Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M600526200 on March 17, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13574-13580, May 12, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/19/13574    most recent
M600526200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hua, H.
Right arrow Articles by Sarvetnick, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hua, H.
Right arrow Articles by Sarvetnick, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

BMP4 Regulates Pancreatic Progenitor Cell Expansion through Id2*

Hong Hua, You-Qing Zhang, Sandrine Dabernat, Marcie Kritzik, Daisy Dietz, Lori Sterling, and Nora Sarvetnick1

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, January 18, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate pancreatic development. Moreover, bone morphogenetic proteins (BMPs) regulate the expression of Ids. We hypothesized that BMP4 and Id proteins play a role in the expansion and differentiation of epithelial progenitor cells. We demonstrate that BMP4 induces the expression of Id2 along with the expansion of AR42J pancreatic epithelial cells. Furthermore, neutralization of BMP4 significantly reduced duct epithelial cell expansion in a mouse model of islet regeneration. BMP4 stimulation promotes Id2 binding to the bHLH transcription factor NeuroD, which is required for the differentiation of pancreatic islet cells. Therefore, our results indicate that BMP4 stimulation blocks the differentiation of endocrine progenitor cells and instead promotes their expansion thereby revealing a novel paradigm of signaling explaining the balance between expansion and differentiation of pancreatic duct epithelial progenitors. Understanding the mechanisms of BMP and Id function elucidates a key step during pancreas embryogenesis, which is important knowledge for expanding pancreatic progenitors in vitro.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The progression of pancreatic progenitors into the differentiated endocrine or exocrine lineage is governed by the sequential expression of key transcription factors. Several basic helix-loop-helix (bHLH)2 transcription factors, which bind to regulatory E box segments (CANNTG) of target genes, are important for pancreas development. For example, Ngn3 is specifically required for the development of the endocrine cells, as was demonstrated in null mice (1). Hes-1, another bHLH factor, is a critical repressor of endocrine differentiation, because in its absence accelerated differentiation of endocrine cells occurs (2, 3). Moreover, NeuroD regulates the survival and terminal differentiation of pancreatic beta cells (4). However, the mechanisms regulating the biological activities of these bHLH transcription factors are not completely understood.

Inhibition of differentiation proteins (Ids), comprised of four members (Id1-Id4), are a family of proteins that are implicated in a number of cellular processes, including control of proliferation and differentiation (5, 6). The major role of Id proteins is to bind to and inhibit the function of bHLH transcription factors such as those described above that are critical for pancreatic development. Id proteins lack basic DNA-binding domains and heterodimers between Id and bHLH proteins cannot bind DNA, allowing them to inhibit the biological activity of these transcription factors (7, 8). In this sense, Ids inhibit transcription factor binding and may serve to regulate the timing of key events during pancreatic ontogeny. Indeed, the expression of Ids has been detected in the fetal pancreas in a microarray study (9). Importantly, Id2 has been shown to prevent NeuroD from binding to its DNA target (10). Because NeuroD is required for pancreatic islet development, we hypothesized that Id proteins may be important in regulating its biological function, thereby affecting the kinetics of cell growth and differentiation.

Bone morphogenetic proteins (BMPs) are pleiotropic proteins that regulate proliferation, differentiation, and migration of various cell types (reviewed in Ref. 11). BMPs signal through heteromeric complex combinations of types I and II serine/threonine kinase receptors and their nuclear effectors, SMADs (11). Binding of BMP to at least one type I and type II receptor is necessary for signaling (12). Interestingly, reverse transcription (RT) PCR analysis of mouse embryonic pancreas demonstrates expression of BMP4, -5, and -7, as well as BMP type I and II receptors (13). In addition, BMP signaling molecules, including SMADs 1, 2, and 4, are expressed in neonatal and adult pancreatic islets (14), and BMP4, -5, and -6 promote the proliferation and development of epithelial islet-like structures in vitro (15). Several studies also implicate BMPs as propluripotency factors. In particular, BMP4 has been reported to enhance mouse embryonic stem cell self-renewal (16, 17) and is necessary for the production of hematopoietic progenitors (18, 19). As SMAD-binding elements are found in the promoter of Id proteins (20), BMPs have been shown to regulate the expression of Id proteins in several cell types, including embryonic stem cells (20-23), This indicates that BMPs can control the expression of Ids, which in turn, could regulate the biological activity of bHLH transcription factors. However, the role of BMP and Id protein signaling in regulating expansion of pancreatic progenitors has not been explored. Clearly, a more complete knowledge of the function of this family of proteins in pancreatic islet development would allow us to understand the biology behind the maintenance of progenitor cells and the regulation of differentiation in the pancreas.

