Expression of ?1 Integrin Receptors during Rat Pancreas Development—Sites and Dynamics
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内分泌学杂志 2005年第4期
Departments of Physiology and Pharmacology (N.K.Y., J.L., R.W.) and Medicine (R.W.), University of Western Ontario, London, Ontario, Canada N6C 2V5; and Department of Physiology, University of Toronto (M.B.W.), Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Dr. Rennian Wang, Victoria Research Laboratories Room A5 140, 800 Commissioners Road, East London, Ontario, Canada N6C 2V5. E-mail: rwang@uwo.ca.
Abstract
The integrin receptors link to extracellular matrix proteins and exert a dynamic role in development by providing the physical basis for cell adhesion and controlling cell growth. In the present study, we examined changes in the expression of ?1 integrins and its associated -subunits to islet cell development in the rat pancreas. A significant increase in protein expression of integrin 3, 6, and ?1 was observed from fetal to postnatal life. High mRNA levels of these integrin subunits was detected at embryonic d 18 and dropped significantly after birth with relatively low expression throughout postnatal life. Integrins 3, 5, 6, and ?1 were expressed in a cell-specific manner in the pancreas with high integrin immunoreactivity in duct and islet regions during fetal life, and a progressive increase later into postnatal life. The coexpression with islet and putative islet precursor markers during fetal and postnatal development suggest a role for these integrin subunits in differentiation and maturation of islets. Functional studies in vitro showed that anti-?1 antibody treatment inhibited islet cell adhesion to extracellular matrices and disrupted islet architecture. Blockade of ?1 integrin receptor and knockdown ?1 mRNA resulted in a decrease in the expression of insulin mRNA and increased islet cell death. These results suggest that progression in islet cell development is accompanied by and dependent upon cell adhesion via ?1 integrin and its respective -subunits and suggest that the ?1 family of integrins may play a critical role in islet cell architecture, development, integrity, and function.
Introduction
DIABETES MELLITUS ARISES from an inadequate mass of ?-cells leading to hyperglycemia and a multitude of disease complications. Given the pivotal role of ?-cells in the control of glucose homeostasis, a growing number of studies have focused on exploring the molecular signals that control morphogenesis and cell differentiation of the mammalian pancreas. In recent years, focus has shifted to therapeutic strategies aimed to induce organ regeneration or the production of replacement ?-cells and islets for the treatment of diabetes, as well as for other diseases. However, such an undertaking requires thorough knowledge of the developmental biology of the pancreas and the factors that control pancreatic organogenesis and tissue maintenance.
Of particular interest is the extracellular matrix (ECM) and their receptors, integrins, which exert a profound role during development controlling morphogenetic decisions and maintain homeostasis during adulthood. The ECM-integrin connection provides a physical substratum for the spatial organization of cells, but also regulates cell growth, proliferation, migration, and differentiation by interacting with growth factors (1). Integrins are a family of (120–180 kDa) and ? (90–110 kDa) heterodimeric cell adhesion glycoproteins that link the ECM proteins and the intracellular cytoskeleton (2, 3, 4). In addition to mediating cell-matrix and cell-cell interactions, integrins transduce extracellular signals (3, 4, 5), providing a dynamic interaction of environmental cues and intracellular events. The ?1 family of integrins is the largest and they orchestrate a plethora of cellular processes that include changes in gene expression (6, 7, 8), intracellular calcium levels (9), activation of cytoplasmic kinases including ERK1/2, and organization of actin cytoskeleton (5, 10, 11) leading to changes in cell growth (12), cell death (11, 13, 14), migration (15), and differentiation (16, 17).
Multiple functions of ?1 integrins in other organ systems other than the pancreas have been previously elucidated; however, research on pancreatic integrins and ECM is limited. In the pancreas, integrins have been studied in cancer and pancreatitis, but little is known of their expression and function (18, 19, 20). To date, it is known that pancreatic ductal morphogenesis requires the ECM laminin-1 during embryonic life and is inhibited by the blockade of integrin 6?1 or laminin (21). In addition, ?1 integrin may be involved in early motile processes that are required for the formation of new islets by supporting motility and migration of human fetal ?-cells (22). Thus, these studies suggest a role for integrin receptors in early pancreatic developmental events.
Integrin receptors and the ECM have also been shown to be important determinants of islet cell biology, influencing survival, proliferation, and differentiation. In regards to survival, disruption of the islet-matrix relationship induces some islet apoptosis, and leads to a loss of stability, resulting in transdifferentiation of an islet to a ductal phenotype (23). Integrin-ECM interactions may also be important in maintaining ?-cell function, as observed when human islets embedded in type I collagen gels have a greater insulin secretatory response (24) or when human ?-cells cultured on bovine corneal endothelial cell matrix have an increase in both basal and stimulated insulin release (25). Furthermore, rat islets cultured on matrices of laminin-5 or a matrix produced by the rat carcinoma cell line 804G have increased insulin secretatory response (26) and the expression of 6?1 is regulated by insulin secretagogues and highly expressed in spreading cells that can be inhibited by antibodies against this receptor. The study by Bosco et al. (26) suggests that islet cell matrix interactions improves sensitivity of insulin cells to glucose, which can be mediated by 6?1 integrins, suggesting that outside-in signaling through 6?1 integrins plays a major role in the regulation of ?-cell function. Integrin 3 and ?1 have been shown to mediate islet cell attachment and spreading to the ECM (27). Taken together, there is considerable evidence that ?1 integrins may play an important role in islet cell function and pancreatic biology.
Given that the integrin family of receptors plays a prominent role in a variety of cellular events and tissue development, one might expect this family of cell adhesion receptors and the signaling pathways that they activate to be involved in numerous aspects of cell behavior and development in the pancreas. The role of integrin-mediated interactions on islet cell development before and after birth is poorly understood, despite the potential importance of these molecules in pancreatic development and function. As a baseline for further investigation, our objective was to examine the coordinated expression patterns of integrins 3, 5, 6, and ?1 in the fetal and postnatal rat pancreas and correlate their expression to the appearance of the putative stem cell markers, mature ?-cell markers and to the substantial amount of pancreatic restructuring that is occurring. The information of the regional and temporal expression of ?1 integrin associated with its -subunits is of limited use in understanding the roles of integrins in development. Therefore, to obtain a greater understanding of ?1 integrin function in islet development, two specific function-blocking analyses were also used: gene silencing by small interfering RNA (siRNA) and functional-blocking monospecific antibody for ?1 integrin.
Materials and Methods
Animals
Fetal or Newborn Wistar rats were obtained (Charles River, Quebec, Canada) and kept in an environment of constant temperature, humidity, and day-night cycle. Rats were euthanized by CO2 asphyxia on embryonic d 18, 20 (e18 and e20), the day of birth [postnatal d 0 (p0)], p3, 7, 14, and 28 with at least twelve rats per time point. Pancreata were dissected and immediately processed for immunohistochemistry, RNA and protein extraction. The Animal Care Committee at the University of Western Ontario approved all protocols, in accordance to the guidelines of the Canadian Council on Animal Care.
Immunohistochemistry and immunofluorescence
Pancreata were fixed in 4% paraformaldehyde overnight at 4 C followed by a standard protocol of dehydration and paraffin embedding, and 5-μm sections were cut throughout the length of the pancreas, as described previously (28). Sections were incubated overnight at 4 C with primary antibodies for integrin subunits directed at their cytoplasmic domains including rabbit antiintegrin 3 and 5, mouse antiintegrin ?1 and 6?1 (Chemicon, Temecula, CA); rabbit antiintegrin 6 and c-Kit (C-19, C-terminal domain) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse antirat nestin (PharMingen, Mississauga, Ontario, Canada); cytokeratin 20 (CK20, Dako, Mississauga, Ontario, Canada); Guinea-pig antihuman insulin (Zymed, San Francisco, CA) and mouse antihuman glucagon (Sigma, St. Louis, MO). Using the AB complex (streptavidin-biotin-horseradish peroxidase complex) method (Zymed Histostain plus Kit, Zymed), staining was visualized using 4-amino-9-ethylcarbazole (Zymed) as chromogens. Controls were performed by omitting the primary or secondary antibody. No staining was observed under the negative control conditions.
To identify the coexpression of integrin receptors with other cell-specific or progenitor markers, double immunofluorescence staining was performed (28). Sections that had been reacted for integrin receptors were subsequently stained with progenitor or islet cell markers. The fluorescent antibodies, obtained from Jackson ImmunoResearch (West Grove, PA) were fluorescein isothiocyanate (FITC) antimouse and tetramethyl rhodamine isothiocyanate (TRITC) antirabbit antibody. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). The double-labeled images were recorded by a Leica DMIRE2 fluorescence microscope with the Openlab image software (Improvision, Lexington, MA).
