- Open Access
Regulation of Laminin 1-Induced Pancreatic β-Cell Differentiation by α6 Integrin and α-Dystroglycan
© Picower Institute Press 2001
- Accepted: 2 October 2000
- Published: 1 February 2001
The ability to manipulate the development of pancreatic insulin-producing β cells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes β-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, α6 integrin, and α-dystroglycan (α-DG).
Materials and Methods
Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or α-DG. Fetuses fixed in Bouin’s solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake.
Blocking the binding of laminin-1 to α6 integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of β cells. Inhibition of molecules involved in α6 integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, β cells appeared to develop normally in α6 integrin-deficient fetuses. Blocking the binding of laminin-1 to α-DG with a monoclonal antibody, IIH6, dramatically decreased the number of β cells. Heparin, also known to inhibit laminin-1 binding to α-DG, had a similar effect. In the presence of heparin, the increase in β cells in response to blocking α6 integrin with GoH3 was abolished.
These findings reveal an interplay between α6 integrin and α-DG to regulate laminin-1-induced β-cell development. Laminin-1 had a dominant effect via α-DG to promote cell survival and β-cell differentiation, which was modestly inhibited by α6 signaling.
Lineage differentiation of pancreatic β cells has been extensively studied (1–3). However, regulation of β-cell development by extracellular factors remains poorly understood and is a barrier to the cure of type 1 diabetes. Generally, two categories of extracellular factors cooperatively mediate cell growth, differentiation, and survival: soluble hormones and growth factors and cell-associated extracellular matrix (ECM) proteins (4). Previously, we found that laminin-1, a major ECM protein in the basement membrane, promotes differentiation of fetal pancreatic cells into β cells in vitro (5). Two types of receptors for laminin-1, α6 integrins and α-dystroglycan (α-DG), have been identified in epithelial tissues (6). Integrins are a well-characterized family of heterodimeric cell adhesion molecules composed of noncovalently bound α (120–180 kDa) and β (90–110 kDa) subunits, of which 16 and 8 isoforms, respectively, are known. The α6 integrin subunit is expressed in a wide range of tissues including the pancreas (7–10), although its expression in fetal mouse pancreas has not been reported. The α6 subunit dimerizes with the β1 or β4 subunit to form α6β1, or α6β4 integrin (6). ECM proteins including laminin-1 bind to integrin receptors to trigger receptor aggregation, actin cytoskeleton polymerization, and activation of tyrosine kinases and signaling cascades leading to growth, differentiation, or apoptosis (11–13). Focal adhesion and actin polymerization following integrin ligation may align signaling molecules in the phosphotidylinositol 3-kinase and the mitogen-activated protein (MAP) kinase pathways to facilitate signal transduction (11,12).
α-DG is a nonintegrin, highly glycosylated peripheral membrane protein identified initially in muscle (14) and subsequently in other tissues, including adult pancreas (15). It is associated with a membrane-spanning protein, β-DG, in the dystrophin-glycoprotein complex. In muscle, the complex is structurally organized into three distinct subcomplexes: the dystroglycans (α-DG and β-DG), the sarcoglycans (SGs), and the cytoskeletal proteins dystrophin, syntrophin, and dystrobrevin (16,17). α-DG associates with the F-actin cytoskeleton through dystrophin (18,19). However, SGs are not expressed in epithelial cells (20) and signaling downstream of α-DG has not been characterized. In the present study, we investigated the roles of α6 integrins and α-DG in laminin-1-induced β-cell development.
Mouse Fetal Pancreas Cell Culture
Pancreata were dissected from 13.5 days postcoitum (dpc) fetuses and dissociated into single cells as described (5). Cells were counted in a hemocytometer and viability determined by Trypan blue dye exclusion. Each fetal pancreas yielded approximately 25,000 viable cells (25,694 6 1324; n 5 20). Dissociated cells were plated in eight-chamber slides (Nunc, Naperville,) at 1.5 3 104 cells/well in 0.3 ml HYBRIDOMA medium supplemented with 500 UI/ml penicillin and 500 µg/ml streptomycin (GibcoBRL Life Technologies, Gaithersburg, Md) and 200 µg/ml laminin-1 purified from murine Engelbreth-Holm-Swarm tumor basement membrane (GibcoBRL), with or without various concentrations of antibodies and reagents. They were cultured in 10% CO2 90% air at 378C for 4 days.