We hypothesize that BMP4 and Id2 proteins play prominent roles in pancreatic ontogeny and regeneration by affecting islet progenitor cell expansion during the differentiation of pancreatic islet cells. In this report, we show that BMP4 and Id proteins regulate the expansion of AR42J cells, a pancreatic epithelial cell line with the potential to differentiate into endocrine cells in vitro (24, 25). Furthermore, we found that BMP4 is necessary for the hyperplasia of duct cells during pancreatic islet regeneration. To define the functional mechanism of BMP4 regulation of progenitor cell expansion, we demonstrate that BMP4 exposure enhances binding of Id proteins to the bHLH transcription factor NeuroD. Our work dissects a critical signaling pathway for the regulation of cell growth and differentiation of endocrine progenitors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Housing and Cell Lines—IFN{gamma}NOD mice were backcrossed onto the NODShi background and were maintained in the specific pathogen-free barrier facility at The Scripps Research Institute. AR42J cells were a generous gift from Dr. Itaru Kojima (Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan) and were maintained in Dulbecco's modified Eagle's medium at 37 °C and 5% CO2. All studies were conducted in strict accordance with the TSRI Animal Care and Use Committee guidelines.


Figure 1
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1.
BMP4 regulates Id2 expression and proliferation in AR42J cells. A, Western immunoblot of Id2 protein expression in total cell lysates of AR42J cells in the presence of BMP4 (1-100 ng/ml, 18 h) stimulation with and without noggin (0.5-5 µg/ml). Protein loading was normalized by stripping the membrane and reprobing with actin antibody, followed by densitometric analysis. B, densitometric analysis of at least three independent experiments (using the 100 ng/ml concentration) was performed. Data are presented as the fold over control (in the absence of BMP4). C, AR42J cells were growth-arrested and stimulated with recombinant BMP4 (100 ng/ml) for 18 h with the addition of [3H]thymidine in the last 2 h. Data are presented as raw counts, in the absence of BMP4 and with BMP4 treatment (p < 0.0001). D and E are representative confocal micrographs depicting AR42J cells stained with anti-BrdUrd antibody (FITC label) with and without BMP4 (100 ng/ml, 18 h) stimulation. Nuclei were stained with TOPRO3. Bar represents 50 µm on all confocal images.

 
Immunocytochemistry—Tissue sections (4 µm) were subjected to immunohistochemistry using commercial polyclonal antibodies including Id2 (1:600, 1:1000), BMPR2 (1:50), BMP4 (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA), insulin (1:1000) (Dako, Denmark), BrdUrd (1:50) (Accurate Chemical, San Diego, CA), PAX6 (1:100) (Convance, CA), and NeuroD (1 µg/ml) (Cemines, La Jolla, CA and Chemicon, Temecula, CA). An avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain ABC Kit, Vector laboratories, Burlingame, CA), and 3,3-diaminobenzedine (Sigma) was used as the chromagen. Samples were counterstained with hematoxylin. In some cases, images were acquired using a Zeiss 2100 confocal microscope with a 60x objective, and RGB micrographs were compiled with Image J software.

Western Immunoblotting—AR42J cells were lysed in 2x sample buffer (0.13 mol/liter Tris-base, pH 6.8, 20% glycerol, and 4% SDS). Aliquots were taken for protein determination using the Bradford protein assay (Bio-Rad). The remaining cell extracts were denatured in 4x sample buffer (0.13 mol/liter Tris, 40% glycerol, 8% SDS, 4% beta-mercaptoethanol, and 0.02% bromphenol blue). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose or Immobilon polyvinylidene fluoride membranes (Bio-Rad) overnight in transfer buffer (25 mmol/liter tris-base, 192 mmol/l glycine, pH 8.3, and 20% methanol). The membranes were blocked in 5% skim milk powder in Tris buffer containing 0.05% Tween 20, then probed with the indicated antibody and visualized with enhanced chemiluminescence. Equal loading of protein was assessed by reprobing the membrane for actin. The densitometry analysis was carried out using Image J software. Briefly, the intensity of images of scanned Western blots was determined, and a ratio of each band to its actin control was calculated.

Co-immunoprecipitation—AR42J cells were growth-arrested in medium lacking fetal calf serum, stimulated with BMP4 (100 ng/ml) for 18 h, and the cells were lysed in radioimmune precipitation assay buffer. Equal amounts of protein (400-600 µg) were precleared with 50 µl of TrueBlot anti-rabbit Ig IP beads (eBioscience, San Diego, CA), immunoprecipitated with 10 µg of Id2 antibody (2h, 4 °C), and then pulled down with TrueBlot anti-rabbit Ig IP beads. The samples were lysed in 2x sample buffer, and equal volumes of protein were separated by SDS-PAGE and probed for the indicated antibody. The protein levels were determined by reprobing the blot with the Id2 antibody.

IFN{gamma} Duct Cell Isolation—Pancreatic duct cell isolation was performed as described previously (26). Briefly, each IFN{gamma}NOD pancreas was treated with 1 mg/ml collagenase P in Dulbecco's modified Eagle's medium F-12 with slight agitation at 37 °C for 30 min. The duct preparation was then filtered through a 200-µm mesh, washed in cold phosphate-buffered saline, and treated with 0.05% trypsin, EDTA at 37 °C for 10-15 min. Fetal calf serum was added to stop the trypsin treatment, and the duct preparation was washed in cold phosphate-buffered saline. Finally, duct cells were filtered over a 70-µm cell strainer and cultured in 10% fetal calf serum Dulbecco's modified Eagle's F-12 medium overnight. For BMP4 treatment, duct cells were growth-arrested in 0.5% fetal calf serum Dulbecco's modified Eagle's F-12 medium for 24 h before the addition of BMP4 (1-100 ng/ml) for 18 h. For some of the experiments, the duct epithelial cells isolated from IFN{gamma}NOD pancreas were pretreated with the neutralizing antibody to BMP4 (1-10 µg/ml) for 2 h before the addition of recombinant BMP4.


