Role for 1 Integrin and Its Associated 3, 5, and 6 Subunits in Development of the Human Fetal Pancreas
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糖尿病学杂志 2005年第7期
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
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 (8eC20 weeks fetal age) and the relevance of 1 integrin function for insulin gene expression and islet cell survival. Its subunits 3, 5, and 6 1 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 3, 5 and 6 1 integrins with endocrine cell markers was frequently observed in 8- to 20-week fetal pancreatic cells. When the 1 integrin receptor was functionally blocked in cultured islet-epithelial clusters with a 1 immunoneutralizing antibody or following transient 1 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 1 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 (1eC,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 -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 (3eC5).
The 1 integrin family is believed to play a critical role in morphogenesis (6eC7), cell differentiation, and proliferation (8eC9) 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 1-deficient embryos (7).
Multiple functions of 1 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) 31 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 61 integrin or laminin; 4) 61 is believed to enhance and regulate the insulin secretory response of rat islets (2); and 5) 1 integrin may be involved in early motile processes required for the formation of new islets by supporting migration of human fetal -cells (16). More recently, Hammer et al. (17) determined that 804G matrix protects -cells against apoptosis via the integrin 1/focal adhesion kinase pathway and that blocking 1 integrin function induces cell death. Thus, these studies suggest a role for 3, 5, 6, and 1 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 8eC20 weeks of fetal age using immunofluorescence, Western blot, and real-time RT-PCR. We also examined the role of 1 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 1 integrin and its associated 3, 5, and 6 subunits during early pancreatic development. Our data also provide evidence for a major role for the 1 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 celleCbased therapies for the treatment of diabetes.
RESEARCH DESIGN AND METHODS
Tissues.
Human fetal pancreata (8eC20 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 e were cut throughout the length of the pancreas with two sets of six serial sections at 50-e 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-1 and anti-61 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 (18eC19). The integrin immunoreactive area within the ductal and endocrine cell compartments was traced manually. In each pancreatic section, 8eC12 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 eC80°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 e of pancreatic lysate proteins were separated by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) (19eC20). The membranes were washed in Tris buffereCsaline 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 -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 e 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 1 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 1 integrin and insulin were performed on 0.1 e 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 (14eC16 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 2eC10% 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 e/ml) or laminin (50 e/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-1-integrin (CD29, 5 e/ml; Pharmingen, Mississauga, ON, Canada), with hamster IgM isotype (5 e/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-e 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, -cells, and -cells was determined.
1 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 1 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 1 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 1 siRNA and control siRNA transfected groups were plated in triplicate and cultured for 72 h. The clusters were harvested in 500 e蘬 of culture medium, and then 50 e蘬 of stock MTT (5 mg/ml, Sigma) was added for 2-h incubation at 37°C. Cells were washed and lysed by 200 e蘬 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
Expression of 1 integrin and its associated 3, 5, and 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 8eC20 weeks of fetal age. Using immunohistochemistry, we observed 1, 3, 5, and 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 1 integrin and its associated -subunits revealed an increased expression of 1, 3, and 6 integrin that was statistically significant (P < 0.05) by 16 weeks, whereas the expression of 5 was relatively constant from weeks 8eC20 (Fig. 1AeCD). Quantitative analysis of 1 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 1 protein levels (Fig. 1A).
To examine the distribution and colocalization of integrin 3, 5, and 61 with the epithelial (CK19) and mature endocrine cell (insulin and glucagon) markers, dual immunofluorescence experiments were conducted. Morphometric analysis of the expression patterns of 3, 5, and 61 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 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 3 (P < 0.05) and for 61 (P < 0.005) by week 20 (Fig. 2).
