Role for ß1 Integrin and Its Associated {alpha}3, {alpha}5, and {alpha}6 Subunits in Development of the Human Fetal Pancreas

Rennian Wang1,2, Jinming Li1, Kristina Lyte1, Nina K. Yashpal1, Fraser Fellows3, and Cynthia G. Goodyer4

1 Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
2 Department of Medicine, University of Western Ontario, London, Ontario, Canada
3 Department of Obstetrics and Gynecology, University of Western Ontario, London, Ontario, Canada
4 Department of Pediatrics, McGill University, Montreal, Quebec, Canada


    ABSTRACT
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The integrin receptors play a major role in tissue morphogenesis and homeostasis by regulating cell interactions with extracellular matrix proteins. We have examined the expression pattern of integrin subunits in the human fetal pancreas (8–20 weeks fetal age) and the relevance of ß1 integrin function for insulin gene expression and islet cell survival. Its subunits {alpha}3, {alpha}5, and {alpha}6 ß1 integrins are expressed in ductal cells at 8 weeks, before glucagon- and insulin-immunoreactive cells bud off; their levels gradually increase in both ductal cells and islet clusters up to 20 weeks. Colocalization of {alpha}3, {alpha}5 and {alpha}6 ß1 integrins with endocrine cell markers was frequently observed in 8- to 20-week fetal pancreatic cells. When the ß1 integrin receptor was functionally blocked in cultured islet-epithelial clusters with a ß1 immunoneutralizing antibody or following transient ß1 integrin small interfering RNA treatment, there was inhibition of cell adhesion to extracellular matrices, decreased expression of insulin, and increased cell apoptosis. These data offer evidence for dynamic and cell-specific changes in integrin expression during human pancreatic islet neogenesis. They also provide an initial insight into a molecular basis for cell-matrix interactions during islet development and suggest that ß1 integrin plays a vital role in regulating islet cell adhesion, gene expression, and survival.

Islets of Langerhans contain a remarkable cellular organization that is ideal for rapid, yet precisely controlled, responses to changes in blood glucose levels. Any permanent disturbance of this regulatory system leads to diabetes, one of the most common metabolic diseases affecting millions of people throughout the world. Determining the factors that control islet cell development and maintain survival and function is essential to help develop viable strategies for any cell-based approach toward the repopulation of islets for the treatment of diabetes. Therefore, recent efforts have concentrated on exploring the molecular signals that control morphogenesis in the normal human pancreas. One important research focus has been the integrin receptors, a family of cell adhesion molecules that mediate cell-cell and cell-matrix interactions. They have been shown to regulate the proliferation, maturation, and function of rodent islets in vitro (1–,2). However, the role of integrin-mediated interactions with the extracellular matrix (ECM) on the formation and function of the islets of Langerhans before birth, especially in the human, is poorly understood.

Integrins are a large family of heterodimeric transmembrane adhesion molecules composed of noncovalently bound {alpha}- and ß-subunits that possess the unique ability to regulate cell adhesiveness through a process called "inside-out signaling." In addition, after binding to their ligands at the cell surface, these receptors integrate the cues from their external environment to the cell by generating specific intracellular signals, in a process termed "outside-in signaling" (3). This results in modifications of cell structure and functions such as cell adhesion, motility, cell proliferation, differentiation, and gene transcription (35).

The ß1 integrin family is believed to play a critical role in morphogenesis (67), cell differentiation, and proliferation (89) as well as cell survival (10) by binding selectively to collagen, fibronectin, and laminin extracellular matrices (11). The importance of this receptor is evident from the embryonic lethality that ensues in homozygous ß1-deficient embryos (7).

