Differential Expression of Neural Cell Adhesion Molecule and Cadherins in Pancreatic Islets, Glucagonomas, and lnsulinomas

Claus J. Maker, Stephan Christgau, Michael Ft. Williamson, Ole D. Madsen, Niu Zhan-Po, Elisabeth Bock, and Steinunn Baekkeskov

Research Center for Medical Biotechnology (C.J.M., E.B.) The Protein Laboratory University of Copenhagen K-2200 Copenhagen N, Denmark Departments of Microbiology/Immunology Hormone Research Institute (SC., S.B.) University of California San Francisco, California 94143-0534

and Medicine and the

Hagedorn Research Laboratory (M.R.W., O.D.M., N.Z.-P., S.B.) DK-2820 Gentofte, Denmark

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagonproducing cells (cr-cells) and then cells that express insulin (B-cells), somatostatin (&cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and Ecadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. lnsulinoma cells express E-cadherin but differ 0688~8809/92/1332-1342$03,00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine

from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed (Ycell type has converted to an immature phenotype with strong neuronal traits, reflecting the early place of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits. (Molecular Endocrinology 6: 1332-1342, 1992)

INTRODUCTION

Pancreaticendocrinecells bud from the pancreaticduct during development and assembleinto a microsociety of cells, the islet. The four endocrinecell types assembled in the islet probably develop from the same precursor (1) with glucagon-producingcells appearing at embryonic day 10 (2, 3) and the other three cell types later (4, 5). Islet cellsexpressseveral neuronalmarkersincluding the catecholaminebiosynthetic enzymes tyrosine hydroxylase (3) and phenylethanolamineN-methyltransferase (6), neuron-specificenolase(7) glutamic acid as well as decarboxylase (8) and dopa decarboxylase (9). Furthermore, pancreatic /3-cellshave synaptic-like microvesiclesthat store y-aminobutyric acid (GABA) (10). Dispersedislet cells can form neurites in culture and express neurofilaments, the neuron-specific form of intermediate filament (11). Yet experiments in quail/ chicken (12) and rat embryos (2) indicate that although islet cellsexpress neuronalmarkers,they originatefrom

Scmety

1332

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Cell Adhesion

Molecules

in Islet Ceils

endodermal precursors and not from the neuroectoderm. To explain these observations, it has been suggested that cells containing neuronal traits can arise not only from the ectoderm but also from the endoderm and that the latter differentiate into the endocrine islet cells under the influence of epigenetic signals (1, 11). In the absence of such signals neuron-specific traits may develop in embryonic cells of endoderm/mesoderm origin and reappear in differentiated islet cells dispersed in culture but not in intact islets (11). The organization of pancreatic endocrine cell types into the islet may thus not only play a role for their interaction and function but may also be important for maintenance of the differentiated endocrine phenotype. In each islet nutrient factors and hormones are received via the blood, and neuropeptides via afferent axons (13). In addition, paracrine influences within the islet play an important role in modulation of function of the different endocrine cells (14). The function of P-cells (15) a-cells and a-cells (16) have been shown to differ significantly in dispersed cells and intact islets. The information to assemble into a nonrandomized cluster where P-cells form a central core and the (Y-, a-, and PP-cells attach in the periphery is maintained in dissociated islet cells (17, 18). Islet cells must therefore express adhesion molecules that contain the necessary information for this construction. The 135kilodalton (kDa) isoform of the neural cell adhesion molecule NCAM (NCAM-B) is expressed predominantly in the non-p-cells of the islet (18). NCAM is likely to play a role in the assembly of the non-/I-cells in the periphery, whereas expression of E-cadherin may play a role in Ca”-dependent formation of the P-cell core (19). In addition to islet cells, NCAM is expressed by several nonneural cells, including muscle cells (20) kidney epithelial cells (21) and several cell types in the gonads and ducts (22). NCAM promotes cell-cell adhesion via a homophilic binding mechanism (23, 24). NCAM may also bind to heparin as well as heparan sulfate and thus possess two alternative or cooperative mechanisms for interaction with surrounding cells and extracellular matrix (25). One of the unusual features of NCAM in neurones is its polysialylation which is developmentally regulated (26) and plays a role as a regulator of cell-cell interactions (27). NCAM can be expressed as three main isoforms of the approximate molecular sizes of 190 kDa (NCAM-A), 135 kDa (NCAM-B), and 115 kDa (NCAM-C). NCAM-A and -B are both integral membrane proteins, whereas NCAM-C is linked to the plasma membrane via a phosphatidyl-inositol anchor (28) (for review see Ref. 29). The cadherin family of proteins consists of several calcium-dependent cell adhesion molecules with extensive amino acid homology. The well characterized cadherin molecules include E-cadherin [also known as uvomorulin, cell-CAM 120/80 (human), and L-CAM (chicken)] (30); N-cadherin (31) and P-cadherin (32; for review see Ref. 33). Cadherin molecules mediate homophilic cell-cell interactions but may also react with other