Morphometrical analysis
Quantitative evaluation was performed using computer-assisted image analysis. A Leica light microscope was connected via a camera to a MacIntosh computer and, using the Openlab version 3.1 morphometric analysis software (Improvision). Integrin immunopositive areas within the islet region were traced manually (29). The percentage of integrin immunoreactive area in the duct and islet regions was determined under high magnification (x400). Ten random fields per pancreas were chosen with a total of 12 islets per pancreas and at least 500 ductal cells were counted per pancreas. A minimum of four rat pancreata per time point was analyzed. For all morphometric analyses, out of focus and extended, nondiscrete regions of staining were excluded from quantification. To reduce interobserver variation, one person was responsible for all the manual tracing of islets.
Protein extraction and Western blotting
Total protein was extracted from whole pancreata at each time point using lysis buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 100 mM vanadate, and 1 mM phenylmethylsulfonylfluoride. The protein concentration in each sample was measured with a spectrophotometer by using the Micro-Lowry protein assay. An equal amount of protein samples, 50 μg, from each time point was resolved by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). After transfer, the membrane was blocked with 5% fat-free milk in Tris-buffered saline plus 0.05% Tween 20 overnight at 4 C. Primary antibodies were incubated with the membrane as described above and detected by peroxidase-linked goat antirabbit or antimouse IgG conjugates (Santa Cruz Biotechnology). Peroxidase moieties reacted with enhanced chemiluminescence (ECL) reagents (ECL, Amersham, Arlington Heights, IL) and were exposed to X-OMAT Blue film (Eastman Kodak, Rochester, NY), which was then developed with a Kodak X-OMAT processor. Densitometric quantification of bands at subsaturating levels was performed using the Syngenetool gel analysis software (Syngene, Cambridge, UK) and normalized to band intensities at e18 (30). Loading controls of presumably constantly expressed proteins such as ?-actin were used; however, their variability and increase in development precluded their use. For negative controls, the primary antibody was omitted.
RNA extraction, RT and PCR
Total RNA was extracted from six pancreata at each time point with TRIZOL reagent (Invitrogen Life Technologies, Burlington, Ontario, Canada), according to the manufacturer’s instructions. The quality of the RNA was verified by agarose gel electrophoresis using ethidium bromide staining. For each PCR, 2 μg DNA-free total RNA with oligo(deoxythymidine) primers and reverse transcriptase were used. PCR was performed in 25-μl reactions containing 25 ng of cDNA, 0.2 nM of each primer pair, and 0.3 μl of Taq DNA polymerase (QIAGEN, Valencia, CA). PCR was carried out in a T-gradient Biometra PCR thermal cycler (Montreal Biotech Inc., Kirkland, Quebec, Canada) to determine the annealing temperature for each paired integrin primers (28). The primer pairs used are as follows: ?1 integrin, forward: 5'-GACCTGCCTTGGTGTCTGTGC-3', reverse: 5'-AGCAACCACACCAGCTACAAT-3' (313 bp); 3 integrin, forward: 5'-GTCTGGAAACCT TCTCAACCC-3', reverse: 5'-CAACCACAGCTCAATCT CAGC-3' (436 bp); 5 integrin, forward: 5'-AGCCAAAGTCTGCAGTTGCATTT-3', reverse: 5'-ATGAAGAGGGTATGTGTAAACAAG-3' (561 bp); 6 integrin, forward: 5'-CCCAAGGAGATTAGCAATGGC-3', reverse: 5'-CAGTCTTT GAGGGAAACACCG-3' (452 bp); and 18S, forward: 5'-GTAACCCG TTGAACCCCATT-3', reverse: 5'-CCATCCAATCGGTAGTAGCG-3' (131 bp). The amplified products were analyzed on 1% agarose gels and visualized by ethidium bromide staining. Controls involved omitting reverse transcriptase, cDNA or DNA polymerase and showed no reaction bands. Real-time quantitative PCR of ?1 integrin and its subunits was performed on 0.1 μg cDNA using the SYBR green quantitative PCR kit and the DNA Engine Option (MJ Research, South San Francisco, CA), according to the manufacturer’s instructions. The data were normalized by 18S RNA subunit (31).
Tissue culture and islet adhesion assay
An adhesion assay is a common method used to assess the functional role of integrins. We therefore adapted and developed an islet adhesion assay to examine whether integrin ?1 mediates the attachment and spreading of islet units on ECM (32). Islet isolation was carried out on 7-d-old rat pancreata, as described previously (33). Freshly isolated islets were then handpicked and preincubated with monoclonal antiintegrin 3, ?1 (PharMingen) or 5 (Chemicon) immunoneutralizing antibodies (5 μg/ml) before plating on matrix-coated dishes in RPMI 1640 medium (Invitrogen Life Technologies) for 1 h at 37 C. The experimental groups included treatment with a monoclonal anti-?1 antibody (Anti-?1), or control groups that included no antibody (Ctrl) or hamster IgM antibody (IgM, 5 μg/ml) treatment. After treatment, 100 islets per well from each experimental group were plated in duplicate on a type I collagen (rat), fibronectin (human), or laminin (mouse) matrices coated 12-well tissue culture dishes (VWR, Mississauga, Ontario, Canada) and cultured with RPMI 1640 supplemented with 10% FBS, containing IGF-I (50 ng/ml, ID Lab, London, Ontario, Canada) at 37 C for 2 h and 24 h. At the end of the incubation, nonadhered islets were carefully washed out, and the number of adhered islets to the various matrices was counted and reported as the percentage of islets attached with each experiment repeated a total of six times per time point. RNA samples were also harvested from freshly isolated islets and islets 2 h and 24 h after culture.
In situ cell death detection [terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL)]
TUNEL assay was performed on islets harvested after 24 h in suspension culture in the presence or absence of integrin ?1 immunoneutralizing antibodies. Briefly, harvested islets embedded in 2% agarose were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections from both controls and anti-?1-treated groups were deparaffinized and pretreated with 0.1% trypsin, and incubated with the TUNEL reaction mixture conjugated with fluorescein-deoxyuridine triphosphate (Roche, Montreal, Quebec, Canada) for 60 min at 37 C, as described previously (32). The sections were subsequently stained with mouse monoclonal antihuman insulin labeled with rhodamine (TRITC). The percentage of TUNEL-positive islets or ?-cells was determined.
Transfection of ?1 integrin siRNA in rat islet cells
Four different siRNA were designed corresponding to the rat ?1 integrin gene (34, 35), using the Ambion’s Silencer siRNA Construction Kit (Ambion, Austin, TX) with a 5' AA dinucleotide sequence and an eight-nucleotide 3' end (as highlighted). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA transfection was used as a control for nonspecific effects on gene expression. A set of four oligos designed for rat ?1 integrin siRNA were as follows: 1) ?1a, sense 5'-AATTTGTACACCACC CACAATCCTGTCTC-3', antisense 5'-AAATTGTGGGTGGTGTACAAACCTGTCTC-3'; 2) ?1b, sense 5'-AAGACTGTTCTTTGATTCTGTCCTGTCTC-3', antisense 5'-AAACAGAATC AAAGAACAGTCCCTGTCTC-3'; 3) ?1c, sense 5'-AAAATCAAATTCTTCAATTCC CCTGTCTC-3', antisense 5'-AAGGAATTGAAGAATTTGATTCCTGTCTC-3'; and 4) ?1d, sense 5'-AAAGTAATCCTCCTCATTTCACCTGTCTC-3', antisense 5'-AATGAAATGAG GAGGATTACTCCTGTCTC-3'. The freshly isolated islets after 2 h recovery culture were transient transfected with 0.1 μM ?1 integrin siRNAs, GAPDH siRNA, and control without siRNA by using Oligofectamine reagent (Invitrogen Life Technologies) for 4 h in serum-free medium followed by adding 3x serum into the medium, according to the manufacturer’s instructions. Islet cells were harvested 48 h post transfection and assessed the expression of ?1 integrin, preproinsulin, and glucagon mRNA, for which ?1 integrin siRNA could knock down its gene (36). The efficiency of transfection was determined using Cy3 Silencer siRNA Labeling Kit (Ambion) with approximately 70% of the islet cells being transfected. Cell death was examined in parallel using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (37). Fifty islets per transfected group of ?1a and ?1d siRNAs, GAPDH siRNA, and controls were plated in triplicate and cultured for 48 h. At the end of the incubation, the clusters were harvested in sterile Eppendorf (Fisher, Toronto, Ontario, Canada) tubes with 200 μl of culture medium and 20 μl of stock MTT (5 mg/ml, Sigma) was added for a 2 h incubation at 37 C. Cells were washed and lysed by 200 μl dimethylsulfoxide (Sigma). The samples were assayed for absorbance at 595 nm using a Multiskan Spectrum spectrophotometer (Thermo Labsystems, Franklin, MA) and data are expressed as the percentage of the controls for triplicate samples from the three independent experiments.
