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The use of polarized salivary gland cell monolayers has contributed to our understanding of salivary gland physiology. However, these cell models are not representative of glandular epithelium in vivo, and, therefore, are not ideal for investigating salivary epithelial functions. The current study has developed a three-dimensional (3D) cell culture model for rat Par-C10 parotid gland cells that forms differentiated acinar-like spheres on Matrigel. These 3D Par-C10 acinar-like spheres display characteristics similar to differentiated acini in salivary glands, including cell polarization, tight junction (TJ) formation required to maintain transepithelial potential difference, basolateral expression of aquaporin-3 and Na+/K+/2Cl− cotransporter-1, and responsiveness to the muscarinic receptor agonist carbachol that is decreased by the anion channel blocker diphenylamine-2-carboxylic acid or chloride replacement with gluconate. Incubation of the spheres in the hypertonic medium increased the expression level of the water channel aquaporin-5. Further, the proinflammatory cytokines tumor necrosis factor-α and interferon-γ induced alterations in TJ integrity in the acinar-like spheres without affecting individual cell viability, suggesting that cytokines may affect salivary gland function by altering TJ integrity. Thus, 3D Par-C10 acinar-like spheres represent a novel in vitro model to study physiological and pathophysiological functions of differentiated acini.
Mammalian salivary glands are composed of acinar and ductal cells (Fig. 1A). To achieve unidirectional saliva secretion, acinar cells must polarize to differentiate distinct apical and basolateral membranes (Fig. 1B). In current models of saliva secretion, the transepithelial movement of Cl− is the primary driving force for fluid and electrolyte secretion by salivary acinar cells (Fig. 1B).1 In salivary epithelium, agonist-induced stimulation of the basolateral muscarinic M3 receptor initiates activation of Ca2+-dependent Cl− channels on the apical surface and K+ channels on the basolateral surface.1 The stimulated efflux of K+ and Cl− down their electrochemical gradients produces a transepithelial potential difference (PD) that drives Na+ transport and water diffusion across the tight junction (TJ), creating an isotonic primary saliva secretion in the gland lumen2,3 (Fig. 1B).
TJs consist of a narrow belt-like structure in the apical region of the lateral plasma membrane and circumferentially bind each cell to its neighbor, thereby contributing to epithelial polarity.4 The major TJ proteins are occludin, claudins, and junctional adhesion molecules.5 These proteins associate with intracellular scaffold proteins called zonula occludens (ZO), which anchor TJ proteins to the actin cytoskeleton5 (Fig. 1B).
Salivary cell lines have been useful for studying salivary gland function and differentiation, most commonly the human salivary gland (HSG) cell line that differentiates into an acinar-like phenotype when grown on a basement membrane, such as Matrigel.6 However, HSG cell cultures on Matrigel are unable to differentiate into polarized monolayers,6 most likely due to inappropriate expression and localization of TJ proteins. A promising alternative to the use of HSG cells is the rat parotid gland (Par-C10) cell line, which forms TJs when grown as two-dimensional (2D) cell monolayers on permeable membranes7,8; however, there are some limitations with 2D cultures, including difficulties pertaining to experimental manipulation, detailed microscopic and biochemical analyses, and detection of essential structural features of glandular epithelium in vivo. The aim of the current study was to develop an in vitro three-dimensional (3D) cell culture model using Par-C10 cells that more closely resembles salivary gland morphology in vivo regarding study of TJs and formation of transepithelial gradients for investigations of physiological and pathological responses in salivary gland acini.
