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Originally published In Press as doi:10.1074/jbc.M411202200 on October 12, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53762-53769, December 17, 2004
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Regulation of Human {beta}-Cell Adhesion, Motility, and Insulin Secretion by Collagen IV and Its Receptor {alpha}1{beta}1*

Thomas Kaido, Mayra Yebra, Vincenzo Cirulli, and Anthony M. Montgomery{ddagger}

From the Department of Pediatrics, Islet Research Laboratory at The Whittier Institute for Diabetes, University of California at San Diego, La Jolla, California 92037

Received for publication, September 30, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Collagens have been shown to influence the survival and function of cultured {beta}-cells; however, the utilization and function of individual collagen receptors in {beta}-cells is largely unknown. The integrin superfamily contains up to five collagen receptors, but we have determined that {alpha}1{beta}1 is the primary receptor utilized by both fetal and adult {beta}-cells. Cultured {beta}-cells adhered to and migrated on collagen type IV (Col-IV), and these responses were mediated almost exclusively by {alpha}1{beta}1. The migration of cultured {beta}-cells to Col-IV significantly exceeded that to other matrix components suggesting that this substrate is of unique importance for {beta}-cell motility. The interaction of {alpha}1{beta}1 with Col-IV also resulted in significant insulin secretion at basal glucose concentrations. A subset of {beta}-cells in developing islets was confirmed to express {alpha}1{beta}1, and this expression co-localized with Col-IV in the basal membranes of juxtaposed endothelial cells. Our findings indicate that {alpha}1{beta}1 and Col-IV contribute to {beta}-cell functions known to be important for islet morphogenesis and glucose homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Collagens are ubiquitous structural proteins responsible for maintaining the mechanical resilience of multicellular organisms (1). To date more than 20 genetically distinct collagen types have been described (1, 2). Collagens not only provide structural support but also serve as signaling molecules that control a myriad of cellular responses from migration and morphogenesis to proliferation and differentiation (3–5). Communication between collagens and cells is achieved through the intermediary of receptors that include members of the integrin superfamily and discoidin domain receptors (3, 5, 6).

The integrin superfamily currently contains 24 integrin heterodimers, 5 of which function as collagen receptors (5, 6). These collagen receptors consist of the {beta}1 integrin subunit paired with {alpha}1, {alpha}2, {alpha}3, {alpha}10, or {alpha}11 (6). Four of these integrins, namely {alpha}1{beta}1, {alpha}2{beta}1, {alpha}10{beta}1, and {alpha}11{beta}1, comprise a distinct subgroup by virtue of sharing an extra "inserted" domain, which serves as the main collagen-binding region ({alpha}I domain) (6, 7). Despite structural similarities, these integrins all differ in their individual collagen subtype specificities, and variations in their cytoplasmic domains suggest distinct signaling functions (6, 8). Adding to such complexity, all four of these collagen receptors have different patterns of expression in vivo (5). Although integrin {alpha}3{beta}1 is described as a collagen receptor it does not have an {alpha}I domain and may only play an accessory role in collagen recognition (9).

Several studies have described the distribution of common collagen subtypes in the human pancreas (10–13). Col-IV1 has been reported in basement membranes (BMs) associated with pancreatic ducts and acini (10–12), and peri-insular Col-IV staining has been reported in rat, porcine, and human pancreata (11, 13). Moderate peri-insular and interstitial collagen type I (Col-I) staining has also been described (11, 13). Other collagen subtypes reported in the pancreas include collagen types V, III, and VI (11, 13).

The expression and distribution of collagen-binding integrins in normal human pancreas have also been documented (14–17). Pancreatic ducts and acini have been shown to express {alpha}2{beta}1 but not {alpha}1{beta}1 (14, 15). Integrin {alpha}3{beta}1 has been reported on ductal epithelia (14) and on human and rat islets (16, 17). Expression of {alpha}2{beta}1 or {alpha}1{beta}1 by human islets or {beta}-cells has not, to our knowledge, been detected or reported. The expression of {alpha}10{beta}1 and {alpha}11{beta}1 in human pancreas remains to be determined; however, existing studies suggest that these integrins are primarily involved in the metabolism or formation of bone and cartilage (6).

Prior reports suggest that collagens can have a significant impact on {beta}-cell development and function. Several studies have shown that hydrogels composed of Col-I can promote {beta}-cell proliferation (18) or maintain long term islet viability and insulin secretion (19–22). Such responses may ultimately depend on the pliant three-dimensional environment of collagen gels because a more recent study has shown that monolayer culture on Col-I results in a loss of insulin secretion (23). Gels composed of rat Col-I have also been reported to promote the transdifferentiation of islets into ductal cysts (24–26).