Figure 2
View larger version (109K):
[in this window]
[in a new window]
  		FIGURE 2.
Id2 and BMP immunoreactivities in the developing and regenerating pancreas. A-C, representative confocal micrograph of E16 embryo sections demonstrated that insulin (FITC label, A and C) does not colocalize with Id2 (Cye3 label, B and C). D-F, BMP4 immunoreactivity in adult IFN{gamma}NOD (D-L) is localized to "islet-like" areas of the IFN{gamma}NOD pancreas (D), whereas BMPR2 (E) and Id2 (F) are localized to both large and small ducts of the IFN{gamma}NOD pancreas. Insulin-positive cells (FITC label, G) for the most part are not Id2 immunoreactive (H-I), whereas some Id2-positive cells are BrdUrd-positive (J-L). Confocal images were acquired with a 60x objective, whereas 3,3-diaminobenzedine images were acquire using a 40X objective.

 
BMP4 Neutralizing Antibody Treatment and BrdUrd Count—To ascertain whether BMP4 signaling affects pancreatic duct cell expansion, we injected mice intravenously with a neutralizing antibody to BMP4 (200 µg) (R&D Systems, Inc., Minneapolis, MN) three times a week for 2 weeks. These mice were injected with BrdUrd (100 µg/g) intraperitoneally on day 15 and sacrificed on day 16, and the tissue was harvested and fixed for histology. After sections were cut, the tissues were analyzed by immunohistochemistry. Every fifth section was stained with antibody to BrdUrd, and the cells were counted for BrdUrd-positive duct cells in at least 10 fields per section. The labeled cells were evaluated as the percentage of BrdUrd-labeled cells out of the total number of epithelial cells per duct (27, 28).

Statistics—Data were expressed as mean ± S.D. and analyzed using Instat (GraphPad Software Inc.). Comparisons were made by Student's t test. A value of p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
BMP4 Regulates Id Expression in AR42J Cells and Induces Cell Proliferation—The AR42J cell line is a pluripotent cell line derived from a rat pancreatic acinar carcinoma, which are acinar-like cells when exposed to dexamethasone (29), and in contrast, convert into insulin-producing cells with betacellulin and activin A treatment (24). Therefore, AR42J cells have the potential to become exocrine or endocrine cells, making them a valuable in vitro correlate to study the mechanisms of beta cell differentiation as well as for expansion of pancreatic progenitors. We tested the hypothesis that BMP4 regulates the expression of Id protein and is required for cell expansion in AR42J pancreatic cells. To this end, AR42J cells were growth-arrested and then treated with recombinant BMP4 for 18 h, and the expression of Id was quantitated by Western immunoblotting (Fig. 1A). Id1 and Id3 were not detected (data not shown), whereas Id2 was present, and its expression was inducible by recombinant BMP4. Densitometric analysis showed that BMP4 increased Id2 expression by 2 ± 0.6-fold over untreated control cells (Fig. 1B). BMP antagonists, such as noggin, regulate the activities of BMPs by forming a complex with them and preventing their binding to BMP receptors (30, 31). We found that pretreatment of AR42J cells with noggin (5 µg/ml) significantly reduced BMP4-dependent stimulation of Id2 protein expression (Fig. 1, A and B). Therefore, our data are consistent with the hypothesis that Id protein expression is induced by BMP4 signaling in pancreatic epithelial cells.


Figure 3
View larger version (89K):
[in this window]
[in a new window]
  		FIGURE 3.
BMP4 stimulates Id2 expression and BrdUrd incorporation. Epithelial cells isolated from IFN{gamma}NOD pancreas were growth-arrested and then stimulated with recombinant BMP4 (100 ng/ml, 18 h) with and without the neutralizing BMP4 antibody, as described under "Experimental Procedures." A, total cell lysates were probed for Id2 and actin (n = 3). B and C are representative micrographs depicting BrdUrd immunoreactivity of IFN{gamma}NOD pancreas sections from control (B) and mice treated with neutralizing BMP4 antibody (C). D is the numerical summary of the BrdUrd counts. C = no BMP4 treatment. Micrographs were taken at 20x.

 
To determine whether BMP4 regulates AR42J cell proliferation, we growth-arrested the cells and stimulated them with BMP4 for 18 h with the addition of [3H]thymidine during the last 2 h. The growth index of the cultured AR42J cells was determined by quantitatively measuring [3H]thymidine uptake. We found that BMP4 increased [3H]thymidine counts after 18 h of BMP4 stimulation compared with control cells grown without BMP4 (p < 0.0001) (Fig. 1C). We confirmed these results utilizing BrdUrd incorporation experiments. After 2 h of pulsing with BrdUrd, we observed an increased proportion of AR42J cells stimulated with BMP4 that were BrdUrd positive as determined by immunofluorescence staining (Fig. 1, D and E). These results demonstrate that BMP4 stimulates the expansion of pancreatic progenitor cells.