The Par-C10 cell line was derived from freshly isolated rat parotid gland acinar cells by transformation with simian virus 40 (SV40) and exhibits characteristics of freshly isolated acinar cells.9 Fifty microliters of growth-factor-reduced (GFR) Matrigel (8mg/mL; 2:1 GFR Matrigel: Dulbecco's modified Eagle's medium [DMEM]–Ham's F12 [1:1] medium; Becton Dickinson Labware, Franklin Lakes, NJ) was allowed to solidify in a 37°C incubator for 1h in eight-well chambers mounted on #1.5 German borosilicate coverglasses (Nalge Nunc International Corporation, Naperville, IL). Then, Par-C10 cells (1×104 cells/well; passage 40–60) were plated on the GFR Matrigel in DMEM–Ham's F12 (1:1) medium with supplements, as defined previously.7 Differentiated 3D cultures of Par-C10 acinar-like spheres were used for assays after incubation in a 37°C incubator with 5% air and 95% CO2 for 2 days. Osmotic stress was induced in some Par-C10 3D cultures to upregulate aquaporin-5 (AQP5) expression, as described previously.10,11 Accordingly, cells were incubated for 24h in hypertonic serum-free DMEM–Ham's F12 medium containing 200mM sorbitol.
Intralumenal recordings from 3D Par-C10 acinar-like spheres grown on a 35×10mm tissue culture dish (Becton Dickinson Labware) precoated with GFR Matrigel were made using 20–30MΩ glass microelectrodes filled with 0.3M KCl with a tip diameter of ~1μm and an Axoclamp 2A intracellular amplifier (Molecular Devices, Downingtown, PA). Measurements were made of PD between the lumen of the spheres and the bath before, during, and after stimulation of the cells with carbachol (10 or 100μM, as described in figure legends). Data were acquired with a Digidata 1332A data acquisition board (Molecular Devices) and analyzed with Clampfit software (version 9; Molecular Devices).
The intracellular free Ca2+ concentration ([Ca2+]i) was quantified in single cells using an InCyt Dual-Wavelength Fluorescence Imaging System (Intracellular Imaging, Cincinnati, OH), as described previously.7 Increases in [Ca2+]i are expressed as the peak response obtained under the indicated conditions (see Fig. 5C legend) or after subtracting basal [Ca2+]i from the peak [Ca2+]i (see Fig. 5D legend).
Par-C10 acinar-like spheres were fixed in 4% paraformaldehyde for 30min at room temperature, incubated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5min, and washed thrice with PBS. Then, the acinar-like spheres were incubated with 5% goat serum containing 10μM digitonin for 2h at room temperature and washed thrice with PBS. The spheres were incubated overnight at 4°C with the following primary antibodies at 1:500 dilution in 5% goat serum containing 10μM digitonin: rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA), mouse anti-occludin (Invitrogen), rabbit anti-Na+/K+-ATPase (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ATP7A (a gift from Professor Betty Eipper, University of Connecticut Health Center), rabbit anti-muscarinic-3 receptor (Abcam, Cambridge, MA), rabbit anti-Na+/K+/2Cl− cotransporter-1 (NKCC1; Santa Cruz Biotechnology), rabbit anti-aquaporin-3 (AQP3; Millipore, Bedford, MA), rabbit anti-AQP5 (Millipore), or rabbit anti-claudin-3 antibodies. The next day, spheres were warmed to room temperature for 20min and washed thrice for 5min with PBS. Spheres were incubated for 45min with AlexaFluor 488–conjugated goat anti-rabbit or AlexaFluor 594–conjugated goat anti-mouse secondary antibodies (1:500 dilution in 5% goat serum containing 10μM digitonin) and washed thrice with PBS. Spheres were stained for 5min with 1:10,000 dilution in PBS of Hoechst nuclear stain and 1:500 dilution in PBS of phalloidin F-actin stain (Sigma, St. Louis, MO). Images were obtained and analyzed using a Carl Zeiss 510 confocal microscope.
Par-C10 cells were transfected with cDNA encoding the green fluorescent protein (GFP)–tagged human P2Y2 receptor (GFP-P2Y2R; cDNA was a gift from Dr. Fernando A. González, University of Puerto Rico) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's recommendation. After 7 days, G418-resistant cells were pooled and maintained in media containing 0.5mg/mL G418. Further selection was achieved by fluorescence activated cell sorting for GFP.