Multiple functions of {alpha}ß1 integrins in a number of organ systems have been described previously; however, research on their expression and interactions during pancreatic development is limited. Thus far, studies have shown that only a few members of the integrin family affect islet cell survival, maturation, and insulin production (2,12). In particular, {alpha}3, {alpha}5, and {alpha}6 integrins have been reported to mediate certain pancreatic developmental events: 1) {alpha}3ß1 mediates the attachment and spreading of primary rat islet cells to ECMs (12) and regulates the migration of CK19+/PDX-1+ putative pancreatic progenitors of human fetal pancreatic epithelial cells on netrin-1 (13); 2) {alpha}5 expression has been shown to decrease during culture of rat islets, which parallels increased islet apoptosis, implicating this particular integrin in controlling signaling events that protect against cell death (14); 3) Crisera et al. (15) reported that mouse pancreatic ductal morphogenesis requires the ECM laminin-1 during embryonic life and is inhibited by the blockade of {alpha}6ß1 integrin or laminin; 4) {alpha}6ß1 is believed to enhance and regulate the insulin secretory response of rat islets (2); and 5) ß1 integrin may be involved in early motile processes required for the formation of new islets by supporting migration of human fetal ß-cells (16). More recently, Hammer et al. (17) determined that 804G matrix protects ß-cells against apoptosis via the integrin ß1/focal adhesion kinase pathway and that blocking ß1 integrin function induces cell death. Thus, these studies suggest a role for {alpha}3, {alpha}5, {alpha}6, and ß1 integrin receptors in early pancreatic developmental events in multiple species.

Based on the above findings, the goal of the current study was to examine the expression pattern of integrin subunits in situ during islet growth in the human fetal pancreas from 8–20 weeks of fetal age using immunofluorescence, Western blot, and real-time RT-PCR. We also examined the role of ß1 integrin in cultured islet-epithelial clusters, in mediating islet cell adhesion to extracellular matrices, insulin gene expression, and cell death, using immunoneutralizing antibodies and small interfering RNAs (siRNAs). Here we report that human fetal ductal and islet cells express ß1 integrin and its associated {alpha}3, {alpha}5, and {alpha}6 subunits during early pancreatic development. Our data also provide evidence for a major role for the ß1 integrin receptor in mediating adhesion, insulin gene expression, and survival of human fetal islet-epithelial clusters. Furthermore, this study provides a molecular connection between cultured islets and the ECM that can be manipulated and is thus highly useful information for future investigations that seek to improve islet cell–based therapies for the treatment of diabetes.


    RESEARCH DESIGN AND METHODS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissues.
Human fetal pancreata (8–20 weeks fetal age) were collected according to protocols approved by the Health Sciences Research Ethics Board at the University of Western Ontario and the Research Ethics Board of the Royal Victoria Hospital at the McGill University Health Centre, in accordance with the Canadian Council on Health Sciences Research Involving Human Subjects guidelines. Tissues were immediately processed for immunohistochemistry, RNA, and/or protein extraction, with at least three pancreata per age or experimental group.

Immunofluorescence.
Pancreata were fixed in 4% paraformaldehyde overnight at 4°C followed by a standard protocol of dehydration and paraffin embedding (18). Sections of 5 µm were cut throughout the length of the pancreas with two sets of six serial sections at 50-µm intervals. The tissue sections were incubated overnight at 4°C with appropriate dilutions of the following primary antibodies: rabbit anti-{alpha}3 and anti-{alpha}5 integrins (cytoplasmic domains), mouse anti-ß1 and anti-{alpha}6ß1 integrins (Chemicon, Temecula, CA), rabbit anti-{alpha}6 integrin (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anticytokeratin 19 (CK19; Dako, Mississauga, ON, Canada), guinea pig anti-human insulin (Zymed, San Francisco, CA), mouse anti-human glucagon (Sigma, St. Louis, MO), rabbit anti-PDX-1 (gift from Dr. Wright, University of Vanderbilt, Nashville, TN), and antibodies to laminin, fibronectin, and collagen IV (Chemicon) as described previously (19). To identify colocalization of integrins with epithelial and endocrine cell markers, double immunofluorescence staining was performed. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Images were recorded by a Leica DMIRE2 fluorescence microscope with the Openlab image software (Improvision, Lexington, MA). Negative controls included the omission of the primary antibodies.