BMP Signaling Molecules and Id2 Are Expressed in the Pancreatic Epithelium of Embryonic, Adult, and Regenerating IFN{gamma}NOD Pancreas—BMP4 and its receptors are expressed in the embryonic pancreas (13, 15). Because we observed that BMP4 exposure promotes Id2 expression in AR42J progenitor cells, we next asked whether these factors are expressed in the developing and adult pancreas. We performed immunolocalization experiments and found that Id2-expressing cells were located in the embryonic pancreas but not strikingly colocalized with insulin (Fig. 2).

Next, we studied the expression of BMP signaling proteins in mice that express IFN{gamma} (IFN{gamma}NOD) driven by the insulin promoter. These mice are ideal for studying pancreatic regeneration as they demonstrate ductal hyperplasia and recapitulate several aspects of pancreas development (32, 33). Immunoreactive BMP4 and BMPR2 expression were found to be localized to "islet-like" regions and in the small ducts (Fig. 2, D and E) of this transgenic strain. Likewise, in the regenerating IFN{gamma}NOD pancreas, Id2 expression was strong in duct epithelial cells (Fig. 2F). Moreover double-labeling experiments demonstrated that the majority of Id2-positive cells do not express insulin (Fig. 2, G-I). To ascertain the potential relevance of Id2 in epithelial cell expansion in the IFN{gamma}NOD pancreas, we stained the pancreas for BrdUrd and Id2 and performed confocal analysis. We found that a subset of Id2 positive cells also incorporated BrdUrd (Fig. 2, J-L). Therefore, cells harboring BMP, its receptor, and Id2 are found within the epithelial regions of the developing and regenerating mouse pancreas, suggesting that these molecules may regulate expansion of duct epithelial progenitor cells.


Figure 4
View larger version (100K):
[in this window]
[in a new window]
  		FIGURE 4.
BMP4 regulates Id2 binding to NeuroD. IFN{gamma}NOD pancreas immunostained with NeuroD antibody for control (A) and BMP4-neutralizing antibody-treated mice (B). BMP4 treated and nontreated cell lysates from AR42J cells were immunoprecipitated (IP) with the Id2 antibody and immunoblotted (IB) for NeuroD, the membrane was stripped and reprobed for Id2 (C). D is the compiled densitometric data for 8 experiments. Pancreas tissues from control mice (E) show reduced numbers of positive cells and intensity of immunoreactivity for PAX6 compared with mice that received BMP4-neutralizing antibody (F). C = no BMP4 treatment. Micrographs were taken at 40x.

 
BMP4 Regulates Id Protein Expression and Pancreatic Duct Cell Expansion in IFN{gamma}NOD—We asked whether Id proteins are induced by BMPs in pancreatic duct cells. We isolated IFN{gamma}NOD duct epithelial cells (because this strain expressed the BMPR2 in the epithelium), growth arrested the cultures, and then stimulated them with recombinant BMP4 protein. We focused on the effect of BMP4 on Id2, because it is found exclusively in duct epithelial cells of the IFN{gamma}NOD pancreas. We found that Id2 expression was enhanced in the presence of BMP4 by 2-fold (Fig. 3A). We further found that when these duct epithelial cells were treated with a neutralizing BMP4 antibody, Id2 expression was reduced, supporting the notion that Id2 propagates BMP4 signals in the pancreatic epithelium (Fig. 3).

We next asked whether BMP4 regulates epithelial cell expansion in vivo. Because the IFN{gamma}NOD strain displays ductal hyperplasia, it represents a model to test whether BMP4 regulates cellular proliferation in the pancreas. To ascertain whether BMP4 signaling affects pancreatic duct epithelial cell expansion, we injected mice intravenously with the neutralizing antibody to BMP4 (34). Fig. 3, B and C, depicts a representative histological section of the IFN{gamma}NOD pancreas tissue stained for BrdUrd in the control and BMP4 neutralizing antibody-treated groups, demonstrating a reduction in BrdUrd-positive epithelial cells following anti-BMP treatment. We determined the percentage of BrdUrd-positive cells and found that in control animals, the average percentage of BrdUrd-positive cells is 17 ± 7, whereas treatment with the BMP4 neutralizing antibody reduced expansion to 8 ± 3% (n = 5 animals/condition, p = 0.003) (Fig. 3D). Our cumulative data demonstrate that BMP4 regulates the expansion of pancreatic duct epithelial cell progenitors in vivo.


Figure 5
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5.
Model of BMP4 and Id protein signaling in the expansion of pancreatic epithelial progenitors. In the presence of BMP4 ligand, BMP receptors transmit the signal that leads to phosphorylation (P) of SMAD1 and -5 as well as co-SMAD4. Activated SMADs bind to SMAD-binding elements (SBE) on the Id promoter resulting in transcription of Id proteins. These bind to bHLH transcription factors such as NeuroD, preventing it from binding to the E-box elements of endocrine-specific genes. Therefore, pancreatic epithelial progenitors do not differentiate along the NeuroD-specific endocrine lineage but may instead expand, as Id proteins are involved in cell proliferation. In the absence of BMP4 or the presence of noggin, SMAD-dependent expression of Id does not occur, allowing NeuroD to activate transcription of endocrine-specific genes.