Par-C10 cells grown on GFR Matrigel were lysed in 200μL of 2× Laemmli buffer, and lysates were sonicated for 5s with a Branson Sonifier 250 (microtip; output level 5; duty cycle 50%) and boiled for 3min. The lysates were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis on mini-gels and transferred to nitrocellulose membranes. Membranes were blocked for 1h with 5% nonfat dry milk in Tris-buffered saline (0.137M NaCl, 0.025M Tris [hydroxymethyl]-aminomethane, pH 7.4) containing 0.1% Tween-20 (TBST) and immunoblotted overnight with rabbit anti-AQP5 antibody (1:1000 dilution; Millipore) at 4°C in TBST containing 3% bovine serum albumin and 0.02% sodium azide. After incubation with the primary antibody, membranes were washed thrice for 15min each with TBST and incubated with peroxidase-linked goat anti-rabbit immunoglobulin G antibody (1:2000 dilution; Santa Cruz Biotechnology) at room temperature for 1h. The membranes were washed thrice for 15min each with TBST and then treated with chemiluminescence detection reagent containing 20mM Tris buffer (pH 8.5), 250mM Luminol, and 90mM coumaric acid (Sigma), and protein bands were observed on X-ray film. For signal normalization, membranes were treated with stripping buffer (0.1M glycine, pH 2.9, and 0.02% sodium azide) and reprobed with rabbit anti-extracellular signal–regulated kinase antibody (1:1000 dilution; Santa Cruz Biotechnology). All experiments were performed in duplicate and repeated at least thrice.
Tumor necrosis factor-α (TNFα; 10ng/mL) and/or interferon-γ (IFNγ; 10ng/mL) (Becton Dickinson Pharmingen, San Diego, CA) were added to 3D Par-C10 acinar-like spheres and incubated for 48h at 37°C, as described previously for Par-C10 cell monolayers.7 The anion channel blocker diphenylamine-2-carboxylic acid (DIDS; 100μM) was incubated with the 3D Par-C10 acinar-like spheres for 30min at 37°C before the PD measurements. In some experiments, chloride in the medium was replaced with gluconate, and the Ca2+ concentration was increased to 4mM to counteract Ca2+ chelation by gluconate.
Data are means±standard error of the mean of results from three or more experiments. p-Values <0.05 calculated from a two tailed t-test represent significant differences. Results from experiments with 100μM carbachol alone were analyzed by a one-sample t-test. Data from subsequent PD measurements for spheres incubated with multiple agents were analyzed by one-way analysis of variance followed by pair-wise post hoc Tukey's t-test where p<0.001 represents significant differences between experimental groups.
Par-C10 cells plated on top of a thin layer of GFR Matrigel formed multicellular acinar-like spheres of ~30–60μm diameter after 2 days in culture. Confocal microscopy sections of acinar-like spheres stained for actin (Fig. 1C) indicated a hollow luminal space that resembled a donut hole whose size increased over 2 days to a sphere diameter of approximately 20μm (Fig. 1C). The luminal region of 3D Par-C10 acinar-like spheres was encircled by actin filaments that were stained intensely with the F-actin fluorescent dye phalloidin (Fig. 1C, green; and Fig. 2H, K, P, S, T, W, X, Z1, red). Fainter actin staining was detected at the plasma membrane (Fig. 1C, green; Fig. 2H, K, P, S, T, W, X, Z1, red). These results indicate that 3D Par-C10 acinar-like spheres form an apical peri-junctional actin ring typical of polarized acinar epithelium. The 3D Par-C10 acinar-like spheres also expressed the TJ proteins occludin (Fig. 2A, red); ZO-1 (Fig. 2B, green) and claudin-3 (Fig. 2E) at the tips of cell–cell contacts surrounding the lumen (i.e., the apical membrane), another indicator of cell differentiation into polarized acinar-like spheres.