1333

members within the family (34). During development cadherins are involved in the sorting out of cells and pattern formations (33). In this study we have analyzed the expression of cadherins and the expression and polysialylation of the different isoforms of NCAM in primary, as well as transformed, rat islet cells. We show that both rat islet cells and insulinoma cells express two forms of NCAM and that they are not polysialylated. NCAM in glucagonoma cells, however, is polysialylated as NCAM in the neonatal brain. Whereas rat islet cells express E-cadherin, glucagonoma cells predominantly express a cadherin similar to the major cadherin of brain, N-cadherin. Insulinoma cells express E-cadherin but also a second Ncadherin-like cadherin molecule. The results suggest that the glucagonoma cells and, to a lesser degree, the insulinoma cells have escaped some of the signals necessary to maintain the differentiated endocrine phenotype and have reverted to cells that contain both endocrine and neuronal traits. The appearance of polysialylated NCAM, which is the normal NCAM form expressed in the embryonal/neonatal stages of brain development, and the loss of E-cadherin expression in the glucagonoma but not in insulinoma cells may reflect the sequential order of appearance of glucagon- and insulin-producing cells during development of the endocrine pancreas. Thus glucagon-producing cells appear first in the lineage and may revert as a result of tumorigenesis to a cell with stronger neuronal traits and closer to a precursor cell than the insulinoma cell.

RESULTS Expression Cells

of NCAM and Cadherins

in Normal

Islet

Sections of pancreas from El9 and from adult rats were analyzed by immunohistochemical methods using antibodies to NCAM, E-cadherin, and a peptide antiserum to a common epitope found in all cadherin molecules. lmmunostaining of El9 rat pancreas with a monoclonal antibody specific for the exocrine tissue (Fig. IA) was complementary to the staining with the NCAM antibody (Fig. lB), demonstrating that NCAM expression is restricted to the endocrine pancreas. This is confirmed by staining for insulin immunoreactivity, which is comprised within the NCAM positive area (Fig. 1C). In adult islets NCAM immunoreactivity was also restricted to the endocrine pancreas. Furthermore, the cells in the periphery of the islet, which consists of nonP-cells, stained very strongly with the NCAM antibody. In contrast, cells in the central core stained weakly for NCAM (Fig. 1, F and G). Staining of consecutive sections for insulin and glucagon respectively showed similar localization of glucagon and the strongly NCAMpositive cells (Fig. 1E) and identified the cells weakly positive or negative for NCAM in the central core as insulin-producing P-cells (Fig. 1 D). Some additional cells in the peripherv, which were stronalv oositive for NCAM

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Serial frozen Sections of El9 rat pancreas were stained by indirect immunofluorescence using a mon~clonal antibody, H37, which reacts with enxrine pancreatic cells (A), a polyclonal rabbit antiserum to NCAM (S), and a monoclonal antibody, 36a Ct 0, to insulin (C). Pancreatic sections from adult rat were stained for insulin (D). glucagon (monoclonal antibody Glu-001; E), and NCAM (F) by indirect immunofluorescence and stained for NCAM by indirect peroxidase staining (0). Peroxidase-stained sections were counterstained with hematoxylin-eosin to obtain blue nuclei. The use of nonfixed frozen sections is required for the detection of

the NCAM and H37 epitopes which are destroyed upon fixation. Insulin immunocytochemistw, however, results in a low sicnal on frozen sections. The staining for insulin is therefore not representative for the ins&n content;n 8.cells. Dilution of antibodie-s were as follows: H37, l:lO; NCAM, 1:tOOO; 36a ClO, 1~50; Glu-001. 1:iO.