Despite these studies little is known about collagen receptor utilization by {beta}-cells, and the functional outcome of ligating individual receptors remains to be determined. Studies indicate that Col-IV, the main structural component of BMs, is a major peri-insular matrix component, yet the functional significance of {beta}-cell-Col-IV interaction remains to be determined. In this study we assess the impact of Col-IV and individual collagen receptors on human {beta}-cell functions including adhesion, motility, and insulin secretion. Our findings indicate that {alpha}1{beta}1 is the primary collagen receptor utilized by cultured {beta}-cells and demonstrate that an interaction between this integrin and Col-IV is uniquely important for {beta}-cell motility. Based on the observation that {alpha}1{beta}1 ligation potentiates insulin secretion, it is proposed that this collagen receptor may also contribute to glucose homeostasis. Immunohistochemical studies indicate that restricted {alpha}1{beta}1 expression and Col-IV availability will serve to regulate the activity of these molecules in developing islets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Function-blocking monoclonal antibodies (mAbs) to human integrin subunits {alpha}1 (MAB1973Z), {alpha}2 (MAB1950Z), and {alpha}3 (MAB1952Z) were obtained from Chemicon (Temecula, CA). A polyclonal antibody (pAb) to human Col-IV (AB748) was also from Chemicon. A mAb to human insulin (2D11-H5) and a goat pAb to platelet endothelial cell adhesion molecule-1 (PECAM-1) (C-20) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Guinea pig pAb to insulin (A0564) was from DakoCytomation (Carpinteria, CA). Secondary donkey anti-guinea pig, anti-rabbit, and anti-mouse immunoglobulin (Ig) antibodies conjugated to alkaline phosphatase, biotin, or fluorescein isothiocyanate (FITC) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Streptavidin conjugated to R-phycoerythrin (PE) and all normal animal sera were also from Jackson ImmunoResearch. Mouse PE- or FITC-conjugated mAbs to human integrin {alpha}1 (SR84), {alpha}2 (12F1-H6), and fluor-conjugated isotype controls were obtained from Pharmingen. Purified human vitronectin (Vn) and fibronectin (Fn) were from Chemicon. Mouse Col-IV and ultrapure (entactin-free) laminin-1 (Lm-1) were purchased from BD Biosciences. Rat-tail collagen type-I (Col-I) was from Upstate Cell Signaling (Lake Placid, NY). Fibronectin {alpha} and plasminogen-free fibrinogen (Fbg) was from DiaPharma (West Chester, OH). MatrigelTM (reconstituted BM) was from BD Biosciences.

Tissue Procurement, Processing, and Cell Culture—Institutional approval was obtained for the use of fetal and adult human pancreata. Human adult cadaveric pancreata (ages 45–72 years) were obtained via the Juvenile Diabetes Research Foundation Human Islet Distribution Program, and human fetal pancreata (12–24 weeks gestational age) were provided by Advanced BioResources (Alameda, CA).

Fetal islet-like cell cluster (ICCs) were generated by mincing pancreata with scissors and digesting them with collagenase (2.5 mg/ml Hanks' balanced salt solution; collagenase-P, Sigma) essentially as described (27). Resulting cell clusters were washed and resuspended in RPMI 1640 containing 10% normal human serum (Omega Scientific, Tarzana, CA). These cells were subsequently incubated overnight on non-tissue culture-treated plastic to allow the formation of ICCs (28). ICCs derived from individual pancreata (~100–200) were seeded into Petri dishes (140 mm, Nalge Nunc) coated with a HTB-9 matrix as described (27). These cells were then cultured as monolayers in RPMI 1640, 10% fetal bovine serum, and 10 ng/ml hepatocyte growth factor (Genentech, San Francisco, CA). Cells were expanded for 3–4 days prior to use and were confirmed to contain 85–90% pancreatic epithelial cells (PEC) and 1–2% insulin-positive {beta}-cells. Human adult islet preparations were isolated in-house using a modified semiautomated method essentially as described (29). Islet clusters were subsequently expanded on HTB-9 matrix for 4–5 days as described for fetal ICC preparations. These cells typically contained 30–50% insulin-positive {beta}-cells and 50–60% PEC.

Migration Assays—Migration was assessed using 6-well Transwell migration plates from Costar essentially as described (30). The underside of the insert membranes (8.0 µm pore size) were coated with 75 nM Col-I, Col-IV, Fn, Fbg, Lm-1, or Vn for 3–4 h at 37 °C. Adult or fetal pancreatic cells expanded on HTB-9 matrix (70–80% confluence) were harvested with 0.025% trypsin/Versene and were resuspended in fibroblast basal media (BioWhittaker) supplemented with 0.5% BSA and 0.4 mM MnCl2 (pH 7.4). The cells were then added to migration plates at 5 x 105 cells per insert. Directed migration from the upper to lower chamber of the transwell was determined after 8 h. The contribution of integrins to migration to Col-IV was assessed using function-blocking antibodies as specified in the text. The cells were pretreated with the antibodies (40 µg/ml) for 20 min prior to the addition of both cells and antibodies to migration plates.

Migrant {beta}-cells on the underside of the membrane inserts were detected by staining for insulin as described (30). The top side of the membrane inserts was swabbed extensively to remove cells that failed to migrate, and the entire underside of the insert was then scanned and counted for insulin-positive cells using a Nikon inverted microscope equipped with a 10x objective. Migrant pancreatic epithelial cells (PEC) on the underside of the inserts were identified and counted after staining with 1% toluidine blue as described (30). PEC were counted per 20x field, and a minimum of 8 fields were scored per membrane. Percent {beta}-cell migration was calculated based on the number of {beta}-cells migrating ÷ total {beta}-cells added x100 as described (30).

Cell Adhesion Assays—The adhesion of {beta}-cells to extracellular matrix components was assessed as described (30). Adult and fetal pancreatic cell monolayers, expanded on HTB-9 matrix (70–80% confluence), were harvested with a 0.025% trypsin/Versene solution. Single cell populations were then added to non-tissue culture-treated 96-well high binding enzyme immunoassay plates (Costar, Corning, NY) coated with equimolar amounts of Col-I, Col-IV, Fn, Fbg, Lm-1, or Vn (75 nM) for 3–4 h at 37 °C. Wells were blocked with 5% BSA for 45 min prior to use. Control wells received BSA alone. Fetal or adult cells were added at 5 x 104 and 1 x 104 cells/well, respectively, in serum-free fibroblast basal media supplemented with 0.5% BSA and 0.4 mM MnCl2 (pH 7.4). Cells were allowed to adhere at 37 °C for 90 min, and at the end of the assay the wells were carefully washed, and non-adherent cells were removed under a constant vacuum. Remaining adherent cells were fixed with 3.7% paraformaldehyde. The contribution of integrins to cell adhesion on Col-IV was assessed using function-blocking antibodies as specified in the text. The cells were pretreated with the antibodies (40 µg/ml) for 20 min prior to the addition of both cells and antibodies to precoated wells.

