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Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro
Biochemical and Biophysical Research Communications xxx (2017) 1e5
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Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro Katharina S. Mießler a, Alexander G. Markov b, Salah Amasheh a, * a b
€t Berlin, Oertzenweg 19b, 14163 Berlin, Germany Institute of Veterinary Physiology, Freie Universita Department of General Physiology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2017 Accepted 17 November 2017 Available online xxx
During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation. © 2017 Elsevier Inc. All rights reserved.
Keywords: Barrier function Tight junction Ussing chamber Hydrostatic pressure Mammary gland HC11
1. Introduction Mammary gland (MG) epithelium is functionally transformed during physiological differentiation, namely mammogenesis, lactogenesis, and involution [1]. This functional adaptation from an undifferentiated state to a secretory epithelium and subsequent involution is dependent on an orchestrated action of hormones, such as cortisol and prolactin [2e4]. Lactation includes secretion of milk ingredients such as electrolytes, lactose, casein and milk fat [5]. Apart from the direct movement of ions dependent on distribution and transmembrane transport mechanisms, prerequisite for a controlled vectorial transcellular transport in epithelia is an intact barrier function [6,7]. Thus, differentiation into a secretory epithelium is dependent on an assembly and structural integrity of the tight junction (TJ) [8]. TJs are located in the apicolateral membrane of the epithelial cells, and
* Corresponding author. E-mail address: [email protected] (S. Amasheh).
are organized in strands. These strands interconnect neighboring epithelial cells and determine permeability of the paracellular pathway. Within these strands, a number of tetraspan TJ proteins have been identified, namely occludin, tricellulin, and the family of claudins [9e11]. The latter family is regarded to primarily determine organ- and tissue specific paracellular permeability [12]. Extracellular loops of these proteins mediate interaction within TJ strands in trans, and an interaction with the actin filaments is provided via PDZ-domain binding scaffolding protein, ZO-1 [13,14]. Molecular TJ analysis was initiated by the identification of occludin [9]. Surprisingly, a knock out of the protein did not induce a change of intestinal barrier function [15]. Comparable results could be observed in tricellulin analyses [16]. In contrast, claudin deficiency has been described to induce major perturbation of paracellular permeability, and thus has been described in context with a wide variety of hereditary and acquired diseases [17]. Among the family of claudins, crucial function of single members for organ and tissue physiology has been demonstrated, e.g. claudin-1 in skin [18], claudin-5 within the capillary endothelium providing the blood brain barrier [19], and claudin-16
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Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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in kidney [20]. Moreover, knockout of ZO-1 has lethal consequences for the organism during embryogenesis [21]. Although a primarily barrier determining function of TJ proteins is assumed, known as the gate function, additional functions such as fence function, and mechanical adhesive functions have been described [22,23]. In vivo, reactions of mouse MGs to high intraglandular pressures and the influence on TJ proteins have been studied recently [24]. In vitro, first results were accomplished in analyzing effects of increased pressure on epithelial reactions in lung tissue [25]. In lactating MGs, pressure analysis became accessible by establishment of a mammary monolayer model exposed to hydrostatic pressure (HP) in a modified Ussing chamber [26]. The method provided the application of up to 10 kPa HP induced by bathing solution, which was located in an attached tube system. Based on the outcome of this study, we hypothesized that pressure induces a regulation of barrier properties and TJ proteins in cultured monolayers of the mammary epithelial cell line HC11, in vitro. 2. Material and methods 2.1. Cell lines and handling HC11 is a mouse MG epithelial cell line [27]. If not stated otherwise, cells were maintained in an atmosphere of 5% CO2 at 37 C and cultivated in growth medium, which comprises RPMI 1640 medium (PAN, Biotech, Aidenbach, Germany) supplemented with 5 mg insulin per ml, 10 ng Epithelial Growth Factor (EGF) per ml (Biochrom, Berlin, Germany), 10% fetal calf serum (FCS, Biochrom, Berlin, Germany) and 100 U/mg penicillin/streptomycin per ml (Biochrom, Berlin, Germany). Monolayers were seeded on PCF cell culture inserts with 12 mm diameter and 0.4 mm pore size (Merck Millipore, Darmstadt, Germany) and used between passages 50 and 75. After reaching confluency with growth medium cells were maintained until they reached an age of 14 ± 1 day without supplementation of EGF (induction medium). Hormone supplementation was performed by adding 100 nM dexamethasone (Sigma-Aldrich, Munich, Germany) and 5 mg prolactin (Sigma-Aldrich, Munich, Germany) per ml to the induction medium (differentiation medium). Cells were cultivated with differentiation medium during the last three days before the experiments. An EVOM (World Precision Instruments, Sarasota, USA) was used to manually measure the transepithelial resistance (RT). Data were converted to U $ cm2 after blank value was subtracted.
