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RECEPTORS AND SIGNAL TRANSDUCTION

Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

Departments of ¹Medicine and ²Surgery, Vanderbilt University, Nashville, Tennessee

Submitted 27 June 2007 ; accepted in final form 10 September 2007

	ABSTRACT

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Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK^rat5aa cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK^human5aa cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK^rat5aa cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK^rat5aa cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na⁺ in MDCK^rat5aa cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca²⁺-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK^rat5aa cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK^rat5aa cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.

heparin-binding-epidermal growth factor; membrane-anchored; epidermal growth factor receptor

EGF receptor (EGFR) activation plays fundamental roles in development, proliferation, and differentiation (44). By contrast, constitutive activation of the EGFR is implicated in the development and progression of numerous types of human cancers (3, 44, 49). Loss of cell polarization is a key event on transformation of the epithelial cells, and loss of functional tight junctions has been correlated with tumorigenesis (27, 30). Therefore, a possible correlation between EGFR activation and regulation of tight junctions can be postulated.

Among the EGF family of growth factors, heparin-binding EGF-like growth factor (HB-EGF) is of special interest, as it is synthesized as a type 1 transmembrane protein that can be shed enzymatically to release a soluble 14- to 20-kDa growth factor (19, 20). Both the transmembrane (proHB-EGF) and the mature soluble form may serve as ligands for EGFRs (32, 35, 45, 46). The soluble form of HB-EGF can interact with its receptors in an autocrine and/or paracrine manner, is a potent mitogen, and induces proliferation and migration in a variety of cells (24, 41). Under physiological conditions, the majority of HB-EGF is found as the uncleaved transmembrane precursor (proHB-EGF) that is suggested to activate EGFRs in a juxtacrine fashion (17). In addition, proHB-EGF interacts with other integral membrane proteins, especially integrins, via the tetraspanin, CD9/DRAP27 (26, 31). It is noteworthy that the HB-EGF-CD9-integrin complex and EGFR are both targeted to cell-cell junctions in polarized epithelial cells (13, 31). In the mammalian kidney, HB-EGF expression is primarily localized to the collecting duct, the nephron segment with the highest TER (21).

The Madin-Darby canine kidney (MDCK) cell line has been widely used as an in vitro model of polarized epithelium, including the study of mechanisms underlying formation and regulation of tight junctions (15, 29, 43, 50). Four strains of MDCK cells have been established, MDCK strains I, II, 7, and 11 (16, 38). These established strains of MDCK cells exhibit functional and biochemical differences, including a marked difference in TER (4, 15, 28). MDCK strains I and 7 exhibit 10- to 100-fold higher TER than MDCK strains II or 11. The former strains model a "tight" epithelium, whereas the latter strains model a "leaky" epithelium. All four MDCK strains express claudin-1, claudin-3, claudin-4, claudin-7, and occluding, as well as zonula occludens-1 (ZO-1), the tight junction-related protein (4, 15). The differences between TER in MDCK strains I or 7 vs. II or 11 are due, at least in part, to differences in expression of claudin-2, with expression only in the strains with lower TER (4, 15). Furthermore, MDCK strain I or 7 cells can be converted to a leaky epithelium, if exogenous claudin-2 is expressed in these cells (4, 15). In addition, exogenous claudin-2 expression in MDCK-7 cells also induces cation-selective paracellular current in the tight junctions (4). In the mammalian kidney, claudin-2 expression is restricted to nephron segments with lower TER (proximal tubule and thin descending limb of Henle) and is absent in the remaining distal nephron segments (25).

We have previously determined that the expression of noncleavable, membrane-anchored rat HB-EGF isoform in MDCK II cells enhanced cell-matrix adhesion, spreading, and TER (45). Current studies were designed to determine the details of the mechanism underlying the increased TER in cells expressing noncleavable, membrane-anchored HB-EGF.

