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Keywords:

  • air-liquid interface;
  • airway epithelial cell;
  • aquaporin;
  • lipopolysaccharide;
  • signalling pathway

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Background and objective:  The aim of this study was to investigate the changes in expression of aquaporins (AQP) during differentiation of human bronchial epithelial cells and the role of lipopolysaccharide (LPS) in AQP expression.

Methods:  The levels of AQP3, AQP4 and AQP5 transcripts in human primary cultured bronchial epithelial cells were evaluated by real-time polymerase chain reaction at different time points before and after treatment with LPS. Western blotting was performed to assess the effects of LPS on AQP3, AQP4 and AQP5 expressions in normal human bronchial epithelial cells. Using pharmacological tools, the pathways involved in the regulation of LPS-induced changes in AQP5 were further explored.

Results:  The levels of AQP3, AQP4 and AQP5 transcripts were increased during differentiation of human bronchial epithelial cells. Expression of AQP5, but not AQP3 or AQP4, was downregulated by LPS. LPS-induced downregulation of AQP5 was inhibited by p38 and c-Jun N-terminal kinase (JNK) inhibitors.

Conclusions:  This study demonstrated that LPS decreases AQP5, but not AQP3 or AQP4, expression in human primary bronchial epithelial cells. The downregulation of AQP5 expression is mediated through a p38/JNK signalling pathway.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

The respiratory epithelium acts as a barrier that protects the lungs from inhaled substances.1 It regulates airway surface liquid volume and composition, mucin secretion and hydration, and ciliary beating, thereby maintaining a sterile lung through effective mucociliary clearance.2,3

The aquaporins (AQP) are a family of small (30 kDa monomer) integral membrane proteins that function as selective water transporters. Recently, AQP-mediated promotion of cell migration was also identified in colon, pancreatic and brain cancer cells.4–7 Three AQP are expressed in the airways:8–10 AQP3 is expressed at the basolateral membrane of basal epithelial cells in the large airways, nasopharynx and small airways. AQP4 is expressed at the basolateral membrane of ciliated columnar cells of bronchial, tracheal and nasopharyngeal epithelium. AQP5 is expressed at the apical membrane of acinar epithelial cells in submucosal glands and large airway epithelia.9 Increased protein and mucus secretion were detected in the upper airways of mice after deletion of AQP511 possibly due to impaired fluid transport in the absence of AQP5 expression, as previously observed in salivary glands.12 In addition, previous studies indicated that AQP5 may be involved in the regulation of MUC5AC production.13

Bacterial infection is believed to be a common cause of lung inflammation. Bacterial endotoxins, including lipopolysaccharides (LPS), constitute the major outer surface membrane components of almost all Gram-negative bacteria and are associated with mucin overproduction in patients with chronic obstructive pulmonary disease.14

This study was undertaken in order to identify changes in the expression of AQP in cultured normal human bronchial epithelial cells (NHBE), the effects of LPS on AQP3, AQP4 and AQP5 expression, and the possible signalling pathways leading to alterations in AQP5 expression.

METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Human primary bronchial epithelial cell culture

Bronchi were obtained from macroscopically normal lung tissue from subjects who underwent tumour resection after providing informed consent in accordance with Zhongshan Hospital Ethics Committee guidelines. Tissues were only collected from patients who were non-smokers, did not consume alcohol and were non-asthmatic. Bronchial tissue was immediately transferred to Dulbecco's modified Eagle's medium containing penicillin (100 U/mL), streptomycin (100 µg/mL) and fungizone (0.1%), and refrigerated at 4°C within 2 h. Bronchial epithelial cells were isolated, as described in detail in Appendix S1.

Air-liquid interface culture

Bronchi epithelial cells at passage two were seeded at a density of 1 × 105 cells on the upper surface of 24-mm Transwell collagen membranes (Corning, Inc., New York, NY, USA) in serum-free, hormone- and growth factor-supplemented medium, as described previously. The medium was changed every 2 days. When the epithelial cells reached 95–100% confluence at the air-liquid interface (on average, after 5 days), no medium was added to the upper space of the filter. The transmembrane resistance was monitored using an epithelial ohm-volt meter (Millicell Electrical Resistance System, Millipore, Billerica, MA, USA). A polarized and highly differentiated airway epithelium was obtained 2 weeks after the air-liquid interface was established.

Light and electron microscopy

Proliferating cells were examined daily by phase contrast light microscopy (Leica, Wetzlar, Germany). Cells were fixed with 3.7% neutral-buffered formalin and stained with haematoxylin-eosin. For scanning electron microscopy (Leica), NHBE cells were fixed in chilled 2.5% glutaraldehyde for 4–6 h, washed with 0.1 mol/L phosphate-buffered saline (Gibco, Carlsdad, CA, USA) and post-fixed with 1% osmium tetroxide for 2 h. After critical point drying and gold coating, the samples were examined by scanning electron microscopy.