The 3D Par-C10 acinar-like spheres expressed M3 muscarinic receptors at the basolateral surface (Fig. 2I, K, green), whereas GFP-P2Y2Rs expressed in Par-C10 spheres were localized to the apical surface (Fig. 2L, green), a receptor distribution consistent with previous studies with polarized lung and colonic epithelium,12,13 as well as Par-C10 cells grown on permeable supports.7 Par-C10 acinar-like spheres expressed AQP3 at the basolateral membrane (Fig. 2M, O, red), consistent with previous studies with rat14 and HSGs.15
The 3D Par-C10 acinar-like spheres also expressed the copper transporter ATP7A in the peri-nuclear region (Fig. 2Q, S, green), consistent with the localization of ATP7A to the Golgi apparatus,16 whereas Na+/K+-ATPase and NKCC1 localized to the lateral membrane (Fig. 2U, W and 2Y, Z1, respectively, green), as described previously for salivary acini.17,18 These findings strongly suggest that the 3D Par-C10 acinar-like spheres are well-differentiated structures.
Par-C10 acinar-like spheres express low levels of the water channel AQP5 (Fig. 3). However, when Par-C10 acinar-like spheres were exposed to the hypertonic medium for 24h, AQP5 protein expression was upregulated (Fig. 3), consistent with previous studies with human airway epithelium,10 SV40 transformed submandibular cells,11 rat lung, and salivary and lacrimal glands.19 The localization of AQP5 could not be determined given that commercially available anti-AQP5 antibodies do not recognize the protein in 3D acinar-like spheres.
Using a microelectrode inserted into the lumen of a 3D Par-C10 acinar-like sphere, a PD of approximately −6 to −10mV was recorded between the lumen and the basolateral medium (Fig. 4B), demonstrating that the acinar-like spheres maintain transepithelial ion gradients required for fluid secretion. Further, there was a significant decrease in PD within ~2min after addition of 100μM carbachol to the basolateral (outer) surface of the spheres (p<0.001; one-sample t-test; Fig. 4B). A similar decrease in PD was seen with 10μM carbachol (Fig. 4B, C). These results demonstrate that 3D Par-C10 acinar-like spheres exhibit muscarinic receptor-mediated changes in transepithelial PD required for saliva secretion in intact acini. The carbachol-induced decrease in PD was abolished in spheres pretreated with the anion channel inhibitor DIDS or when chloride in the medium was replaced with gluconate (Fig. 4B, C), suggesting the presence of muscarinic-receptor-mediated apical chloride efflux into the lumen, the major mechanism underlying fluid secretion in salivary glands.
We tested whether TNFα and/or IFNγ affected TJ integrity or M3 receptor signaling in 3D Par-C10 acinar-like spheres. As shown in Figure 5A, the TJ protein ZO-1 (green) was located at intercellular junctions near the apical surface in untreated Par-C10 acinar-like spheres, indicative of localization to TJs. However, a 48h treatment with TNFα and/or IFNγ caused ZO-1 protein re-distribution throughout the apical membrane, which correlated with the generation of disorganized lumens of decreased size, as compared to untreated controls (Fig. 5A). Luminal disorganization was most pronounced when acinar-like spheres were treated with both TNFα and IFNγ (Fig. 5A). The 48h treatment with TNFα and/or IFNγ also caused a significant decrease in carbachol-induced PD (Fig. 5B). These results suggest that pro-inflammatory cytokines disrupt TJ integrity in 3D Par-C10 acinar-like spheres. Carbachol (100μM) induced an increase in [Ca2+]i in Par-C10 acinar-like spheres in the absence of cytokines (Fig. 5C), consistent with results from previous studies with Par-C10 cells.20 In contrast to the effects of cytokines on TJ integrity or PD in Par-C10 acinar-like spheres (Fig. 5A, B), TNFα and/or IFNγ treatment for 48h had no effect on increases in [Ca2+]i induced by 100μM carbachol in individual cells of 3D Par-C10 acinar-like spheres (Fig. 5D), indicating that intracellular calcium signaling is unaffected by these cytokines.
