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Molecular Endocrinology, doi:10.1210/me.2007-0045
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Molecular Endocrinology 21 (6): 1467-1477
Copyright © 2007 by The Endocrine Society

Transforming Growth Factor ß Is a Critical Regulator of Adult Human Islet Plasticity

Stephen Hanley and Lawrence Rosenberg

Department of Surgery, McGill University, and Centre for Pancreatic Diseases, McGill University Health Centre, Montreal, Quebec, Canada H3G 1A4

Address all correspondence and requests for reprints to: Dr. Lawrence Rosenberg, Montreal General Hospital C9-128, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4. E-mail: lawrence.rosenberg{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue plasticity is well documented in the context of pancreatic regeneration and carcinogenesis, with recent reports implicating dedifferentiated islet cells both as endocrine progenitors and as the cell(s) of origin in pancreatic adenocarcinoma. Accordingly, it is noteworthy that accumulating evidence suggests that TGFß signaling is essential to pancreatic endocrine development and maintenance, whereas its loss is associated with the progression to pancreatic adenocarcinoma. The aim of this study was to examine the role of TGFß in an in vitro model of islet morphogenetic plasticity. Human islets were embedded in a collagen gel and cultured under conditions that induced transformation into duct-like epithelial structures (DLS). Addition of TGFß caused a dose-dependent decrease in DLS formation. Although it was demonstrated that collagen-embedded islets secrete low levels of TGFß, antibody-mediated neutralization of this endogenously released TGFß improved DLS formation rates, suggesting local TGFß concentrations may in fact be higher. Time course studies indicated that TGFß signaling was associated with an increase in ERK and p38 MAPK phosphorylation, although inhibitor-based studies were consistent with an islet endocrine-stabilizing effect mediated by p38 alone. Localization of TGFß signaling molecules suggested that the action of TGFß is directly on the ß-cell to inhibit apoptosis and thus stabilize endocrine phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UNTIL QUITE RECENTLY, the consensus was that adult pancreatic cells were terminally differentiated, being neither stable nor labile. However, increasing evidence suggests that, given the appropriate conditions, mature pancreatic cells can be directed to transdifferentiate into one of the other pancreatic cell types: endocrine, acinar, or ductal (1, 2, 3).

With respect to pancreatic endocrine regeneration, there is compelling evidence that ß-cell mass exhibits morphogenetic plasticity in its adaptation to prevailing physiological demands. For example, insulin resistance during pregnancy (4) or in obesity (5, 6) causes as much as a doubling of ß-cell mass. Conversely, an argument could be advanced that type 2 diabetes is the result of defective ß-cell mass dynamics, given the observed step-wise decline in ß-cell mass from the nondiabetic state to impaired fasting glucose to clinically apparent type 2 diabetes (6).

Although the proliferation of existing endocrine cells has been proposed as the major mechanism of endocrine pancreatic regeneration (7), the role of islet neogenesis, that is the de novo formation of endocrine cells from nonendocrine progenitors (8, 9), cannot be discounted. Although islet neogenesis has principally been attributed to islet precursors in the duct epithelium (8, 9, 10, 11, 12), acinar cells (13, 14) and even intra-islet progenitors (15, 16, 17) have been proposed as alternate sources of new islets. Moreover, several recent reports have focused on the ability of adult islets to undergo in vitro dedifferentiation into proliferative cells/structures that can then redifferentiate into functional islet-like structures (18, 19, 20, 21), suggesting that the islet itself may be implicated in morphological transformation in the pancreas through a process of transdifferentiation.

It is therefore noteworthy that transdifferentiation has also been associated with pancreatic carcinogenesis (22). Thus, whereas pancreatic ductal epithelium constitutes less than 15% of all pancreatic cells, adenocarcinomas account for 90% of pancreatic cancer, suggesting the adoption of a ductal phenotype during the course of dysplasia (23). Accordingly, both acinar-to-ductal (24, 25, 26) and islet-to-ductal transformation (22) have been proposed as early stages in carcinogenesis. Likewise, several groups have reported on the phenotypic instability of cultured islets (27, 28, 29, 30, 31) as well as that of transplanted islet-derived cells (32), suggesting that local factors are critical to the maintenance of the mature adult cellular phenotype.

One such factor, TGFß, appears to play a crucial role in normal pancreatic development because it may be required to control the relative balance of pancreatic cell types. Furthermore, TGFß has been demonstrated to limit the proliferation of acinar (33) and ductal cells, (34), whereas expression of a dominant-negative type II TGFß receptor (TGFßRII) results in increased acinar proliferation and differentiation (35). Conversely, TGFß has been proposed as a key mediator of islet morphogenesis (36) because the addition of TGFß to mouse embryonic explants induces a reversal in relative frequencies to favor endocrine cell types over acinar and ductal (37). Likewise, a recent report has indicated that inhibition of TGFß signaling in the adult pancreas causes diabetes via the disruption of islet function (38). TGFß also appears to be an islet survival factor, given that it acts to protect islets from cytokine-mediated cell death (39).

