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
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ABSTRACT |
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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 - 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 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,
3,
5, and
6 integrins have been reported to mediate certain pancreatic developmental events: 1)
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)
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
6ß1 integrin or laminin; 4)
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
3,
5,
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 820 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 3,
5, and
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 cellbased therapies for the treatment of diabetes.
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RESEARCH DESIGN AND METHODS |
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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-3 and anti-
5 integrins (cytoplasmic domains), mouse anti-ß1 and anti-
6ß1 integrins (Chemicon, Temecula, CA), rabbit anti-
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, 812 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 buffersaline 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 manufacturers 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 (1416 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 210% 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 -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 Students 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.
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RESULTS |
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DISCUSSION |
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Immunohistochemical, morphometrical, RNA, and protein analyses showed a specific temporal and spatial pattern for ß1 integrin expression associated with 3,
5, and
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 911 weeks, newly forming single endocrine cells or small islets frequently expressed
3,
5, and
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
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
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 3,
5, and
6ß1 are expressed in cells that localize in proximity to immunoreactive areas for these matrix molecules. Dissecting the functional significance of the individual
subunitECM 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
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
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 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 - 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 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 3,
5, and
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 cellbased therapies for the treatment of ß-cell destruction in insulin-dependent diabetes.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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
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REFERENCES |
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