Morphometric analysis.
Both single- and double-labeled images were recorded under a high magnification (400x). Endocrine and ductal regions were defined through staining of consecutive sections with a cocktail of antibodies for pancreatic hormones and an antibody for the ductal cell, as previously described (1819). The integrin immunoreactive area within the ductal and endocrine cell compartments was traced manually. In each pancreatic section, 8–12 random fields were chosen with a minimum of three pancreata per age or experimental group, and data are expressed as the percentage of integrin immunoreactivity in both endocrine and duct regions. To determine the percentage of integrin colocalization with insulin or glucagon, the double-labeled cells are expressed as a percentage of the total number of insulin- or glucagon-positive cells.

Western blots.
Pancreatic tissues were homogenized in a Nonidet-P40 lysis buffer (Nonidet-P40, phenylmethylsulfonyl fluoride, sodium orthovanadate [Sigma] and complete protease inhibitor cocktail tablet [Roche, Montreal, QC, Canada]) and centrifuged at 12,000 rpm for 20 min. The supernatant was recovered and frozen at –80°C. The protein concentration was measured by Bradford protein dye (Bio-Rad, Mississauga, ON, Canada), using bovine serum albumin (fraction V) as standard. As described previously, 25 µg of pancreatic lysate proteins were separated by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) (1920). The membranes were washed in Tris buffer–saline containing 0.1% Tween 20 and blocked with 5% nonfat dry milk overnight at 4°C. Immunoblotting was performed with the integrin antibodies at the concentrations recommended by the manufacturer for 1 h at room temperature. Secondary antibody was anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz) diluted at 1:1,000. Proteins were detected by enhanced chemiluminescence reagents (Amersham, Oakville, ON, Canada) and exposed to BioMax MR Film (Kodak, Rochester, NY). Densitometric quantification of bands at subsaturating levels was performed using the Syngenetool gel analysis software (Syngene, Cambridge, U.K.) and normalized to band intensities at 8 weeks of fetal age (19,21). Loading controls of calnexin (BD Biosciences) and ß-actin (Sigma) were tested; however, their variability during development precluded their use (19). For negative controls, the primary antibody was omitted.

RT-PCR and real-time RT-PCR.
Total RNA was extracted from pancreas tissues with TRIZOL reagent (Invitrogen, Burlington, ON, Canada), according to the manufacturer’s instructions. Quality of the RNA was verified by agarose gel electrophoresis using ethidium bromide staining. For each RT reaction, 2 µg DNA-free RNA were used with oligo(dT) primers and Superscript reverse transcriptase. PCRs were carried out in a T-gradient Biometra PCR thermal cycler (Montreal Biotech, Kirkland, QC, Canada) to determine the annealing temperature for each pair of primers (19). The PCR primers used include ß1 integrin, F, 5'-GACCTGCCTTGGTGTCTGTGC-3' and R, 5'-AGCAACCACACCAGCTACAAT-3' (313 bp); insulin, F, 5'-TCACACCTGGTGG AAGCTC-3' and R, 5'-ACAATGCCACGCTTCTGC-3' (179 bp); and 18S, F, 5'-GTAA CCCGTTGAACCCCATT-3' and R, 5'-CCATCCAATC GGTAGTAGCG-3' (131 bp). Controls involved omitting RT, cDNA, or DNA polymerase and showed no reaction bands. Real-time PCR analyses of ß1 integrin and insulin were performed on 0.1 µg cDNA using the SYBR green qPCR kit in DNA Engine Option (MJ Research, South San Francisco, CA). Data were normalized to the 18S RNA subunit with at least three pancreata per age or experimental group (19). Similar results were obtained if the data were normalized to glyceraldehyde-3-phosphate dehydrogenase (data not shown). Both housekeeping genes showed stable mRNA expression in the 8- to 20-week fetal pancreatic tissues and cultured islets.