	MATERIALS AND METHODS

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Cell culture and transfections. MDCK II control and cells expressing either noncleavable, membrane-anchored rat HB-EGF or noncleavable, membrane-anchored human HB-EGF were grown in Dulbecco modified Eagle's medium containing Earle's balanced salt solution supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B in 5% CO₂/95% air at 37°C. The details of rat HB-EGF constructs and generation of stable populations of HB-EGF mutants and characterization have been described previously (45). Two separate clones of MDCK^rat5aa cells with similar expression levels were used for the studies, unless otherwise stated. For stable reexpression of claudin-2 in the MDCK^rat5aa cells, full-length canine claudin-2 cDNA subcloned in pcDNA-4-Zeocin mammalian expression vector was used, as the original selection was done using neomycin (G418 1 mg/ml). The resultant cells were named MDCK^rat5aaCl2 cells. No clonal selection was performed to ensure similar expression levels of noncleavable, membrane-anchored HB-EGF between MDCK^rat5aa and MDCK^rat5aaCl2 cells.

For generation of MDCK II cells stably expressing the noncleavable, membrane-anchored human HB-EGF mutant construct, a human HB-EGF mutant with deletion of the potential cleavage site, PVENP, was generated by PCR using human HB-EGF as template and primers with custom restriction sites. A COOH-terminal hemagglutinin tag was added to this construct to aid in immunodetection. Transfections and further selection were performed using neomycin (G418 1 mg/ml), as described previously (45). The resultant cell line was named MDCK^human5aa cells, and two separate clones were studied.

Antibodies and reagents. The rabbit anti-rat HB-EGF polyclonal antibody was a generous gift from Dr. Li Feng (Department of Immunology, The Scripps Research Institute, La Jolla, CA). The anti-claudin-1, -2, -3, and -4, occludin, and ZO-1 antibodies were purchased from Invitrogen (Carlsbad, CA). The anti-EGFR, anti-phosphorylated-EGFR (Y1173), and anti-hemagglutinin tag antibodies were purchased from Santa Cruz biotechnology (Santa Cruz, CA). E-cadherin and β-catenin antibodies were purchased from Transduction Laboratories (San Jose, CA). Anti-mouse or rabbit-rhodamine-X or FITC were purchased from Jackson Laboratories (West Grove, PA). The EGFR kinase inhibitor PD-153035, phosphatidylinositol 3 (PI3) kinase inhibitor LY-294002, and mitogen-activated protein (MAP) kinase (ERK1/2) inhibitor U-0126 were obtained from EMD Biosciences (San Diego, CA). The β₁-integrin blocking antibody was obtained from the Developmental Studies Hybridoma Bank (University of Iowa), and the CD9 blocking antibody was purchased from abcam.com.

TER. TER was measured as described previously (43, 45). In brief, cells were plated on transwell filters (12 mm, 0.4-µm pore size; Corning Costar, Cambridge, MA) at confluence and allowed to attach overnight to form a confluent monolayer in normal Ca²⁺-containing (NC) medium. TER was measured after 12 h of plating (day 1) and every 24 h thereafter using a Millipore (Bedford, MA) electrical resistance measurement system, and the results are expressed in x cm².

Measurement of paracellular flux. To determine the possible changes in paracellular flux, medium containing [³H]inulin was added to the top (inner) chamber of the transwell. Samples were collected from the bottom (outer) chamber after 4 h, and counts were measured using a scintillation counter (Beckmann). The data are presented as total counts per minute collected in the bottom chamber after 4 h.

Ca²⁺ switch. Cells were plated on 6-mm polycarbonate filters (Transwell; Costar) and allowed to establish confluence over 24 h in NC medium. Medium was changed to low-Ca²⁺ medium [minimal essential media with 5% dialyzed fetal calf serum, Ca²⁺ (1–4 µM)] for 16–24 h. At the start of each experiment, cells were switched to NC medium containing 1.8 mM Ca²⁺, and cells from two separate transwells were collected in RIPA (cell lysis buffer) and pooled together.

Determination of paracellular ionic selectivity. Control MDCK, MDCK^rat5aa, and MDCK^rat5aaCl2 cells were grown on transwell filters, and TER was measured as described above. When the cells achieved maximum TER, cells were washed, and both top (inner) and bottom (outer) chambers of the transwell were filled with buffer P: 140 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 10 mM glucose, pH 7.3. In buffers A and B, NaCl was replaced by arginine-HCl or lysine-HCl, respectively, to determine the effect of Cl^– without Na⁺, and in buffer C NaCl was replaced by sodium aspartate to eliminate the effect of Cl^–. TER was measured following replacements of various buffers to assess the contribution of specific ion in the increased TER in the MDCK^rat5aa cells.