Adherent {beta}-cells were visualized by staining for insulin as described (30). Insulin-positive cells per field were counted using a Nikon inverted microscope equipped with a 20x objective and an ocular grid. Treatments were performed in triplicate, and the entire well surface area was counted for {beta}-cells. Percent {beta}-cell adhesion was calculated based on the number of {beta}-cells adhering ÷ total {beta}-cells added x 100 as described (30).

Analysis of Insulin Release—Fetal pancreatic cells expanded on HTB-9 matrix for 4 days were harvested, and single cell preparations were added to non-tissue culture-treated 96-well high binding enzyme immunoassay plates (Costar, Corning, NY) previously coated with 75 nM Col-IV, Fn, Lm-1, or poly-D-lysine at 5 µg/ml. Cells were added at 5 x 104 cells/well in glucose-free Dulbecco's modified Eagle's medium supplemented with 4 mM glucose (Sigma), 0.5% BSA, and 0.4 mM MnCl2 (pH 7.4). Culture supernatants were collected 1.5, 5, and 12 h after adhesion. Culture supernatants were centrifuged at 14,000 x g for 5 min to remove cells and debris and then assayed for insulin using a Mercodia insulin ELISA kit according to the manufacturer's instructions (ALPCO, Windham, NH). The contribution of {alpha}2{beta}1 or {alpha}1{beta}1 to insulin secretion following adhesion to Col-IV or poly-D-lysine was assessed using mAbs to {alpha}2 or {alpha}1 (MAB1973Z and MAB1950Z, Chemicon). The cells were pretreated with the mAbs (40 µg/ml) for 20 min prior to the addition of both cells and antibodies to coated wells. Culture supernatants were harvested after 1.5 h.

Immunofluorescence Staining and Flow Cytometry—Double immunofluorescence staining was performed on fetal pancreatic cells grown on HTB-9 matrix or MatrigelTM for 4 days or on freshly isolated pancreatic cells (22–28 h postpancreatectomy). Cells were cultured on HTB-9 matrix as described (27) or were cultured under identical conditions on 6-well plates precoated with MatrigelTM at 78 µg/ml according to the manufacturer's recommendations (BD Biosciences). Further cells were maintained in suspension for 4 days as ICCs without seeding onto HTB-9 matrix or MatrigelTM. Cell monolayers on HTB-9 matrix or MatrigelTM were harvested with a 0.025% trypsin/Versene solution (1:5000, Invitrogen) as described (30). Freshly isolated cells were generated from minced pancreata digested with collagenase-P (Sigma) at 2.5 mg/ml in Hanks' balanced salt solution. Digestion was performed three times for 10 min in a shaking water bath at 37 °C. Large cell clumps were removed using a 70-mm nylon cell strainer (BD Falcon, Bedford, MA). Single cell populations from the different treatments were incubated for 45 min on ice with anti-{alpha}1 or -{alpha}2 mAbs (MAB1973Z and MAB1950Z, Chemicon) at 1 µg/106 cells in PBS containing 0.02% NaN3 and 0.1% BSA. These cells were subsequently fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were then incubated in a blocking buffer (2% normal donkey serum, 1% BSA in PBS) for 30 min prior to incubation with guinea pig anti-insulin pAb (A0564 at 1:40, DakoCytomation) for 45 min. After washing the cells were incubated for 45 min with a FITC-conjugated donkey anti-guinea pig-Ig antibody (Jackson) and a PE-conjugated donkey anti-mouse Ig antibody (Jackson). Cells were photographed using an inverted Nikon Eclipse E800 fluorescent microscope and a 20x or 40x objective. Single and double staining cells were counted in order to determine the percentage of insulin-positive cells expressing either {alpha}2{beta}1 or {alpha}1{beta}1.

Two-color flow cytometric (FACS) analysis was performed on fetal pancreatic cell monolayers grown on HTB-9 matrix for 4 days. Cells were stained as described above except they were incubated with directly labeled PE-conjugated mAbs to {alpha}1 (SR84) or {alpha}2 (12F1-H6) or with fluor-conjugated isotype controls (Pharmingen). FACS was performed using a FACscan flow cytometer (BD Biosciences).