Cell adhesion assay.
To examine integrin function in regulating cell adhesion to ECM, human fetal pancreata (14–16 weeks) were digested with collagenase V (2 mg/ml) for 30 min at 37°C. Islet-epithelial clusters, which contained mostly undifferentiated epithelial cells and 2–10% endocrine cells (22), were washed in cold 1x Hanks’ balanced salt solution and recovered in CMRL 1066 supplemented with 10% fetal bovine serum for 2 h at 37°C. Adhesion assays were carried out in 12-well plates (Corning/VWR, Toronto, ON, Canada) coated with fibronectin (50 µg/ml) or laminin (50 µg/ml); rat tail collagen (1 mg/ml) was also used by applying neutralized collagen onto the surface of each well to form a thin gel (14). Cell clusters were pretreated for 1 h with hamster monoclonal anti-ß1-integrin (CD29, 5 µg/ml; Pharmingen, Mississauga, ON, Canada), with hamster IgM isotype (5 µg/ml) or vehicle (control), plated onto coated wells (100 clusters/well) and cultured with CMRL 1066 supplemented with 10% fetal bovine serum for 24 h at 37°C in 5% CO2. At the end of the incubation period, unattached cell clusters were washed off by repeated rinses in Hanks’ balanced salt solution. The attached cell clusters were counted using an inverted microscope. The number of cell clusters adhered to coated matrix wells was calculated as a percentage of total cell clusters plated; each experiment used triplicate wells/group and was repeated five times (14).

Transferase-mediated dUTP nick-end labeling assays and insulin mRNA expression.
To analyze for cell death and insulin gene expression, clusters of the three experimental groups were cultured in suspension. RNA samples were harvested after 2 and 24 h of treatment followed by RT-PCR and real-time RT-PCR analyses for insulin mRNA (19). For the cell death assays, 24-h treated cell clusters embedded in 2% agarose were fixed in 4% paraformaldehyde followed by paraffin embedding. As described previously, 5-µm sections were deparaffinized, pretreated with 0.1% trypsin and incubated with the transferase-mediated dUTP nick-end labeling (TUNEL) reaction mixture (Roche) for 60 min at 37°C (14,19). The sections were subsequently stained with guinea pig anti-human insulin or mouse anti-human glucagon labeled with rhodamine (tetramethylrhodamine isothiocyanate [TRITC]). The percentage of total TUNEL-positive islet-epithelial cluster cells, ß-cells, and {alpha}-cells was determined.

ß1 integrin siRNA transfections.
Freshly isolated clusters, after a 1-h calibration culture in antibiotic-free medium, were transiently transfected as suspensions for 30 h with 60 nmol/l ß1 integrin siRNA (proprietary sequence; accession #NM_002211) or control siRNA (proprietary sequence) commercially produced by Santa Cruz Biotechnology using an siRNA transfection kit (Santa Cruz Biotechnology). Islet-epithelial clusters were harvested 72 h after transfection and assessed for the expression of ß1 integrin and insulin protein as well as insulin mRNA (19). Transfection efficiency was monitored using fluorescein-conjugated control siRNA (Santa Cruz Biotechnology) with ~60% of the islet-epithelial cluster cells being transfected during each experiment. Cell viability was examined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (19,23) and 100 islet-epithelial clusters from both ß1 siRNA and control siRNA transfected groups were plated in triplicate and cultured for 72 h. The clusters were harvested in 500 µl of culture medium, and then 50 µl of stock MTT (5 mg/ml, Sigma) was added for 2-h incubation at 37°C. Cells were washed and lysed by 200 µl DMSO (Sigma). The samples were assayed for absorbance at 595 nm using a Multiskan Spectrum spectrophotometer (Thermo Labsystems, Franklin, MA).

Statistical analysis.
Data are expressed as means ± SE. Statistical significance was determined using a two-tailed unpaired Student’s t test or one-way ANOVA followed by the Student-Newman-Keuls group comparison test. Differences were considered to be statistically significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of ß1 integrin and its associated {alpha}3, {alpha}5, and {alpha}6 subunits in the developing human pancreas.
To examine when and where integrins appear during pancreas development, we first screened for several possible integrin receptors in the human pancreas from 8–20 weeks of fetal age. Using immunohistochemistry, we observed ß1, {alpha}3, {alpha}5, and {alpha}6 integrin immunoreactivity in the pancreatic ductal cells at 8 weeks, before glucagon- and insulin-immunoreactive cells bud off. Western blot analysis of the protein level of ß1 integrin and its associated {alpha}-subunits revealed an increased expression of ß1, {alpha}3, and {alpha}6 integrin that was statistically significant (P < 0.05) by 16 weeks, whereas the expression of {alpha}5 was relatively constant from weeks 8–20 (Fig. 1AD). Quantitative analysis of ß1 integrin mRNA by real-time RT-PCR determined that its mRNA signal increased significantly by 12 weeks (P < 0.05) (Fig. 1E), before the increase observed in ß1 protein levels (Fig. 1A).