TGFß may be equally important in pancreatic cancer (40) because the vast majority of pancreatic adenocarcinomas are defective in TGFß signaling (41). Although TGFß receptor mutations have also been identified in pancreatic cancers (41, 42), far and away the most frequent mutation in pancreatic cancer is that of mothers against decapentaplegic homolog-4 (SMAD4) (43), a key effector of TGFß signaling.

Here, we report novel findings on the important role of TGFß in regulating islet plasticity in an in vitro model of islet-to-duct transdifferentiation. Although we (27, 30, 31) and others (44, 45) have already described the ability of islets to transform into duct-like structures (DLS), we now provide evidence that TGFß regulates islet plasticity by stabilizing ß-cell phenotype and inhibiting dedifferentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Embedded Islets Form DLS
After embedding in collagen and culture for 8 d in differentiation medium (DM), adult human islets formed cystic DLS lined by a layer of flattened or low cuboidal epithelium of one to several cells thickness (Fig. 1Go). Double immunofluorescence and electron microscopy studies indicated that islet-to-DLS conversion is characterized by the transdifferentiation of mantle islet cells into cells expressing duct epithelial markers, whereas ß-cells in the islet core are removed as a result of apoptosis (20, 31). Recent lineage tracing studies confirmed the mantle cell origins of DLS (46). We have previously characterized these islet-derived DLS as expressing high levels of epithelial and progenitor markers, low levels of endocrine markers, being highly proliferative and possessing the ability to regenerate islet-like structures given appropriate stimulation (20).


Figure 1
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  		Fig. 1. Embedded Islets Form DLS

Inverted (A–C) and immunofluorescence (D–F) microscopy demonstrates that fresh islets are solid spherical structures (A) comprised of insulin+, glucagon+, somatostatin+, or pancreatic polypeptide+ endocrine cells (D). By d 2 of the culture period, foci of DLS formation appear and expand (B and E), eventually replacing the islet. By d 8, these DLS are hollow, cystic structures (C) that are a heterogeneous population of cytokeratin+ duct epithelial cells (F). G, Morphological evaluation of cultures, including TGFß-treated, shows that DLS formation correlates positively with ductal (green, cytokeratin+) cell frequency (r2 = 0.74, P < 0.001) and correlates negatively with endocrine (red, insulin+, glucagon+, somatostatin+, pancreatic polypeptide+) cell frequency (r2 = 0.64, P < 0.001, n = 7 individual donors).

 
DLS formed from 64.2 ± 2.5% of embedded islets, as determined visually. Morphological scoring was established as an appropriate indicator of endocrine (hormone+) and epithelial (cytokeratin+) cell frequency, as evaluated by immunofluorescence microscopic studies (Fig. 1GGo) (endocrine: r2 = 0.64, P < 0.001; cytokeratin: r2 = 0.74, P < 0.001).

TGFß Inhibits DLS Formation
Given the importance of TGFß signaling in regulating the development of the endocrine pancreas (36, 37), as well as the during pancreatic carcinogenesis (41, 42, 47, 48, 49), seemingly from islet source tissue (22), we sought to determine the role of TGFß in islet-to-DLS transdifferentiation. Although embedded islets formed DLS with relatively high frequency, the addition of TGFß1 post embedding blocked DLS formation (evaluated at d 8) in a concentration-responsive manner (Fig. 2AGo) (P < 0.001), with an EC50 of approximately 34 pM. This concentration is similar to that reported for other cultured human cells (50, 51). For subsequent experiments, a concentration of 100 pM TGFß was chosen based on the inhibition of DLS formation by approximately 70%.


Figure 2
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  		Fig. 2. TGFß Inhibits DLS Formation

A, Culture medium was supplemented with TGFß, causing a dose-dependent reduction in DLS formation, as evaluated morphologically at d 8 (§, P < 0.001 vs. control; n = 7). B, Insulin content is reduced in islets cultured for 8 d, either forming DLS (DM; 64.2 ± 2.5% DLS formation), or remaining as islets (CM; 16.7 ± 5.5% DLS formation). In both cases, culture with added TGFß partially maintains insulin content, as assessed by ELISA (*, P < 0.05; §, P < 0.01 vs. baseline; n = 5).