Staining of Tissue Sections—Fetal pancreata (12 to 24 weeks) were snap-frozen immediately upon receipt (22–28 h postpancreatectomy). Cryostat sections (10 µm thick) were fixed in ice-cold methanol for 30 s and were then blocked in PBS containing 1% BSA, 2% normal donkey serum, 50 mM glycine, and 1% cold water fish gelatin (pH 7.4) for 1 h at room temperature. After washing with 0.05% Tween 20 in PBS, sections were incubated for 1–2 h with a combination of three different primary antibodies. Some sections were stained with a guinea pig pAb to insulin (1:40) (A0564, DakoCytomation), a goat pAb to PECAM (1:200) (C-20, Santa Cruz), and a mouse mAb to either the {alpha}1 integrin subunit (5 µg/ml) (MAB1973Z, Chemicon) or the {alpha}2 integrin subunit (5 µg/ml) (MAB1950Z, Chemicon). Other sections were stained similarly except that they were incubated with a rabbit pAb to Col-IV (1:100) (AB748, Chemicon) in lieu of the pAb to PECAM. Adjacent serial sections were incubated with control antibodies including isotype-matched mouse IgG1 (5 µg/ml) (PP100, Chemicon), normal guinea pig IgG, and normal goat or rabbit IgG (Jackson ImmunoResearch). After washing the sections were incubated for 1 h at room temperature with F(ab') fragments of Cy5-conjugated donkey anti-guinea pig Ig, FITC-conjugated donkey anti-goat or anti-rabbit Ig, and biotin-conjugated donkey anti-mouse Ig (all at 1:250) (Jackson ImmunoResearch). Binding of biotin-labeled antibodies to tissue was visualized by further incubating sections for 10 min in PE-conjugated avidin (1:100) (Jackson ImmunoResearch). After washing sections were examined and photographed using an inverted Nikon Eclipse E800 fluorescent microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Type IV Collagen Is a Unique Substrate for the Migration of Cultured {beta}-Cells—Provisional matrix components (Vn and Fbg) and primary constituents of the extracellular matrix (Fn, Col-IV, Lm-1, Col-1) were assessed for their ability to support fetal and adult {beta}-cell migration. Transwell migration inserts were precoated with equimolar concentrations of the individual matrix components (75 nM), and the migration of {beta}-cells previously cultured on HTB-9 matrix was assessed after 8 h.

In a direct comparison, fetal {beta}-cell migration to Col-IV was 8–10-fold greater than migration observed to the other substrates tested including Vn, Lm-1, Fbg, and Fn (Fig. 1A). Intermediate, but variable, migration to Col-I was observed (Fig. 1A). Adult {beta}-cells also displayed preferential migration to Col-IV (Fig. 1B). Despite this preference, adult {beta}-cells were significantly less motile to Col-IV than their fetal counterparts. Thus, the percentage of adult {beta}-cells migrating to Col-IV was only 2–3% compared with 40–50% by fetal {beta}-cells (Fig. 1C).



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  		FIG. 1.
{beta}-Cell migration and adhesion to matrix components. A and B, migration of fetal or adult {beta}-cells to Col-IV (C-IV), Col-I (C-I), Vn, Fn, Fbg, or Lm-1. {beta}-Cell migration was assessed after 8 h by counting the number of insulin-positive cells that had migrated per insert. Treatments were performed in duplicate, and results are expressed as the mean number of migrating cells per insert ± S.D. *, Student's t test confirmed a significantly greater migration to Col-IV compared with the other substrates (p < 0.05). C, percentage of fetal and adult {beta}-cells migrating to Col-IV. D and E, adhesion of fetal or adult {beta}-cells to Col-IV, Col-I, Vn, Fn, Fbg, or Lm-1. {beta}-Cell adhesion was assessed after 90 min by counting adherent insulin-positive cells per well. Treatments were performed in triplicate, and results are expressed as the mean number of adherent cells per well ± S.D. F, percentage {beta}-cell adhesion to Col-IV.

 
Adhesion assays were performed to determine if enhanced {beta}-cell migration to Col-IV is a result of preferential attachment to this substrate. Adult and fetal {beta}-cells attached equally to Col-IV, Col-I, Vn, and Fn (Fig. 1, D and E). Enhanced migration to Col-IV relative to these substrates must, therefore, depend on a mechanism other than initial attachment. Lm-1 and Fbg were relatively poor substrates for {beta}-cell adhesion (Fig. 1, D and E), and this could account for reduced motility to these constituents. Adult and fetal {beta}-cells attached equally well to Col-IV (Fig. 1F). The marked discrepancy in Col-IV migration observed between adult and fetal {beta}-cells (Fig. 1C) cannot, therefore, be attributed to a failure of adult {beta}-cell attachment.

Together these data demonstrate that Col-IV is a uniquely important substrate for fetal {beta}-cell motility and suggest that there is maturational loss of adult {beta}-cell motility to Col-IV and other substrates. It is important to note that the predilection of {beta}-cells for Col-IV is an attribute of the cells rather than a function of the assay. Using the same Transwell migration assay, other cell types have been shown to preferentially migrate to alternative substrates such as Fn or Lm (31, 32).

Integrin {alpha}1{beta}1 Is Responsible for Enhanced {beta}-Cell Migration to Col-IV—The receptor responsible for {beta}-cell adhesion and migration to Col-IV was determined using function-blocking antibodies to known collagen receptors (i.e. {alpha}1{beta}1, {alpha}2{beta}1, and {alpha}3{beta}1). Antibodies to the {alpha}1 or the {beta}1 integrin subunit markedly reduced {beta}-cell adhesion and migration to Col-IV (Fig. 2, A–D). In contrast, antibodies to {alpha}2{beta}1 or {alpha}3{beta}1 were either ineffective or had only a marginal impact (Fig. 2, A–D). Fetal and adult {beta}-cells were found to be equally dependent on {alpha}1{beta}1 (Fig. 2, A–D). These data implicate {alpha}1{beta}1 as the primary collagen receptor used by {beta}-cells irrespective of maturity. It is important to note that although {alpha}1{beta}1 can recognize both Col-IV and Col-I, migration to the former collagen subtype was consistently higher (Fig. 1, A and B).