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FIG. 1. Western blot analyses of human fetal pancreatic tissue using anti-ß1 (A), anti-{alpha}3 (B), anti-{alpha}5 (C), and anti-{alpha}6 (D) integrin antibodies. Data are expressed as means ± SE (n = 3 per age-group) relative to the 8-week group. Representative blots are shown. E: Real-time RT-PCR analysis of ß1 integrin mRNA expression in human fetal pancreatic tissue. Data are normalized to 18S RNA subunit and expressed as means ± SE (n = 3 per age-group). *P < 0.05 vs. 8-week group.

 
To examine the distribution and colocalization of integrin {alpha}3, {alpha}5, and {alpha}6ß1 with the epithelial (CK19) and mature endocrine cell (insulin and glucagon) markers, dual immunofluorescence experiments were conducted. Morphometric analysis of the expression patterns of {alpha}3, {alpha}5, and {alpha}6ß1 integrins revealed that there was a significant increase in integrin immunoreactive area at 20 weeks in the ductal regions (P < 0.05) (Fig. 2), except for the {alpha}5 subunit. Expression in endocrine cells was detected as early as when single endocrine cells began to bud off from the ducts (9 weeks); a gradual increase in their immunoreactive area in islets was significant for {alpha}3 (P < 0.05) and for {alpha}6ß1 (P < 0.005) by week 20 (Fig. 2).



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FIG. 2. Morphometric analysis of {alpha}3, {alpha}5, and {alpha}6ß1 integrin immunoreactivity in duct and endocrine regions of the developing human pancreas. Data are expressed as means ± SE (n = 4) *Duct. #Endocrine region. *#P < 0.05 and ##P < 0.01 vs. respective 12- to 13-week group.

 
It was readily apparent that many single insulin- or glucagon-positive cells or newly formed small islets costained with the three integrins (Fig. 3). A high proportion of {alpha}3 or {alpha}5 integrin and insulin costaining was observed at 12 weeks with slightly decreased coexpression by week 18 (Fig. 4A). In contrast, {alpha}6ß1 integrin colocalization with insulin increased slightly from 12–18 weeks (63 ± 9% vs. 78 ± 12%) (Fig. 4A). The percentage of cells coexpressing {alpha}3 or {alpha}6ß1 with glucagon showed no difference during development; however, there was a significant increase in frequency of coexpression of {alpha}5 with glucagon at 18 weeks (P < 0.01) (Fig. 4B), indicating that {alpha}5 may play a prominent role in glucagon cell maturation and functional maintenance. Colocalization of {alpha}6ß1 integrin with PDX-1, an early pancreatic developmental transcription factor, was frequently observed in both duct and islet regions (Fig. 3).



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FIG. 3. Coexpression patterns of {alpha}3/insulin, {alpha}5/glucagon, {alpha}6ß1/insulin, and {alpha}6ß1/PDX-1 in a 14-week human fetal pancreas. Integrins are labeled by fluorescein isothiocyanate (green) and insulin, glucagon, or PDX-1 by TRITC (red). Nuclei were counterstained by 4',6-diamidino-2-phenylindole (blue). Arrows indicate double-labeled cells, with integrins (green) located primarily at cell borders and endocrine cell markers (red) within the cytoplasm. *Nonspecific staining of blood cells. Original magnification 400x; insert 630x.

 


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FIG. 4. The percentage of cells demonstrating coexpression of {alpha}3, {alpha}5, and {alpha}6ß1 integrins with insulin (A) or glucagon (B) in 12- and 18-week human fetal pancreata. Data are expressed as means ± SE (n = 4). **P < 0.01 for 18 vs. 12 weeks.

