibody (1:500) and one of 141 (96 in ␣,45in␤) and fell into different subgroups between the antibodies against ATF3, HIF1␣, and FHL1 together for the two cell types (for further details, see Tables 4 and 5, which another 2 h. Then, the second antibodies (donkey anti-guinea pig are published as supporting information on the PNAS web site). IgG conjugated with Cy2 1:200; donkey anti-rabbit or goat IgG Large numbers of bZip, bHLH, ZF, high mobility group, and conjugated with Cy3 1:1,000; The Jackson Laboratory) were other family members were highly represented in ␣ cells; by applied for 1–2 h after washing. Fluorescent images were exam- contrast, HD factors accounted for 51% of the TFR members ined with a BX51 (Olympus, Melville, NY) microscope. enriched in ␤ cells but for only 14% in the ␣ cells. In addition, two forkhead factors were expressed highly in ␤, but not in ␣, Results cells. Intriguingly, compared with the stem cell data reported Transcripts Expressed Differentially in ␣ or ␤ Cells. The statistical recently by Melton and colleagues (29), 30 TFR molecules results of analysis of Ͼ11,000 transcripts showed that the total present in stem cells (including the common members of the numbers of transcripts enriched in ␣TC1.6 or MIN6 cells are similar Tead2, Etl1, and FHL1 family in all three stem cells) were (Fig. 1). The percentage of transcripts interrogated that had 3-fold enriched in ␣ cells and only eight in ␤ cells. These findings or greater difference of averaged expression change in duplicate further suggest that the ␣ cell lineage may preserve more experiments between ␣TC1.6 and MIN6 cells is Ϸ9–10%, the properties of its progenitors. Their possible contributions to ␣ or percent Ն5-fold is Ϸ4%, and the percent Ն10-fold is Ϸ1.3%. ␤ cell unique profiles will be discussed below (see Discussion). Among enriched known transcripts (Ͼ700 in either ␣ or ␤ cells) in various functional categories, the TFR category showed RT-PCR. To further validate the microarray data, Ͼ100 transcripts the most significant differences (comprising 14% of total in ␣ from several categories were further analyzed by RT-PCR in

Wang et al. PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12661 Downloaded by guest on September 30, 2021 Table 1. Summary of the enriched TFR members predominantly in a TC1.6 or MIN6 cells Subgroups aTC1.6 MIN6

bZip domain 10 0 c-Fos, Chop-10, ATF3, c-Maf, Jun B*, Fra-1, Maf B, Maf K, JDP-1 homolog, c-Jun bHLH domain 15 2 E2A, Id2, N-myc, Id1, Tcfeb, ITF-2b, NeuroD3, ITF-2a, MOP4, Ngn3, Hed Stral3, Mxil, Id3, HIF1a, HIF2a, NeuroD1 HD 13 23 Brain-4, OG-12, Oct-1, Meislb, Arx, Hoxa9, Oct-3, HNF-1b, PDX1, PBX3b, Oct-2b, OG-2, Mox-2a, Lim1, Six1, Dlx7, Pem, Hlx, HNF-6, unex-4.1 Hoxb-13, TTF-1, Hoxd12, Hox-4.4 (5), Pax-4, Hoxa2, Sax1, Nkx-3.1 Pax-5, Mox-1, Cux, Og9x, Pmx, Meis2, Cux-2, Pax-1 ZF domain 26 10 GATA2, FHL1, GKLF͞KLF4, FOG-1, Sall3, COUP-TF1, tisl 1d, SP1, GATA1, MTF-1, Gli3, Emzf1, Zfp-29, PPAR deltaEF1, KLF3͞BKLF, Slugh, Murr1, Efp-97, c-Krox, All-1, ␣, lkaros, CAR2, RAR␣1 MOK2, Png-1, YY1, E4F, Ozrf1, Zfp-30, WT1, Jumonji, Krox-20, Zfp-35, GATA5, REX-1 High mobility group domain 5 2 Sox5, Lef1, HMG-2, Sox1, Sox3 Sox15, Sox10 Forkhead domain 0 2 Foxa3, Foxf1 Other family members 27 6 Fli-1, Msg1, TF IID, oxyR, Et11, NAB1, Tbx6, NF-YA, Tead2, NFiX1, C-myb, Bcl-3, NFiC1A, AP-2.2, ERF AP-2, Bright, P113, IFF3, Tbx2, IRF4, Tead3, ER81, MEF2b, Enx-1, relB, SIN3B, CDEBP, Tead1, DMP1, Ahch, Eya2, Start1