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  		FIG. 2.
{beta}-Cell adhesion and migration to Col-IV are mediated by {alpha}1{beta}1. A and B, adhesion of fetal and adult {beta}-cells to Col-IV in the presence or absence of function-blocking antibodies to {alpha}1, {alpha}2, {alpha}3, or {beta}1 integrin subunits. After 90 min, the number of adherent {beta}-cells was determined by staining for insulin and counting the number of positive cells per well. C and D, migration of fetal and adult {beta}-cells to Col-IV in the presence or absence of function-blocking antibodies to {alpha}1, {alpha}2, {alpha}3, or {beta}1 integrin subunits. {beta}-Cell migration was assessed after 8 h by counting the number of insulin-positive cells per insert. E and F, migration of fetal and adult PEC to Col-IV in the presence or absence of function-blocking antibodies to {alpha}1, {alpha}2, {alpha}3, or {beta}1 integrin subunits. Migrant PEC and were detected by staining with toluidine blue. A–F, results are expressed as mean adhesion or migration as a percent of control (no antibody treatment) ± S.E. (n = 3). *, Student's t test confirmed a significant inhibition of adhesion or migration in the presence of antibodies to the {alpha}1, {alpha}2, or {beta}1 integrin subunit (p < 0.05).

 
Fetal and adult PEC, present in the same pancreatic preparations, were also observed to migrate to Col-IV; however, in marked contrast to the {beta}-cells, the migration of these cells was found to depend primarily on {alpha}2{beta}1 without a significant role for {alpha}1{beta}1 (Fig. 2, E and F).

The Interaction between Col-IV and {alpha}1{beta}1 Promotes Insulin Secretion—Prior reports have shown that an interaction between isolated rat {beta}-cells and a tumor-derived BM (804G matrix) can potentiate insulin secretion (33, 34). Because Col-IV is a major structural constituent of BMs we determined if ligation of Col-IV (via {alpha}1{beta}1) also influences insulin secretion. Cultured fetal {beta}-cells were plated on Col-IV, poly-D-lysine (an inert adhesive substrate), or BSA-blocked plastic. Levels of insulin secretion at a basal glucose concentration (4 mM) were subsequently assessed over a 5-h time frame. Relative to the control substrates, Col-IV induced a 4- to 5-fold increase in insulin secretion after 5 h (Fig. 3A). The secretion observed on Col-IV also exceeded that observed on other primary constituents of BM, including Fn and Lm-1 (Fig. 3B). After additional analysis, it was determined that the amount of insulin secreted after 90 min on Col-IV was 8–9% of the total insulin content of the cells added. These data suggest that Col-IV present in peri-insular or intraislet BMs (11, 13) could influence insulin secretion and subsequently glucose homeostasis. Importantly, the enhanced insulin secretion observed on Col-IV could be prevented by the addition of a function-blocking antibody to {alpha}1{beta}1, but not by an antibody to {alpha}2{beta}1 (Fig. 3C). The antibody to {alpha}1{beta}1 did not affect insulin secretion in the absence of Col-IV (Fig. 3D). These data are the first to implicate a collagen-binding integrin in the regulation of insulin secretion.



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  		FIG. 3.
{alpha}1{beta}1-mediated adhesion to Col-IV promotes insulin secretion. A, comparison of insulin release from fetal {beta}-cells after plating on Col-IV, poly-D-lysine (PDL), or BSA-blocked plastic. Culture supernatants were collected after 1.5 and 5 h and assayed for insulin by ELISA. Results are expressed as mean µIU of insulin/ml ± S.E. (n = 3). B, comparison of insulin release from fetal {beta}-cells after plating on Col-IV, poly-D-lysine, Fn, or Lm-1. Culture supernatants were collected after 12 h and assayed for insulin by ELISA. Results are expressed as mean µIU of insulin/ml ± S.E. (n = 3). C, insulin release from fetal {beta}-cells after plating on Col-IV in the presence or absence of function-blocking antibodies to {alpha}1 or {alpha}2 integrin subunits. *, Student's t test confirmed a significant inhibition of secretion in the presence of an antibody to the {alpha}1 integrin subunit (p < 0.05). D, insulin release from fetal {beta}-cells after plating on poly-D-lysine in the presence or absence of a function-blocking antibody to the {alpha}1 integrin subunit. C and D, results are expressed as mean insulin levels as a percent of control (no antibody treatment) ± S.E. (n = 3).

 
It is important to note that adhesion to Col-IV was not observed to have an adverse effect on {beta}-cell viability. Double staining for NCAM (a marker of endocrine cells) (35) and annexin-V (a marker of apoptotic cells (36)) did not reveal any difference in the percentage of double positive cells between the treatment groups after 5 h (data not shown).

BM Preparations Induce {alpha}1{beta}1 Expression by Cultured {beta}-Cells—Fetal pancreatic cells that had been expanded on HTB-9 matrix were harvested and double-stained for insulin and either {alpha}1{beta}1 or {alpha}2{beta}1. The integrin status of insulin-positive {beta}-cells was then determined by FACS analysis. Using this approach it was determined that over 80% of cultured fetal {beta}-cells expressed {alpha}1{beta}1 whereas only 18% express {alpha}2{beta}1 (Fig. 4, A and B, upper quadrants). In marked contrast, PEC present in the same preparations had high levels of {alpha}2{beta}1 and little or no {alpha}1{beta}1 (Fig. 4, A and B, lower quadrants). Such divergent expression can explain the differential contributions of {alpha}1{beta}1 and {alpha}2{beta}1 to {beta}-cell and PEC migration (Fig. 2, C–F). Moderate levels of {alpha}1{beta}1 expression on insulin-positive cells and high levels of {alpha}2{beta}1 on PEC were further confirmed by fluorescent microscopy (Fig. 5, A and B).