 
TGFß Maintains Islet Phenotype
In vitro dithizone staining suggested that cultures treated with TGFß also maintained islet insulin content. To quantify this effect of TGFß on islet cultures, insulin content was compared between freshly isolated and islets cultured for 8 d. Embedding and culture in DM decreased insulin content by 78.9 ± 12.9% (P < 0.05 vs. baseline), consistent with our previous reports (Fig. 2BGo) (27, 30). In cultures supplemented with TGFß (DM plus TGFß), insulin content was decreased relative to freshly isolated islets, but was preserved relative to DM cultures (Fig. 2BGo). It should be noted that embedded islets cultured in control medium (CM), with minimal DLS formation (16.7 ± 5.5%), also had reduced insulin content (Fig. 2BGo) (P < 0.01), confirming our previous report that prolonged culture reduces islet insulin content (30). Addition of TGFß to CM improved overall insulin content (CM plus TGFß) (Fig. 2BGo).

TGFß Inhibits Islet Dedifferentiation
Evaluation of DLS formation over the course of culture suggested that the majority of DLS formation occurred by d 2 post embedding (Fig. 3AGo), consistent with previous observations regarding the timing of the loss of endocrine phenotype (31) and function (27, 30). TGFß-treated cultures showed similar dynamics, although the overall DLS formation rate was reduced (Fig. 3AGo).


Figure 3
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  		Fig. 3. TGFß Maintains Islet Phenotype

A, DLS formation occurs primarily within the first 2 d of culture, as determined morphologically. Furthermore, supplementing with TGFß (- - -) reduces DLS formation relative to control (···), with this effect occurring within the first 2 d of culture (§, P < 0.01 vs. time-matched control; n = 4). B, DLS formation, evaluated morphologically at d 8, is not affected if cultures are exposed to TGFß for only the first 2 d, or the final 4 d of culture (n.s.; n = 3). C, Supplementing with TGFß results in an increased frequency of insulin+ ß-cells and decreased frequency of cytokeratin (CK)+ ductal cells, determined by immunofluorescence at d 8 (*, P < 0.05 vs. DM; n = 5). D, The islet-stabilizing effect of TGFß is mediated via inhibition of ß-cell apoptosis, as assessed by immunofluorescence and terminal deoxynucleotidyl transferase-mediated TMR-dUTP nick end labeling (TUNEL)-based detection of apoptosis at d 2 (§, P < 0.01 vs. DM; n = 3).

 
To determine whether the effect of TGFß was to prevent islet dedifferentiation, embedded islets were cultured in DM for the first 4 d, then TGFß was added for the final 4 d of culture. Under these conditions, TGFß had no significant effect on the overall DLS formation rate (Fig. 3BGo), suggesting that TGFß administered after 4 d acted only on those islets that had not yet undergone DLS formation.

To assess whether the effect of TGFß was transient or whether acute TGFß treatment conferred resistance to dedifferentiation, embedded islets were exposed to TGFß for the first 2 d of culture (the period of maximal DLS formation) followed by a 6-d washout period. Under these conditions, DLS formation rates matched those of untreated islet cultures (Fig. 3BGo), suggesting that the effect of TGFß is transient, and thus would require chronic exposure to stabilize islet phenotype.

Quantification of cell numbers after 8 d confirmed that TGFß-treated cultures had an increased insulin+ cell and decreased cytokeratin+ cell frequency relative to DM cultures (Fig. 3CGo) (P < 0.05 vs. baseline). Moreover, this effect of TGFß appeared to be mediated by a reduction in the frequency of ß-cell apoptosis, as assessed at d 2, the point of maximal DLS formation (Fig. 3DGo) (P < 0.01 vs. DM).

TGFß Adversely Affects Islet Function
Based on the above observations regarding the effect of TGFß on islet cell phenotype and insulin content, we hypothesized that TGFß-treated cultures would also preserve islet function, as assessed by glucose-stimulated insulin secretion on d 8. As expected, islets cultured in DM had a stimulation index close to unity, indicating a lack of glucose-stimulated insulin secretion, especially when compared with the 2.4-fold glucose-stimulated increase in insulin secretion observed in freshly isolated islets (Fig. 4AGo) (P < 0.05). Interestingly, DM plus TGFß cultures exhibited a stimulation index of 0.36 ± 0.07, suggesting a TGFß-mediated dysregulation of glucose sensing and insulin secretion in the remaining islets, to the extent that insulin secretion was in fact reduced in response to elevated glucose concentrations. Investigation of islets cultured in CM confirmed these results; whereas CM cultures had a somewhat reduced stimulation index of 1.50 ± 0.30, reflecting the effect of 8 d of culture, CM plus TGFß cultures also displayed an inverted stimulation index, at 0.40 ± 0.24 (Fig. 4AGo) (P < 0.05).


Figure 4
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  		Fig. 4. TGFß Adversely Affects Islet Function

A, Although cultured islets (CM) have a reduced stimulation index relative to freshly isolated islets (fresh), islets cultured to produce DLS (DM) have further reduced function. However, culture with TGFß reduces glucose-stimulated insulin secretion in both CM and DM cultures (*, P < 0.05 vs. fresh islets; n = 3). B, This effect of TGFß is observed as an inversion of normal secretion kinetics (CM+TGFß), compared with freshly isolated (fresh) and cultured islets (CM; *, P < 0.05; §, P < 0.01 vs. equivalent fresh islet value; n = 3).