The full names and GenBank accession nos. of these factors are shown in Tables 4 and 5.

duplicate. The results on the bZip protein subgroup are shown environmental changes in vivo and in vitro as immediate–early in Fig. 3. All 10 bZip factors enriched in ␣TC1.6 cells were also response genes. The observed expression differences between ␣ detected at higher levels in the PC2 null ␣ cell line (an ␣ cell line; and ␤ cells appear to be intrinsic and not due to in vitro artifacts. G.W., unpublished data) compared with two ␤ cell lines, MIN6 The RT-PCR results thus indicate that the enriched molecules and ␤TC3. Furthermore, they were all detected in adult islets detected by microarray are importantly implicated in the unique from PC2 null mice (23) and also in control islets, although profile of either ␣ or ␤ cell type. Fra-1, c-Fos, and JDP-1 homologue levels were lower in both type islets; Maf K, Maf B, c-Jun, and JDP were detected at higher ATF3, HIF1␣, and FHL1 Are Present at Higher Levels in ␣ Cells than in levels in PC2 null islets than in control islets, consistent with ␣ ␤ Cells Within Islets. To further validate our data in vivo,we cell hyperplasia in this model (23). This subgroup deserves examined the distribution (Fig. 4) in pancreatic sections of attention not only due to increasing evidence of their involve- ATF3, HIF1␣, and FHL1, which belong to the bZip, bHLH, and ment in the regulation of glucagon and insulin gene transcription ZF subgroups, respectively. Their transcripts have all been (30–32), glucose homeostasis (33), differentiation, and devel- described to be present in various stem cells (29), and they have opment (34, 35); but also because of their rapid responses to been implicated in murine development (33, 36, 37). Immuno-

Fig. 3. RT-PCR analysis of the expression of the bZip proteins in various ␣ or ␤ cell lines and wild-type or PC2 null islets. Lanes 1 and 7, PC2 null ␣ cell line (␣TCPC2(Ϫ/Ϫ)); lanes 2 and 8, ␣TC1.6; lanes 3 and 9, MIN6; lanes 4 and 10, ␤TC3; lanes 5 and 11, the control littermate islets; lanes 6 and 12, PC2 null islets. Lanes 1–6, 30 cycles; lanes 7–12, 35 cycles. Markers, 100-bp DNA ladder (Biolabs, Northbrook, IL).

12662 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1735286100 Wang et al. Downloaded by guest on September 30, 2021 Fig. 4. Double immunofluorescent detection of glucagon with ATF3, HIF1␣, or FHL1 in the pancreatic islets of PC2 null or wild-type littermate mice. Pancreas sections of PC2 null (d–f, j–l, and p–r) and wild-type (a–c, g–i, and m–o) mice were immunostained with antiglucagon (a, d, g, j, m, and p), anti-ATF3 (b and e), anti-HIF1␣ (h and k), and anti-FHL1 (n and q) antibodies. c, f, i, l, o, and r are images of double staining of a and b, d and e, g and h, j and k, m and n, and p and ϭ ␮