2.3. Solutions and drugs The bathing solution used in Ussing chamber experiments was adjusted to pH 7.4 ± 0.1 with HCl and contained (in mM): 113.6 NaCl, 5.4 KCl, 0.6 NaH2PO4, 2.4 Na2HPO4, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, 5 Glucose, 5 HEPES. Solution was gassed with 95% O2 and 5% CO2, and temperature was maintained at 37 C using water bath. 2.4. Quantification of TJ proteins by Western blot technique To determine the expression of TJ proteins, cell filters were taken from the Ussing chambers. Cells were lysed in total lysis buffer containing 25 mM HEPES (pH 7.6), 2 mM EDTA, 25 mM NaF, 1% SDS, and protease inhibitors (Complete, Boehringer, Mannheim, Germany) and homogenized by 20 s of ultrasonic incubation and passage through a 29-gauge 1/2 needle. Whole cell lysate of 1e3 filters was analyzed as pool-sample by Western blotting. Protein concentrations were determined by Biorad DC protein assay (BioRad, München, Germany) and measured in an EnSpire Multimode Plate Reader (PerkinElmer, Rodgau, Germany). Aliquots of 18.9 mg were separated by gel electrophoresis with mini-Protean TXG stain free gradient gels 4e20% (Bio-Rad, München, Germany) for succeeding electrophoresis. Blots were incubated with primary mouse monoclonal IgG antibodies directed against occludin, claudin-4, ZO-1 and b-actin, and with primary rabbit polyclonal IgG antibodies directed against claudin-3 (Life technologies GmbH, Darmstadt, Germany). POD conjugated goat anti-rabbit IgG or goat antimouse IgG antibodies and the Clarity Western ECL (Bio-Rad, München, Germany) chemiluminescence detection system were used to detect bound antibodies. Chemiluminescence signals were detected using a ChemiDoc MP (Bio-Rad, München, Germany) luminescence imager. 2.5. Validation of TJ proteins by immunofluorescence staining To proceed immunofluorescent staining of TJ proteins, immediate ethanol fixation, permeabilization with 0.5% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) and blocking of unspecific proteins with 5% goat serum diluted in PBS was performed. Primary antibodies mouse anti-occludin, rabbit anti-claudin-3, mouse anticlaudin-4 and mouse anti-ZO-1, and secondary antibodies (dilution 1:500) Alexa fluor 488 goat anti-mouse (green) and Alexa fluor 594 goat anti-rabbit (red) were applied for detection of TJ proteins; (Life technologies GmbH, Darmstadt, Germany). Staining with 4’.6diamidino-2-phenylindole (DAPI) was employed for nuclei and a Leica DMI6000 B microscope (Leica, Wetzlar, Germany) was used for immunofluorescence microscopy.
2.2. Bilateral HP incubation 2.6. Statistical analysis Modified Ussing chambers were mounted with cell inserts and the base level of 1 kPa bathing solution was filled to both sides of the cell monolayer. Voltage clamp conditions were used, and equilibration was performed until RT was stable. In a first approach cells grown without hormone supplementation (undifferentiated) were used. Bathing solution was added on both sides of the cell inserts to a HP of 10 kPa and monolayers were incubated under these conditions for 4 h. In a second approach the same experimental protocol was used with hormone supplemented (differentiated) cells. Both series were repeated with the addition of 1 mM barium chloride (BaCl2) to the bathing solution to inhibit transcellular transport mechanisms. Controls were maintained at the base level of 1 kPa and otherwise treated equally. Prerequisite for the current outcome of the study were stable parameters such as pH, temperature, buffer composition, and defined pressure application, as reported in detail recently [26].
For the starting point and the last minute of every hour average of RT and ISC were calculated. For Western blot quantification the Image Lab Software (Bio-Rad, Hercules, USA) was used to perform background subtraction and normalization to the inter-run calibrator, followed by normalization to b-actin as standard protein. Statistics were done with the unpaired Student's t-test and p-values < 0.05 were considered to be significant. Results are presented as means ± SEM (standard error of the mean) of the obtained data. 3. Results 3.1. Transepithelial electrical resistance Differentiated cells showed a higher RT than undifferentiated cells after 14 ± 1 days of cultivation: 1365 ± 37 U cm2 versus
Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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890 ± 50 U cm2; (p < 0.001, n ¼ 34 and 36). RT remained stable during pressure incubation in all experimental setups, compared to controls. After pressure incubation undifferentiated monolayers revealed RT values of (sample versus control) 486 ± 68 U cm2 versus 479 ± 55 U cm2 without BaCl2 (n ¼ 11) and 458 ± 117 U cm2 versus 617 ± 92 U cm2 with BaCl2 (n ¼ 6). After induction of differentiation by dexamethasone and prolactin, RT values of 879 ± 53 U cm2 versus 954 ± 38 U cm2 without BaCl2 (n ¼ 12), and 890 ± 90 U cm2 versus 1017 ± 72 U cm2 with BaCl2 (n ¼ 6) were measured. In all experimental setups bilateral HP of 10 kPa had no marked effect on RT during 4 h of incubation compared to controls (n ¼ 11). 3.2. Short circuit current During HP incubation with 10 kPa no significant change of ISC was observed in undifferentiated cells (n ¼ 11; Fig. 1), however, with addition of 1 mmol/l BaCl2 (n ¼ 6), a strong deviation of ISC was observed. In differentiated monolayers, ISC decreased significantly after 2 h (n ¼ 12), which was blocked by BaCl2 (n ¼ 6), indicating inhibition of chloride secretion. After 4 h, the effect was still observed, but not inhibited by BaCl2. This effect could not be observed in undifferentiated cells. 