 
After incubation in high glucose, cultures were washed and incubated in low glucose to confirm that insulin secretion was glucose concentration dependent. TGFß-supplemented cultures also displayed increased insulin secretion in this low glucose environment (Fig. 4BGo), suggesting that TGFß causes a dysregulation of glucose-stimulated insulin secretion.

Endogenous TGFß Acts on Islet Cultures
Based on previous reports that islets and acinar tissue produce TGFß (52, 53), we hypothesized that endogenous TGFß may be acting to inhibit DLS formation. To determine the effect of endogenously produced TGFß, cultures were treated with either a control or pan-TGFß neutralizing antibody. Whereas the control antibody had no effect on DLS formation, antibody-mediated neutralization of endogenous TGFß resulted in a significant increase in DLS formation (Fig. 5AGo) (P < 0.05).


Figure 5
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  		Fig. 5. Endogenous TGFß Acts on Islet Cultures

A, Supplementing the culture medium with a panneutralizing {alpha}-TGFß antibody increases DLS formation, whereas a control antibody has no effect (*, P < 0.05 vs. control; n = 6). B, Islet cultures secrete TGFß at low levels, and this secretion is decreased by inhibition of the ERK (U0126) or JNK (SP600125) signaling pathways, whereas p38 (SB203580) or TGFßRI (SB431542) inhibition has no effect (*, P < 0.05 vs. vehicle; n = 6).

 
Given the significant effect of endogenous TGFß neutralization on DLS formation rates, it was surprising that the ELISA-determined concentration of secreted TGFß1, at 0.85 ± 0.22 pM, was 40-fold less than the concentrations required to affect DLS formation rates (Fig. 2AGo). However, given our previous observation of increased intra-islet concentrations of islet secretory products (54), the local concentration of TGFß could in fact be significantly higher.

To identify the signaling pathways regulating TGFß production and/or secretion, we undertook inhibitor-based studies. These experiments determined that the ERK and c-Jun N-terminal kinase (JNK) MAPK pathways were required for TGFß secretion (Fig. 5BGo), whereas p38 signaling was not. TGFß release may be regulated in an autocrine manner, because inhibition of TGFß signaling via TGFßRI inhibition caused a slight increase in TGFß secretion that was not statistically significant (Fig. 5BGo).

Analysis of conditioned media samples also confirmed that significant levels of exogenously added TGFß were still present for up to 2 d in culture (data not shown).

TGFß Induces ERK and p38 Phosphorylation
Our previous inhibitor-based studies established that islet-to-DLS conversion is characterized by JNK-mediated ß-cell apoptosis, followed by ERK-mediated duct cell proliferation (31). We sought to determine what pathways were involved in transducing the TGFß signal in this model.

Western blot analysis of islet cultures confirmed our previous report that embedding and culture led to an early spike in JNK phosphorylation, followed by a late rise in ERK phosphorylation (Fig. 6Go). Evaluation of p38 and SMAD2 phosphorylation states indicated that p38 followed the same pattern as ERK, with limited phosphorylation early in the cystic progression, followed by a late increase in phosphorylation, whereas there was limited SMAD2 phosphorylation throughout (Fig. 6Go).


Figure 6
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  		Fig. 6. TGFß Induces ERK and p38 Phosphorylation

Assessment of protein phosphorylation in embedded islets by Western blot suggests that 1 d of TGFß exposure leads to an increase in ERK, p38, and SMAD2 phosphorylation levels, whereas JNK phosphorylation is decreased. These effects are maintained as far as 6 d in culture.

 
Addition of TGFß had a dramatic effect on all signaling pathways studied; SMAD2, p38, and ERK phosphorylation were all increased substantially at d 1 post embedding, and this increase in phosphorylation followed through until at least d 6 in culture (Fig. 6Go). Conversely, JNK phosphorylation was decreased by TGFß treatment, although this effect was less pronounced at d 6 (Fig. 6Go).

TGFß Activates p38 Downstream of TGFßRI
Observation of phosphorylation states at 1 and 6 d post embedding provides an indication of the signaling pathways involved in DLS formation, but gives little information as to the signaling pathways activated directly downstream of TGFß treatment. To assess the immediate downstream effects of TGFß, cultured islets were examined by Western blot and kinase assay 0, 15, 30, 60, and 120 min after administration of TGFß. In this context, TGFß was observed to have no discernable effect on ERK phosphorylation. However, a significant increase in SMAD2 phosphorylation was observed 15–30 min after TGFß treatment (Fig. 7AGo). JNK phosphorylation was subsequently observed to decrease from 30–60 min after treatment, and p38 kinase activity was observed to increase from 60–120 min post treatment (Fig. 7Go). Coadministration of a TGFßRI kinase inhibitor completely abrogated the observed increase in p38 kinase activity, suggesting that p38 phosphorylation is downstream of TGFßRI (Fig. 7BGo).