q, respectively. (Bar 50 m.) BIOCHEMISTRY

staining of pancreas sections from normal wild-type controls protein-1 (c-Fos, ATF3, Jun B, Fra-1, JDP-1 homologue, and c-Jun) (Fig. 4) revealed that these factors are expressed predominantly (34, 38); and CCAAT enhancer-binding protein (Chop10) families in ␣ cells within islets compared with ␤ cells, despite some (39). One of the major features of this group is their ability to uneven staining of ATF3 or FHL1 in ␤ cells at lower levels. The function as activators͞repressors either as homodimers and selec- distribution found in controls was also clearly demonstrated in tive heterodimers within the group (34, 35, 38, 39) or with members islets of PC2 null mice, which are characterized by a thick mantle of some other groups such as the HD proteins (30, 40, 41). ␣ of cells (23). In addition, ATF3 was strongly stained in ductal Increasing numbers of reports indicate that c-Maf and Maf B ␣ cells, whereas HIF1 and FHL1 staining also extended beyond contribute to differentiation and development (34). In pancreas, the islets in adult pancreas (data not shown), although weakly in c-Maf and Maf B have been reported to contribute synergistically the case of FHL1. These findings validated the higher expression ␣ ␤ with Pax6 to the activation of the glucagon promoter (30). in vivo of these factors in cells compared with cells within Activated protein-1 proteins are implicated in many biological islets and provided more evidence supporting the hypothesis that processes such as cell differentiation, proliferation, and apopto- factors associated with progenitor or stem-type cells are en- sis (35, 38). Because disruption of the essential genes (Jun B, riched in differentiated ␣ cells. Fra-1, and c-Jun) leads to early (E8.5–12.5) embryonic lethality Discussion (35), their putative roles in pancreatic development are unclear. However, ATF3 overexpression in pancreas leads to defects in In this study, we have focused on the area of transcription factors endocrine cell number and islet morphology (33). Our data and their regulators to gain more insight into the molecules that ␣ define the unique phenotypes of ␣ and ␤ cells. This information suggest that ATF3 may have a prominent physiological role in forms a basis for further studies of their developmental origins, cells of murine islets (32). Because c-Jun can inhibit insulin gene transcription by interfering with E2A products (40), differences mode of differentiation, functional regulation, and defects that ␣ ␤ may contribute to the pathogenesis of diabetes mellitus. We have in expression of c-Jun and E2A between and cells may ␤ described a pool containing 141 TFRs, which are differentially contribute to the cell-specific expression of the insulin gene. ␣ expressed between ␣ and ␤ cell types. The following review Chop10 is expressed at a 26.8-fold higher level in TC1.6 cells ͞ attempts to highlight the differences that may contribute to ␣ and (Table 4). As a dominant negative inhibitor of C EBP proteins ␤ cell ‘‘uniqueness’’ in both function and development. This by forming heterodimers, it has been implicated in differentia- evaluation has been organized to focus on the contribution of tion of epidermis and fat cells (39, 42) and in endoplasmic important TFR subgroups and their members. reticulum stress-mediated ␤ cell apoptosis and diabetes (43). It is rapidly induced by low glucose in MIN6 cells (13). How it The bZip Factors. The 10 bZip factors that are enriched in ␣ cells functions in ␣ cells and whether it is regulated by glucose are not belong to the Maf (c-Maf, Maf B, and Maf K) (34); activated known.