3.3. Western blotting Western blot analysis was used to verify the presence of TJ proteins and to quantify their expression, compared to controls. Western blot revealed signals for occludin, claudin-3, claudin-4 and ZO-1 in both, samples and controls of the experimental setups with and without BaCl2. However, significant effects were only observed for occludin in undifferentiated cells (Fig. 2A) and for ZO-1 in differentiated cells (Fig. 2B). In undifferentiated cells, after pressure incubation a decrease in the concentration of occludin could be observed. In differentiated cells no change of occludin could be found. However, a marked increase of ZO-1 was observed, which on
Fig. 2. Relative Western blot intensity after 4 h of 10 kPa bilateral HP incubation in undifferentiated (Ud, A) and differentiated (D, B) cells with and without the addition of 1 mM BaCl2, compared to controls. Bars represent means ± SEM, (C) representative images of Western blots (*p < 0.05, n ¼ 3). A: In undifferentiated cells, occludin showed a significant lower intensity after pressure incubation compared to controls, (0.47 ± 0.27 versus 1.50 ± 0.06). This effect could not be observed after the addition of BaCl2. No marked changes in the expression level of claudin-3, claudin-4 and ZO-1 could be observed. B: In differentiated cells, ZO-1 showed a significant higher intensity after pressure incubation compared to controls, (2.06 ± 0.37 versus 0.91 ± 0.11). On the contrary, with addition of BaCl2 a significant lower intensity was observed in ZO-1 compared to controls, (1.50 ± 0.12 versus 2.20 ± 0.16). No marked changes in the expression level of occludin, claudin-3 and claudin-4 could be observed. (C) Representative Western blot signals.
Fig. 1. ISC at the beginning and after 2 and 4 h of 10 kPa bilateral pressure incubation in undifferentiated (Ud) and differentiated (D) cells with and without the addition of 1 mM BaCl2, compared to controls. Bars represent means ± SEM, (*p < 0.05, n ¼ 6 to 12). In undifferentiated cells, no significant changes of short circuit current (ISC) could be observed, compared to controls. In differentiated cells, ISC decreased from the initial 4.7 ± 0.4 mA/cm2 to 2.9 ± 0.5 mA/cm2 after 2 h and ended with 2.8 ± 0.6 mA/cm2 after 4 h of pressure incubation, compared to controls with 5.4 ± 0.4 mA/cm2 (start), 4.8 ± 0.5 mA/cm2 (2 h) and 5.0 ± 0.6 mA/cm2 (4 h). With the addition of 1 mM BaCl2, ISC started at 5.4 ± 0.8 mA/cm2, followed by 3.6 ± 0.8 mA/cm2 after 2 h and 2.9 ± 0.5 mA/cm2 after 4 h of pressure incubation, compared to controls with 5.9 ± 0.8 mA/cm2 (start), 4.4 ± 0.5 mA/cm2 (2 h) and 4.2 ± 0.3 mA/cm2 (4 h).
the contrary, was changed to a decrease after addition of BaCl2, (n ¼ 3).
3.4. Immunofluorescence microscopy Immunofluorescence microscopy was used to localize TJ proteins and to examine structural condition of the HC11 monolayers after 4 h of 10 kPa bilateral HP incubation compared to controls. Integrity of the monolayers was preserved throughout the experiments. In controls, monolayer integrity was visible by occludin,
Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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claudin-3, claudin-4 and ZO-1, which were continuously located in TJs of undifferentiated and differentiated cells (not shown). In accordance with Western blots, in undifferentiated cells, occludin showed a marked reduction after pressure incubation. Claudin-3, claudin-4 and ZO-1 showed no marked changes in distribution or concentration and all proteins could be detected in TJs after pressure incubation and were still located in the apicolateral membrane areas. The same observations were made in differentiated cells with the difference that the reduction of occludin could not be detected, (n ¼ 3). 4. Discussion Recent analysis of TJ regulation in MGs revealed (i) formation of a tight barrier during lactogenesis [28,29], and (ii) changes of TJ protein expression and localization during accumulation of milk [24]. Moreover, a disappearance of adhesive contacts and desmosomes between cells is induced in the alveolar epithelium by the formation to lactation period [23]. Consequently, integrity of the epithelial structure and paracellular transport is determined by TJ proteins, which highlights the relevance of their regulation during maturation and lactation. Accordingly, an epithelial cell model was established representing the mammary epithelial monolayer, namely HC11 [27,30]. The influence of pressure on mammary epithelial cells was recently studied in a mouse model, in vivo [24]. In our previous study, pressure was generated by accumulation of milk in the MG during 20 h of interrupted suckling. Molecular analysis revealed an increased expression of the tightening TJ proteins claudin-1 and -3 and a reduction of claudin-2 and -16, which are pore-forming proteins. The change in TJ expression was interpreted as a mechanism against a back-leak of milk components and might have been induced by mechanical stimuli [24]. However, it is known that milk can affect epithelial barrier properties, too [31]. In this context, previous analyses have demonstrated that several milk components have effects on intestinal barrier function, e.g. laurate, glutamate and lactoferrin [32e34], and therefore might also affect mammary TJs. Whereas the mouse