Immunoblotting. Equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with the antigen-specific antibodies. Signals were detected using an enhanced chemiluminescence detection kit (Amersham Biosciences). Equal protein loading was assessed by immunoblotting with anti-β actin antibody (Sigma-Aldrich, St. Louis, MO) after stripping the respective membrane.

Immunofluorescence microscopy. Immunofluorescence staining and confocal analysis were performed as described previously (43). In brief, filters were rinsed with ice cold x1 PBS and fixed with ice-cold methanol. Cells were further permeabilized with 0.2% Triton X-100 in PBS for 10 min. Thereafter, cells were washed three times with ice-cold PBS. After being blocked in PBS containing 5% normal goat serum and 1% bovine serum albumin, the samples were incubated with primary antibodies for 1 h in a moist chamber at 37°C or overnight at 4°C. The samples were then washed three times with PBS, followed by incubation for 30 min with the respective conjugated secondary antibody. The samples were again washed five times with PBS, embedded in glycerol/PBS-based mounting medium, and examined using a fluorescent microscope. Confocal images were obtained with Zeiss 510 confocal microscope available at the Vanderbilt University Medical Center Cell Imaging Core Resource. Image analysis was performed using the Metamorph cell-imaging program (Universal Imaging, Downingtown, PA).

Statistics. Graphic data are presented as means ± SE. Statistical analysis was performed, where appropriate, using the Student t-test. Differences with P < 0.05 were considered statistically significant.

	RESULTS

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Epithelial cells polarize upon culture on permeable filter supports, and TER across the confluent epithelial cell layer is a measure of tight junction function (9). Our laboratory has previously shown that a confluent monolayer of MDCK cells stably expressing a noncleavable form of the transmembrane form of rat proHB-EGF (MDCK^rat5aa cells) developed significantly higher TER compared with control MDCK cells (45). In agreement with our laboratory's published findings, in the present studies TER values in MDCK^rat5aa cells were approximately threefold higher compared with the control MDCK cells (Fig. 1A). To determine whether the observed effects were unique to rat HB-EGF, we developed analogous noncleavable human HB-EGF construct (PVENP, the cleavage site, was deleted) and stably expressed it in MDCK II cells (MDCK^human5aa cells). We confirmed that the human HB-EGF mutant lacking the putative cleavage site (PVENP) was not cleaved under normal conditions or in response to stimulation with phorbol myristate acetate ester (1 µM) (data not shown). Similar to the MDCK^rat5aa cells, TER in MDCK^human5aa cells was also 2.5- to 3-fold higher than that in the control cells (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Changes in transepithelial resistance (TER) and paracellular permeability. A: TER in Madin-Darby canine kidney (MDCK) II cells stably expressing noncleavable, membrane-anchored rat heparin-binding EGF (HB-EGF) mutant construct. Cells (2 x 105) were plated in transwell filters (0.4-µm pore size), and TER was measured as described in MATERIALS AND METHODS. Values represent means ± SE of peak TER values from each cell line. **P < 0.01. B: TER in MDCK cells stably expressing noncleavable, membrane-anchored human HB-EGF mutant construct. Cells were plated and TER was measured as described above. D1–D7, days 1–7. Values represent means ± SE. *P < 0.05, **P < 0.01, *** P < 0.001. C: change in the paracellular permeability. Cells (2 x 105) were plated in transwell filters (0.4 µm pore size), and [3H]inulin (4 kDa) was added to the top chamber when cells achieved peak TER values (day 4). Determination of [3H]inulin leakage was performed as described in the MATERIALS AND METHODS. Values represent mean of total counts per minute (CPM) collected from the lower chamber ± SE. *P < 0.05.

View larger version (56K): [in this window] [in a new window]   Fig. 2. A: representative phase-contrast images of confluent cells expressing noncleavable, membrane-anchored rat HB-EGF mutant construct and control MDCK II cells cultured on transwell filters. Cells expressing membrane-anchored HB-EGF (MDCKrat5aa) retained epithelial morphology similar to the control MDCK II cells. Cells expressing noncleavable membrane-anchored human HB-EGF mutant construct (MDCKhuman5aa) demonstrated similar cell morphology (data not shown). Original magnification x100. B, I: comparison of the expression profile of tight and adherens junction proteins between MDCKrat5aa and control MDCK cells. Total cell lysates from confluent cells were electrophoresed on appropriate percentage of SDS-PAGE and immunoblotted with antigen-specific antibodies. Equal protein loading was determined by probing for β-actin following stripping of the respective membranes. II: densitometric analysis of the expression of the proteins under investigation. Densitometric values for each protein were normalized with respective actin values, and data are presented as relative increase or decrease compared with the control MDCK cells. ZO-1, zonula occludens-1.