Figure 7
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  		Fig. 7. TGFß Activates p38 Downstream of TGFßRI

A, Short-term TGFß treatment of islets does not affect ERK or JNK phosphorylation levels, whereas p38 kinase activity and SMAD2 phosphorylation are induced, with peaks at 120 and 15 min, respectively. B, TGFß treatment of islets causes a 4-fold increase in p38 kinase activity, as assessed by in vitro kinase assay, and this effect is blocked by SB431542-mediated TGFßRI inhibition (*, P < 0.05 vs. baseline; n = 3).

 
DLS Formation Requires ERK and JNK, Whereas TGFß Signaling Requires p38
The aforementioned studies established the effects of TGFß on the signaling pathways involved in DLS formation; however, these studies did not establish the dependence of the process on these pathways, nor the pathways by which TGFß mediates its effect. To this end, studies were undertaken using pharmacological inhibitors, either alone or in combination, with and without TGFß coadministration.

Inhibition of the ERK and JNK pathways by U0126 and SP600125, respectively, confirmed our previous observation that DLS formation is dependent on ERK phosphorylation and JNK activity (Fig. 8Go) (31). Furthermore, the effect of TGFß was observed to be additive to inhibition of either the ERK or JNK pathways (Fig. 8Go), suggesting that TGFß does not inhibit DLS formation via inhibition of ERK or JNK signaling.


Figure 8
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  		Fig. 8. DLS Formation Requires ERK and JNK, Whereas TGFß Signaling Requires p38

Administration of inhibitors [U0126 (ERK), SP600125 (JNK), SB203580 (p38), SB431542 (TGFßRI)], alone or in combination, with ({blacksquare}) or without ({square}) TGFß coadministration, demonstrates that inhibition of ERK and/or JNK abrogates DLS formation, as does TGFß treatment. The effect of TGFß is additive with ERK and/or JNK. TGFß-mediated inhibition of DLS formation is abrogated by inhibition of p38 and/or TGFßRI (*, P < 0.05 vs. TGFß alone; §, P < 0.01 vs. untreated control; ¶, P < 0.01 vs. same treatment without TGFß; n = 7).

 
Under culture conditions without TGFß, inhibition of p38 or TGFßRI kinase activity had no effect on DLS formation rates (Fig. 8Go). However, inhibition of either p38 or TGFßRI kinase activity did abrogate the effect of TGFß on DLS formation, as did coinhibition of p38 and TGFßRI (Fig. 8Go). Thus, it would appear that TGFß stabilizes islet phenotype via activation of p38, downstream of TGFßRI.

TGFß Signaling Molecules Are Expressed by Islet ß-Cells
Although others have confirmed the expression of TGFß receptors and SMAD signaling molecules in islets (55), the cellular localization of these molecules remains to be elucidated. Likewise, although we have observed significant effects of TFGß on stabilizing islet phenotype, the cellular targets of TGFß also remain to be identified. Thus, we sought to examine, by immunofluorescent studies, the cellular localization of TGFßRI, TGFßRII, and SMAD2, key mediators of TGFß signaling. Double immunofluorescence studies localized these molecules almost exclusively to insulin+ ß-cells within the islet (Fig. 9Go), suggesting that TGFß elicits its effects via direct effects on islet ß-cells.


Figure 9
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  		Fig. 9. TGFß Signaling Molecules Are Expressed by Islet ß-Cells

Immunofluorescent colocalization studies demonstrate that TGFßRI, TGFßRII, and SMAD2 are expressed predominantly in insulin+ ß-cells, rather than glucagon+ {alpha}-cells. Immunofluorescence also demonstrates that cytokeratin+ ductal cells of the DLS express TGFßRI and TGFßRII, but do not appear to express significant levels of SMAD2. DAPI, 4',6-Diamidino-2-phenylindole.