but negative for glucagon, are presumably a- and/or PP-cells (Fig. IF). lmmunostaining for NCAM using the indirect peroxidase technique clearly labeled the central core of the islet and also showed strings of NCAMpositive tissue among the exocrine tissue, representing the peripheral nerves and ganglia in the pancreas (Fig. 1G). These results show that the expression of NCAM in pancreas is restricted to the endocrine cells both in late embryonic life and in the adult pancreas and confirm the observation of Rouiller et al. (18) that the expression is several-fold higher in non-p- than in &cells. In contrast

to the differential staining with NCAM antibodies, an antiserum to E-cadherin and an antiserum toacommon cadherin epitope stained islets uniformly yet much weaker than the surrounding exocrine tissue (not shown). Thus, all endocrine islet cells seem to express low levels of E-cadherin and perhaps other cadherin molecules. Islet Cells Predominantly Fnrm _.... “f_. NCAM ..-. .

Express

the 135-kDa

B-

To further assess the expression of NCAM in islet cells and to analyze its molecular composition, [%]methio-

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Cell Adhesion Molecules in Islet Cells

1335

-200

1 -94

-69

-43

-30 1

2

3

Fig. 2. lmmunoprecipitation Analysis of NCAM Expression in Rat Islets lmmunopracipitation of Triton X-l 14 detergent phase-purified membrane proteins prepared from [35S]methionine-labeled neonatal rat islets using a polyclonal rabbit antiserum to rat NCAM (dilution 1:200) in the absence (lane 1) or presence (lane 2) of 5 pg purified NCAM. lmmunoprecipitation with a normal rabbit serum (dilution 1:200) is shown in lane 3. The mobility of molecular size standards is indicated in kilodaltons.

2

3

4

Fig. 3. lmmunoblot Analysis of NCAM Expression in Rat Islets at Different Ages lmmunoblotting of total cellular rat islet and brain proteins with a polyclonal rabbit antiserum to rat NCAM (Dilution 1:2000). Lane 1, NCAM in neonatal rat islets; lane 2, NCAM in 4-5 week rat islets; lane 3, NCAM in adult rat islets; lane 4, NCAM in p40 rat brain membranes. Approximately 10 ug islet cell protein were loaded in each of the lanes l-3; 2.5 pg brain protein were loaded in lane 4. The molecular sizes of the different brain NCAM forms are indicated in kilodaltons.

nine-labeledislet cell proteins were subjected to immunoprecipitationand immunoblottingexperimentsusing an antiserumto NCAM. This antiserumspecifically immunoprecipitateda 135kDa protein from [35S]methionine-labeledneonatal (Fig. 2, lane l), 4-5 week, and adult islets (result not shown). The A and C forms of NCAM were not detected by immunoprecipitation.The presenceof an excess amount of purified NCAM from brain during the incubation with the NCAM antiserum abolishedthe immunoprecipitationof the 135kDa band (Fig. 2, lane 2) further verifying its identity as NCAM. The 135kDa NCAM protein was also detected in immunoprecipitatesof proteins from both P-celland nonP-cellfractions isolated from adult dispersedrat islets by fluorescence-activated cell sorting before labeling with [35S]methionine(results not shown), consistent with the results of the immunohistochemicalanalysis shown here and with the results of Rouilleret al. (18) that NCAM is expressedin both P-cellsand non-&cells. lmmunoblottingof islet and brain material with the NCAM antiserumshowed that the 135kDa protein in isletscorrespondedto the NCAM B-componentin brain (Fig. 3, lanes l-4). Furthermore, immunoblottingrevealed a significantlyweaker but specificimmunostaining of an additional component in islets with mobility similarto the 190-kDa A-form of NCAM in brain. This form was detected in neonatal, 4- to 5-week-old, and adult islets but was most abundant in 4-5 week islets (Fig. 3). The 190-kDa A-form was not detected in im-

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MOL ENDO. 1992

Vol6 No. 8

1336

m

u-s

A

\

\ \

\ \

-200

120 ---,

-94

munoblots using a nonimmune rabbit serum (not shown). In addition, some specific immunostaining was detected of bands which had a mobility intermediate between the B- and C-forms for NCAM in brain and which may represent degraded NCAM (Fig. 3, lanes 2 and 3). The detection of the NCAM A-form in islet cells by Western blotting adds to the growing list of neuronal markers expressed in those ceils (11 and Refs. therein). The lack of detection of the NCAM-A form by immunoprecipitation of [35S]methionine-labeled material (Fig. 2 and results not shown) may reflect a low rate of biosynthesis of this rare form in islets. The rare NCAMA form was also not detected in the analysis of NCAM in rat islet cells by Rouiller et a/. (18). Quantification of NCAM in 4-5 week and adult islets by an enzyme-linked immunosorbent assay (ELISA) showed an average level of 1.6 f. 0.8 pg (mean + SD) NCAM/mg protein, which is severalfold lower than in total brain (35).