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 3 or 5 integrin and insulin costaining was observed at 12 weeks with slightly decreased coexpression by week 18 (Fig. 4A). In contrast, 61 integrin colocalization with insulin increased slightly from 12eC18 weeks (63 ± 9% vs. 78 ± 12%) (Fig. 4A). The percentage of cells coexpressing 3 or 61 with glucagon showed no difference during development; however, there was a significant increase in frequency of coexpression of 5 with glucagon at 18 weeks (P < 0.01) (Fig. 4B), indicating that 5 may play a prominent role in glucagon cell maturation and functional maintenance. Colocalization of 61 integrin with PDX-1, an early pancreatic developmental transcription factor, was frequently observed in both duct and islet regions (Fig. 3).
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 31 integrin (11). Fibronectin is a known adhesive site for 51 and 61 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 3 integrin-positive cells colocalizing with collagen IV, which highlights the basal membrane of pancreatic ducts and islets. There was also localization of 3 integrineCpositive cells at sites where fibronectin and laminin were observed in the developing human pancreas (data not shown). Expression of 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 61 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.
1 integrin receptor mediates fetal islet-epithelial cluster adhesion, insulin gene expression, and cell survival.
We subsequently tested the effects of blocking 1 integrin activity in islet-epithelial cell clusters using a 1 integrin immunoneutralizing antibody. The percentage of clusters in both control and IgM antibodyeCtreated 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 antieC1-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 1 integrin expression: quantitative analysis demonstrated a 37% reduction in 1-integrineCexpressing cells after 24 h of antieC1-antibody treatment (35 ± 5% vs. 55 ± 4% of controls, P < 0.001). Cells coexpressing 1 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 1 integrin receptor affects insulin gene expression using quantitative RT-PCR assays (Fig. 6C). There was no change in preproinsulin expression after 2 h of 1 integrin blockade. However, after 24 h, there was an 18% reduction in insulin mRNA (P < 0.05) compared with the control group (Fig. 6C).
Blockade of 1 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 - and -cells in the antieC1-antibody group (P < 0.01) (Fig. 7).
Furthermore, we examined the effects of suppressing 1 integrin expression in islet-epithelial clusters using a specific 1 integrin siRNA. After the transfection and 72 h of culture, a significant downregulation of 1 integrin protein expression was observed (43 ± 3.5% vs. 72 ± 1.2%, P < 0.01) (Fig. 8A). Knockdown of 1 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).
DISCUSSION
The present study demonstrates that 1 integrin and its associated -subunits are expressed in the early to mid-gestation human fetal pancreas in a dynamic, temporally regulated fashion. Furthermore, blockade of 1 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 1 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 9eC11 weeks, newly forming single endocrine cells or small islets frequently expressed 3, 5, and 61, 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 61 subunit has been previously reported to mediate -cell differentiation and the -cell secretory response in dissociated fetal mouse pancreatic epithelium on a laminin-1 matrix (1eC2). Our results indicate that 1 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 61 are expressed in cells that localize in proximity to immunoreactive areas for these matrix molecules. Dissecting the functional significance of the individual subuniteCECM 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 -cell differentiation mediated by 6 integrin when placed on a matrix of laminin-1.
To examine the functional role of 1 in developing islets, we used an immunoneutralizing monoclonal antibody. Blockade of 1 integrin resulted in impairment of islet-epithelial cluster adhesion on several ECM, highlighting that the 1 subunit plays a critical role during pancreatic development. Treatment with an equal amount of IgM antibody had no effect, indicating that the interference with 1 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 V1 integrin may be responsible for early motile processes that regulate human fetal islet formation.
We also demonstrated that blockade of 1 integrin receptor in islet-epithelial clusters is associated with an increase in the number of cells undergoing apoptosis, with a specific increase in - and -cell death. These data are in line with the previously described role for the 1 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 1 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 (2,28eC29). Whether maturation of the glucose-induced insulin response will occur if fetal cells are cultured in the presence of 1 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 (30eC32). 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 1 integrin siRNA, demonstrated a significant decrease in islet cell survival (19). We therefore examined the effect of 1 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 1 integrin protein correlated with a reduction in insulin mRNA and protein as well as cell viability. These data support the hypothesis that 1 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 1 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 celleCbased therapies for the treatment of -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.
