ARTICLE |
Correspondence to: Leonard C. Harrison, Walter & Eliza Hall Inst. of Medical Research, Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. E-mail: harrison@wehi.edu.au
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Summary |
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Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin ß1- and 1-chains, the
1-chain, unique to laminin-1, was not detected. Laminin-10 (
5ß1
1) was expressed in acinar tissue, whereas laminins-2 (
2ß1
1) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin ß1 and
1, whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits
6 and
3 were detected in acinar cells and duct epithelial cells, but
6 was absent in islet cells. Integrin
6ß4 was detected only in duct cells,
6ß1 in both acinar and ductal cells, and
3ß1 in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.
(J Histochem Cytochem 50:16251632, 2002)
Key Words: laminin, nidogen, fibulin, integrin, pancreas
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Introduction |
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THE PANCREAS is derived from the foregut endoderm and comprises three main elements: exocrine tissue, which produces and secretes digestive enzymes; a duct system that transports the digestive enzymes to the intestinal tract; and endocrine tissue, the islets of Langerhans, which secrete hormones, predominantly insulin, to regulate blood glucose metabolism and homeostasis. Pancreatic development and function, as for other organs, depends on cooperative extracellular signals, both soluble hormones and growth factors and cell-associated insoluble extracellular matrix (ECM) proteins ( (400 kD)-, ß (210 kD)- and
(200 kD)-chains (
15-, ß13-, and
13-chains, and 12 laminin isoforms have been identified (
Integrins, one type of receptor for laminins, comprise a family of heterodimeric cell adhesion glycoprotein molecules composed of non-covalently bound (120180 kD)- and ß (90110 kD)-subunits, of which there are 16 and at least 22 isoforms, respectively. In addition to mediating cellmatrix and cellcell interactions, integrins transduce extracellular signals. All known epithelial integrin receptors for laminins comprise isoforms of
6-,
3-, ß1-, and ß4-subunits (
3 and
6 being the only subunits with significant (40%) identity (
The expression and localization of laminins and integrins in the pancreas are poorly documented, despite the potential importance of these molecules in pancreatic development and function. As a baseline for further investigations, we analyzed the expression of laminins and associated molecules, and of integrins, in adult mouse pancreas.
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Materials and Methods |
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Animals and Tissues
Adult (812-week) CBA mice bred under specific pathogen-free conditions were sacrificed by cervical dislocation and their pancreases harvested and snap-frozen in liquid nitrogen.
Primary Antibodies
Primary antibodies available for use for indirect immunofluorescence, immunoblotting, and immunoprecipitation are listed in Table 1.
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Indirect Immunofluorescence Microscopy
Cryostat sections (8 µm) were air-dried for 4060 min and fixed in cold (-20C) acetone for 10 min before indirect immunofluorescence staining, or were stored at -80C if not used immediately. Before antibody staining, nonspecific protein binding was blocked by incubation for at least 30 min with warm MTPBS containing 2% bovine serum albumin or 2% normal rabbit serum. Controls were performed by replacing the first antibody with isotype control antibodies [for monoclonal antibodies (MAbs)] or with pre-immune serum from the appropriate species (for polyclonal antibodies). Tissues were incubated with primary antibodies for 90 min at 25C, followed by three thorough washes with MTPBS. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse or anti-rat immunoglobulins (DAKO; Glostrup, Denmark) were incubated for 30 min at 25C, followed by three thorough washes. Slides were observed and photomicrographed under a Zeis Axiophot fluorescent microscope.
Immunoprecipitation and Immunoblotting
To further characterize integrin expression in islet cells, immunoprecipitation and immunoblotting analysis was undertaken. A ß-cell line, rat insulinoma RIN A12 cells (6 or -
3 integrin antibodies and rotated at 4C for 2 hr. The beads were washed five times with the lysis buffer, resuspended in reducing SDS sample buffer, and boiled for 3 min. The lysate was electrophoresed in a 1020% SDS-PAGE gel (Novex; Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membrane. After blocking for 1 hr in 5% milk powder in MTPBS, the membrane was incubated with anti-ß1 or -ß4 integrin antibodies diluted in 1% milk powder in MTPBS with 0.05% Tween-20 (PBS-T) for 2 hr at RT, washed five times for 10 min with PBS-T, and then incubated with secondary antibodies diluted in 1% milk powder in PBS-T for 1 hr at RT. After five washes with PBS-T, the immunoblot was developed for 5 min with Lumi-Light Western Blotting Substrate (Roche; Indianapolis, IN) and exposed to Hyperfilm MP (Amersham Pharmacia Biotech; Poole, UK).