Islets of Langerhans Express Only E-Cadherin -69

-43

-30 1

2

3

4

5

6

B

+ CADHERIN

--+

NCAM

An E-cadherin-specific antiserum which recognizes the N-terminal region of the E-cadherin molecule (36) specifically immunoprecipitated a 120-kDa component from TX-l 14 detergent phase-purified membrane proteins isolated from [35S]methionine-labeled islets (Fig. 4A, lane 2) and P-cell and non-p-cell fractions isolated from islets (results not shown). Three peptide antibodies to conserved epitopes in the cytoplasmic domain of the proteins in the cadherin family also immunoprecipitated only a 120-kDa protein (Fig. 4A, lanes 3-5) suggesting that E-cadherin is the only cadherin molecule expressed in normal islet cells. This finding was confirmed by the immunoblotting experiments (Fig. 5, A, lane 3; and B, lanes 3, 7, and 11). Thus, the only cadherin detected with the pan-cadherin antibody in neonatal islets was a band of a slightly increased mobility compared to the major band in neonatal rat brain (N-cadherin) (Fig. 5A, lanes 3 and 4). This band was recognized by the polyclonal E-cadherin-specific antibody, which did not

Fig. 4. lmmunoprecipitation Analyses of Cadherin Expression in Rat islets A, lmmunoprecipitation of Triton X-l 14 detergent phasepurified membrane proteins prepared from [35S]methioninelabeled neonatal rat islets with cadherin antibodies. Lane 1, Normal rabbit serum (dilution 1:20); lane 2, polyclonal rabbit antihuman E-cadherin serum (dilution 1:20); lanes 3-4, polyclonal rabbit antisera to common cadherin epitopes (dilution 1 :I 0), which react with all cadherins; lane 5, polyclonal rabbit antiserum CADCYTO 2, which recognizes most cadherins but not N-cadherin (dilution 1 :lO); lane 6, an irrelevant monoclonal antibody to rat major histocompatibility complex la antigens (dilution 1 :lO). The mobility of molecular size standards is indicated in kilodaltons. B, lmmunoprecipitation of cadherins and NCAM from equal aliquots of [35S]methionine-labeled rat islet membrane proteins prepared as in A. Lane 1, polyclonal rabbit antiserum to a common cadherin epitope (dilution 1:lO); lane 2, normal rabbit serum (dilution 1:lO); lane 3, polyclonal rabbit antibody against rat SCAM (dilution 1:lOO).

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Cell Adhesion Molecules in Islet Cells

1337

-1 16

-66

E-Cadherin

1

2

N-Cadherin

Pan-Cadherin

3

4

5

6

7

6

9

10

11

12

Fig. 5. lmmunoblot Analyses of Cadherin Expression in Rat Islets, Glucagonomas, and lnsulinomas A, lmmunoblot (short SDS gels) of total cellular proteins from rat insulinoma (lane 1, 20 pg), rat glucagonoma (lane 2, 20 rg), neonatal rat islet (lane 3, 20 pg), and neonatal rat brain (lane 4, 8 pg) with a polyclonal rabbit antiserum against a common cadherin epitope (dilution 1 :lOOO). The mobility of molecular size standards is indicated in kilodaltons. B, lmmunoblot (long SDS gels) of membrane proteins isolated from brain (lanes 1, 5, and 9, 100 Pg protein/lane), glucagonoma (lanes 2, 6, and 10, 51 rg protein/ lane), islet (lanes 3, 7, and 11, 28 fig protein/lane), and insulinoma (lanes 4, 8, and 12, 48 rg protein/lane), and probed with a polyclonal rabbit antiserum to human E-cadherin (dilution 1:2000) (lanes I-4) a polyclonal rabbit antiserum to a common cadherin epitope (dilution 1 :lOOO; lanes 5-8) and a polyclonal rabbit antiserum to Ncadherin (dilution 1:250; lanes 9-l 2).

stain cadherins in neonatal brain (Fig. 5B, lanes 2 and 3). No difference in cadherin expression was detected between neonatal (Fig. 4A), 4 week, and 12 week islets (results not shown). Consistent with the low staining of tissue sections of islets with the cadherin antibodies as compared to the intense staining of peripheral islet cells with NCAM antibodies, the E-cadherin band had a significantly lower intensity on fluorograms than the NCAM 135kDa band (Fig. 4B, lanes 1 and 3).