Antibodies and Reagents
Rat monoclonal antibody (clone NKI-GoH3, IgG2a) that specifically blocks laminin-1 binding to α6 integrins (9,21,22) was from Chemicon International (Temecula,). Blocking mouse monoclonal antibodies to integrin α3 (clone P1B5) (23) and integrin β4 (clone 3E1) (24) were from GibcoBRL. Blocking mouse monoclonal IgM antibody to α-DG, IIH6, was generously provided as hybridoma supernatant by Dr. Kevin Campbell, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City. The IgM concentration of IIH6 hybridoma supernatant was estimated from Coomassie staining in SDS-PAGE against serial dilutions of purified mouse IgM standard. Rat monoclonal IgG2a (control for NK1-GoH3), mouse IgM (control for IIH6), and blocking rat monoclonal IgG2a to integrin β1 (clone 9EG7) (8,9,25) from Pharmingen (San Diego, Calif) were dialyzed against HYBRIDOMA medium at 48C prior to use. Guinea pig antiserum to porcine insulin was from Dako (Glostrup, Denmark). Mouse monoclonal IgG2a to bromodeoxyuridine (BrdU, clone BU-1) was from Amersham Life Science (Buckinghamshire, UK). Rabbit antiserum to porcine glucagon and to human somatostatin and pancreatic polypeptide were from Dako. Fractionated rabbit antiserum to human α-amylase, a marker of acinar cells, and to laminin, were from Sigma Chemicals (St. Louis, Mo).
Heparin, a known blocker of laminin-1 binding to α-DG (18,26), was from Sigma. Wortmannin and Ly294002, inhibitors of PI3K (27,28), genistein and herbimycin, inhibitors of Src family tyrosine kinases (29) associated with focal adhesion kinase (FAK) (30,31), and PD98059, an inhibitor of the MAP kinase kinase, MEK1 (32), were from Calbiochem (La Jolla, Calif). Cytochalasin D, an inhibitor of actin polymerization, was from Sigma.
Immunoperoxidase Staining and Cell Quantitation
Fetuses at 15.5 and 18.5 dpc from homozygous (2/2) or heterozygous (1/2) α6 integrin gene targeted (33) or wild-type mice were fixed overnight in Bouin’s solution. After standard dehydration processing, fetuses were embedded into paraffin and sectioned at 7 µm. Cultured pancreatic cells were washed three times with warm mouse tonicity phosphate-buffered saline (MT-PBS) and fixed with 4% paraformaldehyde (PFA) for 10 min. Endogenous peroxidase was blocked by 3% H2O2 in methanol for 8 min. Prior to antibody staining, nonspecific protein binding was blocked by incubation for at least 30 min with MT-PBS containing 2% bovine serum albumin or 2% normal rabbit serum. Controls were performed by replacing first antibody with preimmune serum from the appropriate species. Cells were incubated with primary antibodies for 90 min at room temperature, followed by three thorough washes with MT-PBS. Horseradish peroxidase-conjugated rabbit anti-guinea pig or swine anti-rabbit immunoglobulins (Dako) were added for 30 min at room temperature followed by thorough washes. Immunoperoxidase was detected with 3,3 9-diaminobenzidine/H2O2 for 4–8 min, and slides counterstained with hematoxylin.
Immunoperoxidase-positive and -negative cells were counted in the central strip of each culture chamber (90 3 90 mm square) under a microscope equipped with an eyepiece graticule (Olympus, Japan) at 3 40 power and calibrated with a micrometer (Olympus).
In some cultures, 100 µM BrdU (Sigma) was included to label proliferating cells; cells were fixed with 4% PFA at days 1, 2, 3, and 4 for insulin and BrdU double immunofluorescence staining. Pretreatment and primary antibody incubations were as described above, followed by incubation with Texas Red-conjugated goat anti-guinea pig immunoglobulins (Vector Laboratories, Burlingame,) or fluorescein isothiocyanate-conjugated rabbit anti-mouse, -rat, or -goat or swine anti-rabbit immunoglobulins (Dako) for 30 min at room temperature and three thorough washes. Slides were observed and photomicrographed under a Zeis Axiophot fluorescence microscope.