In addition to the changes in total cellular expression, appropriate cellular localization of cell-cell adhesion proteins is essential for their normal cellular functioning. Therefore, we examined the cellular distribution of the proteins under investigation in MDCK^rat5aa and control MDCK cells. As shown in Fig. 3, distinct membrane localization of tight junction proteins claudin-1, -3, and -4 and ZO-1 and adherens junction protein E-cadherin was observed in MDCK^rat5aa cells, with relatively stronger expression compared with the control MDCK cells. Also, Z-sectioning showed interesting lateral membrane distribution for claudins-1 and -3 in the MDCK^rat5aa cells compared with the exclusively apical expression in the control MDCK cells. While claudin-2 was expressed in distinct apical membrane localization in the control MDCK cells, expression levels as well as the membrane localization were severely compromised in MDCK^rat5aa cells (Fig. 3).

View larger version (91K): [in this window] [in a new window]   Fig. 3. Immunofluorescent localization of tight and adherens junction proteins. Control MDCK and MDCKrat5aa cells were plated on transwell filters, and TER was measured. Cells were fixed when they achieved peak TER values and processed for immunofluorescence staining, as described in MATERIALS AND METHODS. All images were treated identically. The bottom panels show the XZ sectioning of the corresponding panels. Z-sectioning of the confocal image stack showed distinct apical membrane staining for all tight junction proteins and lateral membrane staining for E-cadherin in the control MDCK cells. In the MDCKrat5aa cells, a more lateral distribution for claudin-1 and claudin-3 was observed. Original magnification x400.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Reexpression of claudin-2 in MDCKrat5aa cells. A, I: Western blot analysis of the expression of claudin-2 in control MDCK, MDCKrat5aa, and MDCKrat5aaCl2 cells. Total cell lysates from confluent cells were subjected to appropriate percentage of SDS-PAGE and immunoblotted with antigen-specific antibodies. II: densitometric analysis of the expression of the proteins under investigation. Densitometric values for each protein were normalized with respective actin values, and data are presented as relative increase or decrease compared with the control cells. B: immunofluorescent localization of claudin-2 in MDCKrat5aa and MDCKrat5aaCl2 cells. Original magnification x400.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of reexpression of claudin-2 in MDCKrat5aa cells upon TER and paracellular permeability for [3H]inulin and charged ions. A: TER in control MDCK, MDCKrat5aa, and MDCKrat5aaCl2 cells. Values represent means ± SD. ***P < 0.001, **P < 0.01. B: change in the paracellular permeability upon reexpression of claudin-2 in MDCKrat5aa cells. Cells were plated on transwell filters, and determination of [3H]inulin flux was performed as described in the MATERIALS AND METHODS. Data are presented as relative change compared with the control MDCK cells ± SE. *P < 0.05. C: TER in response to alterations in extracellular ionic composition. TER measurements were performed in cells grown on transwell filters. Buffer P contained 140 mM NaCl, and TER in buffer P was no different compared with the TER in normal culture medium. In buffer A, 140 mM NaCl was replaced by 140 mM arginine-HCl. In buffer B, 140 mM NaCl was substituted by 140 mM lysine-HCl. In buffer C, 140 mM NaCl was replaced with 140 mM sodium aspartate. Data are presented as means ± SE. *P < 0.01; **P < 0.001.

Increased activation of EGFR and PI3 kinase were observed in MDCK^rat5aa cells upon Ca²⁺ switch. The membrane-anchored HB-EGF is a functional ligand of EGFR receptors and is potentially capable of juxtacrine activation (17, 32). Therefore, we examined signaling mechanisms underlying development of increased TER in MDCK^rat5aa cells. We used the Ca²⁺-switch model to distinguish signaling changes upon initiation of cell-cell contact. As shown in Fig. 6, an early and time-dependent increase in the activation of EGFR (Fig. 6A, I and II) and Akt phosphorylation (Fig. 6B, I and II) was observed in MDCK^rat5aa cells compared with control MDCK cells. However, no major changes were observed in the activation of ERK1/2 in MDCK^rat5aa cells compared with control MDCK cells (Fig. 6C, I and II). There was decreased expression of claudin-2 but increased expression of claudin-1 in MDCK^rat5aa cells compared with control MDCK cells at all time points after the switch (Fig. 6D, I and II).

View larger version (58K): [in this window] [in a new window]   Fig. 6. Phosphorylation (P) of EGF receptor (EGFR; A), Akt (B), and ERK (C) and expression of claudin-1 and -2 (D) following Ca2+ switch. Cells (0.8 x 106) were plated in 6-well transwell filters, and Ca2+ switch was induced as described in MATERIALS AND METHODS. Samples were collected after indicated time points, and total cell lysates were prepared. Immunoblotting was performed using antigen-specific phospho-antibodies. A, I–C, I: immunoblot analysis for P-EGFR, P-Akt, and P-ERK1/2. Blots were stripped and reprobed with nonphospho-antibodies to confirm equal protein loading. A, II–C, II: densitometric analysis of the respective blots. Values for phospho-proteins were normalized to the values from nonphospho-protein, and data are presented as relative change compared with control MDCK cells. D, I: expression of claudin-1 and -2 was examined in the same cell lysates using antigen-specific antibodies. Actin was used for normalization. D, II: densitometric analysis of the respective blots. Values were normalized to actin values from the same blot and presented as relative change compared with the control MDCK cells.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effects of inhibition of signaling pathways and molecules on TER. Cells were incubated with PD-153035 (inhibitor of EGFR phosphorylation, 1 µM), U-0126 (inhibitor of mitogen-activated protein kinase, ERK1/2, 10 µM), LY-294002 (inhibitor of phosphatidylinositol 3-kinase, 20 µM), A2B2 (functional blocking antibody for β1-integrin, 4 µl/ml), or MEM-61 (functional blocking antibody for CD9, 2 µg/ml) before switching cells to normal Ca2+ medium for 30 min. An IgG of the same class (2 µg/ml) was used as control. All inhibitors were also present in the normal Ca2+ medium used for the switch and remained present throughout the study. Values represent the means ± SE of the peak TER from each cell line. *P < 0.01.

	DISCUSSION

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Studies that have genetically manipulated expression of specific claudin family members in cultured cells have confirmed that changes in the expression and cellular localization of claudins are central to the observed alterations in tight junction structure and function. Overexpression of various claudins, including claudins-1 and -4, leads to increased TER (2, 10, 23). By contrast, overexpression of claudin-2 results in remarkable decreases in TER in high-TER clonal variants of MDCK cells (4, 15). Conversely, RNA interference knockdown of claudin-2 in low-TER MDCK II cells results in significant increases in TER. Interestingly, similar knockdown of claudin-1 in the same cell line did not result in substantial changes in TER, although knockdown of claudins-4 and -7 did decrease TER to low levels (22). In vivo, claudin-2 expression is restricted to leaky epithelia, including the kidney proximal tubule and thin descending limb, and is absent from tight epithelia (25). We have previously reported a role for EGFR activation in the regulation of TER, whereby acute EGF administration resulted in a time-dependent and significant increase in TER in MDCKII cells (43). This increase in TER was accompanied by specific decreases in claudin-2 expression (43). Recent studies have identified EGF as the factor in human urine that increases TER in MDCK II cells (12). In the present studies, expression of noncleavable, membrane-associated HB-EGF not only significantly decreased expression of claudin-2, but also increased expression of other claudins (1, 3, and 4) known to increase TER. In addition, Z-sectioning demonstrated interesting lateral membrane distribution of claudins-1 and -3 compared with the apical membrane localization in the MDCK cells. Similar lateral membrane localization for claudins-1 and -3 have been described in other studies (18, 36, 39), but the physiological importance remains unknown.

As with certain other EGFR ligands, the transmembrane HB-EGF precursor (proHB-EGF), as well as its secreted mature product (soluble HB-EGF), are both potentially capable of activating EGFR, although the mode of receptor activation is different. While transmembrane HB-EGF precursor activates EGFR in a juxtacrine fashion, the secreted soluble HB-EGF activates EGFR in an autocrine/paracrine fashion. In fact, simple substitution of the transmembrane domain of the human proEGF with the corresponding domain of human HB-EGF renders human proEGF capable of activating EGFR in a juxtacrine fashion (11). The current studies confirm a role for EGFR activation in the regulation of tight junction composition and function and provide evidence regarding a possible physiological function of the membrane-anchored HB-EGF and hence juxtacrine activation of EGFR, in vivo. Of note, during normal physiological conditions, HB-EGF is predominantly expressed as the membrane-anchored precursor (17), and, in this regard, in the kidney, HB-EGF is expressed predominantly in the collecting tubules (21), the nephron segment that demonstrates the highest TER among nephron segments and does not express claudin-2 (25).

The possibility that the observed effects on tight junctions in MDCK^rat5aa cells may represent more than simple juxtacrine activation of EGFR cannot be ruled out, as the ability of membrane-anchored HB-EGF to form a complex with accessory proteins, such as CD9, seems to be crucial for full activity (26). Accessory proteins may be required to remove a steric constraint imposed by another structural feature of the ligand, such as the heparin-binding domain. Alternatively, the accessory protein could be required to "present" the ligand to the receptor by forming part of a multiprotein complex. In this regard, functional blocking antibodies against either CD9 or β₁-integrin decreased TER in MDCK^5aa cells. Both of these proteins are known to associate with membrane-anchored HB-EGF and have been colocalized with EGFR on lateral cell borders (31).

Taken together, we postulate that the juxtacrine activation and protein complexing by the membrane-anchored HB-EGF provide local and tightly regulated control of cell-matrix and cell-cell adhesion and maintenance of the normal epithelial architecture. In contrast, pathological disruption of cell-cell junctions will induce cleavage of HB-EGF, resulting in the release of mature secreted soluble HB-EGF, which may be involved in proliferation, migration, and dedifferentiation. Dedifferentiation of the epithelial cells has been recognized as a necessary and important step in regeneration following epithelial injury (33). Our laboratory has previously shown that, in contrast to acute EGFR activation, which, as mentioned above, resulted in increased TER, stable expression of secreted HB-EGF resulted in transformed cells with decreased cell-matrix, cell-cell adhesion, increased cell proliferation, migration, and complete loss of TER (45). These results suggested different responses with acute vs. chronic EGFR activation. Indeed, chronic administration of exogenous EGF to MDCK II cells also resulted in dedifferentiation and loss of functional tight junctions (unpublished data). Normal functioning of EGFR is essential for epithelial growth and differentiation, whereas dysregulated expression/activation of EGFR induces dedifferentiation of epithelial cells and is correlated with neoplastic transformation. However, it is important to note that constitutive expression of the noncleavable, membrane-anchored HB-EGF and hence juxtacrine signaling resulted in a contrasting phenotype characterized by increased cell-matrix and cell-cell adhesion, decreased migration, and significantly increased TER, suggesting qualitative/quantitative differences between the juxtacrine vs. autocrine/paracrine signaling.

In summary, we demonstrate that the presence of transmembrane HB-EGF in MDCK II cells results in tighter epithelium and enhanced TER. MDCK II cells display certain properties of proximal tubular epithelium, including low TER and claudin-2 expression. The presence of membrane-anchored HB-EGF changed the characteristics of MDCK II cells from a more proximal tubular epithelial cell (low TER) toward a phenotype more consistent with distal nephron epithelia (higher TER). These findings, combined with the fact that, in the kidney, HB-EGF expression is largely restricted to the distal nephron segments and is expressed predominantly as membrane-anchored precursor under normal physiological conditions, suggest a possible physiological function of the membrane-anchored HB-EGF in vivo. However, further studies are required to confirm this hypothesis.

	GRANTS

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This work was supported by funds from the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK51265 to R. C. Harris, and American Heart Association Scientist Development Grant 0435471N to A. B. Singh.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. B. Singh, C-3102 Medical Center North, Dept. of Medicine, Vanderbilt Univ., Nashville, Tennessee 37232–4794 (e-mail: amar.singh{at}vanderbilt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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