Statistical analysis
Values are expressed as mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test, and differences were considered to be statistically significant when P < 0.05.
Results
Patterns of integrin expression in fetal through postnatal transition
To investigate the spatiotemporal expression of the integrin subunits within the rat pancreas through fetal to postnatal life, we examined integrin expression through immunohistochemistry. We find that integrins ?1, 3, 5, and 6 were expressed in a cell-specific manner such that each integrin was expressed within the rat islets of Langerhans and ducts, but only integrin 5 and ?1 expressed in the acinar cells of the pancreas. Integrin 3 and 6 were also observed to be expressed in centroacinar cells. Examination of the integrin expressed patterns of the -subunits that commonly associate with ?1 revealed that there was a significant increase in integrin immunoreactive area at e18 (Fig. 1) and 1 month after birth in comparison to the expression area at birth in both duct and islet regions. Fetal integrin -subunit expression suggests that each integrin may support endocrine cell development and differentiation from immature cell types. A later postnatal integrin expression increase may support a role for integrins in the maturation and function of mature islet cell types.
FIG. 1. The ratio of integrin 3, 5, and 6 immunoreactive area over the total islet area (A) and the percentage of cells positive for integrin 3, 5 and 6 in the ductal region (B) at e18, p0, and 1 month after birth (p28). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4).
Examining the abundance of integrin receptors at the protein level during rat pancreatic development from embryonic to postnatal life in whole pancreata by Western blot analysis (Fig. 2) revealed that integrins 3, 6, and ?1 significantly increased by 1 month after birth, with a 3-fold increase in integrin 3 (P < 0.01), a 4-fold increase in integrin 6 (P < 0.001), and a 6-fold increase in ?1 expression (P < 0.01). No statistical difference in the expression of integrin 5 was observed from prenatal to postnatal development, although there was a slight increase in the protein levels of this integrin after embryonic life. The mRNA expression levels of the integrins in the whole pancreas in the developing rat were examined through real-time quantitative PCR. Highest integrin 3, 6, and ?1 mRNA levels was observed at e18, with a 7-fold decrease in 3 (P < 0.01) and 10-fold decrease in both 6 and ?1 integrin after birth (P < 0.01). Subsequently throughout postnatal development, there was decreased mRNA level for each of these integrin subunits (Fig. 3). In contrast, integrin 5 mRNA expression was constant throughout embryonic to postnatal life (Fig. 3).
FIG. 2. Western blot analysis of whole pancreatic tissue using anti-?1 (A) and anti-3 (B), anti-5 (C), anti-6 (D) integrin antibodies. Densitometric quantification of Western blots with integrin subunit abundance is expressed as a ratio, relative to e18. Progressively increased levels of integrin ?1 and 3 and 6 subunit intensity through the transition from fetal to postnatal life is observed; (n = 4). Representative blots for expression of each integrin during development are shown. *, P < 0.05; **, P < 0.01.
FIG. 3. Real-time PCR analysis of ?1 integrin (A) and its subunits (B) mRNA expression from whole pancreatic tissue. Data are normalized with 18S. **, P < 0.01 vs. p0 (n = 6).
Integrin coexpression patterns with potential islet progenitors and pancreatic endocrine immunophenotypes
Early examination of integrin expression in the pancreas during embryonic life revealed that ?1 integrin and its -subunits are expressed in islet clusters or in single cells budding from the ducts that coexpressed insulin (Fig. 4) and glucagon, and continuously colocalized into the postnatal life. The colocalization of integrin ?1, 3, 5, and 6 was observed frequently with the ductal cell marker cytokeratin 20 throughout the pre- and postnatal development of the pancreas (data not shown). Along with the substantial amount of development that occurs during embryonic to postnatal life, it is, therefore, interesting to examine the interrelationship between integrin and potential islet progenitor cell markers expression. Two stem cell markers in other cell lineages have been identified during the pre- and postnatal development of the rat pancreas in our previous study (28): the hematopoietic stem cell marker, c-Kit (38), and the neural stem cell marker, nestin (39, 40). The coexpression of 6?1 integrin with c-Kit in the fetal pancreas was frequently observed in the core of islets during the e18 development (Fig. 5A). Subsequently, there is a postnatal shift observed at d 7 of postnatal pancreas, where 6?1 is restricted only to the core of islets, and c-Kit expression shifts to the periphery (Fig. 5B). None of the integrin subunits are observed to colocalize with nestin (Fig. 5C).
FIG. 4. Colocalization of integrin ?1 (A) and 3 (B), 5 (C), 6 (D) subunits with insulin in islets of an e18 rat pancreas. Integrin are labeled by FITC, whereas insulin is labeled by TRITC, the arrows indicate costained integrin and insulin-positive cells. Original magnification, x400.
FIG. 5. Colocalization of 6?1 with c-Kit at e18 (A) shows frequent colocalizing cells in the islets of Langerhans; however, a postnatal shift in both integrin 6?1 and c-Kit expression is observed by d 7 of postnatal life such that integrin 6?1 is restricted to the core of islets, and c-Kit is localized in the islet periphery (B). There is no costaining between nestin and integrin 6 observed during pancreatic development at e18 (C). Integrin subunits are labeled by TRITC, whereas c-Kit and nestin are labeled by FITC. Arrows indicate the costained integrin with c-Kit cells. Original magnification, x400.
Integrin ?1 blockade effects islet cell adhesion on ECMs
Single dispersed islet cells have been shown to have alterations in their interactions with matrix proteins as a possible consequence of altered integrin expression patterns or function due to the dispersive process (21). Alternatively, a high proportion of islet cells stained for integrin 3, 5, 6, and ?1 subunits after islet isolation (data not shown) (32, 41). Therefore, we examined the role of integrins in regulating whole islet adhesion to various ECMs. Immunoneutralizing monoclonal antibodies directed at the integrin subunits were used. Islets without integrin blocking treatment as controls were plated on type I collagen and fibronectin-coated dishes began to spread from the periphery of the islets, and had a high adhesive capacity, 52 ± 7% and 65 ± 6%, respectively (Fig. 6A). Islets of control groups when cultured on laminin-coated dishes adhered as 48 ± 5% (Fig. 6B). The adhesion rate was not hindered by IgM antibody. There was, however, a greater inhibition of islet adhesion to all matrices observed in the groups treated with anti-?1 integrin antibody, with 17-, 26, and 5-fold decrease in attachment to collagen I, fibronectin, and laminin compared with the controls (Fig. 6A; P < 0.01). At a structural level, a loss of islet cell integrity was clearly observed (Fig. 6B). When islets were treated with anti-3 or 5 integrin antibodies, adhesion of islets to collagen I and fibronectin matrices was inhibited partially with less than a 25% reduction in islet adhesion, and there was no effect on the attachment to laminin.
FIG. 6. Quantitative analysis of the percentage of islets attached to matrices of collagen I, fibronectin, and laminin (A), and phase contrast micrography of islets in culture after 24 h in the absence or presence of monoclonal anti-?1 integrins antibody on a laminin-coated dish (B). **, P < 0.01 (n = 6).
Integrin ?1 blockade effects on islet cell survival and gene expression
Loss of attachment to ECM is known to induce apoptosis, also referred to as anoikis in many cells (14, 42). ?1 Integrin signaling is suggested to provide protection against apoptosis (43, 44). We therefore examined the role of integrin ?1 blockade on cell death of islets, specific on ?-cells. Because of culturing for only 24 h, there was no visible evidence of central necrosis (32), but a significant increase in the total percentage of apoptotic islet cells was observed in islets cultured in the presence of immunoneutralizing anti-?1 integrin antibody (Fig. 7A). Furthermore, there was a significant increase in the total percentage of TUNEL-labeled ?-cells in anti-?1 integrin antibody treatment group (Fig. 7, A and B; P < 0.04).
FIG. 7. Quantitative analysis reveals an increased percentage of ?-cells and non-?-cells in the islets undergoing apoptotic treated with anti-?1 integrin immunoneutralizing antibody cultured after 24 h (A), apoptotic ?-cells are labeled through TUNEL staining by FITC with insulin staining by TRITC (B), and (C) decreased preproinsulin and glucagon mRNA expression of islets treated with anti-?1 integrin immunoneutralizing antibody are observed in islets cultured after 2 and 24 h (n = 3). Arrows, Apoptotic ?-cells. *, P < 0.05. RT–, Omitting reverse transcriptase.