 
BMP4 Promotes the Binding of Id2 with NeuroD and Increases PAX6 Expression—Id proteins regulate the biological activity of bHLH transcription factors. NeuroD is a bHLH transcription factor that is important for the terminal differentiation of the endocrine lineage in the pancreas (4). Because NeuroD is required for the terminal differentiation of pancreatic endocrine cells, we asked whether it was found in the pancreatic epithelium during regeneration, and whether Id proteins might affect the expansion of pancreatic epithelial cells by inhibiting the differentiation program promoted by NeuroD. We found that NeuroD immunoreactivity was present in ductal cells, and in islet-like areas of the regenerating pancreas, but the overall level of immunoreactivity was not altered by inhibiting BMP4 signaling (Fig. 4, A and B). We next asked whether BMP treatment could enhance the binding of Id2 and NeuroD. Using immunoprecipitation experiments, we determined that recombinant BMP4 treatment increased the amount of Id2 bound to NeuroD. The ratio of bound NeuroD to the immunoprecipitated Id2 was 0.8 compared with 0.5 in untreated samples (Fig. 4, C and D). Interestingly, we did not observe any increase in NeuroD levels in the total cell lysate (data not shown), suggesting that regulation is enacted through protein-protein interactions or through induction of Id2 expression, as demonstrated above. These data therefore reveal that BMP4 regulates the accumulation and expansion of ductal progenitor epithelial cells through the activity of Id proteins and their bHLH targets.

PAX6 is an important transcription factor in the regulation of pancreatic morphogenesis (35). Importantly, it has been demonstrated that NeuroD binds specifically to E boxes in the promoter of PAX6, regulating its expression (36). We asked whether neutralization of BMP4 affected PAX6 expression. We stained the BMP4-neutralized IFN{gamma}NOD mouse pancreas with an antibody to PAX6. We found increased numbers of PAX6-positive cells and enhanced intensity of PAX6 expression in the BMP4-neutralized pancreata compared with controls (Fig. 4, E and F). These results demonstrate the BMP pathway affects the expression of this critical factor for islet differentiation through NeuroD regulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Id proteins are important in the regulation of cell proliferation and inhibition of differentiation. Our studies demonstrate the importance of BMP4 induction of Id2 in ductal progenitor cell expansion in the pancreas. We showed that BMP4 stimulated Id2 expression and induced cellular proliferation in AR42J cells. Furthermore, BMP signaling proteins and Id2 are expressed in the developing and regenerating mouse pancreas. The importance of BMP4 in epithelial duct cell expansion was ascertained by neutralization of BMP4, which significantly reduced BrdUrd incorporation during pancreatic regeneration. Therefore, our results are consistent with the paradigm that BMP4 signaling is important for ductal expansion. Moreover, we unraveled a link between BMP4 and Id2, which contributes to endocrine-specific differentiation in the pancreas. We have observed that BMP4 induces Id2 expression and its binding to NeuroD, a bHLH transcription factor critical for endocrine differentiation during pancreatic development. Furthermore, in the IFN{gamma}NOD pancreas, neutralizing BMP4 enhanced PAX6 immunoreactivity, a transcription factor that has been previously demonstrated to be a target of NeuroD, thereby indicating enhanced NeuroD activity in the absence of BMP4. Our results support the hypothesis that BMP4 is critical for the expansion of pancreatic duct epithelial progenitors and perhaps in the maintenance of their predifferentiated state (Fig. 5).

BMPs and Ids in Cell Growth—The importance of BMP signaling in growth has been amply demonstrated. For example, SMAD1 regulates lung morphogenesis in an explant culture model by reducing lung epithelial branching, cell proliferation, and differentiation (37). In addition, exposure to low concentrations of BMP4 promoted proliferation of subventricular zone progenitors (38). Furthermore, BMP4 promotes proliferation of committed neuronal progenitors, as well as facilitating their commitment into astrocytes (38). Indeed, we found that BMP4 is essential for the growth of pancreatic epithelial cells in AR42J cells in vitro as well as in a regenerating pancreas model.

Id protein expression correlates with cell proliferation and inhibition of differentiation in many cell types. In the pancreas, Id2 expression was demonstrated to be elevated in both cancerous pancreatic tissues and in PANC-1 pancreatic cancer cell line (39). In mammary epithelial cells and breast cancer cells, ectopic expression of Id1 allowed for continued proliferation (40). In addition, Id protein signaling is known to intersect with cell cycle regulatory functions, and deregulation of Ids may lead to tumorigenesis (7, 41, 42). However, the function of Id proteins in the growth of progenitor cells in the pancreas has not previously been studied. Here, using AR42J cells and in vivo studies, we demonstrated that BMP4 acts upstream of Id2 and that these signaling molecules are required for pancreatic epithelial cell expansion.