In this study, we demonstrate that rat parotid Par-C10 cells are capable of differentiating into 3D acinar-like structures that exhibit numerous features of glandular epithelium in vivo. These features include the formation of cyst-like spheroids with a hollow lumen, polarization of the cells to differentiate basolateral and apical surfaces (Figs. 1C and and2),2), and the establishment of a transepithelial PD upon muscarinic receptor activation (Fig. 4B, C) that is essential for saliva secretion in vivo.21
The apical versus basolateral distribution of several ion transporters and receptors within the 3D Par-C10 acinar-like spheres resembles their distribution in intact salivary glands. Apical expression of the P2Y2 nucleotide receptor (Fig. 2L) and basolateral expression of the M3 muscarinic receptor (Fig. 2I, K) mimic their distribution in Par-C10 cells grown on permeable supports.8 Our previous studies indicate that P2Y2R mRNA or activity is barely detectable in native salivary glands; however, the P2Y2R is upregulated in isolated salivary gland cells cultured for 24h,22 in vivo 3 days after ligation of salivary gland ducts in rats,23 and in established salivary gland cell lines, including Par-C10 cells,7,24,25 indicating that the P2Y2R is upregulated in acinar cells undergoing dedifferentiation. Although the expression levels of the P2Y2R have not been quantitated in Par-C10 3D cell cultures that express the P2Y2R on the apical membrane only, it is highly likely that P2Y2R expression levels are lower in the acinar-like spheres than in undifferentiated Par-C10 cells. These studies suggest that Par-C10 cells are capable of transitioning between an undifferentiated and differentiated state in 3D culture and that P2Y2R distribution could be used as a marker to determine the state of differentiation.
The expression levels of the water transporter AQP5 in Par-C10 acinar-like spheres were very low under basal conditions (Fig. 3), although expression of AQP5 was upregulated in Par-C10 acinar-like spheres exposed to the hyperosmotic medium (Fig. 3). These data suggest that the Par-C10 acinar-like spheres are capable of mediating increased water permeability associated with saliva secretion. These results are consistent with previous studies with other tissues and species demonstrating that hyperosmotic stress induces increases in aquaporin expression at the mRNA and protein levels.10,11,19 Par-C10 acinar-like spheres also express AQP3 on the basolateral membrane (Fig. 2M), consistent with previous studies with rat14 and HSGs.15 These results indicate that Par-C10 acinar-like spheres represent a valuable tool for studying mechanisms underlying fluid secretion.
Par-C10 acinar-like spheres in the present study lack amylase expression (data not shown), consistent with previous observations using 2D cultures of Par-C10 cells grown on permeable supports where it was suggested that the activities of signaling molecules downstream of the β-adrenergic receptor may be defective, including Gs, adenylyl cyclase, and CFTR.8 This would result in a decrease in cyclic adenosine monophosphate (cAMP)-dependent cellular responses and may relate to the culture conditions or medium constituents used, including the presence of cholera toxin routinely employed to promote a differentiated state of Par-C10 cells. Therefore, it is predicted that the conditions described here for the culture of Par-C10 acinar-like spheres are not conducive to monitoring amylase secretion. However, a later study demonstrated that SV40-transformed rat parotid gland cells treated with rat serum showed a 16-fold increase in amylase content.26 These studies suggest that Par-C10 3D acinar-like spheres may be capable of amylase secretion in the presence of factors provided by rat serum.