To test the hypothesis that integrin ?1 receptors regulate endocrine gene expression, we cultured postnatal rat islets in suspension culture in the presence of immunoneutralizing antibody. The results indicated that upon integrin ?1 blockade, at 2 h, no significant difference in insulin and glucagon gene expression was observed. However, islets cultured in the presence of ?1 immunoneutralizing antibody up to 24 h resulted in a dramatic down-regulation in preproinsulin and glucagon gene expression (Fig. 7C). Furthermore, we transfected islets with a set of rat ?1 integrin siRNAs by using gene silencing technique. Only ?1a and ?1b integrin siRNAs nearly knocked down the expression of integrin ?1 mRNA and decreased insulin gene expression. Rat ?1a integrin siRNA also down-regulated the expression of glucagon and pancreas duodenum homeobox-1 (Pdx-1) gene, whereas ?1b siRNA-treated islets had no expression of glucagon and Pdx-1 (Fig. 8A). However, both ?1c and ?1d did not have an effect on ?1 mRNA expression compared with the control siRNA group (Fig. 8A). It was also noted that ?1d siRNA treatment showed weak insulin gene expression and no expression of glucagon and Pdx-1 gene, and the treatment with ?1c siRNA was associated with no glucagon gene expression (Fig. 8A). Silencing ?1 integrin gene expression led to increased cell death with a 54% and a 35% decrease in islet cell viability after culture for 48 h in ?1a and ?1d siRNA-treated groups, respectively (P < 0.01), as measured by the MTT assay (Fig. 8B). The cells treated with GAPDH siRNA showed a slight increase in cell death with only a 13% decrease in cell viability compared with the controls (Fig. 8B).
FIG. 8. Silencing of rat ?1 integrin mRNA led to decreased expression of preproinsulin, Pdx-1, and glucagon mRNA (A) and increased cell death with a decrease in islet cell viability, as determined by the MTT assay (B), in islets cultured after 48 h. *, P < 0.05; **, P < 0.001 (n = 3). RT–, Omitting reverse transcriptase.
Discussion
The present study demonstrates that pancreatic development is accompanied by a specific spatiotemporal pattern of protein and mRNA expression of integrin ?1 and its associated 3, 5, and 6 subunits, suggesting a role for this family of adhesion receptors during pancreatic development. At a cellular level, we also demonstrated a potential role for integrins in islet cell development and ?-cell differentiation based on the coexpression of integrins with c-Kit, an islet progenitor marker and with insulin and glucagon. In vitro antagonization of the integrin ?1 subunit and ?1 gene silencing revealed that this integrin modulate of islet cell adhesion, structural integrity, insulin gene expression and cell death. Taken together, our data suggests that integrin ?1 and its partners, 3, 5, and 6 may play an important role in numerous aspects of pancreatic biology.
The protein and mRNA expression of the integrin subunits in the whole pancreas determined a specific temporal pattern of expression during embryonic to postnatal development. High mRNA expression along with low protein level of integrin 3, 6, and ?1 was observed in the embryonic pancreas. Subsequently into postnatal life, decreased integrin mRNA expression was paralleled to increased protein levels in postnatal life. The expression of these integrins at both the mRNA and protein level indicates that these specific integrin subunits may be critical in mediating the biological functions of the developing pancreas. Regardless, these studies demonstrate that integrins are expressed at the protein and mRNA level throughout development, indicating potential molecular signals that may control various aspects of pancreatic function and development. The coordinated expression pattern of these integrin receptors in the endocrine and exocrine pancreas suggests a role in key events of differentiation, function, and maturation. Immunohistochemical staining of 3, 5, 6, and ?1 demonstrated the expression of these ?1 integrin within the ducts and islets, and only integrin 5 and ?1 are expressed within the acinar cells. These subunits, therefore, may have a potential role in duct and islet cell development but only integrin 5 and ?1 may orchestrate acinar cell biology.
Within the islet, integrin immunoreactive area is high during embryonic pancreatic development, decreasing at birth and increasing postnatally 1 month after birth. Increased expression in embryonic and postnatal life highlights a potential role of ?1 integrin-mediated differentiation or neogenesis of new islets from potential precursors before birth, and integrin activity in maturation of islets postnatally. Interestingly, we observe the coexpression of 6?1 with c-Kit, a potential precursor marker of islets that we have shown in the rat pancreas to possess an immature phenotype, transiently coexpress with insulin during embryonic life, and capable of giving rise to insulin-expressing cells in vitro (28, 33). Postnatally, integrin 6?1 and c-Kit are no longer coexpressed and the shift in their immunolocalization within the islets suggest that the early patterning of integrin 6?1 may be important in early embryonic ?-cell formation and subsequent ?-cell function and maturation. This interesting find highlights that integrin receptors may be powerful targets for inducing the differentiation of precursor cell types.
Blockade of integrin ?1 resulted in impaired islet cell adhesion, poor structural integrity on various ECMs, increased islet and ?-cell apoptosis and decreased insulin gene expression in vitro. Alternatively, the treatment of islets with an equal amount of IgM antibody did not compromise islet attachment or insulin gene expression, indicating that the interference with ?1 function by blocking antibody is not the result of nonspecific interactions. Such observational differences between islet adhesion, viability, and gene expression between cells treated with a monoclonal antibody to integrin ?1 and control groups can be attributed to the fact that integrin ?1 acts though multiple signaling pathways that control a variety of cellular functions (6, 7, 8). Integrin ?1 offers both a protective factor from cell death (43, 45) and is a critical mediator of gene expression and cell adhesion (6, 7, 8). Given that ?1 integrin induces the assembly of multicomponent complexes containing kinases, adapters, substrates, and scaffolding proteins (11, 12, 13, 14), it is not surprising that blockade of the ?1 integrin results in a dramatic loss of islet integrity, increased cell death and reduced insulin gene expression. In regards to cell death, integrin ?1 has been shown to stimulate phosphatidylinositol-3-kinase (PI3K)-mediated protein kinase B (PKB)-AKT activity and suppress caspase levels in mammary epithelial cells, thus protecting these cell types from apoptosis. Furthermore, activation of the ?1 integrin subunit has also been shown to increase the bcl-2/Bax ratio, and suppress p53, which is known to induce apoptosis (11, 13, 14, 45). More recently, Hammar et al. (46) determined that the ?1 integrin subunit is involved in the effects of the ECM (804G) on ?-cell survival and spreading. Blocking ?1 integrin function resulted in significantly increased ?-cell death on 804G matrix. Furthermore, this ECM was shown to protect the ?-cells against apoptosis via an integrin ?1/focal adhesion kinase pathway and subsequent activation of the downstream effectors ERK and Akt/protein kinase B in the phosphoinositol 3-kinase and MAPK pathways, respectively (46). Integrin ?1 also activates several tyrosine kinase components of signaling pathways that are known to control insulin gene expression. Thus, we can only speculate that the ?1 receptor in concert with numerous tyrosine kinases control intracellular signaling pathways that regulate adhesion, viability, and gene transcription within islets.
The advent of siRNA silencing systems has allowed for the identification of the functional importance of several genes (34, 35, 36, 47, 48). In mammalian cells, siRNA’s directed at a target gene are capable of suppressing gene expression transcriptionally, and thus allowing for a means to inhibit mammalian gene function (48). However, this technique has been confined to studies on cell lines, with limited information on the effects of gene silencing on primary islets (36). Therefore, we examined the effects of various siRNAs against the ?1 integrin sequences on the expression of insulin, and its transcript factor Pdx-1 and glucagon in pancreatic islets to explore the role of this receptor in primary cell cultures. Interestingly, a novel finding is that insulin gene expression is suppressed across various ?1 integrin siRNAs in comparison to control siRNA treatment and that Pdx-1 and glucagon down-regulation is also present within experimental groups. These studies highlight that ?1 integrin may not only be involved in cellular resistance to apoptotic stimuli but may also be an important regulator of various pancreatic islet genes expression. However, it must be noted that the level of down-regulation of insulin, glucagon, Pdx-1, and ?1 itself varies among the siRNAs used. These differences may be associated with the specific sequences targeted and reflect functional differences intrinsic within the ?1 subunit (35).
In summary, our study is the first to describe the spatial and temporal pattern of expression of integrin ?1 associated with 3, 5, and 6 subunits during pancreatic development and their involvement in islet cell adhesion, death, and a possible mode of insulin gene expression regulation. These findings highlight primary information in regards to the possible roles that members of the ?1 subfamily and ?1 itself may play in the potential lineage differentiation and function of various pancreatic cell types. Further investigation into the role of integrins and pancreatic development must be pursued. Understanding the factors that regulate cellular differentiation and identification of factors that are important to islet cell survival and function are critical to approaches that could lead to the treatment of ?-cell destruction in insulin-dependent diabetes.