Id Proteins Are Regulated by BMPs—We found that exposure to BMPs caused the induction of Id protein expression in AR42J cells as well as in isolated IFN{gamma}NOD epithelial duct cells. Several other interesting studies have also linked BMPs with the expression of Ids (reviewed in Ref. 11). Importantly, SMAD-binding elements are found on Id gene promoters (21, 22, 42). In addition, BMP4 contributes to astrocyte lineage commitment through Id-mediated inhibition of OLIG and E2A transcription factors (43). Id1, Id2, and Id3 are up-regulated following BMP stimulation in osteogenic cells. Furthermore, microarray gene analysis of epithelial cells stimulated with BMP7 showed induction of Ids, which prevented TGF-beta-induced growth and epithelial-mesenchymal transition (44). Similar to our studies in the pancreas, the up-regulated Id genes promoted cell proliferation and inhibited differentiation. Clearly, BMP-dependent Id activity is a positive regulator of proliferation and a modulator of cell differentiation. Our data using AR42J cells and isolated IFN{gamma}NOD ductal epithelial cells showed that Id2 is induced by BMP4 signaling. Thus BMPs may promote expansion via their ability to suppress terminal differentiation, a process that is mediated by bHLH transcription factors.

BMPs Are Important Factors in Cell Fate Determination—BMPs are important for directing the cell fate of several cell types, including mesenchymal cells (45). NeuroD is a bHLH transcription factor expressed in pancreatic, intestinal, and neural tissue, and plays a prominent role in endocrine lineage commitment and regulation of insulin gene transcription (4, 46). Clinically, mutations in NeuroD cause maturity-onset diabetes of the young (MODY). NeuroD null mice have a striking reduction in the number of beta cells, develop severe diabetes, and die perinatally (4, 47), although the genetic background of the mice determines the severity of the phenotype, as neogenesis of beta cells can occur in NeuroD null mice (48). It is possible that the neogenesis observed in this knock-out strain may arise from the propensity for expansion, which results from the absence of strong signals promoting terminal differentiation. Here, we show the net outcome of BMP4 on the regulation of endocrine differentiation, in which Id2 binding to NeuroD is regulated by BMP4. This relationship may underlie the distinct homeostasis of pancreatic progenitor cells compared with differentiated endocrine cells. NeuroD was previously shown to form heterodimers with E47, resulting in binding to the E-box of the insulin gene promoter and its transactivation (47). Moreover, overexpression of Id2 in COS M6 cells inhibited NeuroD from binding to its E-box target (10). It has been demonstrated that one of the targets of NeuroD in determining pancreas morphogenesis and maintaining endocrine cell type numbers is PAX6 (35, 36). In agreement with this, our data show an enhanced PAX6 immunoreactivity when BMP4 signaling is neutralized and NeuroD binding to Id2 is reduced. Therefore, Id proteins may regulate beta cell-specific gene expression through NeuroD. Overall, this is the first study to demonstrate that BMP4 and Id2 regulate expansion and differentiation of the endocrine lineage in pancreatic duct epithelial progenitors by inducing Id2 binding to NeuroD. These results will enhance our knowledge of the signaling pathways that are necessary for expansion of pancreatic progenitors and, subsequently, for their differentiation into beta cells for future therapeutic treatment of type I diabetes.


   FOOTNOTES
 
* This work was supported by Grants DK066511 and DK060746 from the National Institutes of Health (to N. S.) and by a Larry L. Hillblom Foundation Fellowship (to H. H.). The Scripps Research Institute manuscript number 17811-IMM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Immunology (IMM-23), The Scripps Research Institute, La Jolla, CA 90237. Tel.: 858-784-9066; Fax: 858-784-9096; E-mail: noras{at}scripps.edu.