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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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
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 (8eC20 weeks fetal age) and the relevance of 1 integrin function for insulin gene expression and islet cell survival. Its subunits 3, 5, and 6 1 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 3, 5 and 6 1 integrins with endocrine cell markers was frequently observed in 8- to 20-week fetal pancreatic cells. When the 1 integrin receptor was functionally blocked in cultured islet-epithelial clusters with a 1 immunoneutralizing antibody or following transient 1 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 1 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 (1eC,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 -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 (3eC5).
The 1 integrin family is believed to play a critical role in morphogenesis (6eC7), cell differentiation, and proliferation (8eC9) 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 1-deficient embryos (7).
Multiple functions of 1 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) 31 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 61 integrin or laminin; 4) 61 is believed to enhance and regulate the insulin secretory response of rat islets (2); and 5) 1 integrin may be involved in early motile processes required for the formation of new islets by supporting migration of human fetal -cells (16). More recently, Hammer et al. (17) determined that 804G matrix protects -cells against apoptosis via the integrin 1/focal adhesion kinase pathway and that blocking 1 integrin function induces cell death. Thus, these studies suggest a role for 3, 5, 6, and 1 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 8eC20 weeks of fetal age using immunofluorescence, Western blot, and real-time RT-PCR. We also examined the role of 1 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 1 integrin and its associated 3, 5, and 6 subunits during early pancreatic development. Our data also provide evidence for a major role for the 1 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 celleCbased therapies for the treatment of diabetes.
RESEARCH DESIGN AND METHODS
Tissues.
Human fetal pancreata (8eC20 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 e were cut throughout the length of the pancreas with two sets of six serial sections at 50-e 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-1 and anti-61 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 (18eC19). The integrin immunoreactive area within the ductal and endocrine cell compartments was traced manually. In each pancreatic section, 8eC12 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 eC80°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 e of pancreatic lysate proteins were separated by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) (19eC20). The membranes were washed in Tris buffereCsaline 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 -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 e 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 1 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 1 integrin and insulin were performed on 0.1 e 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 (14eC16 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 2eC10% 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 e/ml) or laminin (50 e/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-1-integrin (CD29, 5 e/ml; Pharmingen, Mississauga, ON, Canada), with hamster IgM isotype (5 e/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-e 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, -cells, and -cells was determined.
1 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 1 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 1 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 1 siRNA and control siRNA transfected groups were plated in triplicate and cultured for 72 h. The clusters were harvested in 500 e蘬 of culture medium, and then 50 e蘬 of stock MTT (5 mg/ml, Sigma) was added for 2-h incubation at 37°C. Cells were washed and lysed by 200 e蘬 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
Expression of 1 integrin and its associated 3, 5, and 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 8eC20 weeks of fetal age. Using immunohistochemistry, we observed 1, 3, 5, and 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 1 integrin and its associated -subunits revealed an increased expression of 1, 3, and 6 integrin that was statistically significant (P < 0.05) by 16 weeks, whereas the expression of 5 was relatively constant from weeks 8eC20 (Fig. 1AeCD). Quantitative analysis of 1 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 1 protein levels (Fig. 1A).
To examine the distribution and colocalization of integrin 3, 5, and 61 with the epithelial (CK19) and mature endocrine cell (insulin and glucagon) markers, dual immunofluorescence experiments were conducted. Morphometric analysis of the expression patterns of 3, 5, and 61 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 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 3 (P < 0.05) and for 61 (P < 0.005) by week 20 (Fig. 2).