model of interrupted suckling revealed important insights into the physiological regulation of mammary TJ during lactation, it was not suitable for a differentiation between regulative effects induced by hormones, milk components, and pressure. The present study however, focuses on the identification of pressure effects, which turned out to have effects on ZO-1 in differentiated cells, whereas in an undifferentiated state occludin was affected, which was also reflected in immunostainings. Transepithelial resistance of differentiated mammary monolayers, most accurately representing the situation in MGs in vivo, was not significantly affected by pressure alone. One important finding of the study is the observation, that claudin-3 and -4 were not affected by pressure incubation in undifferentiated and differentiated monolayers. This might indicate that (i) barrier function is not actively regulated by pressure effects on claudins alone, and (ii) alterations in MG claudin expression may not be dependent on pressure. The latter finding may also indicate that further functions of claudin-3 and -4, which have been also described as tumor markers relevant for proliferation, apoptosis, migration and in vivo growth of mammary epithelial cells [35e37], may not be changed by pressure alone. During lactogenesis, MG epithelium is transformed into a secretory tissue. As ion transport has been reported to be highly dependent on mechanical stimuli in a variety of organs and tissues, including lung epithelium, the urinary bladder, and intestine [25,38,39], we also addressed the question whether an effect of pressure on secretion might occur. Surprisingly, with increasing HP,
BaCl2 sensitive Cl secretion could be observed in differentiated monolayers. However, a limitation of our experimental approach might be the high volumes in Ussing chambers reservoirs necessary for pressure incubation, which limit the application of inhibitors. BaCl2 has been reported to provide a high selectivity for analysis of chloride secretion via inhibition of inward rectifier potassium channels as a requirement for the driving force of Cl secretion, though [40,41]. Using BaCl2, chloride secretion was blocked in differentiated cells after 2 h of pressure incubation, inhibiting a decrease of ISC. However, chloride secretion was not continuously blocked until end of pressure incubation, which might indicate a pressure adaptation mechanism. On molecular level, the expression of ZO-1 was upregulated without BaCl2, but decreased after addition of BaCl2. This mechanism might reflect the functional and regulatory interaction of barrier and transport mechanisms, as demonstrated for single TJ proteins and transport before [42e44]. In conclusion, our current study revealed that bilateral HP incubation in vitro induces changes to both, paracellular barrier and transcellular transport, which represent major physiological mechanisms during lactation. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments €llig, Uwe Tietjen, and We thank Martin Grunau, Katharina So Susanne Trappe for excellent technical assistance. This work was supported by the Center for International Cooperation of the Freie €t Berlin [grant number FSP2016-300, and 1.60.221.2017], Universita the Deutsche Forschungsgemeinschaft [grant number AM 141/111], SPbU grant No. 1.40.486.2017, and the Sonnenfeld Stiftung [doctoral studies grant to K.S. Miebler]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.110. References [1] F. Borellini, T. Oka, Growth control and differentiation in mammary epithelial cells, Environ. Health Perspect. 80 (1989) 85e99. [2] C. Brisken, S. Kaur, T.E. Chavarria, et al., Prolactin controls mammary gland development via direct and indirect mechanisms, Dev. Biol. 210 (1999) 96e106. [3] M. Ono, T. Oka, The differential actions of cortisol on the accumulation of alactalbumin and casein in mid-pregnant mouse mammary gland in culture, Cell 19 (1980) 473e480. [4] W. Schneider, G. Shyamala, Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol, Endocrinology 116 (1985) 2656e2662. [5] J. Cerbulis, H.M. Farrell Jr., Composition of milks of dairy cattle. I. Protein, lactose, and fat contents and distribution of protein fraction, J. Dairy Sci. 58 (1975) 817e827. [6] J.L. Linzell, M. Peaker, Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism, J. Physiol. 216 (1971) 683e700. [7] S. Amasheh, C. Barmeyer, C.S. Koch, et al., Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis, Gastroenterology 126 (2004) 1711e1720. [8] M. Itoh, M.J. Bissell, The organization of tight junctions in epithelia: implications for mammary gland biology and breast tumorigenesis, J. Mammary Gland. Biol. Neoplasia 8 (2003) 449e462. [9] M. Furuse, T. Hirase, M. Itoh, et al., Occludin: a novel integral membrane protein localizing at tight junctions, J. Cell. Biol. 123 (1993) 1777e1788. [10] M. Furuse, K. Fujita, T. Hiiragi, et al., Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to
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Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro Katharina S. Mießler a, Alexander G. Markov b, Salah Amasheh a, * a b
€t Berlin, Oertzenweg 19b, 14163 Berlin, Germany Institute of Veterinary Physiology, Freie Universita Department of General Physiology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2017 Accepted 17 November 2017 Available online xxx
During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation. © 2017 Elsevier Inc. All rights reserved.