Expression of NCAM and Cadherins in lnsulinomas and Glucagonomas lmmunoblotting analysis of NCAM in rat insulinomas demonstrated that those tumors predominantly express the NCAM B-form but also low levels of the NCAM A-form. Thus the NCAM expression is similar in insulinomas and primary islet cells (Fig. 6A, lane 1). In

contrast, rat glucagonomas presented a broad staining between 135-200 kDa (Fig. 6A, lane 2), which is typical of the polysialylated NCAM expressed in newborn rat brain (37) (Fig. 6B, lanes 1 and 2) but not adult brain (Fig. 6, A, lane 4; and B, lane 3). Thus the NCAM expression in glucagonomas is different from primary islets and resembles that of polysialylated neurons in the developing brain. The pan-cadherin antiserum immunostained two bands at 120-l 30 kDa in insulinomas (Fig. 5A, lane l), and one major band, and one minor band in the same molecular size area in glucagonomas (Fig. 5A, lane 2). The cadherins in glucagonomas and insulinomas were analyzed on long polyacrylamide gels optimized to give a maximum resolution in the high molecular size area and immunoblotted using the pan-cadherin antiserum, the E-cadherin antiserum, the NIcadherin antiserum (Fig. 5B), and the CADCYTO 2 antiserum which rec-

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MOL ENDO. 1992 1338

1

VoI6

2

3

4

B -190 -135 -115

NO. 6

between N-cadherinand the larger cadherin molecule in insulinomascould be due to differences in posttranslational modification. The cloning of an additionalcadherin with a very high homology to N-cadherin from brain (38) however, suggeststhat there are additional N-cadherin-likeproteins. The insulinomacadherin may represent such a molecule.The major cadherin in glucagonomashad a mobility similarto N-cadherinin brain on long SDS gels (Fig. 58, comparelanes5 and 6 with 9 and 10). This cadherin was immunostainedwith the N-cadherinantibody (Fig. 58, lane 10) but not with the CADCYTO 2 antibody (not shown) and is, therefore, either N-cadherin or an N-cadherin-like protein. The minor band stained with the pan-cadherinantibody in glucagonomashad a similarmobility to E-cadherin in insulinomasand islets (Fig. 58, compare lane 6 with lanes7 and 8). This band was, however, not detected by immunostainingwith the E-cadherin antibody (Fig. 5B, lane 2) either becauseof ‘a lower sensitivity of this antibody or becauseit represents a different cadherin or a degradation product of the larger cadherin. The results show that insulinomasexpress the same cadherin as primary islet cells (E-cadherin)and, in addition, a N-cadherin-likecadherin. The glucagonomas,however, either express very low levels or are negative for E-cadherin and instead predominantly express a cadherin that is similaror identical to N-cadherinin brain. The results of analysisof NCAM and cadherinexpression in rat islet and brain material are summarized in Table 1.

DISCUSSION Fig. 6. Comparative Analysis of NCAM Expression in Glucagonomas, Insulinomas, and Brain at Different Ages A, lmmunoblot of total cellular proteins from insulinoma (lane 1, 19 fig), glucagonoma (lane 2, 19 rg), adult rat brain (lane 3, 1.2 pg), and p5 rat brain (lane 4, 1.2 pg) probed with a polyclonal rabbit antiserum against rat NCAM (dilution 1:2000). The molecular sizes of the different NCAM forms are indicated in kilodaltons. B, lmmunoblot of total cellular proteins from p0 (lane 1). p5 (lane 2) and p40 (lane 3) rat brains probed with a polyclonal rabbit antiserum against rat NCAM (dilution 1:2000). Six micrograms of protein were loaded in each lane. The molecular sizes of the different NCAM forms are indicated in kilodaltons.

ognizes most cadherins

except N-cadherin

(not shown).