 
Double immunofluorescence studies indicated that the cytokeratin+ cells comprising the DLS express TGFßRI and TGFßRII, but do not appear to express SMAD2 to an appreciable degree (Fig. 9Go), suggesting the possibility of a defective TGFß signaling pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We report herein the ability of TGFß to stabilize islet phenotype such that adult human islets do not undergo dedifferentiation into proliferative DLS. Although this observation represents a novel effect attributable to TGFß, it is not altogether unexpected given the role of TGFß in directing islet development (36, 37). However, although it is known that TGFß favors endocrine development, the de novo formation of islets, islet neogenesis, represents the reverse of islet stabilization in that the former represents a precursor-to-islet transition, whereas the latter is characterized by islet-to-precursor dedifferentiation. That being said, our islet-to-DLS model has also been used to study islet neogenesis induced by various growth factors (20, 56). Interestingly, in this context, administration of TGFß was not observed to have any islet neogenic effect on DLS cultures (our unpublished observations). This is somewhat contradictory to previous reports that suggest TGFß favors endocrine differentiation in the developing pancreas (37), although it has been argued that the mechanisms of ß-cell formation are different in the developing and adult pancreas (7). Furthermore, the endocrine potential of developing pancreatic ducts may be very different from that of the DLS formed from dedifferentiating islets (11).

The effect of TGFß on adult pancreatic cell types has been characterized to some extent. TGFß inhibits the growth of ductal (57) and acinar (58, 59) tissue, and has also been suggested to induce apoptosis in the latter. Evidence also indicates that periacinar fibroblastoid cells, the so-called pancreatic stellate cells that act to regenerate the organ after pancreatitis, are activated by TGFß (60, 61, 62) in a self-propagating loop (63). This activation may lead to the extracellular matrix remodeling and fibrosis associated with this disease (64), given that administration (65) or overexpression (66, 67) of TGFß leads to pancreatitis-like fibrotic lesions, whereas administration of neutralizing antibodies limits excessive matrix deposition (68).

TGFß also appears to be an islet survival factor given that it acts to protect islets from cytokine-mediated cell death (39). Furthermore, a recent report has indicated that inhibition of TGFß signaling in the adult pancreas causes diabetes via the disruption of islet function (38). More specifically, targeted expression of SMAD7, a negative regulator of TGFß signaling (69, 70), to pancreatic and duodenal homeobox gene-1+ islet cells leads to a loss of gene expression related to ß-cell function, resulting in decreased insulin content and hyperglycemia. Likewise, reinstatement of normal TGFß signaling reverses the observed diabetes-like state. Other reports also suggest that TGFß improves islet function, as measured by the insulin secretory response to glucose in isolated islets (71, 72).

Interestingly, whereas we observed a TGFß-mediated stabilization of islet phenotype, the effect of TGFß on islet function was in fact the opposite (Fig. 4Go). This is in keeping with a recent report indicating a similar effect of TFGß on isolated mouse islets (73). Thus, there remains some debate as to the effect of TGFß on islet function, whereas we present data indicating a regulatory effect on islet phenotype. Our study is only informative insomuch as it addresses the effect of TGFß on islets cultured in a very specific microenvironment; therefore, more definitive studies are required to establish the effect of TGFß on isolated human islets.

Based on the islet expression profile of TGFß signaling molecules (Fig. 9Go), as well as the mechanism of DLS formation, it appears likely that TGFß elicits an islet stabilization effect by acting directly on ß-cells. In fact, TGFß appears to inhibit the ß-cell apoptosis observed under these specific culture conditions (Fig. 3DGo). As mentioned, TGFß is known to protect islets from cytokine-mediated apoptosis (39). We observed TGFß-induced p38 phosphorylation and activation, both acutely and chronically (Figs. 6Go and 7Go). Furthermore, it appeared that the islet-stabilizing effects of TGFß were p38 dependent (Fig. 8Go). There is no clear consensus on the role of p38 in islet survival and function. Conflicting reports have described p38 as being implicated in both proapoptotic (74, 75, 76) and prosurvival (77) pathways, as well as having positive effects on ß-cell proliferation and gene expression (78, 79), or no effect at all (80, 81). Although our studies cannot be extrapolated beyond the context of the islet dedifferentiation culture model, we can conclude that p38 activity is crucial to the islet-stabilizing effects of TGFß.

The role of the SMAD signaling proteins is equally difficult to elucidate in our model. Although TGFß can signal through the SMADs or the MAPKs, these pathways are not mutually exclusive. In fact, it appears that the interaction between p38 and SMAD2 can be inhibitory or stimulatory, depending on the cellular context (58, 82, 83). Thus, although the lack of pharmacological inhibitors of SMAD activity renders study of these molecules difficult in three-dimensional primary structures such as islets, the observed kinetics of SMAD2 and p38 phosphorylation suggest that TGFß-mediated p38 activation is likely independent of SMAD activity, and as such the islet-stabilizing effects of TGFß are likely SMAD independent. However, the experiments we describe cannot rule out an important role for SMAD signaling in islets.

Likewise, ERK and JNK signaling represent definite effectors of islet dedifferentiation (31), although neither of these MAPKs is affected directly by TGFß. As such, it would appear that the observed TGFß-mediated changes in ERK and JNK phosphorylation may represent changes in the phenotypic state of the islets under study; thus, increased ERK and decreased JNK phosphorylation are related to increased islet phenotypic stability, and TGFß administration is one means of obtaining this effect.