The distribution of TJ proteins to the lateral membrane at the apical surface of acinar-like spheres (Fig. 2A, B, D, E, G) resembles TJ protein distribution in HSGs and rodent salivary glands.27,28 Further, the peri-junctional actin ring encircling the apical pole of cells in the acinar-like spheres (Figs. 1C and 2H, K, P, S, T, W, X, Z1) is structurally similar to native polarized acini.29
The Menkes protein (ATP7A) is a copper-transporting P-type ATPase located in the trans-Golgi network whose dysfunction causes a fatal neurodegenerative disorder, Menkes disease.30 In the 3D Par-C10 acinar-like spheres, ATP7A is restricted to the peri-nuclear region (Fig. 2Q, S), consistent with localization to Golgi and suggesting that the acinar-like spheres delineate intracellular organelles similar to intact rat parotid glands.31 Further, the lateral distribution of Na+/K+-ATPase (Fig. 2U, W) is similar to the distribution of this active transporter in polarized epithelium.32
The acinar NKCC1 has been described as the major Cl− uptake mechanism in acinar cells and is critical for driving saliva secretion in vivo.17 In the Par-C10 acinar-like spheres, NKCC1 was observed at the basolateral membrane (Fig. 2Y, Z1), consistent with its distribution in vivo in parotid glands.17
Although salivary gland cells have been characterized in 3D culture,33–35 this is the first study to demonstrate that 3D acinar-like cultures are capable of establishing a transepithelial PD that is modulated by the muscarinic receptor agonist carbachol (Fig. 4B, C). This response is inhibited by the anion channel blocker DIDS and when chloride in the medium was replaced with gluconate, consistent with carbachol-induced chloride efflux into the lumen, the major mechanism underlying fluid secretion in salivary glands.2,3 Thus, 3D Par-C10 acinar-like spheres apparently exhibit muscarinic-receptor-mediated stimulation of apical Ca2+-dependent Cl− channels, which generates an increase in the transepithelial PD that drives the retrograde diffusion of Na+ and water across the epithelium (Fig. 1B).1
Recently, we have shown that Par-C10 cell monolayers exposed to TNFα and/or IFNγ exhibit a variety of responses, including decreases in transepithelial resistance and agonist-induced Isc, increased paracellular permeability to normally impermeant molecules, and altered TJ morphology associated with reduced expression of the TJ protein claudin-1, suggesting a loss in TJ integrity.7 Similarly, TNFα and/or IFNγ altered ZO-1 distribution in 3D Par-C10 acinar-like spheres (Fig. 5A) and decreased lumen size (Fig. 5A). Additionally, treatment with TNFα and IFNγ inhibited carbachol-induced changes in PD. Because TNFα and/or IFNγ levels are increased in the salivary glands of patients with Sjögren's Syndrome (SS),36,37 the present study supports the hypothesis that cytokines decrease saliva secretion by altering TJ integrity. The 3D Par-C10 acinar-like spheres exhibit increases in [Ca2+]i in response to carbachol, similar to previous studies in Par-C10 cells grown on permeable supports.20 In intact parotid acinar cells or in Par-C10 cells grown on plastic, carbachol-induced intracellular calcium mobilization consists of two phases, transient (which is reached within 1–2s after carbachol addition38) and sustained (in which [Ca2+]i returns toward the basal levels over the next minute and stabilized).9,38 In 3D Par-C10 acinar-like spheres, the intracellular calcium levels do not return completely to basal levels at the times studied (Fig. 5C), indicating a difference with native parotid cells or Par-C10 cells grown on plastic. TNFα and/or IFNγ had no effect on intracellular calcium signaling induced by carbachol in Par-C10 cell monolayers (Fig. 5D), indicating that cytokines are not affecting cell viability, although they alter TJ protein distribution.
In conclusion, the 3D Par-C10 acinar-like spheres described in this study may represent a useful cell culture model for investigating the physiological and pathological functions of salivary glands, including ion and fluid secretion and TJ structure and function. Additionally, 3D Par-C10 acinar-like spheres may be useful for studying acinar formation in 3D biopolymer scaffolds for tissue engineering applications.
The authors are grateful to Esteban Fernandez and Stephanie Boyle of the University of Missouri Molecular Cytology Core Facility for assistance in the imaging of specimens for this study. This work was supported by the NIH-NIDCR grants R01 DE017591, R01 DE07389, and K08 DE017633, and a Sjögren's Syndrome Foundation Grant.
No competing financial interests exist.