KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development

Abstract

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Figure 1

Ep-CAM expression identifies the epithelial compartment of the human fetal pancreas. Triple immunofluorescence, followed by confocal microscopic analysis, was performed on 8-μm cryostat sections from a human fetal pancreas of 18 wk to simultaneously identify Ep-CAM–, insulin-, and glucagon-positive cells. (A) Ep-CAM–specific immunoreactivity that highlights the cell– cell boundaries of epithelial cells. The strongest immunoreactivity for Ep-CAM is detected in cell clusters budding from the ducts (asterisks). Notably, many cell clusters emerging from the ductal epithelium contain large numbers of glucagon- (B) and insulin-positive cells (C). (D) Combined fluorophore spectra. Yellow color results from the colocalization of green and red fluorescences, specific for Ep-CAM and insulin, respectively. Representative of nine independent experiments using five independent donors (18–20 wk of gestation). Bar, 50 μm.

Figure 2

Electron microscopic identification of Ep-CAM at sites of cell–cell contact. Cell monolayers prepared from HFPs were immunolabeled for Ep-CAM by indirect peroxidase method, sectioned either through the horizontal plane (A) or a vertical plane perpendicular to the cell monolayer (B), and then imaged at 300–400 keV using an intermediate voltage electron microscope (JEOL 4000EX). Stereo pair images were obtained by tilting the specimens +5°. As seen in panels A, electron-dense staining specifically localizes to regions of cell–cell contacts, depicting the rim of three cells contacting each other. Panels B reproduce a stereo pair image collected from an Ep-CAM-stained sample sectioned through the z axis. This view identifies basal (left end side), lateral, and apical domains (right end side) of cells in contact. Most of the electron-dense staining appears targeted to the lateral domain of two cells in contact (arrow), and then localizes to interdigitated philopodia-like structures contributed by both cells. Bars, 1 μm.

Figure 3

Identification of E-cadherin in junctional complexes of fetal pancreatic epithelial cells. Samples sectioned through a vertical plane and prepared as in Fig. 2 were immunolabeled for E-cadherin with an anti–E-cadherin antiserum (EcadEC5) (Fannon et al., 1995) to identify functional adhesion complexes in HFP cell monolayers. Panels A represent a stereo pair image of two cells in contact with each other, displaying strong E-cadherin–specific electron-dense staining at the site of contact (arrow). The apical pole of these cells is on the left. Panels B reproduce a stereo pair view of a sample incubated with an isotype-matched antibody used as negative control for KS1/4 mAb. The arrow shows a site of cell–cell contact between two cells. Bars, 1 μm.

Figure 4

Ep-CAM mediates calcium- independent aggregation of fetal pancreatic epithelial cells. Freshly dissociated pancreatic epithelial cells were reaggregated in the presence (A–C) or absence (D–F) of calcium, to discriminate between calcium-dependent and -independent adhesion mechanisms. A shows the qualitative appearance of cell aggregates obtained in the presence of control Fab′ fragments, whereas B depicts clusters formed in the presence of anti–Ep-CAM Fab′ fragments (from KS1/4 mAb). This mAb reduced the size of cell aggregates, causing an inhibition of cell aggregation from 74.6 to 44% (C) (P < 0.001). In the absence of calcium, the aggregation observed in the presence of a control Fab′ (D and F) is decreased from 47 to 18.9% (E and F) (P < 0.001). Values in C and F are expressed as mean ± SEM of four independent experiments, using four independent donors (18–20 wk of gestation).

Figure 5

Developmentally regulated expression of Ep-CAM in the human pancreas. Confocal analysis performed on cryostat sections of a HFP, double immunostained for Ep-CAM (A) and insulin (B), reveals the upregulation of Ep-CAM expression in cell clusters budding from the ductal epithelium (A). The two fluorophore spectra are merged in C, where insulin-positive cells (red) are identified within clusters displaying the brightest immunoreactivity for Ep-CAM (green). D represents a typical histogram obtained by flow cytometric analysis after immunolabeling for Ep-CAM of freshly isolated HFP cells. It shows that two distinct populations of Ep-CAM– positive cells are present in the HFP, an Ep-CAM^low (arrow) and an Ep-CAM^high (arrowhead) population. This pattern, when compared to the in situ detection of Ep-CAM (A and C), suggests that Ep-CAM^low cells comprise ductal cells (C and D, arrows), whereas Ep-CAM^high cells (C and D, arrowheads) correspond to developing islet cells, and clusters of undifferentiated epithelial cells. The white tracing in D represents the autofluorescence of cells incubated with a mouse IgG2a used as a control reference. The data are representative of nine independent immunostainings using five independent donors (18–20 wk of gestation). Triple immunofluorescent localization was also performed on sections of human adult pancreas to simultaneously identify Ep-CAM, insulin, and glucagon. In this set of experiments, the brightest Ep-CAM– specific immunofluorescence was recorded at the cell–cell boundaries of intercalar ductal cells (E, arrowheads), in interlobular ducts (J), and in main ducts (G). Islets of Langerhans, identified by the insulin- (H) and glucagon-specific fluorescence (F), exhibit a significantly less intense Ep-CAM–specific fluorescence (E). Combined fluorophore spectra (I). The data are representative of four independent experiments, using three independent adult donors (20–56 yr old). Bars: (B) 30 μm; (H and G) 50 μm; (J) 40 μm.