Keywords: Barrier function Tight junction Ussing chamber Hydrostatic pressure Mammary gland HC11
1. Introduction Mammary gland (MG) epithelium is functionally transformed during physiological differentiation, namely mammogenesis, lactogenesis, and involution [1]. This functional adaptation from an undifferentiated state to a secretory epithelium and subsequent involution is dependent on an orchestrated action of hormones, such as cortisol and prolactin [2e4]. Lactation includes secretion of milk ingredients such as electrolytes, lactose, casein and milk fat [5]. Apart from the direct movement of ions dependent on distribution and transmembrane transport mechanisms, prerequisite for a controlled vectorial transcellular transport in epithelia is an intact barrier function [6,7]. Thus, differentiation into a secretory epithelium is dependent on an assembly and structural integrity of the tight junction (TJ) [8]. TJs are located in the apicolateral membrane of the epithelial cells, and
* Corresponding author. E-mail address: [email protected] (S. Amasheh).
are organized in strands. These strands interconnect neighboring epithelial cells and determine permeability of the paracellular pathway. Within these strands, a number of tetraspan TJ proteins have been identified, namely occludin, tricellulin, and the family of claudins [9e11]. The latter family is regarded to primarily determine organ- and tissue specific paracellular permeability [12]. Extracellular loops of these proteins mediate interaction within TJ strands in trans, and an interaction with the actin filaments is provided via PDZ-domain binding scaffolding protein, ZO-1 [13,14]. Molecular TJ analysis was initiated by the identification of occludin [9]. Surprisingly, a knock out of the protein did not induce a change of intestinal barrier function [15]. Comparable results could be observed in tricellulin analyses [16]. In contrast, claudin deficiency has been described to induce major perturbation of paracellular permeability, and thus has been described in context with a wide variety of hereditary and acquired diseases [17]. Among the family of claudins, crucial function of single members for organ and tissue physiology has been demonstrated, e.g. claudin-1 in skin [18], claudin-5 within the capillary endothelium providing the blood brain barrier [19], and claudin-16
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Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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in kidney [20]. Moreover, knockout of ZO-1 has lethal consequences for the organism during embryogenesis [21]. Although a primarily barrier determining function of TJ proteins is assumed, known as the gate function, additional functions such as fence function, and mechanical adhesive functions have been described [22,23]. In vivo, reactions of mouse MGs to high intraglandular pressures and the influence on TJ proteins have been studied recently [24]. In vitro, first results were accomplished in analyzing effects of increased pressure on epithelial reactions in lung tissue [25]. In lactating MGs, pressure analysis became accessible by establishment of a mammary monolayer model exposed to hydrostatic pressure (HP) in a modified Ussing chamber [26]. The method provided the application of up to 10 kPa HP induced by bathing solution, which was located in an attached tube system. Based on the outcome of this study, we hypothesized that pressure induces a regulation of barrier properties and TJ proteins in cultured monolayers of the mammary epithelial cell line HC11, in vitro. 2. Material and methods 2.1. Cell lines and handling HC11 is a mouse MG epithelial cell line [27]. If not stated otherwise, cells were maintained in an atmosphere of 5% CO2 at 37 C and cultivated in growth medium, which comprises RPMI 1640 medium (PAN, Biotech, Aidenbach, Germany) supplemented with 5 mg insulin per ml, 10 ng Epithelial Growth Factor (EGF) per ml (Biochrom, Berlin, Germany), 10% fetal calf serum (FCS, Biochrom, Berlin, Germany) and 100 U/mg penicillin/streptomycin per ml (Biochrom, Berlin, Germany). Monolayers were seeded on PCF cell culture inserts with 12 mm diameter and 0.4 mm pore size (Merck Millipore, Darmstadt, Germany) and used between passages 50 and 75. After reaching confluency with growth medium cells were maintained until they reached an age of 14 ± 1 day without supplementation of EGF (induction medium). Hormone supplementation was performed by adding 100 nM dexamethasone (Sigma-Aldrich, Munich, Germany) and 5 mg prolactin (Sigma-Aldrich, Munich, Germany) per ml to the induction medium (differentiation medium). Cells were cultivated with differentiation medium during the last three days before the experiments. An EVOM (World Precision Instruments, Sarasota, USA) was used to manually measure the transepithelial resistance (RT). Data were converted to U $ cm2 after blank value was subtracted.