 
Correlation of ECM proteins and integrin receptors in the developing human pancreas.
Collagen IV, fibronectin, and laminin are important ECM components in basement membranes as well as ligands for {alpha}3ß1 integrin (11). Fibronectin is a known adhesive site for {alpha}5ß1 and {alpha}6ß1 integrin adheres to laminin (11,14). To correlate integrin expression and their ECM ligands in the developing human pancreas, dual immunofluorescent staining was performed on paraffin-embedded fetal pancreatic sections. Figure 5 shows {alpha}3 integrin-positive cells colocalizing with collagen IV, which highlights the basal membrane of pancreatic ducts and islets. There was also localization of {alpha}3 integrin–positive cells at sites where fibronectin and laminin were observed in the developing human pancreas (data not shown). Expression of {alpha}5 integrin was localized to cells that were surrounded by a matrix of fibronectin (Fig. 5) but not collagen IV and laminin (data not shown). Staining of {alpha}6ß1 integrin was associated with cells in close proximity to a matrix of laminin (Fig. 5). These observations suggest that the developing human pancreas has parallel expression patterns of integrins and their respective ligands.



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FIG. 5. Localization of {alpha}3/collagen IV, {alpha}5/fibronectin, and {alpha}6ß1/laminin in a 14-week human fetal pancreas. Integrins are labeled by fluorescein isothiocyanate (green) and extracellular matrices by TRITC (red). Arrows indicate integrin-positive cells associated with labeled ECM. *Nonspecific staining. Original magnification 400x; insert 630x.

 
ß1 integrin receptor mediates fetal islet-epithelial cluster adhesion, insulin gene expression, and cell survival.
We subsequently tested the effects of blocking ß1 integrin activity in islet-epithelial cell clusters using a ß1 integrin immunoneutralizing antibody. The percentage of clusters in both control and IgM antibody–treated groups that adhere to collagen I, fibronectin, or laminin was high (up to 80%) and clusters attached to fibronectin and collagen I began to spread after 24 h of culture (Fig. 6A and B). In contrast, islet-epithelial clusters treated with anti–ß1-integrin had a significantly reduced ability to adhere to ECM, such that the percentages of adhesion dropped to 12 ± 1, 15 ± 2, and 31 ± 6%, respectively, after 24 h (P < 0.001) (Fig. 6A and B). Decreasing adhesion was associated with lost ß1 integrin expression: quantitative analysis demonstrated a 37% reduction in ß1-integrin–expressing cells after 24 h of anti–ß1-antibody treatment (35 ± 5% vs. 55 ± 4% of controls, P < 0.001). Cells coexpressing ß1 integrin with insulin were also significantly decreased (3.3 ± 1% vs. 8.6 ± 3% of controls, P < 0.05), whereas the total percentage of insulin-positive cells was slightly reduced (8.5 ± 4% vs. 11.4 ± 3% of controls). We investigated whether blocking the ß1 integrin receptor affects insulin gene expression using quantitative RT-PCR assays (Fig. 6C). There was no change in preproinsulin expression after 2 h of ß1 integrin blockade. However, after 24 h, there was an 18% reduction in insulin mRNA (P < 0.05) compared with the control group (Fig. 6C).



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FIG. 6. Effect of ß1 integrin loss on adhesion of islet-epithelial clusters on different matrices. A: Phase-contrast micrographs of clusters in culture after 24 h in the absence (Ctrl) or presence of monoclonal anti–ß1-integrin or IgM antibodies on collagen I–, fibronectin-, or laminin-coated dishes. B: Quantitative analysis of cluster adherence rate. Data are expressed as means ± SE (n = 5). **P < 0.01, ***P < 0.001 relative to controls. C: Representative RT-PCR and real-time RT-PCR analyses of preproinsulin expression in the three experimental groups after 2 and/or 24 h of culture. Quantitative RT-PCR data are normalized to 18S RNA subunit and expressed as means ± SE (n = 4). *P < 0.05 vs. controls.