RT-PCR Analysis of Laminin RNAs
Total RNA from mouse pancreas or RIN cells was extracted with RNAzol B (Cinna/Biotecx; Houston, TX) and treated with DNase I (Gibco BRL; Gaithersburg, MD). mRNA was reverse-transcribed with Superscript II reverse transcriptase (Gibco BRL) in 1 x transcription buffer containing 0.5 µM oligo (dT)1618 primer (Gibco BRL) and 400 µM dNTPs. Aliquots of cDNAs were amplified by PCR in 1 x PCR buffer (Roche) containing 200 µM dNTPs, 1 µM of each primer pair, and 1 U Taq polymerase. The primers for laminin 1 (5' GACCGCCATGCCGATTTAGC 3', 5' GACCGCCGTGTTGTTGATGC 3'), laminin
5 (5' CCCTGGGGCCTTGAACTTCTCCTACTC 3', 5' GCATTGCGCCGATC-CACCTCAG 3'), laminin ß1 (5' ACCAGACGGGCCTTGCTTGTGAAT 3', 5' AGTTGTGGCCCGTGGTGTAG-TCCTG 3') and for the control "housekeeping" gene ß-actin (5' GTGGGCCGCCCTAGGCACCA 3', 5' CTCTTTGATGTCACGCACGATTTC 3') all yielded PCR products of 530 bp. PCR reactions were performed for 35 cycles (95C, 30 sec; 60C, 30 sec; 72C, 30 sec), and amplified products were separated in 1.5% agarose gels.
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Results |
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Laminin Expression
Laminin, detected with polyclonal rabbit anti-laminin antibodies, was expressed in the basement membrane (BM) of several tissues in the pancreas. In the basal lamina of exocrine cells, intra-islet capillary plexuses, duct epithelia, and smooth muscle of arteries and veins (Fig 1A1C). This antibody, generated to murine EngelbrethHolmSwarm tumor BM, identifies 1-, ß1-, and
1-chains (
3ß3
2), reported to be preferentially expressed in BM of squamous and transitional epithelia (
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Laminin chains detected with specific monoclonal antibodies (MAbs) had a unique distribution pattern in the pancreas (Table 2). Laminin 1, specific for laminin-1 (
1ß1
1), was not detected in the adult mouse pancreas, consistent with our RT-PCR analysis (see below) and with previous reports (
1 were the same as those revealed with the rabbit anti-laminin antibody (Fig 1D1H). Laminin
2, a component of laminin-2 (
2ß1
1), laminin-4 (
2ß2
1), and laminin-12 (
2ß1
3), and laminin
2, which is present in laminin-5, were detected only in the basal lamina of smooth muscle cells in arteries and veins (Fig 1I and Fig 1J). Laminin
5, a component of laminin-10 (
5ß1
1) and laminin-11 (
5ß2
1), was richly distributed in the BM of endothelia, in the basal lamina of smooth muscle cells in arteries and veins and of myoid cells in interlobular ducts, consistent with other reports (
5 also appeared in the peripheral islet and penetrated into the islets along intra-islet capillaries (Fig 1K1O). The pattern of nidogen-1 expression (Fig 2A2D) was the same as that of laminins ß1 and
1. The distributions of fibulin-1 and -2 were similar (Fig 2E2H), resembling that of laminin
5.
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To provide additional evidence that the laminin 1-chain is not expressed in adult pancreas, RT-PCR analysis of mRNAs was performed. Consistent with the immunofluorescence studies, laminin
1 transcripts were barely detectable, whereas those for laminin ß1 were readily detected (Fig 3). mRNA for laminin
5 was detected only in E17.5 pancreas and RIN cells (Fig 3).
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Integrin Expression
Integrin subunits exhibited a striking cell-specific expression pattern (Table 3). Integrin 6-subunit was strongly expressed basally in acinar cells, where it interacts with laminin, but was not detected in interlobular arteries or in veins and ducts. It also appeared to be expressed in epithelial cells of various orders of pancreatic ducts from interlobular to intercalated ducts, and in endothelial cells (Fig 4A4C). However, islet cells clearly did not express this integrin subunit (Fig 4D). Integrin
3 was expressed weakly in acinar cells but strongly in arteries, veins, and intralobular ducts (Fig 4K and Fig 4L). Integrin ß1 was expressed in various compartments of the pancreas, with strong expression in interlobular duct epithelial cells and endothelial cells (Fig 4E and Fig 4F), and in islet cells (Fig 4G), but with weak expression in acinar cells. ß4 appeared to be expressed in epithelial cells in various orders of the duct system (Fig 4H and Fig 4I), including within islets (Fig 4J) but not in acinar cells.