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Address all correspondence and requests for reprints to: Dr. Rennian Wang, Victoria Research Laboratories Room A5 140, 800 Commissioners Road, East London, Ontario, Canada N6C 2V5. E-mail: rwang@uwo.ca.
Abstract
The integrin receptors link to extracellular matrix proteins and exert a dynamic role in development by providing the physical basis for cell adhesion and controlling cell growth. In the present study, we examined changes in the expression of ?1 integrins and its associated -subunits to islet cell development in the rat pancreas. A significant increase in protein expression of integrin 3, 6, and ?1 was observed from fetal to postnatal life. High mRNA levels of these integrin subunits was detected at embryonic d 18 and dropped significantly after birth with relatively low expression throughout postnatal life. Integrins 3, 5, 6, and ?1 were expressed in a cell-specific manner in the pancreas with high integrin immunoreactivity in duct and islet regions during fetal life, and a progressive increase later into postnatal life. The coexpression with islet and putative islet precursor markers during fetal and postnatal development suggest a role for these integrin subunits in differentiation and maturation of islets. Functional studies in vitro showed that anti-?1 antibody treatment inhibited islet cell adhesion to extracellular matrices and disrupted islet architecture. Blockade of ?1 integrin receptor and knockdown ?1 mRNA resulted in a decrease in the expression of insulin mRNA and increased islet cell death. These results suggest that progression in islet cell development is accompanied by and dependent upon cell adhesion via ?1 integrin and its respective -subunits and suggest that the ?1 family of integrins may play a critical role in islet cell architecture, development, integrity, and function.
Introduction
DIABETES MELLITUS ARISES from an inadequate mass of ?-cells leading to hyperglycemia and a multitude of disease complications. Given the pivotal role of ?-cells in the control of glucose homeostasis, a growing number of studies have focused on exploring the molecular signals that control morphogenesis and cell differentiation of the mammalian pancreas. In recent years, focus has shifted to therapeutic strategies aimed to induce organ regeneration or the production of replacement ?-cells and islets for the treatment of diabetes, as well as for other diseases. However, such an undertaking requires thorough knowledge of the developmental biology of the pancreas and the factors that control pancreatic organogenesis and tissue maintenance.
Of particular interest is the extracellular matrix (ECM) and their receptors, integrins, which exert a profound role during development controlling morphogenetic decisions and maintain homeostasis during adulthood. The ECM-integrin connection provides a physical substratum for the spatial organization of cells, but also regulates cell growth, proliferation, migration, and differentiation by interacting with growth factors (1). Integrins are a family of (120–180 kDa) and ? (90–110 kDa) heterodimeric cell adhesion glycoproteins that link the ECM proteins and the intracellular cytoskeleton (2, 3, 4). In addition to mediating cell-matrix and cell-cell interactions, integrins transduce extracellular signals (3, 4, 5), providing a dynamic interaction of environmental cues and intracellular events. The ?1 family of integrins is the largest and they orchestrate a plethora of cellular processes that include changes in gene expression (6, 7, 8), intracellular calcium levels (9), activation of cytoplasmic kinases including ERK1/2, and organization of actin cytoskeleton (5, 10, 11) leading to changes in cell growth (12), cell death (11, 13, 14), migration (15), and differentiation (16, 17).
Multiple functions of ?1 integrins in other organ systems other than the pancreas have been previously elucidated; however, research on pancreatic integrins and ECM is limited. In the pancreas, integrins have been studied in cancer and pancreatitis, but little is known of their expression and function (18, 19, 20). To date, it is known that pancreatic ductal morphogenesis requires the ECM laminin-1 during embryonic life and is inhibited by the blockade of integrin 6?1 or laminin (21). In addition, ?1 integrin may be involved in early motile processes that are required for the formation of new islets by supporting motility and migration of human fetal ?-cells (22). Thus, these studies suggest a role for integrin receptors in early pancreatic developmental events.
Integrin receptors and the ECM have also been shown to be important determinants of islet cell biology, influencing survival, proliferation, and differentiation. In regards to survival, disruption of the islet-matrix relationship induces some islet apoptosis, and leads to a loss of stability, resulting in transdifferentiation of an islet to a ductal phenotype (23). Integrin-ECM interactions may also be important in maintaining ?-cell function, as observed when human islets embedded in type I collagen gels have a greater insulin secretatory response (24) or when human ?-cells cultured on bovine corneal endothelial cell matrix have an increase in both basal and stimulated insulin release (25). Furthermore, rat islets cultured on matrices of laminin-5 or a matrix produced by the rat carcinoma cell line 804G have increased insulin secretatory response (26) and the expression of 6?1 is regulated by insulin secretagogues and highly expressed in spreading cells that can be inhibited by antibodies against this receptor. The study by Bosco et al. (26) suggests that islet cell matrix interactions improves sensitivity of insulin cells to glucose, which can be mediated by 6?1 integrins, suggesting that outside-in signaling through 6?1 integrins plays a major role in the regulation of ?-cell function. Integrin 3 and ?1 have been shown to mediate islet cell attachment and spreading to the ECM (27). Taken together, there is considerable evidence that ?1 integrins may play an important role in islet cell function and pancreatic biology.
Given that the integrin family of receptors plays a prominent role in a variety of cellular events and tissue development, one might expect this family of cell adhesion receptors and the signaling pathways that they activate to be involved in numerous aspects of cell behavior and development in the pancreas. The role of integrin-mediated interactions on islet cell development before and after birth is poorly understood, despite the potential importance of these molecules in pancreatic development and function. As a baseline for further investigation, our objective was to examine the coordinated expression patterns of integrins 3, 5, 6, and ?1 in the fetal and postnatal rat pancreas and correlate their expression to the appearance of the putative stem cell markers, mature ?-cell markers and to the substantial amount of pancreatic restructuring that is occurring. The information of the regional and temporal expression of ?1 integrin associated with its -subunits is of limited use in understanding the roles of integrins in development. Therefore, to obtain a greater understanding of ?1 integrin function in islet development, two specific function-blocking analyses were also used: gene silencing by small interfering RNA (siRNA) and functional-blocking monospecific antibody for ?1 integrin.
Materials and Methods
Animals
Fetal or Newborn Wistar rats were obtained (Charles River, Quebec, Canada) and kept in an environment of constant temperature, humidity, and day-night cycle. Rats were euthanized by CO2 asphyxia on embryonic d 18, 20 (e18 and e20), the day of birth [postnatal d 0 (p0)], p3, 7, 14, and 28 with at least twelve rats per time point. Pancreata were dissected and immediately processed for immunohistochemistry, RNA and protein extraction. The Animal Care Committee at the University of Western Ontario approved all protocols, in accordance to the guidelines of the Canadian Council on Animal Care.
Immunohistochemistry and immunofluorescence
Pancreata were fixed in 4% paraformaldehyde overnight at 4 C followed by a standard protocol of dehydration and paraffin embedding, and 5-μm sections were cut throughout the length of the pancreas, as described previously (28). Sections were incubated overnight at 4 C with primary antibodies for integrin subunits directed at their cytoplasmic domains including rabbit antiintegrin 3 and 5, mouse antiintegrin ?1 and 6?1 (Chemicon, Temecula, CA); rabbit antiintegrin 6 and c-Kit (C-19, C-terminal domain) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse antirat nestin (PharMingen, Mississauga, Ontario, Canada); cytokeratin 20 (CK20, Dako, Mississauga, Ontario, Canada); Guinea-pig antihuman insulin (Zymed, San Francisco, CA) and mouse antihuman glucagon (Sigma, St. Louis, MO). Using the AB complex (streptavidin-biotin-horseradish peroxidase complex) method (Zymed Histostain plus Kit, Zymed), staining was visualized using 4-amino-9-ethylcarbazole (Zymed) as chromogens. Controls were performed by omitting the primary or secondary antibody. No staining was observed under the negative control conditions.
To identify the coexpression of integrin receptors with other cell-specific or progenitor markers, double immunofluorescence staining was performed (28). Sections that had been reacted for integrin receptors were subsequently stained with progenitor or islet cell markers. The fluorescent antibodies, obtained from Jackson ImmunoResearch (West Grove, PA) were fluorescein isothiocyanate (FITC) antimouse and tetramethyl rhodamine isothiocyanate (TRITC) antirabbit antibody. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). The double-labeled images were recorded by a Leica DMIRE2 fluorescence microscope with the Openlab image software (Improvision, Lexington, MA).