The majorcadherinin insulinomashad a mobility similar to E-cadherin in islets on long sodiumdodecyl sulfate (SDS) gels (Fig. 5B, compare lanes3 and 4 with 7 and 8) and was identifiedas E-cadherinby its reactivity with the E-cadherin-specificserum (Fig. 58, lane 4). The larger cadherin in insulinomashad a slightly lower mobility than N-cadherinin brain (Fig. 5B, compare lanes 5 and 8 with 9 and 12) but was identifiedas N-cadherinlike by its reactivity with the N-cadherinantiserum(Fig. 58 lane 12)and its lack of reactivity with the CADCYTO 2 antibody (not shown). The differences in mobility

NCAM is expressed in derivatives of all three germ layers (39). It has been suggested that polysialylated NCAM present in early (embryonic and fetal) stages of development is involved in cellular migration, whereas the expressionof unsialylatedNCAM in tissuesmay be important for local differentiation and organization (29). Polysialylation of the NCAM molecule decreases its adhesionproperties as demonstratedby binding studies using NCAM inserted in lipid vesicles (37, 40). Analysis of NCAM in rat islets and in the insulinoma MSL-G2-IN showed a predominant expression of the 135-kDa B-isoformas well as low levelsof the 190-kDa A-isoform. Both forms appeared as well defined sharp bands on SDS-polyacrylamide gel electrophoresis (PAGE), indicating that they were unsialylated. In contrast, NCAM in the glucagonomaMSL-G-AN appeared as a broad band of 135-200 kDa, which is consistent with a high degree of glycosylation and polysialylation as observed in the developingbrain (41). We observed an increase in NCAM expression between neonatal islets and older islets, suggestingthat the mature islet is a morestable structure with stronger adhesive properties. Double staining with NCAM and glucagonantiseraconfirmedthat the glucagon-producing a-cellsas well as the other peripheralislet cell types

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Cell Adhesion

Table

Molecules

1. Summarv

in Islet Cells

of Analvsis

1339

of NCAM

and Cadherin

Expression

Brain

NCAM Polysialylated NCAM E-cadherin N-cadherin family

Neonatal

Adult

A, B, C forms X

A, B, C forms

ND X

in Rat Islet and Brain Islets

ND ND X

A~,

B forms ND X ND

@-Cells Adult

Adult

Neonatal A~,

Material

B forms ND X ND

gb

form ND XC ND

Non-@-cells Adult

B” form ND XC ND

Glucagonoma

A, B forms X ND X

lnsulinoma A,

B forms ND X X

ND, Not detected. a A- and B-forms were detected by immunoblotting, but only B-form was detected in immunoprecipitation experiments. b B-form was detected in immunoprecipitation experiments, no immunoblotting data are available. The difference in expression levels between @- and non-P-cells was detected by immunohistochemistry. c Detected by immunohistochemistry.

(a- and PP-cells) have an increased NCAM expression compared to the islet P-cell core. This differential expression within the islet cell types has been suggested to be important for the intraislet organization (18). Expression of cadherins is developmentally regulated, and the switching on and off of the cadherin molecules correlates with a variety of morphogenetic events (42). lmmunocytochemical analysis of islets showed a uniform staining pattern with antibodies recognizing E-cadherin. This result is in agreement with the data of Rouiller et al. (19) who detected E-cadherin expression in p- and non+cells separated by fluorescence-activated cell sorting. Biochemical analysis showed that E-cadherin is the only cadherin molecule expressed in normal islet cells at different ages. In contrast, glucagonomas were either negative or expressed very low levels of E-cadherin. Instead they expressed N-cadherin or a N-cadherin-like molecule as the predominant cadherin. Insulinomas, however, have maintained E-cadherin expression and, in addition, express a N-cadherin-like cadherin. The results demonstrate that glucagonomas are clearly distinct from primary islet cells with regard to both NCAM and cadherin expression, whereas insulinomas are more similar, although not identical, to normal islet cells in their pattern of cell adhesion molecules. The metastatic liver (MSL) tumors were originally derived from tissue cultures established from a liver metastasis of a transplantable insulinoma (43). Individual clones were heterogeneous in culture with multiple hormone expression (43). Such properties are consistent with those of a putative islet stem cell (1). Transformed glucagon- and insulin-producing cells may therefore represent a less differentiated phenotype than normal 01-and /I-cells. The glucagonoma and insulinoma tumor lines used in this study were derived from a common monoclonal origin. The tumor line MSL-G2-IN represents a transplantable insulinoma phenotype which has remained stable for more than 70 successive passages in vivo (44). Similarly, the tumor line MSL-GAN is a highly stable glucagonoma line in vivo. (Madsen, 0. D., unpublished data). The insulinomas uniformly express insulin and islet amyloid polypeptide but not glucagon. The glucagonomas uniformly express gluca-