 
Blockade of ß1 integrin was also associated with a significant increase in the number of cells in the islet-epithelial clusters undergoing apoptosis, as assessed by the TUNEL assay (P < 0.001) (Fig. 7). There was also a significant increase in the total number of apoptotic ß- and {alpha}-cells in the anti–ß1-antibody group (P < 0.01) (Fig. 7).



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FIG. 7. A: The apoptotic index of islet-epithelial clusters cultured in the absence (Ctrl) or presence of anti–ß1-integrin immunoneutralizing or IgM antibodies for 24 h. Data are expressed as means ± SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 relative to controls. Double labeling for TUNEL (green) and insulin (red) in islet-epithelial clusters B: Arrowheads indicate TUNEL-positive cells, arrow indicates an apoptotic ß-cell (green nucleus, red cytoplasm), and asterisks indicate nonspecific staining. Original magnification 400x; insert 630x.

 
Furthermore, we examined the effects of suppressing ß1 integrin expression in islet-epithelial clusters using a specific ß1 integrin siRNA. After the transfection and 72 h of culture, a significant downregulation of ß1 integrin protein expression was observed (43 ± 3.5% vs. 72 ± 1.2%, P < 0.01) (Fig. 8A). Knockdown of ß1 integrin was associated with a decrease in preproinsulin expression and reduced cell viability as determined by the MTT assay (P < 0.02) (Fig. 8B). Decreasing insulin gene expression was correlated with a significant loss in the number of insulin-positive cells in the clusters (8 ± 1% vs. 17 ± 3%, P < 0.05) (Fig. 8A).



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FIG. 8. Transfection of islet-epithelial clusters with ß1 integrin siRNA resulted in decreases in ß1 integrin protein, the number of immunoreactive ß1 integrin and insulin expressing cells (A), ß1 integrin and insulin mRNA expression, and cell viability (RT omitted) (B). Cell viability was assessed by MTT assay. Data are expressed as means ± SE (n = 3). *P < 0.05, **P < 0.01 relative to controls.

 

    DISCUSSION
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrates that ß1 integrin and its associated {alpha}-subunits are expressed in the early to mid-gestation human fetal pancreas in a dynamic, temporally regulated fashion. Furthermore, blockade of ß1 activity results in loss of islet-cluster attachment to various extracellular matrices, diminished insulin gene expression, and decreased cell viability. Taken together, the data from this descriptive and functional investigation demonstrate that integrin-ECM interactions in the developing human pancreas are critical for normal islet formation and endocrine function.

Immunohistochemical, morphometrical, RNA, and protein analyses showed a specific temporal and spatial pattern for ß1 integrin expression associated with {alpha}3, {alpha}5, and {alpha}6 subunits during fetal development. They are detectable within the ducts as early as 8 weeks of fetal age and gradually increase in expression from 12 to 20 weeks. After 9–11 weeks, newly forming single endocrine cells or small islets frequently expressed {alpha}3, {alpha}5, and {alpha}6ß1, suggesting that these receptors are involved in regulating differentiation and migration of endocrine cell types budding from and in close proximity to ductal structures. In addition, within the larger islets observed at mid-gestation, integrin subunit expression is high, supporting a role for these receptors in the formation and function of mature islets. Interestingly, the integrin {alpha}6ß1 subunit has been previously reported to mediate ß-cell differentiation and the ß-cell secretory response in dissociated fetal mouse pancreatic epithelium on a laminin-1 matrix (12). Our results indicate that {alpha}ß1 integrin receptors are also likely to play an important role during ontogeny of the human fetal pancreas.

Proteins such as type IV collagen, laminin, and fibronectin have been previously described as major components of the basement membrane in the postnatal human pancreas (24). Studies from our laboratory as well as others (13,16,25) show that these ECM molecules are also components of the human fetal pancreatic basement membrane and are expressed within the developing pancreas in a specific spatial pattern. The integrin subunits {alpha}3, {alpha}5, and {alpha}6ß1 are expressed in cells that localize in proximity to immunoreactive areas for these matrix molecules. Dissecting the functional significance of the individual {alpha} subunit–ECM interactions will be important in determining their role in mediating pancreatic development. For example, studies of fetal mouse pancreatic epithelia on a commercially available basement membrane gel, Matrigel, have demonstrated that laminin and the {alpha}6 subunit mediate the morphological events of ductal formation (15). Furthermore, Jiang et al. (1) have shown that dissociated pancreatic cells from the 13.5d mouse fetus have increased ß-cell differentiation mediated by {alpha}6 integrin when placed on a matrix of laminin-1.