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 3 or 5 integrin and insulin costaining was observed at 12 weeks with slightly decreased coexpression by week 18 (Fig. 4A). In contrast, 61 integrin colocalization with insulin increased slightly from 12eC18 weeks (63 ± 9% vs. 78 ± 12%) (Fig. 4A). The percentage of cells coexpressing 3 or 61 with glucagon showed no difference during development; however, there was a significant increase in frequency of coexpression of 5 with glucagon at 18 weeks (P < 0.01) (Fig. 4B), indicating that 5 may play a prominent role in glucagon cell maturation and functional maintenance. Colocalization of 61 integrin with PDX-1, an early pancreatic developmental transcription factor, was frequently observed in both duct and islet regions (Fig. 3).
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 31 integrin (11). Fibronectin is a known adhesive site for 51 and 61 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 3 integrin-positive cells colocalizing with collagen IV, which highlights the basal membrane of pancreatic ducts and islets. There was also localization of 3 integrineCpositive cells at sites where fibronectin and laminin were observed in the developing human pancreas (data not shown). Expression of 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 61 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.
1 integrin receptor mediates fetal islet-epithelial cluster adhesion, insulin gene expression, and cell survival.
We subsequently tested the effects of blocking 1 integrin activity in islet-epithelial cell clusters using a 1 integrin immunoneutralizing antibody. The percentage of clusters in both control and IgM antibodyeCtreated 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 antieC1-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 1 integrin expression: quantitative analysis demonstrated a 37% reduction in 1-integrineCexpressing cells after 24 h of antieC1-antibody treatment (35 ± 5% vs. 55 ± 4% of controls, P < 0.001). Cells coexpressing 1 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 1 integrin receptor affects insulin gene expression using quantitative RT-PCR assays (Fig. 6C). There was no change in preproinsulin expression after 2 h of 1 integrin blockade. However, after 24 h, there was an 18% reduction in insulin mRNA (P < 0.05) compared with the control group (Fig. 6C).
Blockade of 1 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 - and -cells in the antieC1-antibody group (P < 0.01) (Fig. 7).
Furthermore, we examined the effects of suppressing 1 integrin expression in islet-epithelial clusters using a specific 1 integrin siRNA. After the transfection and 72 h of culture, a significant downregulation of 1 integrin protein expression was observed (43 ± 3.5% vs. 72 ± 1.2%, P < 0.01) (Fig. 8A). Knockdown of 1 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).
DISCUSSION
The present study demonstrates that 1 integrin and its associated -subunits are expressed in the early to mid-gestation human fetal pancreas in a dynamic, temporally regulated fashion. Furthermore, blockade of 1 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 1 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 9eC11 weeks, newly forming single endocrine cells or small islets frequently expressed 3, 5, and 61, 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 61 subunit has been previously reported to mediate -cell differentiation and the -cell secretory response in dissociated fetal mouse pancreatic epithelium on a laminin-1 matrix (1eC2). Our results indicate that 1 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 61 are expressed in cells that localize in proximity to immunoreactive areas for these matrix molecules. Dissecting the functional significance of the individual subuniteCECM 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 -cell differentiation mediated by 6 integrin when placed on a matrix of laminin-1.
To examine the functional role of 1 in developing islets, we used an immunoneutralizing monoclonal antibody. Blockade of 1 integrin resulted in impairment of islet-epithelial cluster adhesion on several ECM, highlighting that the 1 subunit plays a critical role during pancreatic development. Treatment with an equal amount of IgM antibody had no effect, indicating that the interference with 1 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 V1 integrin may be responsible for early motile processes that regulate human fetal islet formation.
We also demonstrated that blockade of 1 integrin receptor in islet-epithelial clusters is associated with an increase in the number of cells undergoing apoptosis, with a specific increase in - and -cell death. These data are in line with the previously described role for the 1 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 1 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 (2,28eC29). Whether maturation of the glucose-induced insulin response will occur if fetal cells are cultured in the presence of 1 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 (30eC32). 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 1 integrin siRNA, demonstrated a significant decrease in islet cell survival (19). We therefore examined the effect of 1 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 1 integrin protein correlated with a reduction in insulin mRNA and protein as well as cell viability. These data support the hypothesis that 1 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 1 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 celleCbased therapies for the treatment of -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.
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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