2 The abbreviations used are: bHLH, basic helix-loop-helix; BMP, bone morphogenetic protein; FITC, fluorescein isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We greatly appreciate the helpful input and assistance from members of the Sarvetnick Laboratory: N. Hill, A. Kayali, G. Liu, X. Li, A. Marleau, M. Solomon, D. Yadav, M. Cleary, C. Fine, A. Maday, E. Rodriguez, A. Stotland, P. Secrest, S. Weston, and T. Gresham.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gradwohl, G., Dierich, A., LeMeur, M., and Guillemot, F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1607-1611[Abstract/Free Full Text]
  2. Jensen, J., Pedersen, E. E., Galante, P., Hald, J., Heller, R. S., Ishibashi, M., Kageyama, R., Guillemot, F., Serup, P., and Madsen, O. D. (2000) Nat. Genet. 24, 36-44[CrossRef][Medline] [Order article via Infotrieve]
  3. Habener, J. F., Kemp, D. M., and Thomas, M. K. (2005) Endocrinology 146, 1025-1034[Abstract/Free Full Text]
  4. Naya, F. J., Huang, H. P., Qiu, Y., Mutoh, H., DeMayo, F. J., Leiter, A. B., and Tsai, M. J. (1997) Genes Dev. 11, 2323-2334[Abstract/Free Full Text]
  5. Benezra, R. (2001) Trends Cardiovasc. Med. 11, 237-241[CrossRef][Medline] [Order article via Infotrieve]
  6. Norton, J. D., Deed, R. W., Craggs, G., and Sablitzky, F. (1998) Trends Cell Biol. 8, 58-65[CrossRef][Medline] [Order article via Infotrieve]
  7. Lasorella, A., Uo, T., and Iavarone, A. (2001) Oncogene 20, 8326-8333[CrossRef][Medline] [Order article via Infotrieve]
  8. Norton, J. D. (2000) J. Cell Sci. 113, 3897-3905[Abstract]
  9. Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B., and Melton, D. A. (2004) Development (Camb.) 131, 165-179[Abstract/Free Full Text]
  10. Ghil, S. H., Jeon, Y. J., and Suh-Kim, H. (2002) Exp. Mol. Med. 34, 367-373[Medline] [Order article via Infotrieve]
  11. ten Dijke, P., Korchynskyi, O., Valdimarsdottir, G., and Goumans, M. J. (2003) Mol. Cell Endocrinol. 211, 105-113[CrossRef][Medline] [Order article via Infotrieve]
  12. Nohe, A., Keating, E., Knaus, P., and Petersen, N. O. (2004) Cell. Signal. 16, 291-299[CrossRef][Medline] [Order article via Infotrieve]
  13. Dichmann, D. S., Miller, C. P., Jensen, J., Scott Heller, R., and Serup, P. (2003) Dev. Dyn. 226, 663-674[CrossRef][Medline] [Order article via Infotrieve]
  14. Brorson, M., Hougaard, D. M., Nielsen, J. H., Tornehave, D., and Larsson, L. I. (2001) Histochem. Cell Biol. 116, 263-267[Medline] [Order article via Infotrieve]
  15. Jiang, F. X., Stanley, E. G., Gonez, L. J., and Harrison, L. C. (2002) J. Cell Sci. 115, 753-760[Abstract/Free Full Text]
  16. Ying, Q. L., Nichols, J., Chambers, I., and Smith, A. (2003) Cell 115, 281-292[CrossRef][Medline] [Order article via Infotrieve]
  17. Qi, X., Li, T. G., Hao, J., Hu, J., Wang, J., Simmons, H., Miura, S., Mishina, Y., and Zhao, G. Q. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6027-6032[Abstract/Free Full Text]
  18. Park, C., Afrikanova, I., Chung, Y. S., Zhang, W. J., Arentson, E., Fong Gh, G., Rosendahl, A., and Choi, K. (2004) Development (Camb.) 131, 2749-2762[Abstract/Free Full Text]
  19. Sadlon, T. J., Lewis, I. D., and D'Andrea, R. J. (2004) Stem Cells 22, 457-474[Abstract/Free Full Text]
  20. Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U., and Nordheim, A. (1999) J. Biol. Chem. 274, 19838-19845[Abstract/Free Full Text]
  21. Lopez-Rovira, T., Chalaux, E., Massague, J., Rosa, J. L., and Ventura, F. (2002) J. Biol. Chem. 277, 3176-3185[Abstract/Free Full Text]
  22. Korchynskyi, O., and ten Dijke, P. (2002) J. Biol. Chem. 277, 4883-4891[Abstract/Free Full Text]
  23. Nakashima, K., Takizawa, T., Ochiai, W., Yanagisawa, M., Hisatsune, T., Nakafuku, M., Miyazono, K., Kishimoto, T., Kageyama, R., and Taga, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5868-5873[Abstract/Free Full Text]
  24. Zhang, Y. Q., Kanzaki, M., Furukawa, M., Shibata, H., Ozeki, M., and Kojima, I. (1999) Diabetologia 42, 719-727[CrossRef][Medline] [Order article via Infotrieve]
  25. Zhang, Y. Q., Mashima, H., and Kojima, I. (2001) Diabetes 50, Suppl. 1, 10-14
  26. Kayali, A. G., Stotland, A., Gunst, K. V., Kritzik, M., Liu, G., Dabernat, S., Zhang, Y. Q., Wu, W., and Sarvetnick, N. (2005) J. Endocrinol. 185, 45-56[Abstract/Free Full Text]
  27. Kayali, A. G., Van Gunst, K., Campbell, I. L., Stotland, A., Kritzik, M., Liu, G., Flodstrom-Tullberg, M., Zhang, Y. Q., and Sarvetnick, N. (2003) J. Cell Biol. 163, 859-869[Abstract/Free Full Text]
  28. Zhang, Y. Q., Cleary, M. M., Si, Y., Liu, G., Eto, Y., Kritzik, M., Dabernat, S., Kayali, A. G., and Sarvetnick, N. (2004) Diabetes 53, 2024-2033[Abstract/Free Full Text]
  29. Rosewicz, S., Riecken, E. O., and Wiedenmann, B. (1992) Clin. Investig. 70, 205-209[Medline] [Order article via Infotrieve]
  30. Zimmerman, L. B., De Jesus-Escobar, J. M., and Harland, R. M. (1996) Cell 86, 599-606[CrossRef][Medline] [Order article via Infotrieve]