Wang et al. PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12663 Downloaded by guest on September 30, 2021 In contrast to ␤ cells, differentiated ␣ cells are equipped with proposed to be an upstream factor involved in regulatory large numbers of immediate–early response bZip factors at high cascades of important HNF family members in islets, because no levels. These may confer the ability to rapidly and dynamically activation of HNF1␣,-4␣, and -3␥ is observed in HNF-1␤ null regulate such ␣ cell functions as glucagon expression and may mice (56, 57). The higher expression of HNF-1␤ in ␣ cells may contribute to cell-specific features of glucagon and insulin gene contribute to the hyperglycemic phenotype of MODY-5, in transcription in response to cAMP. They may also be involved in addition to its role in islet development. ␣ cell proliferation and apoptosis, because the expression of Of the ␤ cell-enriched HD group members, PDX1 is expressed some of the bZip proteins were altered significantly (Fig. 3) in in the earliest pancreatic progenitor cells, then decreases but the hyperplastic ␣ cells in the islets of PC2 null mice (1, 23). later reappears predominantly in the ␤ cells. It thus plays a vital role in the development of the pancreas, as well as in the The bHLH Factors. Fifteen bHLH factors are enriched in ␣ cells; by differentiation and maintenance of the ␤ cell phenotype (5–7, contrast, only Ngn3 and Hed are enriched in ␤ cells. The bHLH 58). A large body of evidence indicates that PDX1 plays a central factors exert a determinative influence in a variety of develop- role in the transcriptional regulation of ␤ cell-specific genes such mental events, including cellular differentiation and lineage as insulin, IAPP, Glut-2, and glucokinase (27, 58). Intriguingly, commitment (44). PDX1 can form heterodimeric (PDX1͞PBX1b) and trimeric A large body of evidence indicates that the E2A proteins (E12 (PDX1͞PBX1b͞Meis2) complexes, which bind to its sites with and E47 isoforms) are required for the development of B lineage Ͼ10-fold higher affinity (59), and PDX1͞PBX complexes are lymphocytes (45). Disruption of the E2A gene had no significant essential for expansion of pancreatic cell subtypes during devel- effect on pancreas development (46). On the other hand, opment (60). It was reported that PBX and Meis function as NeuroD1 is critical for normal pancreas development (5, 7). non-DNA-binding partners in trimeric complexes with many Both the E2A proteins and NeuroD1 have been reported to Hox proteins to modulate the function of DNA-bound Meis-Hox regulate the expression of glucagon and insulin (27, 47), but E2A and PBX-Hox heterodimers (61). Thus it is plausible to suppose null mice exhibit no defect in insulin gene transcription (27, 46). that dynamic interactions among these ␤ cell-predominant mem- Furthermore, the regulation of glucagon and insulin transcrip- bers vitally contribute to unique features of ␤ cell function and tion by E47 and NeuroD1 differ in ␣ and ␤ cells (47). These differentiation. results and our data indicate that the E2A proteins may mainly Pax4, which is restricted to pancreatic progenitors and is very low play a physiological role in ␣ cells through dynamic interactions in adult ␤ cells, mainly functions in ␤ cell differentiation during with NeuroD1 and other bHLH members. In contrast, NeuroD1 pancreas development (5, 7). Its expression in MIN6 cells suggests is essential for pancreas development (5, 7) and may also play a that the Pax4 may play a role in differentiated ␤ cells or reflects the role in the mature pancreatic endocrine cells. Ngn3, a marker of possible origin of MIN6 cells from ␤ cell precursor cells. pancreatic endocrine progenitor cells (5–7), was recently found Most of the other ␣ and ␤ cell-enriched HD group members have to be present in a few cells residing in adult islets (6, 48). Ngn3 been shown to be involved in the programmed development of is enriched in well differentiated MIN6 cells, suggesting that various cell types. Dissection of their functions in the pancreas may these cells may retain some progenitor characteristics (6). contribute to our understanding of ␣ and ␤ cell differentiation. The Id proteins generally function as positive regulators of cell growth and as negative regulators of cell differentiation by antag- The Forkhead (FH) Factors. Of the FH proteins, Foxa1 (HNF3␣) onizing other bHLH proteins, which drive cell lineage commitment and Foxa2 (HNF3␤) were not differentially expressed in ␣ vs. ␤ and differentiation in diverse cell types (49). Furthermore, Id2, a cells. However, Foxa3 (HNF3␥) was detected at higher levels in dominant negative antagonist of retinoblastoma protein, can be MIN6 cells. Despite fasting hypoglycemia in Foxa3 null mice, no induced by N-myc as its effector (50). On the other hand, Myc and specific pancreatic phenotype was found. That Foxa3 is required Mxi1, which can compete for a common partner, Max (no differ- for hepatic Glut-2 expression and glucose homeostasis during a ence between the two cell types by microarray analysis), may prolonged fast (62) but is not essential for glucagon expression antagonize each other to regulate the switch between differentia- (63) suggests that its role in ␤ cells deserves further attention. tion and proliferation of cells (51). Thus, the higher levels of Id1, Foxo1, a negative regulator of insulin sensitivity in liver, adipo- Id2, Id3, N-myc, and Mxi1 in the ␣ cell type provide a plausible cytes, and pancreatic ␤ cells (64), did not appear in the microar- mechanism to balance its potential for proliferation and differen- ray, and we detected no difference in ␣ vs. ␤ cells by RT-PCR tiation as an early differentiated endocrine cell. (data not shown). Foxf1 (HFH-8), which is expressed in the HIF1␣ is involved in the regulation of many glycolytic enzymes mesoderm in close apposition to the gut endoderm, may play a and development (37, 52). Anaerobic glycolysis is the predom- role in mediating cell-specific transcriptional activation in re- inant source of ATP under limited oxygen conditions (Pasteur sponse to cytokines (65). Its possible role in the endocrine effect), and signal cross-talk can occur between hypoxia and pancreas is unclear. glucose metabolism via HIF1 (53). Our results suggest that HIF1␣ may contribute to ␣ cell function by integrating ␣ cell The ZF, High Mobility Group, and Other Family Factors. For infor- physiology with regulation of blood supply in the islets. mation on the ZF, high mobility group, and other family factors, see Supporting Text, which is published as supporting informa- The HD Factors. The HD protein family, which play major roles in tion on the PNAS web site. developmental processes, also manifest fundamental differences between ␣ and ␤ cell types. Among those enriched in the ␣ cell Summary and Conclusion is brain 4, which is specifically expressed in islet progenitors and In this report, we have identified 141 TFRs that appear likely to differentiating, as well as adult, ␣ cells. It functions as an underlie ␣ and ␤ cell-specific developmental and functional essential transactivator in glucagon gene expression and possibly profiles by comparative microarray analysis of Ͼ11,000 tran- as a dominant regulator of ␣ cell lineages (54). This is the HD scripts. Our data highlight the important contribution of the protein showing the greatest differential in expression, and our bZip, bHLH, HD, ZF, and several other transcription factor data highlight its essential contribution to the ␣ cell’s unique groups for ␣ cells; and the HD and forkhead proteins for ␤ cells. development and functional profile. In addition, 30 and eight TFR members, respectively, that are HNF-1␤, a causative factor in MODY-5, is restricted in its enriched in ␣ and ␤ cells have also been reported to be present expression to the epithelial cells of the pancreas during orga- in various stem cells (29), Immunohistochemical examination of nogenesis and is attenuated in adult pancreas (55). It has been ATF3, HIF1␣, and FHL1 distribution in islets provides further