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  		FIG. 4.
Expression of {alpha}1{beta}1 and {alpha}2{beta}1 by cultured {beta}-cells and PEC. A and B, contour plots showing levels of {alpha}1{beta}1 and {alpha}2{beta}1 expression associated with fetal {beta}-cells and PEC that had been cultured on HTB-9 matrix for 4 days. Fetal pancreatic cell monolayers were harvested, and single cell populations were stained for each integrin (PE) and for insulin (FITC) to identify {beta}-cells. Labeled cells were then analyzed by flow cytometry. The percent of insulin-positive {beta}-cells staining for either {alpha}1{beta}1 or {alpha}2{beta}1 are indicated in upper right quadrants whereas the percent of PEC staining for either {alpha}1{beta}1 or {alpha}2{beta}1 is indicated in the lower right quadrants. Cells in the lower left quadrant are negative for both insulin and either of the integrins. *, note that the PEC populations analyzed contain 5–10% fibroblasts.

 



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  		FIG. 5.
Expression of {alpha}1{beta}1 and {alpha}2{beta}1 by cultured and freshly isolated {beta}-cells A and B, photomicrographs showing {alpha}1{beta}1 and {alpha}2{beta}1 expression by {beta}-cells and PEC that had been cultured on HTB-9 matrix for 4 days (40x). {beta}-Cells are identified based on insulin staining (green), and integrin expression is evident as membrane fluorescence (red). Cultured {beta}-cells expressing both insulin and {alpha}1{beta}1 are shown by arrows (A). Note that PEC present in the same preparations are negative for {alpha}1{beta}1 (A) but are strongly positive for {alpha}2{beta}1 (B). C and D, photomicrographs showing {alpha}1{beta}1 and {alpha}2{beta}1 expression by {beta}-cells stained immediately after isolation and without expansion in culture (20x). C, inset, a subset of {beta}-cells expressing {alpha}1{beta}1. D, isolated {beta}-cells were uniformly negative for {alpha}2{beta}1.

 
Although the majority of cultured {beta}-cells expressed {alpha}1{beta}1 this was not the case for freshly isolated fetal {beta}-cells (19–21 weeks). Under a fluorescent microscope only a small subset of freshly isolated {beta}-cells was observed to express {alpha}1{beta}1, and after careful inspection {alpha}2{beta}1 was found to be entirely absent (Fig. 5, C, inset, and D). Counting of double positive cells confirmed that {alpha}1{beta}1 is expressed by 2–3% of freshly isolated fetal {beta}-cells but is present on 75–85% of cells following culture on HTB-9 matrix (Table I). These data indicate that {alpha}1{beta}1 expression on {beta}-cells is extremely labile, being strongly induced during the course of in vitro culture.


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  		TABLE I
Expression of {alpha}1{beta}1 and {alpha}2{beta}1 by isolated and cultured fetal {beta}-cells Results show the percentage of insulin-positive {beta}-cells expressing either {alpha}1{beta}1 or {alpha}2{beta}1. Fetal pancreatic cell preparations (weeks 19–21) were double-stained for insulin and either {alpha}1{beta}1 or {alpha}2{beta}1 immediately after isolation, after culture in suspension, or after expansion on BM preparations (HTB-9 matrix or Matrigel) for 4 days. Immunofluorescent staining was performed, and single and double positive cells were quantified using a fluorescent microscope.

 
Optimal induction of {alpha}1{beta}1 during culture was found to be dependent upon exposure to BM preparations such as HTB-9 matrix or Matrigel (Table I). Thus a 40–45% reduction in expression was observed if the {beta}-cells were cultured under identical conditions but in the absence of such matrices (Table I). Exposure to such BM preparations also contributed to a modest induction of {alpha}2{beta}1 expression (Table I). Although exposure to BM preparations was found to be important for inducing {alpha}1{beta}1 expression, some induction was noted even when the pancreatic cells were maintained in suspension as ICCs (Table I). This induction may, however, be due to the paracrinal deposition of BM components by PEC or fibroblasts. In this regard, ICCs maintained in suspensions were found to contain significant amounts of Col-IV and other BM constituents (not shown).

{alpha}1{beta}1 Is Expressed by a Subset of {beta}-Cells in Developing Islets—Expression of {alpha}1{beta}1 by a subset of freshly isolated fetal {beta}-cells prompted further studies to determine whether such cells can be detected in situ in developing islets. For this purpose, fetal pancreata (20 week) were sectioned and stained with an antibody to {alpha}1{beta}1. For comparison sections were also stained for {alpha}2{beta}1.

Consistent with results obtained by FACS analysis (Fig. 4B), {alpha}2{beta}1 was detected at high levels on PEC associated with both ducts and acini (Fig. 6A). Double staining with an antibody to PECAM-1 (a marker of endothelial cells) confirmed that {alpha}2{beta}1 is also expressed by pancreatic and islet microvasculature (Fig. 6B, inset). {alpha}2{beta}1 expression was not detected on {beta}-cells in situ (Fig. 6, A and B, inset).



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  		FIG. 6.
Expression of {alpha}2{beta}1 and {alpha}1{beta}1 in fetal pancreas and islets. A and B, photomicrographs showing {alpha}2{beta}1 expression in a 20-week pancreas (20x). A, expression of {alpha}2{beta}1 was found on ductal and acinar epithelial cells (red) but did not co-localize with insulin-positive cells (blue)(A, inset). B, expression of {alpha}2{beta}1 (red) co-localized with microvascular endothelial cells expressing PECAM (green and yellow). Such co-localization was also evident on islet micovasculature (B, inset). C and D, photomicrographs showing {alpha}1{beta}1 expression in a 20-week pancreas (20x). D, expression of {alpha}1{beta}1 (red) was almost exclusively associated with PECAM positive microvascular endothelial cells (yellow) including vasculature associated with islets (D, inset). E and F, photomicrographs showing {alpha}1{beta}1 expression by a small subset of {beta}-cells. {beta}-Cells expressing insulin (blue) and {alpha}1{beta}1 (red) were detected in the islet periphery (arrows) (60x). These cells were in close proximity to microvascular endothelial cells expressing PECAM and {alpha}1{beta}1 (yellow). Note that the {alpha}1{beta}1 expression associated with {beta}-cells was commonly polarized toward adjacent microvascular endothelial cells (arrows).