Morphometrical analysis
Quantitative evaluation was performed using computer-assisted image analysis. A Leica light microscope was connected via a camera to a MacIntosh computer and, using the Openlab version 3.1 morphometric analysis software (Improvision). Integrin immunopositive areas within the islet region were traced manually (29). The percentage of integrin immunoreactive area in the duct and islet regions was determined under high magnification (x400). Ten random fields per pancreas were chosen with a total of 12 islets per pancreas and at least 500 ductal cells were counted per pancreas. A minimum of four rat pancreata per time point was analyzed. For all morphometric analyses, out of focus and extended, nondiscrete regions of staining were excluded from quantification. To reduce interobserver variation, one person was responsible for all the manual tracing of islets.
Protein extraction and Western blotting
Total protein was extracted from whole pancreata at each time point using lysis buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 100 mM vanadate, and 1 mM phenylmethylsulfonylfluoride. The protein concentration in each sample was measured with a spectrophotometer by using the Micro-Lowry protein assay. An equal amount of protein samples, 50 μg, from each time point was resolved by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). After transfer, the membrane was blocked with 5% fat-free milk in Tris-buffered saline plus 0.05% Tween 20 overnight at 4 C. Primary antibodies were incubated with the membrane as described above and detected by peroxidase-linked goat antirabbit or antimouse IgG conjugates (Santa Cruz Biotechnology). Peroxidase moieties reacted with enhanced chemiluminescence (ECL) reagents (ECL, Amersham, Arlington Heights, IL) and were exposed to X-OMAT Blue film (Eastman Kodak, Rochester, NY), which was then developed with a Kodak X-OMAT processor. Densitometric quantification of bands at subsaturating levels was performed using the Syngenetool gel analysis software (Syngene, Cambridge, UK) and normalized to band intensities at e18 (30). Loading controls of presumably constantly expressed proteins such as ?-actin were used; however, their variability and increase in development precluded their use. For negative controls, the primary antibody was omitted.
RNA extraction, RT and PCR
Total RNA was extracted from six pancreata at each time point with TRIZOL reagent (Invitrogen Life Technologies, Burlington, Ontario, Canada), according to the manufacturer’s instructions. The quality of the RNA was verified by agarose gel electrophoresis using ethidium bromide staining. For each PCR, 2 μg DNA-free total RNA with oligo(deoxythymidine) primers and reverse transcriptase were used. PCR was performed in 25-μl reactions containing 25 ng of cDNA, 0.2 nM of each primer pair, and 0.3 μl of Taq DNA polymerase (QIAGEN, Valencia, CA). PCR was carried out in a T-gradient Biometra PCR thermal cycler (Montreal Biotech Inc., Kirkland, Quebec, Canada) to determine the annealing temperature for each paired integrin primers (28). The primer pairs used are as follows: ?1 integrin, forward: 5'-GACCTGCCTTGGTGTCTGTGC-3', reverse: 5'-AGCAACCACACCAGCTACAAT-3' (313 bp); 3 integrin, forward: 5'-GTCTGGAAACCT TCTCAACCC-3', reverse: 5'-CAACCACAGCTCAATCT CAGC-3' (436 bp); 5 integrin, forward: 5'-AGCCAAAGTCTGCAGTTGCATTT-3', reverse: 5'-ATGAAGAGGGTATGTGTAAACAAG-3' (561 bp); 6 integrin, forward: 5'-CCCAAGGAGATTAGCAATGGC-3', reverse: 5'-CAGTCTTT GAGGGAAACACCG-3' (452 bp); and 18S, forward: 5'-GTAACCCG TTGAACCCCATT-3', reverse: 5'-CCATCCAATCGGTAGTAGCG-3' (131 bp). The amplified products were analyzed on 1% agarose gels and visualized by ethidium bromide staining. Controls involved omitting reverse transcriptase, cDNA or DNA polymerase and showed no reaction bands. Real-time quantitative PCR of ?1 integrin and its subunits was performed on 0.1 μg cDNA using the SYBR green quantitative PCR kit and the DNA Engine Option (MJ Research, South San Francisco, CA), according to the manufacturer’s instructions. The data were normalized by 18S RNA subunit (31).
Tissue culture and islet adhesion assay
An adhesion assay is a common method used to assess the functional role of integrins. We therefore adapted and developed an islet adhesion assay to examine whether integrin ?1 mediates the attachment and spreading of islet units on ECM (32). Islet isolation was carried out on 7-d-old rat pancreata, as described previously (33). Freshly isolated islets were then handpicked and preincubated with monoclonal antiintegrin 3, ?1 (PharMingen) or 5 (Chemicon) immunoneutralizing antibodies (5 μg/ml) before plating on matrix-coated dishes in RPMI 1640 medium (Invitrogen Life Technologies) for 1 h at 37 C. The experimental groups included treatment with a monoclonal anti-?1 antibody (Anti-?1), or control groups that included no antibody (Ctrl) or hamster IgM antibody (IgM, 5 μg/ml) treatment. After treatment, 100 islets per well from each experimental group were plated in duplicate on a type I collagen (rat), fibronectin (human), or laminin (mouse) matrices coated 12-well tissue culture dishes (VWR, Mississauga, Ontario, Canada) and cultured with RPMI 1640 supplemented with 10% FBS, containing IGF-I (50 ng/ml, ID Lab, London, Ontario, Canada) at 37 C for 2 h and 24 h. At the end of the incubation, nonadhered islets were carefully washed out, and the number of adhered islets to the various matrices was counted and reported as the percentage of islets attached with each experiment repeated a total of six times per time point. RNA samples were also harvested from freshly isolated islets and islets 2 h and 24 h after culture.
In situ cell death detection [terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL)]
TUNEL assay was performed on islets harvested after 24 h in suspension culture in the presence or absence of integrin ?1 immunoneutralizing antibodies. Briefly, harvested islets embedded in 2% agarose were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections from both controls and anti-?1-treated groups were deparaffinized and pretreated with 0.1% trypsin, and incubated with the TUNEL reaction mixture conjugated with fluorescein-deoxyuridine triphosphate (Roche, Montreal, Quebec, Canada) for 60 min at 37 C, as described previously (32). The sections were subsequently stained with mouse monoclonal antihuman insulin labeled with rhodamine (TRITC). The percentage of TUNEL-positive islets or ?-cells was determined.
Transfection of ?1 integrin siRNA in rat islet cells
Four different siRNA were designed corresponding to the rat ?1 integrin gene (34, 35), using the Ambion’s Silencer siRNA Construction Kit (Ambion, Austin, TX) with a 5' AA dinucleotide sequence and an eight-nucleotide 3' end (as highlighted). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA transfection was used as a control for nonspecific effects on gene expression. A set of four oligos designed for rat ?1 integrin siRNA were as follows: 1) ?1a, sense 5'-AATTTGTACACCACC CACAATCCTGTCTC-3', antisense 5'-AAATTGTGGGTGGTGTACAAACCTGTCTC-3'; 2) ?1b, sense 5'-AAGACTGTTCTTTGATTCTGTCCTGTCTC-3', antisense 5'-AAACAGAATC AAAGAACAGTCCCTGTCTC-3'; 3) ?1c, sense 5'-AAAATCAAATTCTTCAATTCC CCTGTCTC-3', antisense 5'-AAGGAATTGAAGAATTTGATTCCTGTCTC-3'; and 4) ?1d, sense 5'-AAAGTAATCCTCCTCATTTCACCTGTCTC-3', antisense 5'-AATGAAATGAG GAGGATTACTCCTGTCTC-3'. The freshly isolated islets after 2 h recovery culture were transient transfected with 0.1 μM ?1 integrin siRNAs, GAPDH siRNA, and control without siRNA by using Oligofectamine reagent (Invitrogen Life Technologies) for 4 h in serum-free medium followed by adding 3x serum into the medium, according to the manufacturer’s instructions. Islet cells were harvested 48 h post transfection and assessed the expression of ?1 integrin, preproinsulin, and glucagon mRNA, for which ?1 integrin siRNA could knock down its gene (36). The efficiency of transfection was determined using Cy3 Silencer siRNA Labeling Kit (Ambion) with approximately 70% of the islet cells being transfected. Cell death was examined in parallel using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (37). Fifty islets per transfected group of ?1a and ?1d siRNAs, GAPDH siRNA, and controls were plated in triplicate and cultured for 48 h. At the end of the incubation, the clusters were harvested in sterile Eppendorf (Fisher, Toronto, Ontario, Canada) tubes with 200 μl of culture medium and 20 μl of stock MTT (5 mg/ml, Sigma) was added for a 2 h incubation at 37 C. Cells were washed and lysed by 200 μl dimethylsulfoxide (Sigma). The samples were assayed for absorbance at 595 nm using a Multiskan Spectrum spectrophotometer (Thermo Labsystems, Franklin, MA) and data are expressed as the percentage of the controls for triplicate samples from the three independent experiments.