Finally, definitions of endocrine pancreatic plasticity typically focus on ß-cell mass as a unit, responding to the prevailing needs and conditions of the body by increasing or decreasing the net ß-cell mass through a variety of mechanisms. Although not incorrect, this view does not take into account the added plasticity of individual islets and/or other endocrine cell types. Thus, although likely negligible, one cannot discount the contribution of islet dedifferentiation to overall ß-cell mass, especially if islet dedifferentiation, proliferation, and redifferentiation (18, 19, 20, 21) is to be considered as a mechanism of islet regeneration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Islet Isolation and Culture
Pancreata from adult human cadaveric organ donors were obtained through the local organ procurement organization (Table 1Go). Islets were isolated according to established protocols (84). Briefly, after procurement, organs were flushed and stored in cold ViaSpan solution (Barr Laboratories, Pomona, NY) until islet isolation. The main pancreatic duct was cannulated and perfused with a solution of Liberase HI (Roche Diagnostics, Laval, Quebec, Canada). The distended organ was placed in a closed system (Bio-Rep, Miami, FL) and heated to 37 C to activate the enzyme blend. After the appearance of free islets in samples, the system was cooled and free tissues were collected and washed. Tissues were separated by continuous Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation in a cell processor (COBE DCT, Denver, CO). Free islets with diameters ranging from 75–400 µm, determined to be greater than 70% pure by real-time staining with dithizone (Sigma-Aldrich, St. Louis, MO), a Zn2+ chelator, were collected, washed, and counted as islet equivalents (IE, islet with diameter of 150 µm). Immunofluorescent analysis determined that islet preparations contained 71.4 ± 8.8% endocrine (insulin+, glucagon+, somatostatin+, pancreatic polypeptide+) and 3.4 ± 0.9% ductal (cytokeratin+) cells.


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  		Table 1. Donor and Isolation Characteristics

 
Isolated islets were cultured in suspension overnight in serum-free DMEM/F12 (Invitrogen, Burlington, Ontario, Canada) containing penicillin, streptomycin, fungizone, 1 µM dexamethasone, 10 ng/ml epidermal growth factor (Sigma-Aldrich), 24 mU/ml insulin (Lilly, Toronto, Ontario, Canada), and 200 ng/ml cholera toxin (Sigma-Aldrich). Islets were then embedded in type I rat tail collagen, as per previous reports (27, 30, 31), at a density of 2000 IE per 25 cm2, and cultured for 8 d in the above DM, with media changes every other day. To remove tissue from collagen, samples were incubated with 0.25 mg/ml collagenase XI (Sigma-Aldrich) for 30 min at 37 C. CM consisted of the above medium, without cholera toxin, with 10% fetal bovine serum (Montreal Biotech, Dorval, Quebec, Canada).

Resultant structures were scored visually as being either islet (no evidence of DLS formation), DLS (with no residual islet), or islet-DLS (any combination of islet and DLS character). DLS formation was then calculated as a percentage as follows: DLS formation = [(no. islet-DLS/2) + no. DLS]/(no. islet + no. islet-DLS + no. DLS).

Culture Additives
When added, concentrations of culture additives were 100 pM for recombinant human TGFß1 (Sigma-Aldrich), and 10 µg/ml for control and panneutralizing TGFß antibodies (R&D Systems, Minneapolis, MN). U0126, SP600125, SB203580, and SB431542, pharmacological inhibitors of ERK kinase, JNK, p38, and TGFßRI, respectively, were used at 10 µM (Calbiochem, San Diego, CA) (85, 86, 87). Inhibitors were prepared so that the final concentration of dimethylsulfoxide was 0.1%, and vehicle controls were used.

Immunofluorescence
Samples were stored at 4 C in phosphate-buffered formalin until cell pellets were embedded in 2% agarose (Invitrogen). After routine processing and embedding, 4-µm sections were cut and dewaxed in xylene and petroleum ether. Slides were permeabilized by incubation in Tris-buffered saline plus 0.1% Triton X-100 for 5 min, before antigen retrieval by heating for 10 min in 0.1 M citrate buffer (pH 6). Slides were then incubated in blocking buffer (Zymed, San Francisco, CA) for 15 min before overnight incubation with primary antibody in blocking buffer. After washing in PBS, slides were incubated for 1 h with secondary antibody in blocking buffer, and reprobed with a different primary antibody. Again, after washing, slides were incubated with secondary antibody before washing and coverslipping with mounting medium containing 4',6-diamidino-2-phenylindole (Vector, Burlingame, CA). Antibodies used were rabbit {alpha}-insulin, {alpha}-glucagon, {alpha}-somatostatin, and {alpha}-pancreatic polypeptide with mouse {alpha}-cytokeratin-AE1/AE3 (Dako, Carpinteria, CA), and mouse {alpha}-insulin and {alpha}-glucagon (Abcam, Cambridge, MA) with rabbit {alpha}-TGFßRI, {alpha}-TGFßRII (Santa Cruz Biotechnology, Santa Cruz, CA) or {alpha}-SMAD2 (Cell Signaling, Danvers, MA) primary antibodies, along with fluorescein isothiocyanate- and rhodamine-linked species-specific secondary antibodies (Cedarlane, Hornby, Ontario, Canada). To assess apoptotic cell death, the terminal deoxynucleotidyl transferase-mediated TMR-dUTP nick end labeling (TUNEL) (Roche Diagnostics) kit was used per the manufacturer’s instructions.