Figure 6

High levels of Ep-CAM expression mark proliferating pancreatic epithelial cells in situ. A shows a reconstruction of four adjacent microscopic fields from a fetal pancreas (18 wk), immunostained for Ep-CAM (green), insulin (red), and Ki-67 (blue). Numerous proliferating cells identified by the nuclear staining for Ki-67 can be observed in cell clusters budding from pancreatic ducts (asterisks), and within the ductal epithelium (arrowheads). Notably, all Ki-67–positive cells exhibited high levels of Ep-CAM–specific immunoreactivity, including those identified within the monolayered ductal epithelium that normally exhibit low levels of Ep-CAM. Many cycling insulin-positive cells were also observed (arrows). Similar analysis performed on sections of human adult pancreas (B–D) also revealed that expression of Ki-67 is always associated with high levels of Ep-CAM. Although rare cycling β cells were observed (B, arrow; refer to Table II), most Ki-67–positive cells were identified in ducts (C, arrows) and in the acinar tissue (D, arrows). Note that even within the exocrine compartment, which overall expresses low levels of Ep-CAM, cycling cells identified by the Ki-67 staining always showed brighter Ep-CAM–specific fluorescence compared to the surrounding noncycling acinar cells. The data are representative of five independent experiments, using three independent donors of both fetal (18–20 wk of gestation) and adult pancreata (20–56 yr old). Bars: (A) 50 μm; (B and C) 80 μm; (D) 40 μm.

Figure 7

Ep-CAM expression is upregulated by growth stimuli. Western blot analysis of cell extracts from fetal ICCs (lane 1) revealed a band with an apparent molecular mass of 40 kD using the KS1/4 mAb. When cells were plated on the 804G extracellular matrix to promote epithelial cell growth, a remarkable upregulation of Ep-CAM expression was observed (lane 2). In this condition, an additional band of 42 kD was detected. When purified adult islets were cultured either in the absence of growth stimuli (lane 4), or as monolayers on the 804G matrix supplemented with rhHGF/SF (lane 5), a significant upregulation of Ep-CAM expression was observed. Lysates from human insulinomas also showed higher levels of Ep-CAM (lane 8) compared to normal islets (lane 7). Lanes 3, 6, and 9 were loaded with cell lysates from fetal cell monolayers, adult islet monolayers, and human insulinoma, respectively, and then probed with a control mouse IgG2a. The data are representative of seven independent experiments using fetal and adult cells (seven and five independent donors, respectively), and of three independent experiments using human insulinomas (from two independent donors).

Figure 8

Developmentally regulated expression of Ep-CAM during pancreatic islet maturation. Fetal pancreatic cells cultured as floating ICCs (A) were transplanted under the kidney capsule of nude mice, and grafts were analyzed after 12 wk for the presence of islet tissue (B). Hematossilin/ eosin staining of graft sections revealed the presence of numerous ICCs (arrowheads) and some ductal elements (asterisks) immersed in a well-vascularized stroma (arrows indicate blood vessels). K, mouse kidney; KC, kidney capsule. Three-color immunofluorescence for the simultaneous identification of Ep-CAM (green), insulin (red), and glucagon (blue), was performed in sections from ICCs. C shows the Ep-CAM–specific immunofluorescence that highlights cell–cell boundaries of epithelial cells. Few ductal structures can be identified within these cell clusters (arrows), displaying a dimmer Ep-CAM–specific signal, as in fetal pancreas in situ. A small fraction of mesenchymal cells (<10%) lacking Ep-CAM expression can be identified in some ICCs (arrowheads). Merging of the three fluorophore spectra (D), specific for Ep-CAM (green), insulin (red), and glucagon (blue), shows that only a small fraction of the cells forming ICCs express islet hormones (5–10%), whereas most of the other epithelial cells are undifferentiated. Immunostaining of sections from the grafts after 12 wk in vivo shows that high levels of Ep-CAM (E) are found in ductal elements (asterisks), whereas endocrine cells (glucagon, F; insulin, G) exhibit lower levels, as in the adult pancreas in situ. H shows the merging of the three fluorophore spectra specific for Ep-CAM (green), insulin (red), and glucagon (blue). Bars: (A) 100 μm; (B, C, and G) 50 μm.

Figure 9

Blockade of Ep-CAM–mediated cell–cell interactions causes endocrine differentiation of HFP cells. HFP cells cultured as floating ICCs for 5 d in the presence of either Fab′ fragments of KS1/4 mAb or control Fab′ were used for determination of insulin and glucagon transcript levels in a multiprobe RNase protection assay. A shows that treatment with anti–Ep-CAM KS1/4 Fab′ causes a significant increase of both insulin and glucagon gene transcription. Nicotinamide-treated ICCs (lane NIC) were used in the assay as an internal positive control for induction of endocrine differentiation. Yeast tRNA (lane tRNA) was included as a negative control. Lane UP, undigested probes; M, RNA sizing ladder. Quantitation of band intensities performed by scanning densitometry (B) shows that blockade of Ep-CAM produces a 1.9- and 1.4-fold increase, respectively, of insulin and glucagon mRNAs above the levels measured in control ICCs. Note that the insulin protein content is also increased in ICCs treated with anti–Ep-CAM KS1/4 Fab′ (C). A is representative of four independent assays. Data in B and C are expressed as mean ± SEM of four independent experiments. P values for significant differences were: P < 0.002 for both insulin and glucagon transcript levels compared to control in B; P < 0.005 for samples treated with KS1/4 Fab′ compared to control in C.