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To provide additional evidence that the absence of the integrin 6-subunit in mouse islet cells was not species-specific, a rat ß-cell line, RIN-A12, was analyzed by immunoprecipitationblotting. Neither
6ß1 nor
6ß4 integrin was detected in the RIN cells (Fig 5), supporting the findings of our immunocytochemical studies. Immunoprecipitationblotting further demonstrated that the
3-subunit is probably associated with ß1 to form
3ß1-integrin in mouse islet cells.
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Discussion |
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This study has characterized the expression pattern of laminins and their integrin receptors in the adult pancreas. Laminin isoform expression varied with tissue compartments in the pancreas but was not detected extracellularly in islets. Integrin receptor subunit expression had a cell-specific pattern. Laminin-1 was undetectable in the adult pancreas. We previously showed that laminin 1, unique for laminin-1, is expressed in the BM of developing pancreatic duct epithelium between E13.5 and E17.5 (
1 was not detected in any compartment of the adult pancreas, consistent with our RT-PCR analysis and a recent report (
1 is expressed in the epithelial BM during early development but not in the adult (
6 integrins) and differentiation (via
-dystroglycan) of the pancreatic precursor cells (
1-chains and weak staining of laminin
5-chain indicated the existence of laminin-10 (
5ß1
1) in the BM of acinar cells, consistent with the notion that laminin-10 is an adult isoform of epithelial laminin (
2,
5, ß1,
1 and
2 were detected in blood vessels, especially arteries, indicating that laminin-2 (
2ß1
1), -8 (
4ß1
1), and -10 are expressed in the BM of these tissues. The functional significance of these laminin isoforms expressed in the blood vessels remains to be determined.
Our results show that major ECM proteins are absent extracellularly between islet cells, as are nidogen-1 and fibulin-1 and -2. Similarly, a range of collagen isoformsfibronectin, vitronectin, and elastinwere not detected between islet cells (
These studies provide molecular evidence to support a previous electron microscopic study (
Only a limited amount of information on the expression pattern of integrin subunits in the adult pancreas has hitherto been available. We found that the integrin 6-subunit was undetectable in adult mouse islet cells by indirect immunofluorescence and immunoprecipitation, consistent with previous immunochemical studies in several species, including humans (
6 integrin signaling is not required for adult ß-cell function. Because
6 integrins transduce a moderate proliferation signal in the developing pancreas (
6 integrin expression in the adult islet cells may, at least partially, be attributed to the low proliferative potential of these cells. However, our observation contrasts with a report (
6-subunit is present on primary rat ß-cells. This discrepancy may be partially due to differences in experimental techniques and antibody used.
What is the dimerization partner for the integrin ß1 subunit on islet cells? There are six known members of the ß1 integrin subfamily: 1ß1,
2ß1,
3ß1,
6ß1,
7ß1, and
9ß1. Our study has demonstrated that the
6ß1 integrin is absent on mouse islet cells. However, we observed that the
3-subunit was weakly detected on islet cells, suggesting the existence of
3ß1 integrin. This was supported by our immunoprecipitationblotting experiments on RIN cells and by the findings of others on primary and transformed rat islet cells (
3ß1 integrin may also be at least partially responsible for binding to laminin-5 secreted by 804G cells, a rat carcinoma cell line, and for increasing proliferation of human fetal islet cells (
3ß1 integrin may partially overlap with
6 integrins because the islet cell lineage develops normally in homozygous
6 gene knockout (
6-/-) mice (
6-/-/
3-/- mice (
Integrin ß4-subunit expression is primarily restricted to cells of ectoderm- and endoderm-derived tissues, such as skin and intestine (1000 amino acid residues compared to less than 50 residues in other integrin ß-subunits). The expression of
6ß4 in epithelial cells of various orders of pancreatic ducts may be associated with hemidesmosome formation along the basement membrane (
In conclusion, laminin and its integrin receptors exhibit a selective pattern of expression in the adult pancreas. Understanding the functions of these molecules may facilitate the engineering of specific pancreatic cells such as insulin-producing ß-cells.
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Acknowledgments |
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Supported by a Juvenile Diabetes Research Foundation Fellowship and a Diabetes Australia Research Grant (2001) (F-XJ) and by the National Health and Medical Research Council of Australia (LCH).
We thank Dr L.M. Sorokin (Institute of Experimental Medicine, University of ErlangenNurnburg, Germany), who kindly provided rat monoclonal anti-mouse laminin 1 (clone 198),
2 (clone 4H8), and
5 (clone 4G6) antibodies and rabbit anti-nidogen-1 (#913 and 914), fibulin-1 (#1034), and fibulin-2 (#1028) antibodies. We also thank Catherine O'Shea for secretarial assistance.
Received for publication June 10, 2002; accepted June 12, 2002.
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