gon but not insulin or islet amyloid polypeptide (45). Thus both tumor lines represent a cell phenotype that is stable by serial passaging in vivo, and both exhibit pluripotentiality in vitro (Madsen, 0. D., unpublished data). During normal mouse pancreas development the first detectable pancreatic islet cell hormone is glucagon. Later this precursor cell type goes through a phase characterized by coexpression of additional islet hormones within individual cells (1). In the mature islet four distinct cell types are present, each producing only one of the four classical islet hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide. The results in this study show that the glucagonomas have switched to expressing polysialylated NCAM; Ecadherin expression is turned off, and instead, the cells express a cadherin, which seems to be identical to the N-cadherin of neurons. Thus glucagonoma cells express cell adhesion molecules that are consistent with a neuronal trait. In contrast, the insulinoma cells maintain a more islet-like phenotype with regard to expression of cell adhesion molecules but share some features of neurons. Neither of the lines produce aggressive tumors, and both develop metastasis at a similarly very low frequency. The switch to neuronal adhesion molecules in the islet cell tumors does not, therefore, seem to be mandatory for tumorigenesis. Rather it is conceivable that the transformation process, which is accompanied by an escape from some differentiation signals and results in a pluripotent phenotype, can give rise to cells displaying features of different developmental stages. Thus the MSL a-cell phenotype expressed in the glucagonoma may represent a conversion to the early a-like progenitor islet cell with strong neuronal trait. Similarly, the MSL P-cell phenotype may represent a developmental stage in between the progenitor cell and the mature P-cell. If this is correct, it is predicted that the islet stem cells and the first glucagon-expressing cells in development will express N-cadherin and sialylated NCAM. This hypothesis should become testable with the development of suitable reagents.

MATERIALS Islet

Cell

AND METHODS

Material

Islets of Langerhans were isolated from neonatal, 4- to 5week-, and lo- to 14-week-old Wistar rats by collagenase

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MOL 1340

ENDO.

digestion, Percoll gradient purification, and a final purification by selection of each individual islet under a stereomicroscope to yield preparations of 95100% purity (46). Dispersed adult rat islet cells were separated into p- and non-fl-cell populations by fluorescence-activated cell sorting as described (47). Transplantable rat insulinomas, MSL-G2-IN (44) and glucagonomas, MSL-G-AN (Madsen, 0. D., unpublished), were propagated in vivo as described (44). Tumors were excised from anesthetized animals and snap frozen before protein extraction (see below). In vitro culture and radioactive labeling of islet cells with [35S]methionine for 4 h was carried out as described (48).

Islets were homogenized and ultracentrifuged at 100,000 x g, in order to separate a soluble and a particulate fraction (48). The particulate fraction was extracted in Triton X-l 14. Amphiphilic proteins were purified by phase separation (49) and immunoprecipitated and analyzed by SDS-PAGE and fluorography as described (48). In experiments with cadherin antibodies, buffers were supplemented with 1 mM CaCI, during homogenization and immunoprecipitation. Furthermore, incubations with antibodies to a common cadherin epitope were performed in the presence of 0.2% SDS. Purified rat brain NCAM (50) was used for competition of NCAM antibody binding.

For extraction of total cellular proteins, islet cells, tumors, and brain tissue were homogenized in a 70 mM Tris-barbital buffer, pH 8.6, solubilized in Tris-barbital buffer containing 4% Triton X-l 00 and centrifuged for 30 min at 12,000 x g. For isolation of particulate membrane proteins, cells and tissue were homogenized under hypotonic conditions followed by sedimentation of membranes and extraction of membrane proteins as described (51). Protein extracts were subjected to SDS-PAGE on either 7.5% uniform or 4-15% gradient polyacrylamide gels according to Laemmli (52). After SDS-PAGE, proteins were transferred to either a nitrocellulose membrane (0.22 pm, Millipore, Bedford, MA) or a membrane (Immobilon, 0.45 pm, Millipore) by electroblotting. Incubations of membranes with different primary antibodies were carried out as described (53) for either 2 h or overnight and either at pH 7.4 or at a high pH (10.2) for some antisera to reduce background. Immunostaining was visualized using alkaline phosphatase-conjugated second antibodies (Dakopatts, Glostrup, Denmark). lmmunocytochemistry lmmunocytochemistry for NCAM was carried out on frozen serial sections of rat pancreas by indirect immunfluorescence as desribed (54). lmmunocytochemistry for cadherins was carried out on 3.5% paraformaldehyde-fixed rat pancreas. Incubations of sections for cadherin staining were carried out in the presence of 1 mM CaCI,. Photomicrographs were obtained using Agfa 1000 ASA professional films and an Olympus (Tokyo, Japan) BH-2 microscope equipped with fluorescence epiillumination.