 
Expression of {alpha}1{beta}1 was almost exclusively associated with PECAM-1+ blood vessels and was either absent or undetectable on pancreatic epithelium (Fig. 6, C and D). Although the majority of {beta}-cells failed to express {alpha}1{beta}1 (Fig. 6, C and D, inset), a small subset of positive cells was detected (Fig. 6, E and F, arrows). These cells were commonly detected in the periphery of islets in close apposition to PECAM+/{alpha}1{beta}1+ endothelial cells (Fig. 6, E and F). {alpha}1{beta}1 expression by these individual {beta}-cells was often polarized toward the overlying endothelium (Fig. 6, E and F, arrows). Although it is evident that {beta}-cells express {alpha}1{beta}1 in situ the true extent of such expression was often hard to evaluate because of the close apposition of {alpha}1{beta}1+ endothelial cells. Indeed, some separation between the two cell types was usually necessary to confirm expression by {beta}-cells. The number of {alpha}1{beta}1+ {beta}-cells detected in situ is, however, consistent with the low levels of expression detected on freshly isolated {beta}-cells (i.e. 2–3%). Such levels of expression suggest a mechanism for the tight regulation of {alpha}1{beta}1 function in vivo.

{beta}-Cells May Interact with Col-IV Associated with Islet Microvasculature—The distribution of Col-IV in developing islets was assessed using fetal pancreata (weeks 22 and 12). Strong peri-insular Col-IV staining was observed, and this staining was associated with microvascular endothelial cells coexpressing {alpha}1{beta}1 (Fig. 7, A–C). Col-IV was also observed to penetrate into islets in association with {alpha}1{beta}1+ microvasculature but was not detected around or between individual {beta}-cells (Fig. 7, A–C). The staining pattern observed suggests that {beta}-cells in the islet periphery or in contact with intraislet microvasculature will have access to Col-IV (Fig. 7, A–C). Small clusters of {beta}-cells in very young pancreata (12 weeks) were also surrounded by Col-IV (Fig. 7, D–F). The linear Col-IV staining observed, and its association with cells expressing {alpha}1{beta}1, is consistent with localization to subendothelial BMs (Fig. 7, A–F). Prior staining (Fig. 6, E and F) indicates that {beta}-cells expressing {alpha}1{beta}1 are juxtaposed to microvascular endothelial cells. Such positioning should ensure direct contact between {beta}-cells expressing {alpha}1{beta}1 and Col-IV in subendothelial matrix (Fig. 7F, inset).



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  		FIG. 7.
Localization of Col-IV and {alpha}1{beta}1 in fetal islets. A–C, photomicrographs showing Col-IV, {alpha}1{beta}1, and insulin localization in an islet in a 22-week pancreas (20x). A, Col-IV staining (green) surrounds insulin-positive {beta}-cells (blue). B and C, Col-IV staining co-localizes to microvascular endothelial cells expressing {alpha}1{beta}1 (red and yellow). Note that the Col-IV staining is linear in nature consistent with localization to subendothelial BM. D–F, photomicrographs showing Col-IV and {alpha}1{beta}1 localization in a small cluster of {beta}-cells in a 12-week pancreas (20x). D, insulin-positive {beta}-cells (blue) surrounded by Col-IV (green). E and F, the Col-IV staining is associated with {alpha}1{beta}1-positive microvascular endothelial cells (red and yellow). D–F, inset, a {beta}-cell expressing {alpha}1{beta}1 is juxtaposed with {alpha}1{beta}1-positive microvascular endothelial cells and associated Col-IV.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we demonstrate that {alpha}1{beta}1 is the primary collagen-binding integrin utilized by both fetal and adult {beta}-cells and show that this integrin makes a significant contribution to {beta}-cell adhesion, motility, and insulin secretion. Compared with other matrix-integrin interactions, the pairing of {alpha}1{beta}1 and Col-IV is shown to be of unique importance for {beta}-cell migration. Using immunohistochemistry, we confirm that {alpha}1{beta}1 is expressed by a subset of {beta}-cells in vivo and show that these cells reside in close apposition to the islet microvasculature. Based on our findings we propose that subendothelial Col-IV is likely to be an important substrate for the movement and spatial organization of {beta}-cells during islet neogenesis. Based on the observation that {alpha}1{beta}1 ligation promotes insulin secretion it is proposed that this collagen receptor may also influence normal glucose homeostasis.

Expression of {alpha}1{beta}1 by {beta}-cells has not previously been reported and was not anticipated because this integrin is primarily expressed by cells of mesodermal origin such as smooth muscle cells, fibroblasts, and endothelial cells (5, 6). Our findings indicate that {alpha}1{beta}1 expression by {beta}-cells is strongly induced after isolation and culture. Such labile expression has been reported in skin fibroblasts and Schwann cells, which also acquired {alpha}1{beta}1 expression after isolation and culture (37, 38). Our data indicate that exposure to BM preparations (i.e. Matrigel or HTB9 matrix) is a significant factor in the induction of {alpha}1{beta}1 expression in culture. Such matrices may induce integrin expression directly by supporting adhesion, signaling, and gene transcription (39) or indirectly by serving as a scaffold for the deposition and accumulation of growth factors (40). As a reconstituted BM, MatrigelTM is known to contain several growth factors including insulin-like growth factor-1 and transforming growth factor-{beta} (38), and both of these growth factors can induce {alpha}1{beta}1 expression (40).