Dose responses were analyzed by ANOVA and differences between groups were analyzed by the non-parametric Mann-Whitney U test. Data are presented as mean 6 SEM of at least three independent experiments.
Laminin-1 was detected by immunofluorescence on dissociated fetal mouse pancreas cells only after its addition to culture medium (data not shown), indicating that these cells produce little if any laminin-1 in culture. Although both pancreatic epithelial cells and vimentin-positive mesenchymal cells (60% of total initially) attached, the latter did not survive in low-cell density, serum-free conditions [0% vimentin-positive after 4-day culture, see also (5)]. The epithelial cells expressed α6 integrins and α-DG to which the added laminin bound [data not shown; see also (6)].
α6 Integrin Blockade Stimulates β-Cell Differentiation
To measure cell division, 100 µM BrdU was added with laminin-1 (200 µg/ml), with and without GoH3, and BrdU-positive cells analyzed at days 1–4. In the absence of GoH3, 2.5% of cells were BrdU positive; in the presence of GoH3, BrdU-positive cells were not detected.
The α3 integrin subunit is the only α subunit with significant (40%) identity to the α6 subunit (34), but the blocking anti-α3 monoclonal antibody P1B5 (10-40 µg/ml) had no effect on total or β-cell number (data not shown). The α6 integrin subunit dimerizes noncovalently with either β1 or β4 subunits to form α6β1 or α6β4 integrin (6), but neither anti-β1 (9EG7) nor anti-β4 (3E1) monoclonal antibodies influenced total or β-cell number (data not shown).
Inhibition of Molecules Involved in α6 Integrin Signaling Pathways Stimulates β-Cell Differentiation
Islet Cell Development Appears Normal in α6 Integrin-Deficient Mice
α-Dystroglycan Blockade Inhibits β-Cell Development
The increase in laminin-1-induced β-cell differentiation after inhibition of PI3K with wortmannin or Ly294002 confirms a previous report by Ptasznik et al (39) that this enzyme is a negative regulator of β-cell differentiation. These investigators found that wortmannin induces morphologic and functional endocrine differentiation in human fetal undifferentiated pancreas cells. PI3K is upstream of the MEK1 signal cascade (13). Inhibition of MEK1 with PD98059 also enhanced laminin-1-induced β-cell differentiation. This is consistent with reports that MEK1 is activated by ligation of laminin-1 and α6Aβ1 (40) or α6β4 (24) integrins. Inhibition of Srctyrosine kinases had no effect and therefore this family of kinases is probably not involved in laminin-1-induced β-cell differentiation. In summary, these data are consistent with the view that α6 integrin signaling through the MAP kinase ERK module exerts a negative regulatory effect on β-cell differentiation.
On the basis of these findings in vitro, the absence of α6 integrins in vivo might be expected to promote β-cell development. However, this did not appear to be the case. There are a number of possible explanations why β-cell development may be normal in α6 integrin-deficient mice. The first, in agreement with the present findings, is that inhibition through α6 integrin is relatively modest in comparison with the survival-differentiation signal through α-DG. Second, α6 integrin deficiency could be compensated for by other integrin members (41), although we found that blocking the related α3 integrin had no effect on laminin-1-induced β-cell development. Third, laminin-1 signaling through α6 integrins could be antagonized in vivo by signals from other ECM proteins (e.g., collagen IV) (5). Overall, compared with α-DG, α6 integrins may play a contributory but not essential role in β-cell differentiation. A key role for α-DG transducing survival/differentiation signals from laminin-1 is supported by a recent report from Montanaro et al (42) in which transfection of myoblasts with antisense α-DG RNA was shown to decrease the number of myotubes in culture. Our findings indicate that laminin-1 has dual effects on β-cell development via α6 integrins and α-DG, but that its binding to α-DG dominates in promoting survival and β-cell differentiation. Understanding mechanisms by which extracellular factors promote β-cell development should facilitate β-cell replacement therapy for type 1 diabetes.
This study was supported by the Juvenile Diabetes Foundation International (Angelo and Susan Alberti Program Grant) and by the National Health and Medical Research Council of Australia. The authors thank Dr. Jorge Gonez for helpful advice and Margaret Thompson for secretarial assistance.
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