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of N-cadherin was a gift from Drs. H. Semb and R. Kelly (UCSF). This antiserum recognizes N-cadherin but not Ecadherin. A polyclonal rabbit antiserum raised against a 17amino acid peptide corresponding to amino acid residues 665681 in chicken L-CAM (the avian homolog of E-cadherin) was a gift from Dr. B. Gumbiner (UCSF) (55). This antiserum recognizes a conserved region in the intracellular domain of most if not all classical cadherins and is therefore a pancadherin antiserum. Two other rabbit antisera, CADCYTO-1 and CADCYTO-2, raised to conserved peptides in the intracellular domain of L-CAM (56) were a gift from Dr. L. Reichardt (UCSF). The CADCYTO-1 was raised to a 25-amino acid peptide corresponding to amino acid residues 658-682 in LCAM and recognizes most if not all classical cadherins. The CADCYTO-2 antiserum was raised to a 27-amino acid peptide corresponding to the carboxy terminus of L-CAM and recognizes several cadherins but not N-cadherin (56). A monoclonal antibody to insulin, 36a Cl 0 (57) was a gift from Dr. M. Ziegler (Karlsburg Research Institute, Karlsburg, Germany). Glu-001, a monoclonal antibody to glucagon was from Novo Biolabs (Bagsvaerd, Denmark). A monoclonal antibody, H37, specific for exocrine pancreatic tissue, was used for the staining of exocrine tissue (58). Quantification

of NCAM

by ELBA

Islets were harvested and counted in ice-cold PBS containing 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 100 U/ml aprotinin (Bayer, Germany). NCAM was quantified by an ELISA (59) based on inhibition of NCAM antibodv bindina with solubilized rat brain membranes coated on ELISA Gates. Extracts of islets were mixed with an NCAM antibody, and this mixture was incubated with the sensitized solid phase. Bound antibody was visualized using peroxidase-labeled antirabbit immunoglobulin. The titration of NCAM in islets was performed using 2-fold serial dilutions. Purified rat brain NCAM was used as a standard (50).

Acknowledgments We thank Ms. E. Holm Petersen for excellent technical help, Dr. D. Pipeleers, The Free University of Brussels, Belgium, for donation of p- and non-&cell fractions isolated from rat islets, Drs. C. Damsky, L. Reichardt, B. Gumbiner, H. Semb, and R. Kelly, University of California San Francisco, for donation of antisera, and Dr. M. Ziegler, Karlsburg Research Institute.

Received January 3,1992. Revision received April 27,1992. Accepted May 20, 1992. Address requests for reprints to: Dr. Steinunn Baekkeskov, Hormone Research Institute, University of California, 513 Parnassus Avenue. Room HSW 1090. San Francisco, California 94143-0534. This work was supported by NIH Grant 1 PO1 DK-4182201, The Novo-Nordisk Foundation, the Danish Medical Research Council, the Danish Diabetes Foundation, the Danish Research Academy, the Michaelsen Foundation, the J. 0. Madsen Foundation, the Bankdirector Hans Stener and wife Agnes Stener Foundation, and the Hojmosegard Foundation.

Antisera Polyclonal rabbit antiserum against rat brain NCAM was produced and characterized as previously described (50). The specificity of the antibodies was also tested by immunoblotting using adult rat brain homogenate. The antibody recognized the NCAM-A, -B, and -C isoforms. A polyclonal rabbit antiserum raised against human E-cadherin (36) which recognizes the N-terminal domain of E-cadherin and does not recognize other cadherins, was a gift from Dr. C. Damsky, [University of California, San Francisco, CA (UCSF)]. A polyclonal rabbit antiserum raised to a 24-amino acid peptide in the EC 1 domain

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