  31. Canalis, E., Economides, A. N., and Gazzerro, E. (2003) Endocr. Rev. 24, 218-235[Abstract/Free Full Text]
  32. Gu, D., and Sarvetnick, N. (1993) Development (Camb.) 118, 33-46[Abstract]
  33. Gu, D., and Sarvetnick, N. (1994) Recent Prog. Horm. Res. 49, 161-165[Medline] [Order article via Infotrieve]
  34. Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. (1994) J. Cell Biol. 127, 1755-1766[Abstract/Free Full Text]
  35. Ashery-Padan, R., Zhou, X., Marquardt, T., Herrera, P., Toube, L., Berry, A., and Gruss, P. (2004) Dev. Biol. 269, 479-488[CrossRef][Medline] [Order article via Infotrieve]
  36. Marsich, E., Vetere, A., Di Piazza, M., Tell, G., and Paoletti, S. (2003) Biochem. J. 376, 707-715[CrossRef][Medline] [Order article via Infotrieve]
  37. Chen, C., Chen, H., Sun, J., Bringas, Jr., P., Chen, Y., Warburton, D., and Shi, W. (2005) Am. J. Physiol. 288, 1033-1039
  38. Liu, S. Y., Zhang, Z. Y., Song, Y. C., Qiu, K. J., Zhang, K. C., An, N., Zhou, Z., Cai, W. Q., and Yang, H. (2004) Exp. Neurol. 190, 109-121[CrossRef][Medline] [Order article via Infotrieve]
  39. Kleeff, J., Maruyama, H., Ishiwata, T., Sawhney, H., Friess, H., Buchler, M. W., and Korc, M. (1999) Gastroenterology 116, 1202-1216[CrossRef][Medline] [Order article via Infotrieve]
  40. Swarbrick, A., Akerfeldt, M. C., Lee, C. S., Sergio, C. M., Caldon, C. E., Hunter, L. J., Sutherland, R. L., and Musgrove, E. A. (2005) Oncogene 24, 381-389[CrossRef][Medline] [Order article via Infotrieve]
  41. Yokota, Y., and Mori, S. (2002) J. Cell Physiol. 190, 21-28[CrossRef][Medline] [Order article via Infotrieve]
  42. Ruzinova, M. B., and Benezra, R. (2003) Trends Cell Biol. 13, 410-418[CrossRef][Medline] [Order article via Infotrieve]
  43. Samanta, J., and Kessler, J. A. (2004) Development (Camb.) 131, 4131-4142[Abstract/Free Full Text]
  44. Kowanetz, M., Valcourt, U., Bergstrom, R., Heldin, C. H., and Moustakas, A. (2004) Mol. Cell Biol. 24, 4241-4254[Abstract/Free Full Text]
  45. Rajan, P., Panchision, D. M., Newell, L. F., and McKay, R. D. (2003) J. Cell Biol. 161, 911-921[Abstract/Free Full Text]
  46. Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N., and Weintraub, H. (1995) Science 268, 836-844[Abstract/Free Full Text]
  47. Chae, J. H., Stein, G. H., and Lee, J. E. (2004) Mol. Cells 18, 271-288[Medline] [Order article via Infotrieve]
  48. Huang, H. P., Chu, K., Nemoz-Gaillard, E., Elberg, D., and Tsai, M. J. (2002) Mol. Endocrinol. 16, 541-551[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 CarcinogenesisHome page
V. A. Trabosh, K. A. Divito, B. D. Aguda, C. M. Simbulan-Rosenthal, and D. S. Rosenthal
Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis
Carcinogenesis, July 1, 2009; 30(7): 1252 - 1259.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
K. J. Gordon, K. C. Kirkbride, T. How, and G. C. Blobe
Bone morphogenetic proteins induce pancreatic cancer cell invasiveness through a Smad1-dependent mechanism that involves matrix metalloproteinase-2
Carcinogenesis, February 1, 2009; 30(2): 238 - 248.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. Yan, G. Liu, A. Scoumanne, and X. Chen
Suppression of Inhibitor of Differentiation 2, a Target of Mutant p53, Is Required for Gain-of-Function Mutations
Cancer Res., August 15, 2008; 68(16): 6789 - 6796.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
J. M. Oliver-Krasinski and D. A. Stoffers
On the origin of the {beta} cell
Genes & Dev., August 1, 2008; 22(15): 1998 - 2021.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
J. Rajagopal, T. J. Carroll, J. S. Guseh, S. A. Bores, L. J. Blank, W. J. Anderson, J. Yu, Q. Zhou, A. P. McMahon, and D. A. Melton
Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme
Development, May 1, 2008; 135(9): 1625 - 1634.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
P. White, C. Lee May, R. N. Lamounier, J. E. Brestelli, and K. H. Kaestner
Defining Pancreatic Endocrine Precursors and Their Descendants
Diabetes, March 1, 2008; 57(3): 654 - 668.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
I. Serafimidis, I. Rakatzi, V. Episkopou, M. Gouti, and A. Gavalas
Novel Effectors of Directed and Ngn3-Mediated Differentiation of Mouse Embryonic Stem Cells into Endocrine Pancreas Progenitors
Stem Cells, January 1, 2008; 26(1): 3 - 16.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
X. Zhu, Y. Lin, M. Bacanamwo, L. Chang, R. Chai, I. Massud, J. Zhang, M. T. Garcia-Barrio, W. E. Thompson, and Y. E. Chen
Interleukin-1 {beta}-induced Id2 gene expression is mediated by Egr-1 in vascular smooth muscle cells
Cardiovasc Res, October 1, 2007; 76(1): 141 - 148.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/19/13574    most recent
M600526200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hua, H.
Right arrow Articles by Sarvetnick, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hua, H.
Right arrow Articles by Sarvetnick, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement