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Endocrinology Vol. 138, No. 6 2241-2248
Copyright © 1997 by The Endocrine Society

ARTICLES

1,25-Dihydroxyvitamin D3 Stimulates the Assembly of Adherens Junctions in Keratinocytes: Involvement of Protein Kinase C

Robert Gniadecki, Barbara Gajkowska and Michael Hansen

Department of Dermatological Research, Leo Pharmaceutical Products (R.G.), Ballerup; the Department of Dermatology, University of Copenhagen, Bispebjerg Hospital (R.G.), Copenhagen; and the Microbiology Section, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University (M.H.), Frederiksberg, Denmark; and the Electron Microscopy Laboratory, Polish Academy of Sciences (B.G.), Warsaw, Poland

Address all correspondence and requests for reprints to: Robert Gniadecki, M.D., Ph.D., Department of Dermatology D92, University of Copenhagen, Bispebjerg Hospital, Bispebjerg Bakke 23, 2400 Copenhagen NV, Denmark.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Signaling via intercellular junctions plays an important role in the regulation of growth and differentiation of epithelial cells. Loss of cell-cell contacts has been implicated in carcinogenesis, tumor progression, and metastasis. Here, we investigated whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was able to stimulate the assembly of adherens junctions and/or desmosomes in cultured human keratinocytes. After 4-day incubation, 1,25-(OH)2D3 caused assembly of adherens junctions, but not desmosomes. The adherens junctions were identified upon known ultrastructural criteria and evidence of the translocation of specific junctional proteins (E-cadherin, P-cadherin, {alpha}-catenin, and vinculin) to the cell-cell borders. The presence of {alpha}-catenin and vinculin at cell-cell borders indicated that the adherens junctions were functional. This was further supported by showing that anti E-cadherin antibody inhibited the 1,25-(OH)2D3-induced keratinocyte stratification. A relation between protein kinase C and adherens junction regulation was noticed. 1,25-(OH)2D3-dependent formation of junctions was blocked by the inhibitors of protein kinase C, bisindolylmaleimide and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and treatment of keratinocytes with 1,25-(OH)2D3 caused a rapid activation of protein kinase C and its translocation to the membranes. Formation of intercellular contacts may be an important mechanism of 1,25-(OH)2D3 action in hyperproliferative and neoplastic diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
1,25-DIHYDROXYVITAMIN D 1,25-(OH)2D3 PLAYs an important role in regulation of growth of epithelial cells. The effects of 1,25-(OH)2D3 have been particularly well investigated in keratinocytes. 1,25-(OH)2D3 at concentrations 10-8-10-6 M has been reproducibly shown to inhibit proliferation and induce differentiation of murine and human keratinocytes in culture (1, 2, 3, 4). Inhibition of cell growth is also manifested in vivo, where 1,25-(OH)2D3 and its synthetic analogs inhibit excessive proliferation of keratinocytes in psoriasis (5). Recent evidence suggests that 1,25-(OH)2D3 may also be useful in the treatment of skin, breast, and colon cancer (6, 7, 8, 9).

One of the aspects of epidermal cell differentiation is the formation of cell-cell junctions, which enable intercellular communication and are essential for regulation of epithelial morphogenesis, growth, and differentiation (10). In the epidermis, intercellular adhesion is mediated by two major types of junctional structures: the desmosomes and the adherens junctions (AJ) (11, 12). Ultrastructurally, desmosomes consist of two submembranous plaques separated by an electron-lucent 20- to 30-nm wide desmoglea with a distinct electron-dense midline(s) (13). The assembly of a desmosome is mediated by a homophylic interaction between the transmembrane proteins of the cadherin superfamily, desmoglein and desmocolin, the cytoplasmic tails of which bind to desmosome plaque proteins, placoglobin and desmoplakin. AJ are ultrastructurally similar to the desmosome, but are biochemically and functionally different from the latter. Rather than mainly strengthen the epidermis, AJ are dynamic structures capable of signal transduction and facilitate the so-called juxtacrine signaling (10, 14). AJ have been implicated in the regulation of morphogenesis, tissue remodeling, cell migration and stratification, cell spreading, epithelial compactness, and apoptosis (12, 15, 16, 17, 18). AJ are stabilized due to the homophylic binding between N-terminal domains of the classic cadherins, E- and P-cadherin. The cytoplasmic tails of the cadherins interact with the proteins of the catenin family, {alpha}-, ß-, and {gamma}-catenin, and with a number of other accessory proteins, e.g. placoglobin or vinculin. {alpha}-Catenin is required for cadherin-mediated cell adhesion and has an actin-binding activity (19). Thus, AJ are associated with actin cytoskeleton, rather than with the keratin intermediate filaments such as the desmosomes.

Here we investigated whether induction of epidermal cell differentiation by 1,25-(OH)2D3 was associated with assembly of cell-cell junctions. It was found that keratinocytes cultured in the presence of 1,25-(OH)2D3 assemble AJ, but not desmosomes. Since in epithelial cells AJ formation seems to depend on the induction of protein kinase C (PKC) (20, 21, 22), we also studied whether PKC is involved in the mechanism of action of 1,25-(OH)2D3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
1,25-(OH)2D3 was obtained from the Chemical Research Department, Leo Pharmaceutical Products (Ballerup, Denmark), as a 4-mM solution in isopropanol. The stock solution was stored at -70 C, but during the period of the experiment (7–10 days) the aliquots from the stock were kept at 4 C, and further dilutions were made in isopropanol as required. Storage under these conditions did not influence the purity or stability of the compound, as determined by HPLC. The final concentration of isopropanol in culture medium was kept at 0.1% regardless of the 1,25-(OH)2D3 concentration. The PKC inhibitors: bisindolylmaleimide (BIM; Boehringer Mannheim, Mannheim, Germany) and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; Sigma Chemical Co., St. Louis, MO) were used at final concentrations of 5 and 400 µM, respectively. BIM is a specific inhibitor of all PKC isoforms (23), and H-7 may additionally inhibit cyclin nucleotide-dependent kinases (24). Other chemicals were purchased from Sigma.

Keratinocyte culture
Cryopreserved human neonatal keratinocytes were purchased from PromoCell (Heidelberg, Germany) and cultured in the keratinocyte growth medium, which consists of the modified MCDB153 medium containing 0.09 mM CaCl2, 50 µg/liter recombinant human epidermal growth factor, and 0.1% bovine pituitary extract (Life Technologies, Gaithersburg, MD). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 and passaged at 80–90% confluence after trypsinization with 0.2 ml/cm2 0.05% trypsin with 0.02% EDTA solution. Second and third passage cells were used for the studies.

Antibodies
The following monoclonal antibodies were used: anti-E-cadherin (Zymed Laboratories, San Francisco, CA); anti-P-cadherin, anti-{alpha}-catenin, and anti-desmoglein (all from Transduction Laboratories, Lexington, KY); antivinculin and antiinvolucrin (Sigma); and antidesmoplakin I and II (Boehringer Mannheim). As a second antibody for immunofluorescence, the antimouse rabbit isothiocyanate (rodamine)-conjugated antiserum was used (Sigma).

Immunofluorescence staining procedures
Immunofluorescence was performed on cells grown to near confluence in 16-well glass chambers (Lab-Tek Chamber Slide, catalog no. 178599, Nunc, Naperville, IL). After fixation with methanol-acetone, the cells were air-dried, rehydrated in PBS for 5 min, blocked with 1% BSA (fraction V) in PBS for 30 min, and stained with the appropriate monoclonal antibodies. The second antimouse rabbit rodamine-conjugated antiserum was used at a dilution of 1:100. The dilutions of all antibodies were performed in the blocking solution. The samples were investigated with a fluorescent Olympus IX70 microscope or a confocal laser scanning microscope (TCS4d, Leica Laser Technik, Heidelberg, Germany) equipped with an argon krypton laser and two photomultipliers. One recording consisted of a series of 5–20 optical sections reaching from the top to the bottom of the specimen. Control experiments showed negative staining in samples incubated with second antibody only.

Electron microscopy
Keratinocytes were grown on plastic Thermanox coverslips (Nunc) in the standard medium until confluent cultures were obtained. 1,25-(OH)2D3 was added to a final concentration of 10-7 M for 4 days. Coverslips were fixed in 2% glutaraldehyde overnight, postfixed in OsO4, and embedded in Epon. Ultrathin sections were counterstained in uranyl acetate and examined in a JEOL 1200 EX electron microscope (JEOL, Peabody, MA) at 80 kV.

PKC assays
The PKC activity was determined in cultured keratinocytes in situ by the method of Heasley and Johnson (25). Keratinocytes were grown to confluence in 96-well microtiter plates (Nunc) to equalize the number of cells per well. The well to well variability in cell number did not exceed the cell counting error. The cells were washed with prewarmed 100 µl keratinocyte growth medium buffered with 20 mM HEPES to pH 7.2 and incubated with the same medium containing different concentrations of 1,25-(OH)2D3 for various periods of time. The solutions were removed, and the cells were incubated for 10 min with buffered salt solution (137 mM NaCl, 5.4 mM KCl, 2.5 mM CaCl2, 0.3 mM sodium phosphate, 0.4 mM potassium phosphate, 10 mM MgCl2, 1 mg/ml glucose, 5 mM EGTA, 50 µg/ml digitonin, and 25 mM ß-glycerophosphate, buffered with 20 mM HEPES to pH 7.2) containing 100 µM [{gamma}-32P]ATP (5000 cpm/pmol; Amersham International, Aylesbury, UK) and 300 µM KRTLRR peptide as a specific PKC substrate. The reaction was terminated with 10 µl 25% trichloroacetic acid, and 45 µl of the reaction mixture were spotted on Whatman p-81 phosphocellulose paper (Whatman, Clifton, NJ). After extensive washing with phosphoric acid and sodium phosphate, the radioactivity was determined in a Minaxi 443 (United Technologies, Downers Grove, IL) scintillation counter.

To investigate whether the PKC activity was translocated from the cytoplasm to the membrane fraction, cells were cultured in 175-cm2 Nunclon flasks (Nunc) and treated with 1,25-(OH)2D3 as indicated. Keratinocytes were scrapped, homogenized in 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM EGTA, 0.3% (wt/vol) ß-mercaptoethanol, 10 mM benzamidine, and 50 µg/ml phenylmethylsulfonylfluoride with an ultrasound homogenizer. Cytosol and membrane fractions were obtained by ultracentrifugation as previously described (26), and PKC activity was determined with a PKC enzyme assay system (Amersham International).

Statistical analysis
The results are expressed as mean values (n = 3) with SDs. For the experiment described in Fig. 4bGo, the individual membrane PKC activities were subtracted from the corresponding cytosol values to calculate the means for the vehicle-treated and 1,25-(OH)2D3-treated groups. Data were analyzed using Student’s two-sample t test for independent samples or two-way ANOVA, as indicated. For calculations, Minitab statistical software was used (Minitab, State College, PA). P < 0.05 was considered significant. All results were confirmed in at least two other independent experiments.



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  		Figure 4. Activation of PKC by 1,25-(OH)2D3. A, Keratinocytes grown in microtiter plates were incubated with 10-7 M 1,25-(OH)2D3 (•) or 0.1% isopropanol vehicle ({blacksquare}) for the indicated periods of time. PKC activity was determined as described in Materials and Methods. Mean values (n = 3) of labeling per well with SD are shown. *, P < 0.001 compared with the corresponding control value (by t test). B, Keratinocytes were incubated with 10-7 M 1,25-(OH)2D3 for 15 min (closed columns); control keratinocytes were incubated for the same period of time with 0.1% isopropanol (open columns). PKC activity was determined in the cytoplasmic and membrane fractions as described in Materials and Methods. Mean values (n = 3) with SDs are shown. P < 0.01 for the difference in cytosol and membrane activities between the experimental and control groups (by t test).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Incubation of confluent keratinocyte cultures with 10-7 M 1,25-(OH)2D3 for 4 days resulted in cell stratification. Stratification was focal, with emergence of two cell layers; the upper one containing involucrin-positive cells, and the lower one containing undifferentiated involucrin-negative cells (Fig. 1Go, a–c).



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  		Figure 1. 1,25-(OH)2D3-induced stratification, differentiation, and formation of intercellular junctions in cultured keratinocytes. Keratinocytes were cultured in serum-free medium to confluence and exposed for 4 days to 10-7 M 1,25-(OH)2D3. A–C, The cells were fixed and stained with murine antiinvolucrin antibody (1:50) and the rabbit antimouse rodamine conjugate, and observed under confocal laser scanning microscope. A, Keratinocyte stratification with emergence of an involucrin-positive layer of cells. Scans through a deeper layer of keratinocytes did not show any involucrin-positive cells. Scale bar = 100 µm. B, Reconstruction of the image shown in A in the vertical z-axis shows that involucrin-positive cells form a layer above the involucrin-negative basal keratinocytes. The line shows the surface of the coverslip. Scale bar = 10 µm. C, Effects of the anti-E-cadherin antibody. The monoclonal anti-E-cadherin antibody (50 µg/ml) was added 6 h before the addition of 1,25-(OH)2D3. After 4-day incubation (media were changed once daily), the confocal laser scanning microscopy revealed a single layer of cells containing involucrin-positive keratinocytes. Cells incubated with an irrelevant rabbit antimouse antiserum at the same final Ig concentration showed a staining pattern similar to that in A. Scale bar = 50 µm. D, Electron microscopic images of intercellular junctions in cells cultured with 1,25-(OH)2D3. Scale bar = 250 nm.

 
Stratification of keratinocytes has been previously shown to depend on E-cadherin, and it is possible to block keratinocyte and canine kidney cell line stratification with an anti-E-cadherin antibody (15, 27). To investigate whether E-cadherin was involved in 1,25-(OH)2D3-dependent keratinocyte stratification, we added the blocking anti-E-cadherin antibody to the culture medium together with 1,25-(OH)2D3. In the presence of this antibody, the stratification of keratinocytes was disturbed; however, the involucrin-positive cells were still present (Fig. 1cGo).

Keratinocytes treated with 1,25-(OH)2D3 formed ultrastructurally distinct cell-cell junctions (Fig. 1dGo). The junctional structures consisted of two thin subplasmalemmal discs separated by a slit of a moderate electron density. The absence of a midline electron-dense line and no visible connection between the junctions and intermediate cytoskeletal elements indicated that the observed structures were AJ. To further confirm that 1,25-(OH)2D3 induced formation of functional AJ, staining with monoclonal antibodies directed against specific desmosomal and AJ components was performed. The junctional proteins, E-cadherin, P-cadherin, {alpha}-catenin, and vinculin, were diffusely localized throughout the cytoplasm of control keratinocytes (see Fig. 2Go for E-cadherin distribution). Incubation with 1,25-(OH)2D3 resulted in the translocation of these proteins to the cell membrane (Fig. 2Go). In contrast, staining for the desmosome components, desmoplakin and desmoglein, did not reveal the junctional pattern of staining (not shown). This indicated that AJ, but not desmosomes, were induced by treatment with 1,25-(OH)2D3.



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  		Figure 2. 1,25-(OH)2D3-induced formation of AJ. Keratinocyte monolayers were exposed to 10-7 M 1,25-(OH)2D3for 4 days and stained with the antibodies against E-cadherin (10 µg/ml; A), {alpha}-catenin (5 µg/ml; B), P-cadherin (5 µg/ml; C), and vinculin (1:50; D). As a second antibody, an antimouse rodamine conjugate was used. Control cells were treated with isopropanol vehicle and stained with the anti-E-cadherin antibody (E), or treated with 1,25-(OH)2D3 at the above conditions and stained with the second antibody only (F). Scale bars = 50 µm (A and B) and 10 µm (C–F).

 
1,25-(OH)2D3 caused AJ assembly at concentrations from 10-6-10-8 M. At lower concentrations, AJ could not be detected, as assessed by staining with anti-E-cadherin antibody, and at concentrations above 10-6 M, 1,25-(OH)2D3 was cytotoxic for cultured keratinocytes. To assess the dynamics of AJ assembly, the cells were incubated with 10-7 M 1,25-(OH)2D3 and stained with the anti-E-cadherin and antivinculin antibodies at different times. Staining with the anti-E-cadherin antibody was first visible after 1-day incubation with 1,25-(OH)2D3 (Fig. 3aGo). This junction could not be considered functional because no cell-cell border redistribution of vinculin was present. The junctional translocation of E-cadherin and vinculin took place after 4-day incubation with 1,25-(OH)2D3 and persisted throughout the observation period (maximum of 1 week). However, the presence of 1,25-(OH)2D3 was required for the stability of the junction because peripheral anti-E-cadherin staining was lost 24 h after removal of 1,25-(OH)2D3 from the culture medium.



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  		Figure 3. Effects of PKC inhibitors on E-cadherin translocation. Keratinocyte monolayers were incubated for 1 day (A and B) with 10-7 M 1,25-(OH)2D3 (A) or 10-7 M 1,25-(OH)2D3 with 400 µM H-7 (B), or for 4 days (C–F) with 400 µM H-7 alone (C), 10-7 M 1,25-(OH)2D3 (D), 10-7 M 1,25-(OH)2D3, and 5 µM BIM (E), or 10-7 M 1,25-(OH)2D3 and 400 µM H-7 (F). The PKC inhibitors were added 4 h before the addition of 1,25-(OH)2D3. Culture media were changed every day. At termination, cells were fixed and processed for immunofluorescence with the anti-E-cadherin antibody. Scale bar = 15 µm.

 
PKC has been involved both in the formation of AJ in keratinocytes and in mediating the biological effects of 1,25-(OH)2D3 (20, 21, 22, 28, 29, 30, 31). To determine whether 1,25-(OH)2D3 was involved in the formation of AJ, the effects of selective PKC inhibitors, H-7 and BIM, on the formation of AJ was assessed. As shown in Fig. 3Go, a single treatment with PKC inhibitors for 1 day or repeated application for 4 days prevented 1,25-(OH)2D3-induced translocation of E-cadherin to cell-cell borders and formation of AJs. H-7 and BIM alone did not induce any alterations in adherens junction protein distribution in the cells (shown for H-7 in Fig. 3Go). As shown in Fig. 4aGo, 1,25-(OH)2D3 stimulated PKC activity in keratinocyte monolayers (P < 0.01, by ANOVA for the differences between experimental and vehicle-treated groups). The increase in PKC activity was maximal after 15 min, at which time the difference from the control attained statistical significance. These findings were confirmed by showing a translocation of PKC isosymes from the cytoplasm to the membranes (Fig. 4bGo). Thus, 1,25-(OH)2D3 is able to activate PKC-dependent signaling, which may be involved in the assembly of AJ.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides evidence that 1,25-(OH)2D3 causes assembly of AJ in cultured keratinocytes. The junctions were identified by ultrastructural criteria and detecting specific protein components: E-cadherin, P-cadherin, {alpha}-catenin, and vinculin. The characteristic translocation of E-cadherin from the cytoplasm to the membrane took place after 1 day of incubation; however, the fully developed junctions could be observed after 4 days of incubation with 1,25-(OH)2D3. AJ were considered functional because 1) {alpha}-catenin and vinculin were translocated to the membranes; and 2) experiments with the anti-E-cadherin antibody showed that cell stratification depended on E-cadherin, which is a component of AJ. Interestingly, assembly of desmosomal junctions, which normally follows that of AJ, was not induced by 1,25-(OH)2D3. None of the characteristic desmosomal proteins (desmocolin, desmoglein, and desmoplakin) could be detected by immunofluorescence, and typical desmosomal structures were not found by electron microscopy. This does not necessarily imply that 1,25-(OH)2D3 is inherently unable to induce desmosome assembly. The formation of desmosomes is critically dependent on the concentration of extracellular calcium ions (32), and the low calcium concentration used in this study (0.09 mM) might have been insufficient to stabilize desmosomes. Low concentrations of calcium may also explain a relative instability of the 1,25-(OH)2D3-induced AJ, which tended to dissolve within approximately 1 day after 1,25-(OH)2D3 removal from the medium. However, it was impossible to test the effects of 1,25-(OH)2D3 at higher calcium concentrations because elevation of Ca2+ causes a spontaneous assembly of desmosomes and AJ in keratinocytes (33, 34).

There is evidence that in many cell types formation of AJ is mediated by PKC (20, 21, 22, 29, 35). Phorbol esters that directly activate the majority of PKC isoenzymes, induce E-cadherin translocation in cultured keratinocytes (22). 1,25-(OH)2D3 and its analogs may induce PKC in a variety of cells, both in vivo and in vitro (28, 30, 31, 36, 37), and the present results confirmed that this hormone induced PKC in keratinocytes. We, therefore, hypothesized that 1,25-(OH)2D3 induces AJs via PKC. The finding that the effects of 1,25-(OH)2D3 on AJ assembly could be blocked by two independent PKC inhibitors implies an involvement of this kinase. Thus, 1,25-(OH)2D3 could act in a manner similar to phorbol esters by activating PKC, which, in turn, might cause redistribution of E-cadherin and AJ assembly. However, some of our results cannot be explained by this simple model. First, E-cadherin redistribution was first seen after 24 h of 1,25-(OH)2D3 treatment, even though the activation of PKC was observed after 15 min. Second, activation of PKC by phorbol esters does not lead to P-cadherin translocation (22), the opposite of the observed effect of 1,25-(OH)2D3. Thus, the mechanism of action of 1,25-(OH)2D3 in stimulating AJ assembly must involve other effector pathways. 1,25-(OH)2D3 may activate multiple signaling cascades potentially significant in AJ modulation (31, 37, 38, 39, 40, 41, 42), of which PKC is only one of the components. The identification of cellular substrates for PKC and the PKC isosymes activated by 1,25-(OH)2D3 would be helpful for understanding the mechanism of AJ induction in keratinocytes.

AJ have been involved in the regulation of cell differentiation, carcinogenesis, and tumor progression. Disruption of cell-cell contacts is associated with increased proliferation, dedifferentiation, and acquisition of the capacity to invade (18, 43, 44, 45, 46, 47). Expression of E-cadherin and ß-catenin is decreased in a variety of malignant tumors, including basal cell carcinoma, squamous cell carcinoma, and melanoma (48, 49, 50, 51, 52, 53), and this is consistent with the hypothesis that the loss of E-cadherin-mediated adhesion potentates tumor invasiveness. Up-regulation of AJ has been considered a promising therapeutic approach to inhibit the invasion and metastasis of malignant tumors (47). 1,25-(OH)2D3 has been shown to reverse the tumoral phenotype of the Bowen-like lesions in reconstituted epidermis in vitro (7), and the results of some studies indicate that vitamin D compounds may be useful in the therapy of solid tumors (6, 8). It is tempting to speculate that the induction of AJ may constitute a novel mechanism of the antineoplastic and antiproliferative effects of 1,25-(OH)2D3.

Received November 22, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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