Induction of {alpha}1{beta}1 expression by BM preparations in vitro raises the possibility that BM structures also regulate the expression of {alpha}1{beta}1 in developing islets. Our findings indicate that {alpha}1{beta}1 expression is limited to {beta}-cells in close apposition to peri-insular microvasculature and associated BM. Regulation of {alpha}1{beta}1 expression by peri-insular BM would ensure that {alpha}1{beta}1 is only expressed when a suitable matrix is available and may prevent unwanted migration in the islet core. Structural studies suggest that all or most {beta}-cells in islets will be in contact with endothelium (41) so regional variation in the composition or amount of subendothelial matrix may be important in determining whether {alpha}1{beta}1 expression is induced. Local growth factor deposition and accumulation may also have a regional influence on the induction of {alpha}1{beta}1 by BM.

The reported loss of adult {beta}-cell migration to Col-IV and other substrates suggests that there is a generic loss of motility following {beta}-cell maturation. Adult {beta}-cells were consistently less motile on Col-IV than their fetal counterparts, and this deficit was observed despite comparable levels of Col-IV adhesion and {alpha}1{beta}1 expression. Double immunofluorescence staining confirmed that greater than 80% of cultured adult {beta}-cells express {alpha}1{beta}1 (data not shown). A deficit in signaling or cytoskeletal reorganization could account for a general loss of motility following integrin ligation. Tight regulation of {beta}-cell motility, be it through restricted {alpha}1{beta}1 expression or a maturational loss of motility, is expected in order to maintain the architecture and integrity of mature islets. It is also anticipated that only a small percentage of {beta}-cells will need to be motile at any given time during islet neogenesis.

In this study, we show that Col-IV is the optimal substrate for {beta}-cell motility with migration to this BM constituent far exceeding that to Fn, Lm-1, Col-1, Vn, or Fbg. Integrin {alpha}1{beta}1 has a lower affinity for Col-I than for Col-IV, and this difference could account for preferential migration to the latter (42). We did not detect Col-IV between or around {beta}-cells in fetal islets, which supports prior reports that there is an absence of BM constituents around individual islet cells (11, 43). However, we did detect Col-IV in association with the islet microvasculature, and based on this localization we propose that subendothelial BM is likely to be an important scaffold for the migration and spatial organization of {beta}-cells during islet development. Studies performed at the ultrastructural level have confirmed an intimate relationship between individual {beta}-cells and islet capillaries, and direct contact between {beta}-cells and subendothelial BM has been confirmed (41, 44). The contention that endothelium is an important factor in islet neogenesis is supported, not only by a close spatiotemporal relationship between blood vessels and islet cells but also by a recent report that endothelium has an essential role in endocrine pancreatic development (45).

In addition to promoting {beta}-cell migration we provide evidence that {alpha}1{beta}1 potentiates insulin secretion at a basal glucose concentration. This observation is consistent with prior studies showing that adhesion of {beta}-cells to a BM preparation (804G matrix) promotes insulin release (33, 34). In one of these studies it is suggested that enhanced insulin secretion is the result of an interaction between Lm-5 and {alpha}6{beta}1 (33). Our data suggest an additional and important role for {alpha}1{beta}1 and Col-IV. The mechanism responsible remains to be elucidated; however, {alpha}1{beta}1-mediated signaling could result in an increase in intracellular Ca2+ levels, which then serves as a trigger for insulin release (46). This could occur as a result of {alpha}1{beta}1-mediated ERK activation and arachidonic acid release (47). To our knowledge this study is the first to link a collagen-binding integrin to insulin secretion and suggests a potential role for {alpha}1{beta}1 in glucose homeostasis.

Although the focus of this report has been on the contribution of {alpha}1{beta}1 to migration and insulin secretion, it is evident that this integrin could impact other {beta}-cell functions including gene transcription (48) and proliferation (49). As a collagen receptor {alpha}1{beta}1 is unique in its ability to promote proliferation through Shc and mitogen-activated protein kinase (49). Expression of {alpha}1{beta}1 on islet microvascular cells suggests that this integrin may also contribute to islet neovascularization (50). Integrin {alpha}2{beta}1, although largely absent on {beta}-cells, was highly expressed by PEC and in this context could contribute to the development of the pancreatic epithelium. In this regard, {alpha}2{beta}1 has been shown to make an important contribution to branching morphogenesis (51).


   FOOTNOTES
 
* This work was supported by Juvenile Diabetes Research Foundation Grant JDRFI 1-20001-793 (to A. M. M) and by the Larry L. Hillblom Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pediatrics, Islet Research Laboratory at The Whittier Institute for Diabetes, University of California at San Diego, 9894 Genesee Ave., La Jolla, CA 92037. Tel.: 858-550-2909; Fax: 858-558-3495; E-mail: ammontgo{at}ucsd.edu.

1 The abbreviations used are: Col-IV, collagen type IV; Col-I, collagen type I; Fn, fibronectin; Lm-1, laminin-1; Vn, vitronectin; Fbg, fibrinogen; BM, basement membrane; PEC, pancreatic epithelial cells; pAb, polyclonal antibody; mAb, monoclonal antibody; PECAM-1, platelet endothelial cell adhesion molecule-1; ICC, islet-like cell cluster; FACS, fluorescence-activated cell sorting; PE, phycoerythrin; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
Special thanks to Dr. Alberto Hayek and Ana Lopez for providing adult islets and other support.



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 ABSTRACT
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