12664 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1735286100 Wang et al. Downloaded by guest on September 30, 2021 support for the hypothesis that ␣ cells may preserve more development, determination, and cell proliferation) are turned characteristics of their progenitors. off or down-regulated. New approaches to down-regulating ␣ In conclusion, we can estimate that the total number of cell activity in type 2 diabetes may help reduce the excessive transcription factors in the genome that contribute to the production of glucagon thereby allowing an absolute or relative differentiated ␣ and ␤ cell phenotypes may be at least 360–420 deficiency of ␤ cells to provide more adequate control of the and 160–190, respectively, based on this large-scale analysis and blood glucose level. Further analysis of the abundances, expres- current estimates of a total of 30–35,000 genes in the mouse and sion patterns, interactions, and targets in vivo of these and other human genomes (66, 67). The actual number may be larger due TFR factors expressed in ␣ and ␤ cells will further our under- to alternative mRNA splicing. The ␣ cells, or subsets thereof, ␣ standing of their differentiation, function, regulation, and de- such as TC1–6 cells, appear to preserve some multipotential fects in diabetes mellitus. characteristics of their progenitors, which may support their ␤ seemingly greater regenerative capacity compared with the We thank Raymond Carroll, Paul Gardner, Jeff Stein and Margaret cells. Whether they contribute to the differentiation and regen- Milewski for technical assistance; Dynov Hristem at the University of eration of any other islet endocrine cells within fetal and adult Chicago for expert assistance with microarray analysis; and Rosie Ricks ␤ islets is an unresolved issue. The cells, on the other hand, for expert assistance in preparing this manuscript. Our thanks also to although derived from the same progenitors as ␣ cells, appear to Graeme Bell and Louis Philipson for encouragement. This work was have a lower differentiation potential, because many of the TFRs supported by National Institutes of Health Grants DK-13914 and that are enriched in ␣ cells (and that mainly function in early DK-20595 and by the Howard Hughes Medical Institute.

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