2.3. Solutions and drugs The bathing solution used in Ussing chamber experiments was adjusted to pH 7.4 ± 0.1 with HCl and contained (in mM): 113.6 NaCl, 5.4 KCl, 0.6 NaH2PO4, 2.4 Na2HPO4, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, 5 Glucose, 5 HEPES. Solution was gassed with 95% O2 and 5% CO2, and temperature was maintained at 37 C using water bath. 2.4. Quantification of TJ proteins by Western blot technique To determine the expression of TJ proteins, cell filters were taken from the Ussing chambers. Cells were lysed in total lysis buffer containing 25 mM HEPES (pH 7.6), 2 mM EDTA, 25 mM NaF, 1% SDS, and protease inhibitors (Complete, Boehringer, Mannheim, Germany) and homogenized by 20 s of ultrasonic incubation and passage through a 29-gauge 1/2 needle. Whole cell lysate of 1e3 filters was analyzed as pool-sample by Western blotting. Protein concentrations were determined by Biorad DC protein assay (BioRad, München, Germany) and measured in an EnSpire Multimode Plate Reader (PerkinElmer, Rodgau, Germany). Aliquots of 18.9 mg were separated by gel electrophoresis with mini-Protean TXG stain free gradient gels 4e20% (Bio-Rad, München, Germany) for succeeding electrophoresis. Blots were incubated with primary mouse monoclonal IgG antibodies directed against occludin, claudin-4, ZO-1 and b-actin, and with primary rabbit polyclonal IgG antibodies directed against claudin-3 (Life technologies GmbH, Darmstadt, Germany). POD conjugated goat anti-rabbit IgG or goat antimouse IgG antibodies and the Clarity Western ECL (Bio-Rad, München, Germany) chemiluminescence detection system were used to detect bound antibodies. Chemiluminescence signals were detected using a ChemiDoc MP (Bio-Rad, München, Germany) luminescence imager. 2.5. Validation of TJ proteins by immunofluorescence staining To proceed immunofluorescent staining of TJ proteins, immediate ethanol fixation, permeabilization with 0.5% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) and blocking of unspecific proteins with 5% goat serum diluted in PBS was performed. Primary antibodies mouse anti-occludin, rabbit anti-claudin-3, mouse anticlaudin-4 and mouse anti-ZO-1, and secondary antibodies (dilution 1:500) Alexa fluor 488 goat anti-mouse (green) and Alexa fluor 594 goat anti-rabbit (red) were applied for detection of TJ proteins; (Life technologies GmbH, Darmstadt, Germany). Staining with 4’.6diamidino-2-phenylindole (DAPI) was employed for nuclei and a Leica DMI6000 B microscope (Leica, Wetzlar, Germany) was used for immunofluorescence microscopy.
2.2. Bilateral HP incubation 2.6. Statistical analysis Modified Ussing chambers were mounted with cell inserts and the base level of 1 kPa bathing solution was filled to both sides of the cell monolayer. Voltage clamp conditions were used, and equilibration was performed until RT was stable. In a first approach cells grown without hormone supplementation (undifferentiated) were used. Bathing solution was added on both sides of the cell inserts to a HP of 10 kPa and monolayers were incubated under these conditions for 4 h. In a second approach the same experimental protocol was used with hormone supplemented (differentiated) cells. Both series were repeated with the addition of 1 mM barium chloride (BaCl2) to the bathing solution to inhibit transcellular transport mechanisms. Controls were maintained at the base level of 1 kPa and otherwise treated equally. Prerequisite for the current outcome of the study were stable parameters such as pH, temperature, buffer composition, and defined pressure application, as reported in detail recently [26].
For the starting point and the last minute of every hour average of RT and ISC were calculated. For Western blot quantification the Image Lab Software (Bio-Rad, Hercules, USA) was used to perform background subtraction and normalization to the inter-run calibrator, followed by normalization to b-actin as standard protein. Statistics were done with the unpaired Student's t-test and p-values < 0.05 were considered to be significant. Results are presented as means ± SEM (standard error of the mean) of the obtained data. 3. Results 3.1. Transepithelial electrical resistance Differentiated cells showed a higher RT than undifferentiated cells after 14 ± 1 days of cultivation: 1365 ± 37 U cm2 versus
Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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890 ± 50 U cm2; (p < 0.001, n ¼ 34 and 36). RT remained stable during pressure incubation in all experimental setups, compared to controls. After pressure incubation undifferentiated monolayers revealed RT values of (sample versus control) 486 ± 68 U cm2 versus 479 ± 55 U cm2 without BaCl2 (n ¼ 11) and 458 ± 117 U cm2 versus 617 ± 92 U cm2 with BaCl2 (n ¼ 6). After induction of differentiation by dexamethasone and prolactin, RT values of 879 ± 53 U cm2 versus 954 ± 38 U cm2 without BaCl2 (n ¼ 12), and 890 ± 90 U cm2 versus 1017 ± 72 U cm2 with BaCl2 (n ¼ 6) were measured. In all experimental setups bilateral HP of 10 kPa had no marked effect on RT during 4 h of incubation compared to controls (n ¼ 11). 