Statistical analysis
Values are expressed as mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test, and differences were considered to be statistically significant when P < 0.05.
Results
Patterns of integrin expression in fetal through postnatal transition
To investigate the spatiotemporal expression of the integrin subunits within the rat pancreas through fetal to postnatal life, we examined integrin expression through immunohistochemistry. We find that integrins ?1, 3, 5, and 6 were expressed in a cell-specific manner such that each integrin was expressed within the rat islets of Langerhans and ducts, but only integrin 5 and ?1 expressed in the acinar cells of the pancreas. Integrin 3 and 6 were also observed to be expressed in centroacinar cells. Examination of the integrin expressed patterns of the -subunits that commonly associate with ?1 revealed that there was a significant increase in integrin immunoreactive area at e18 (Fig. 1) and 1 month after birth in comparison to the expression area at birth in both duct and islet regions. Fetal integrin -subunit expression suggests that each integrin may support endocrine cell development and differentiation from immature cell types. A later postnatal integrin expression increase may support a role for integrins in the maturation and function of mature islet cell types.
FIG. 1. The ratio of integrin 3, 5, and 6 immunoreactive area over the total islet area (A) and the percentage of cells positive for integrin 3, 5 and 6 in the ductal region (B) at e18, p0, and 1 month after birth (p28). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4).
Examining the abundance of integrin receptors at the protein level during rat pancreatic development from embryonic to postnatal life in whole pancreata by Western blot analysis (Fig. 2) revealed that integrins 3, 6, and ?1 significantly increased by 1 month after birth, with a 3-fold increase in integrin 3 (P < 0.01), a 4-fold increase in integrin 6 (P < 0.001), and a 6-fold increase in ?1 expression (P < 0.01). No statistical difference in the expression of integrin 5 was observed from prenatal to postnatal development, although there was a slight increase in the protein levels of this integrin after embryonic life. The mRNA expression levels of the integrins in the whole pancreas in the developing rat were examined through real-time quantitative PCR. Highest integrin 3, 6, and ?1 mRNA levels was observed at e18, with a 7-fold decrease in 3 (P < 0.01) and 10-fold decrease in both 6 and ?1 integrin after birth (P < 0.01). Subsequently throughout postnatal development, there was decreased mRNA level for each of these integrin subunits (Fig. 3). In contrast, integrin 5 mRNA expression was constant throughout embryonic to postnatal life (Fig. 3).
FIG. 2. Western blot analysis of whole pancreatic tissue using anti-?1 (A) and anti-3 (B), anti-5 (C), anti-6 (D) integrin antibodies. Densitometric quantification of Western blots with integrin subunit abundance is expressed as a ratio, relative to e18. Progressively increased levels of integrin ?1 and 3 and 6 subunit intensity through the transition from fetal to postnatal life is observed; (n = 4). Representative blots for expression of each integrin during development are shown. *, P < 0.05; **, P < 0.01.
FIG. 3. Real-time PCR analysis of ?1 integrin (A) and its subunits (B) mRNA expression from whole pancreatic tissue. Data are normalized with 18S. **, P < 0.01 vs. p0 (n = 6).
Integrin coexpression patterns with potential islet progenitors and pancreatic endocrine immunophenotypes
Early examination of integrin expression in the pancreas during embryonic life revealed that ?1 integrin and its -subunits are expressed in islet clusters or in single cells budding from the ducts that coexpressed insulin (Fig. 4) and glucagon, and continuously colocalized into the postnatal life. The colocalization of integrin ?1, 3, 5, and 6 was observed frequently with the ductal cell marker cytokeratin 20 throughout the pre- and postnatal development of the pancreas (data not shown). Along with the substantial amount of development that occurs during embryonic to postnatal life, it is, therefore, interesting to examine the interrelationship between integrin and potential islet progenitor cell markers expression. Two stem cell markers in other cell lineages have been identified during the pre- and postnatal development of the rat pancreas in our previous study (28): the hematopoietic stem cell marker, c-Kit (38), and the neural stem cell marker, nestin (39, 40). The coexpression of 6?1 integrin with c-Kit in the fetal pancreas was frequently observed in the core of islets during the e18 development (Fig. 5A). Subsequently, there is a postnatal shift observed at d 7 of postnatal pancreas, where 6?1 is restricted only to the core of islets, and c-Kit expression shifts to the periphery (Fig. 5B). None of the integrin subunits are observed to colocalize with nestin (Fig. 5C).
FIG. 4. Colocalization of integrin ?1 (A) and 3 (B), 5 (C), 6 (D) subunits with insulin in islets of an e18 rat pancreas. Integrin are labeled by FITC, whereas insulin is labeled by TRITC, the arrows indicate costained integrin and insulin-positive cells. Original magnification, x400.
FIG. 5. Colocalization of 6?1 with c-Kit at e18 (A) shows frequent colocalizing cells in the islets of Langerhans; however, a postnatal shift in both integrin 6?1 and c-Kit expression is observed by d 7 of postnatal life such that integrin 6?1 is restricted to the core of islets, and c-Kit is localized in the islet periphery (B). There is no costaining between nestin and integrin 6 observed during pancreatic development at e18 (C). Integrin subunits are labeled by TRITC, whereas c-Kit and nestin are labeled by FITC. Arrows indicate the costained integrin with c-Kit cells. Original magnification, x400.
Integrin ?1 blockade effects islet cell adhesion on ECMs
Single dispersed islet cells have been shown to have alterations in their interactions with matrix proteins as a possible consequence of altered integrin expression patterns or function due to the dispersive process (21). Alternatively, a high proportion of islet cells stained for integrin 3, 5, 6, and ?1 subunits after islet isolation (data not shown) (32, 41). Therefore, we examined the role of integrins in regulating whole islet adhesion to various ECMs. Immunoneutralizing monoclonal antibodies directed at the integrin subunits were used. Islets without integrin blocking treatment as controls were plated on type I collagen and fibronectin-coated dishes began to spread from the periphery of the islets, and had a high adhesive capacity, 52 ± 7% and 65 ± 6%, respectively (Fig. 6A). Islets of control groups when cultured on laminin-coated dishes adhered as 48 ± 5% (Fig. 6B). The adhesion rate was not hindered by IgM antibody. There was, however, a greater inhibition of islet adhesion to all matrices observed in the groups treated with anti-?1 integrin antibody, with 17-, 26, and 5-fold decrease in attachment to collagen I, fibronectin, and laminin compared with the controls (Fig. 6A; P < 0.01). At a structural level, a loss of islet cell integrity was clearly observed (Fig. 6B). When islets were treated with anti-3 or 5 integrin antibodies, adhesion of islets to collagen I and fibronectin matrices was inhibited partially with less than a 25% reduction in islet adhesion, and there was no effect on the attachment to laminin.
FIG. 6. Quantitative analysis of the percentage of islets attached to matrices of collagen I, fibronectin, and laminin (A), and phase contrast micrography of islets in culture after 24 h in the absence or presence of monoclonal anti-?1 integrins antibody on a laminin-coated dish (B). **, P < 0.01 (n = 6).
Integrin ?1 blockade effects on islet cell survival and gene expression
Loss of attachment to ECM is known to induce apoptosis, also referred to as anoikis in many cells (14, 42). ?1 Integrin signaling is suggested to provide protection against apoptosis (43, 44). We therefore examined the role of integrin ?1 blockade on cell death of islets, specific on ?-cells. Because of culturing for only 24 h, there was no visible evidence of central necrosis (32), but a significant increase in the total percentage of apoptotic islet cells was observed in islets cultured in the presence of immunoneutralizing anti-?1 integrin antibody (Fig. 7A). Furthermore, there was a significant increase in the total percentage of TUNEL-labeled ?-cells in anti-?1 integrin antibody treatment group (Fig. 7, A and B; P < 0.04).
FIG. 7. Quantitative analysis reveals an increased percentage of ?-cells and non-?-cells in the islets undergoing apoptotic treated with anti-?1 integrin immunoneutralizing antibody cultured after 24 h (A), apoptotic ?-cells are labeled through TUNEL staining by FITC with insulin staining by TRITC (B), and (C) decreased preproinsulin and glucagon mRNA expression of islets treated with anti-?1 integrin immunoneutralizing antibody are observed in islets cultured after 2 and 24 h (n = 3). Arrows, Apoptotic ?-cells. *, P < 0.05. RT–, Omitting reverse transcriptase.