To examine the functional role of ß1 in developing islets, we used an immunoneutralizing monoclonal antibody. Blockade of ß1 integrin resulted in impairment of islet-epithelial cluster adhesion on several ECM, highlighting that the ß1 subunit plays a critical role during pancreatic development. Treatment with an equal amount of IgM antibody had no effect, indicating that the interference with ß1 function by a blocking antibody is the result of a specific interaction. In support of these data, Kaido et al. (16) recently reported that the {alpha}Vß1 integrin may be responsible for early motile processes that regulate human fetal islet formation.

We also demonstrated that blockade of ß1 integrin receptor in islet-epithelial clusters is associated with an increase in the number of cells undergoing apoptosis, with a specific increase in {alpha}- and ß-cell death. These data are in line with the previously described role for the ß1 receptor in offering protection from cell death (26). Adherent cells require integrin signaling for survival; otherwise they undergo a process termed anoiksis (27), evidenced by disengagement of epithelial and fibroblast cells from their microenvironment components.

The perturbation of ß1 integrin in the developing fetal islet clusters was also associated with a decrease in insulin mRNA and protein expression. This is not an unexpected result given that several studies have shown that adult rodent and human islets cultured on or embedded in various ECM have improved insulin secretion and glucose-stimulated secretory responses, potentially mediated by integrin-ECM interactions (2,2829). Whether maturation of the glucose-induced insulin response will occur if fetal cells are cultured in the presence of ß1 integrin and its associated {alpha} subunits is yet unknown. However, given the data from our laboratory as well as others, describing an important role for these integrins during development of the human fetal pancreas (13,16,25), such studies are likely to have positive results.

The siRNA silencing systems are extremely useful tools for studying the functional importance of genes (3032). Most siRNA studies have been carried out on cell lines, with limited information on the effects of gene silencing on primary islets (30). Our recent study of neonatal rat islets, using a nonadenoviral transient transfection of ß1 integrin siRNA, demonstrated a significant decrease in islet cell survival (19). We therefore examined the effect of ß1 integrin siRNA transfection on human fetal islet-epithelial clusters. The results were similar to what we observed with the immunoneutralizing antibody: a significant decrease in ß1 integrin protein correlated with a reduction in insulin mRNA and protein as well as cell viability. These data support the hypothesis that ß1 integrin may be an important regulator of pancreatic endocrine neogenesis as well as being involved in cellular resistance to apoptotic stimuli.

In summary, the present study provides insight into the expression of integrin receptors in the human fetal pancreas and sheds light on how the ß1 receptor, in conjunction with its binding partners {alpha}3, {alpha}5, and {alpha}6, may play multiple roles in islet cell biology, including adhesion, function, and survival. Identifying such factors is a critical first step in developing new islet cell–based therapies for the treatment of ß-cell destruction in insulin-dependent diabetes.


    ACKNOWLEDGMENTS
 
This work was supported by grants from the Department of Medicine at the University of Western Ontario, the Lawson Health Research Institute, and the Canadian Institutes of Health Research. R.W. is supported by a University Faculty Award from the Natural Sciences & Engineering Research Council of Canada.

We thank the Department of Pathology at London Health Science Centre for allowing us to access the Tissue Bank and providing the human fetal pancreas tissue sections.


    FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address correspondence and reprint requests to Dr. Rennian Wang, Victoria Laboratory Centre, Room A5-140, 800 Commissioners Rd. E, London, Ontario, N6C 2V5, Canada. E-mail: rwang{at}uwo.ca

Received for publication November 28, 2004 and accepted in revised form April 4, 2005

ECM, extracellular matrix; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate; TUNEL, transferase-mediated dUTP nick-end labeling


    REFERENCES
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
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