3.2. Short circuit current During HP incubation with 10 kPa no significant change of ISC was observed in undifferentiated cells (n ¼ 11; Fig. 1), however, with addition of 1 mmol/l BaCl2 (n ¼ 6), a strong deviation of ISC was observed. In differentiated monolayers, ISC decreased significantly after 2 h (n ¼ 12), which was blocked by BaCl2 (n ¼ 6), indicating inhibition of chloride secretion. After 4 h, the effect was still observed, but not inhibited by BaCl2. This effect could not be observed in undifferentiated cells. 3.3. Western blotting Western blot analysis was used to verify the presence of TJ proteins and to quantify their expression, compared to controls. Western blot revealed signals for occludin, claudin-3, claudin-4 and ZO-1 in both, samples and controls of the experimental setups with and without BaCl2. However, significant effects were only observed for occludin in undifferentiated cells (Fig. 2A) and for ZO-1 in differentiated cells (Fig. 2B). In undifferentiated cells, after pressure incubation a decrease in the concentration of occludin could be observed. In differentiated cells no change of occludin could be found. However, a marked increase of ZO-1 was observed, which on
Fig. 2. Relative Western blot intensity after 4 h of 10 kPa bilateral HP incubation in undifferentiated (Ud, A) and differentiated (D, B) cells with and without the addition of 1 mM BaCl2, compared to controls. Bars represent means ± SEM, (C) representative images of Western blots (*p < 0.05, n ¼ 3). A: In undifferentiated cells, occludin showed a significant lower intensity after pressure incubation compared to controls, (0.47 ± 0.27 versus 1.50 ± 0.06). This effect could not be observed after the addition of BaCl2. No marked changes in the expression level of claudin-3, claudin-4 and ZO-1 could be observed. B: In differentiated cells, ZO-1 showed a significant higher intensity after pressure incubation compared to controls, (2.06 ± 0.37 versus 0.91 ± 0.11). On the contrary, with addition of BaCl2 a significant lower intensity was observed in ZO-1 compared to controls, (1.50 ± 0.12 versus 2.20 ± 0.16). No marked changes in the expression level of occludin, claudin-3 and claudin-4 could be observed. (C) Representative Western blot signals.
Fig. 1. ISC at the beginning and after 2 and 4 h of 10 kPa bilateral pressure incubation in undifferentiated (Ud) and differentiated (D) cells with and without the addition of 1 mM BaCl2, compared to controls. Bars represent means ± SEM, (*p < 0.05, n ¼ 6 to 12). In undifferentiated cells, no significant changes of short circuit current (ISC) could be observed, compared to controls. In differentiated cells, ISC decreased from the initial 4.7 ± 0.4 mA/cm2 to 2.9 ± 0.5 mA/cm2 after 2 h and ended with 2.8 ± 0.6 mA/cm2 after 4 h of pressure incubation, compared to controls with 5.4 ± 0.4 mA/cm2 (start), 4.8 ± 0.5 mA/cm2 (2 h) and 5.0 ± 0.6 mA/cm2 (4 h). With the addition of 1 mM BaCl2, ISC started at 5.4 ± 0.8 mA/cm2, followed by 3.6 ± 0.8 mA/cm2 after 2 h and 2.9 ± 0.5 mA/cm2 after 4 h of pressure incubation, compared to controls with 5.9 ± 0.8 mA/cm2 (start), 4.4 ± 0.5 mA/cm2 (2 h) and 4.2 ± 0.3 mA/cm2 (4 h).
the contrary, was changed to a decrease after addition of BaCl2, (n ¼ 3).
3.4. Immunofluorescence microscopy Immunofluorescence microscopy was used to localize TJ proteins and to examine structural condition of the HC11 monolayers after 4 h of 10 kPa bilateral HP incubation compared to controls. Integrity of the monolayers was preserved throughout the experiments. In controls, monolayer integrity was visible by occludin,
Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110
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claudin-3, claudin-4 and ZO-1, which were continuously located in TJs of undifferentiated and differentiated cells (not shown). In accordance with Western blots, in undifferentiated cells, occludin showed a marked reduction after pressure incubation. Claudin-3, claudin-4 and ZO-1 showed no marked changes in distribution or concentration and all proteins could be detected in TJs after pressure incubation and were still located in the apicolateral membrane areas. The same observations were made in differentiated cells with the difference that the reduction of occludin could not be detected, (n ¼ 3). 4. Discussion Recent analysis of TJ regulation in MGs revealed (i) formation of a tight barrier during lactogenesis [28,29], and (ii) changes of TJ protein expression and localization during accumulation of milk [24]. Moreover, a disappearance of adhesive contacts and desmosomes between cells is induced in the alveolar epithelium by the formation to lactation period [23]. Consequently, integrity of the epithelial structure and paracellular transport is determined by TJ proteins, which highlights the relevance of their regulation during maturation and lactation. Accordingly, an epithelial cell model was established representing the mammary epithelial monolayer, namely HC11 [27,30]. The influence of pressure on mammary epithelial cells was recently studied in a mouse model, in vivo [24]. In our previous study, pressure was generated by accumulation of milk in the MG during 20 h of interrupted suckling. Molecular analysis revealed an increased expression of the tightening TJ proteins claudin-1 and -3 and a reduction of claudin-2 and -16, which are pore-forming proteins. The change in TJ expression was interpreted as a mechanism against a back-leak of milk components and might have been induced by mechanical stimuli [24]. However, it is known that milk can affect epithelial barrier properties, too [31]. In