To test the hypothesis that integrin ?1 receptors regulate endocrine gene expression, we cultured postnatal rat islets in suspension culture in the presence of immunoneutralizing antibody. The results indicated that upon integrin ?1 blockade, at 2 h, no significant difference in insulin and glucagon gene expression was observed. However, islets cultured in the presence of ?1 immunoneutralizing antibody up to 24 h resulted in a dramatic down-regulation in preproinsulin and glucagon gene expression (Fig. 7C). Furthermore, we transfected islets with a set of rat ?1 integrin siRNAs by using gene silencing technique. Only ?1a and ?1b integrin siRNAs nearly knocked down the expression of integrin ?1 mRNA and decreased insulin gene expression. Rat ?1a integrin siRNA also down-regulated the expression of glucagon and pancreas duodenum homeobox-1 (Pdx-1) gene, whereas ?1b siRNA-treated islets had no expression of glucagon and Pdx-1 (Fig. 8A). However, both ?1c and ?1d did not have an effect on ?1 mRNA expression compared with the control siRNA group (Fig. 8A). It was also noted that ?1d siRNA treatment showed weak insulin gene expression and no expression of glucagon and Pdx-1 gene, and the treatment with ?1c siRNA was associated with no glucagon gene expression (Fig. 8A). Silencing ?1 integrin gene expression led to increased cell death with a 54% and a 35% decrease in islet cell viability after culture for 48 h in ?1a and ?1d siRNA-treated groups, respectively (P < 0.01), as measured by the MTT assay (Fig. 8B). The cells treated with GAPDH siRNA showed a slight increase in cell death with only a 13% decrease in cell viability compared with the controls (Fig. 8B).
FIG. 8. Silencing of rat ?1 integrin mRNA led to decreased expression of preproinsulin, Pdx-1, and glucagon mRNA (A) and increased cell death with a decrease in islet cell viability, as determined by the MTT assay (B), in islets cultured after 48 h. *, P < 0.05; **, P < 0.001 (n = 3). RT–, Omitting reverse transcriptase.
Discussion
The present study demonstrates that pancreatic development is accompanied by a specific spatiotemporal pattern of protein and mRNA expression of integrin ?1 and its associated 3, 5, and 6 subunits, suggesting a role for this family of adhesion receptors during pancreatic development. At a cellular level, we also demonstrated a potential role for integrins in islet cell development and ?-cell differentiation based on the coexpression of integrins with c-Kit, an islet progenitor marker and with insulin and glucagon. In vitro antagonization of the integrin ?1 subunit and ?1 gene silencing revealed that this integrin modulate of islet cell adhesion, structural integrity, insulin gene expression and cell death. Taken together, our data suggests that integrin ?1 and its partners, 3, 5, and 6 may play an important role in numerous aspects of pancreatic biology.
The protein and mRNA expression of the integrin subunits in the whole pancreas determined a specific temporal pattern of expression during embryonic to postnatal development. High mRNA expression along with low protein level of integrin 3, 6, and ?1 was observed in the embryonic pancreas. Subsequently into postnatal life, decreased integrin mRNA expression was paralleled to increased protein levels in postnatal life. The expression of these integrins at both the mRNA and protein level indicates that these specific integrin subunits may be critical in mediating the biological functions of the developing pancreas. Regardless, these studies demonstrate that integrins are expressed at the protein and mRNA level throughout development, indicating potential molecular signals that may control various aspects of pancreatic function and development. The coordinated expression pattern of these integrin receptors in the endocrine and exocrine pancreas suggests a role in key events of differentiation, function, and maturation. Immunohistochemical staining of 3, 5, 6, and ?1 demonstrated the expression of these ?1 integrin within the ducts and islets, and only integrin 5 and ?1 are expressed within the acinar cells. These subunits, therefore, may have a potential role in duct and islet cell development but only integrin 5 and ?1 may orchestrate acinar cell biology.
Within the islet, integrin immunoreactive area is high during embryonic pancreatic development, decreasing at birth and increasing postnatally 1 month after birth. Increased expression in embryonic and postnatal life highlights a potential role of ?1 integrin-mediated differentiation or neogenesis of new islets from potential precursors before birth, and integrin activity in maturation of islets postnatally. Interestingly, we observe the coexpression of 6?1 with c-Kit, a potential precursor marker of islets that we have shown in the rat pancreas to possess an immature phenotype, transiently coexpress with insulin during embryonic life, and capable of giving rise to insulin-expressing cells in vitro (28, 33). Postnatally, integrin 6?1 and c-Kit are no longer coexpressed and the shift in their immunolocalization within the islets suggest that the early patterning of integrin 6?1 may be important in early embryonic ?-cell formation and subsequent ?-cell function and maturation. This interesting find highlights that integrin receptors may be powerful targets for inducing the differentiation of precursor cell types.
Blockade of integrin ?1 resulted in impaired islet cell adhesion, poor structural integrity on various ECMs, increased islet and ?-cell apoptosis and decreased insulin gene expression in vitro. Alternatively, the treatment of islets with an equal amount of IgM antibody did not compromise islet attachment or insulin gene expression, indicating that the interference with ?1 function by blocking antibody is not the result of nonspecific interactions. Such observational differences between islet adhesion, viability, and gene expression between cells treated with a monoclonal antibody to integrin ?1 and control groups can be attributed to the fact that integrin ?1 acts though multiple signaling pathways that control a variety of cellular functions (6, 7, 8). Integrin ?1 offers both a protective factor from cell death (43, 45) and is a critical mediator of gene expression and cell adhesion (6, 7, 8). Given that ?1 integrin induces the assembly of multicomponent complexes containing kinases, adapters, substrates, and scaffolding proteins (11, 12, 13, 14), it is not surprising that blockade of the ?1 integrin results in a dramatic loss of islet integrity, increased cell death and reduced insulin gene expression. In regards to cell death, integrin ?1 has been shown to stimulate phosphatidylinositol-3-kinase (PI3K)-mediated protein kinase B (PKB)-AKT activity and suppress caspase levels in mammary epithelial cells, thus protecting these cell types from apoptosis. Furthermore, activation of the ?1 integrin subunit has also been shown to increase the bcl-2/Bax ratio, and suppress p53, which is known to induce apoptosis (11, 13, 14, 45). More recently, Hammar et al. (46) determined that the ?1 integrin subunit is involved in the effects of the ECM (804G) on ?-cell survival and spreading. Blocking ?1 integrin function resulted in significantly increased ?-cell death on 804G matrix. Furthermore, this ECM was shown to protect the ?-cells against apoptosis via an integrin ?1/focal adhesion kinase pathway and subsequent activation of the downstream effectors ERK and Akt/protein kinase B in the phosphoinositol 3-kinase and MAPK pathways, respectively (46). Integrin ?1 also activates several tyrosine kinase components of signaling pathways that are known to control insulin gene expression. Thus, we can only speculate that the ?1 receptor in concert with numerous tyrosine kinases control intracellular signaling pathways that regulate adhesion, viability, and gene transcription within islets.
The advent of siRNA silencing systems has allowed for the identification of the functional importance of several genes (34, 35, 36, 47, 48). In mammalian cells, siRNA’s directed at a target gene are capable of suppressing gene expression transcriptionally, and thus allowing for a means to inhibit mammalian gene function (48). However, this technique has been confined to studies on cell lines, with limited information on the effects of gene silencing on primary islets (36). Therefore, we examined the effects of various siRNAs against the ?1 integrin sequences on the expression of insulin, and its transcript factor Pdx-1 and glucagon in pancreatic islets to explore the role of this receptor in primary cell cultures. Interestingly, a novel finding is that insulin gene expression is suppressed across various ?1 integrin siRNAs in comparison to control siRNA treatment and that Pdx-1 and glucagon down-regulation is also present within experimental groups. These studies highlight that ?1 integrin may not only be involved in cellular resistance to apoptotic stimuli but may also be an important regulator of various pancreatic islet genes expression. However, it must be noted that the level of down-regulation of insulin, glucagon, Pdx-1, and ?1 itself varies among the siRNAs used. These differences may be associated with the specific sequences targeted and reflect functional differences intrinsic within the ?1 subunit (35).
In summary, our study is the first to describe the spatial and temporal pattern of expression of integrin ?1 associated with 3, 5, and 6 subunits during pancreatic development and their involvement in islet cell adhesion, death, and a possible mode of insulin gene expression regulation. These findings highlight primary information in regards to the possible roles that members of the ?1 subfamily and ?1 itself may play in the potential lineage differentiation and function of various pancreatic cell types. Further investigation into the role of integrins and pancreatic development must be pursued. Understanding the factors that regulate cellular differentiation and identification of factors that are important to islet cell survival and function are critical to approaches that could lead to the treatment of ?-cell destruction in insulin-dependent diabetes.
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