Insulin Content and Glucose-Stimulated Insulin Secretion
For evaluation of insulin content, samples were washed thoroughly in PBS before sonication in lysis buffer [50 mM Tris-HCl (pH 8), 1.37 mM NaCl, 0.1 mM Na3VO4, Complete protease inhibitor tablet (Roche), 1% vol/vol Nonidet P-40, 10% vol/vol glycerol]. Insulin content was assessed by ELISA (Alpco, Salem, NH), per the manufacturer’s instructions, and normalized to protein content, measured using the Bradford method (88).

To assess islet function, cultures were washed thoroughly with Hanks’ buffered salt solution (HBSS) and incubated sequentially with HBSS plus 2.2 mM glucose for two consecutive 60-min periods, HBSS plus 22 mM glucose for 30 min, HBSS plus 22 mM glucose plus 50 µM 3-isobutyl-1-methylxanthine (IBMX) (a cyclic AMP phosphodiesterase inhibitor; Sigma-Aldrich), and finally HBSS plus 2.2 mM glucose for 60 min. Conditioned media were collected and analyzed for insulin content by ELISA. Insulin secretion was normalized to baseline in freshly isolated islets from the same donor. Stimulation index was calculated as the ratio of insulin secretion at 22 mM glucose to that observed at 2.2 mM glucose.

TGFß Secretion
Conditioned medium was collected every other day at the time of media change, and was frozen until analysis. TGFß secretion was quantified by ELISA (Alpco), per the manufacturer’s instructions.

Western Blot and p38 Kinase Assay
Samples were sonicated in lysis buffer and centrifuged to remove cellular debris. After protein quantification, samples of equal protein content were separated by denaturing electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 plus 3% BSA and probed with primary antibody diluted in blocking buffer. After washing, blots were incubated with secondary antibody diluted in blocking buffer. After another wash, blots were developed using enhanced chemiluminescence (GE HealthCare, Piscataway, NJ) and exposed to X-OMAT film (Eastman Kodak, New Haven, CT). Membranes were stripped [62.5 mM Tris-HCl (pH 6.7), 100 mM ß-mercaptoethanol, 2% wt/vol sodium dodecyl sulfate] for 30 min at 65 C, blocked, and reprobed with primary antibodies. Antibodies used were rabbit {alpha}-phospho-ERK, {alpha}-ERK, {alpha}-phospho-p38, {alpha}-p38, {alpha}-phospho-JNK, {alpha}-JNK, {alpha}-phospho-SMAD2, and {alpha}-SMAD2, as well as mouse {alpha}-glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling). Secondary antibodies were {alpha}-rabbit and {alpha}-mouse horseradish peroxidase conjugates (Cell Signaling). p38 kinase activity was assessed by a nonradioactive Western blot-based kinase assay (Cell Signaling), per the manufacturer’s instructions.

Statistical Analysis
All experiments were paired in that control and treatment groups from the same donor were compared. The number of biological replicates is indicated in the figure legends, and results are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc Bonferroni’s test, or paired Student’s t test, when applicable. Differences were considered significant when P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Emily Austin, Mauro Castellarin, Jieping Ding, Deborah Driver, Xinfang Li, Mark Lipsett, Julia Makhlin, and Ryan Scott for technical assistance, as well as Québec-Transplant for coordination of organ availability.


   FOOTNOTES
 
This work was supported in part by the Canadian Institutes of Health Research (CIHR) and the Juvenile Diabetes Research Foundation. S.H. is supported by fellowships from the Canadian Diabetes Association/CIHR and Fonds de la Recherche en Santé Québec (FRSQ). L.R. is a Chercheur National (National Scientist) of the FRSQ.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2007

Abbreviations: CM, Control medium; DLS, duct-like structure; DM, differentiation medium; HBSS, Hanks’ buffered salt solution; IE, islet equivalent; JNK, c-Jun N-terminal kinase; SMAD, mothers against decapentaplegic homolog; TGFßR, TGFß receptor.

Received for publication January 23, 2007. Accepted for publication March 30, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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