this context, previous analyses have demonstrated that several milk components have effects on intestinal barrier function, e.g. laurate, glutamate and lactoferrin [32e34], and therefore might also affect mammary TJs. Whereas the mouse model of interrupted suckling revealed important insights into the physiological regulation of mammary TJ during lactation, it was not suitable for a differentiation between regulative effects induced by hormones, milk components, and pressure. The present study however, focuses on the identification of pressure effects, which turned out to have effects on ZO-1 in differentiated cells, whereas in an undifferentiated state occludin was affected, which was also reflected in immunostainings. Transepithelial resistance of differentiated mammary monolayers, most accurately representing the situation in MGs in vivo, was not significantly affected by pressure alone. One important finding of the study is the observation, that claudin-3 and -4 were not affected by pressure incubation in undifferentiated and differentiated monolayers. This might indicate that (i) barrier function is not actively regulated by pressure effects on claudins alone, and (ii) alterations in MG claudin expression may not be dependent on pressure. The latter finding may also indicate that further functions of claudin-3 and -4, which have been also described as tumor markers relevant for proliferation, apoptosis, migration and in vivo growth of mammary epithelial cells [35e37], may not be changed by pressure alone. During lactogenesis, MG epithelium is transformed into a secretory tissue. As ion transport has been reported to be highly dependent on mechanical stimuli in a variety of organs and tissues, including lung epithelium, the urinary bladder, and intestine [25,38,39], we also addressed the question whether an effect of pressure on secretion might occur. Surprisingly, with increasing HP,
BaCl2 sensitive Cl secretion could be observed in differentiated monolayers. However, a limitation of our experimental approach might be the high volumes in Ussing chambers reservoirs necessary for pressure incubation, which limit the application of inhibitors. BaCl2 has been reported to provide a high selectivity for analysis of chloride secretion via inhibition of inward rectifier potassium channels as a requirement for the driving force of Cl secretion, though [40,41]. Using BaCl2, chloride secretion was blocked in differentiated cells after 2 h of pressure incubation, inhibiting a decrease of ISC. However, chloride secretion was not continuously blocked until end of pressure incubation, which might indicate a pressure adaptation mechanism. On molecular level, the expression of ZO-1 was upregulated without BaCl2, but decreased after addition of BaCl2. This mechanism might reflect the functional and regulatory interaction of barrier and transport mechanisms, as demonstrated for single TJ proteins and transport before [42e44]. In conclusion, our current study revealed that bilateral HP incubation in vitro induces changes to both, paracellular barrier and transcellular transport, which represent major physiological mechanisms during lactation. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments €llig, Uwe Tietjen, and We thank Martin Grunau, Katharina So Susanne Trappe for excellent technical assistance. This work was supported by the Center for International Cooperation of the Freie €t Berlin [grant number FSP2016-300, and 1.60.221.2017], Universita the Deutsche Forschungsgemeinschaft [grant number AM 141/111], SPbU grant No. 1.40.486.2017, and the Sonnenfeld Stiftung [doctoral studies grant to K.S. Miebler]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.110. References [1] F. Borellini, T. Oka, Growth control and differentiation in mammary epithelial cells, Environ. Health Perspect. 80 (1989) 85e99. [2] C. Brisken, S. Kaur, T.E. Chavarria, et al., Prolactin controls mammary gland development via direct and indirect mechanisms, Dev. Biol. 210 (1999) 96e106. [3] M. Ono, T. Oka, The differential actions of cortisol on the accumulation of alactalbumin and casein in mid-pregnant mouse mammary gland in culture, Cell 19 (1980) 473e480. [4] W. Schneider, G. Shyamala, Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol, Endocrinology 116 (1985) 2656e2662. [5] J. Cerbulis, H.M. Farrell Jr., Composition of milks of dairy cattle. I. Protein, lactose, and fat contents and distribution of protein fraction, J. Dairy Sci. 58 (1975) 817e827. [6] J.L. Linzell, M. Peaker, Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism, J. Physiol. 216 (1971) 683e700. [7] S. Amasheh, C. Barmeyer, C.S. Koch, et al., Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis, Gastroenterology 126 (2004) 1711e1720. [8] M. Itoh, M.J. Bissell, The organization of tight junctions in epithelia: implications for mammary gland biology and breast tumorigenesis, J. Mammary Gland. Biol. Neoplasia 8 (2003) 449e462. [9] M. Furuse, T. Hirase, M. Itoh, et al., Occludin: a novel integral membrane protein localizing at tight junctions, J. Cell. Biol. 123 (1993) 1777e1788. [10] M. Furuse, K. Fujita, T. Hiiragi, et al., Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to
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Please cite this article in press as: K.S. Mießler, et al., Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.110