Treatment of cells with LPS and inhibitors

Primary human bronchial epithelial cells, grown for 1 or 2 weeks under air-liquid interface culture conditions, were stimulated with LPS (1, 10 and 20 µg/mL) on both the apical and basal sides for 6 or 24 h. Cells were treated with Pseudomonas aeruginosa LPS (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline, with untreated cells as the control. For inhibitor studies, cells were pretreated with inhibitors for 30 min before exposure to the stimuli. The cells were then washed three times in serum-free medium and cultured for a further 6 or 24 h in medium containing the inhibitors at the same concentrations as those used for pretreatment.

To assess the effects of intracellular mitogen-activated protein kinase activation on the downregulation of AQP5 messenger RNA (mRNA) and protein, cells were treated with (i) the extracellular signal-regulated kinase 1/2 inhibitor, PD98059 (Cell Signaling Technology, Danvers, MA, USA) at 10 µM; (ii) the p38 inhibitor ML3404 (Calbiochem, San Diego, CA, USA) at 5 µmol/L; and (iii) the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (Calbiochem) at 10 µmol/L. After pretreatment with these inhibitors, NHBE cells were stimulated with LPS for 6 h to assess AQP5 gene expression by real-time polymerase chain reaction and for 2 or 24 h to measure protein production by Western blotting.

RNA isolation and real-time polymerase chain reaction

Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions, and RNA was quantified by spectrophotometry. Analysis by real-time polymerase chain reaction was undertaken, as previously described (see Appendix S1 for further details). The primer sequences were: AQP3: (forward) 5′-CAC AGC CGG CAT CTT TGC TA-3′, (reverse) 5′-TGG CCA GCA CAC ACA CGA TA-3′; AQP4: (forward) 5′-CTG TTC TCA GAC CAT GAA ACC AGAC-3′, (reverse) 5′-GAC TTC CAT CTT CAG TTG CCA CA-3′; AQP5: (forward) 5′-CTG TCC ATT GGC CTG TCT GTC-3′, (reverse) 5′-GGC TCA TAC GTG CCT TTG ATG-3′; β-actin: (forward) 5′-CCT GTA CGC CAA CAC AGT GC-3′, (reverse) 5-′ATA CTC CTG CTT GCT GAT CC-3′.

Western blotting

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot were performed, as previously described with further detail in Appendix S1.

Statistical analysis

Data are expressed as means ± standard error of the mean. Differences among multiple groups were assessed for statistical significance by one-way analysis of variance. If the analysis of variance indicated statistical significance, the Student–Newman–Keuls test was used for multiple comparisons. Statistical significance was accepted at P < 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Morphological characterization of bronchial epithelial cells

Confluent NHBE cells were small, polygonal and tightly connected with the typical cobblestone morphology of epithelial cells. Scanning electron microscopy of human epithelia generated at the air-liquid interface demonstrated abundant ciliated and non-ciliated columnar cells (Fig. 1).

image

Figure 1. Microscopic appearance of cultured normal human bronchial epithelial cells. (a) Phase contrast micrograph showing the polygonal shape of the cells in a tightly cohesive monolayer 14 days after reaching confluence on Transwell membranes (magnification 200×). (b) Scanning electron microscopy of cultured human bronchial epithelial cells. Ciliated cells were observed on the 14th day after confluence was reached.

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Changes in AQP mRNA levels over time

To determine whether expression of the AQP genes changed in cultured NHBE cells, real-time polymerase chain reaction was performed on cultured cells at 2, 7 and 14 days. After the cells reached confluence, AQP3 mRNA expression increased progressively up to 2.1-fold on day 7 and then decreased abruptly to 1.1-fold on day 14. Expression of AQP4 mRNA was extremely low on day 2 and day 7 after confluence but increased significantly to 6.5-fold on day 14. Similar to AQP3 mRNA, AQP5 mRNA expression was low on day 2 after confluence but increased significantly to 6.1-fold on day 7 before decreasing to 2.2-fold on day 14 (Fig. 2).

image

Figure 2. Levels of aquaporin (AQP) 3, AQP4 and AQP5 transcripts according to duration of culture. Levels of AQP4 transcript in normal human bronchial epithelial cells increased on day 14 after confluence. The amounts of AQP3 and AQP5 transcripts increased on day 7 after confluence but decreased on day 14. The relative intensities for messenger RNA (mRNA) expression on day 7 and day 14 were compared with the intensity on day 2 after confluence. Bars indicate means ± standard error of the mean, n = 5 for each group. (□) AQP3, (inline image) AQP4, (inline image) AQP5.

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Changes in the levels of AQP3, AQP4 and AQP5 transcripts in LPS-treated NHBE cells

To determine which AQP was induced by LPS, the levels of AQP3, AQP4 and AQP5 transcripts in NHBE cells were measured after the cells were exposed to LPS (10 µg/mL) for 6 h. The levels of AQP5 transcript were significantly decreased by LPS treatment, whereas the levels of AQP3 and AQP4 transcripts remained unchanged (Fig. 3a). Furthermore, AQP5 mRNA levels decreased significantly in a dose-dependent manner when NHBE cells were treated with 1, 10 or 20 µg/mL of LPS (Fig. 3b).

image

Figure 3. (a) Real-time polymerase chain reaction analysis of relative levels of mRNA for aquaporin (AQP) 3, AQP4 and AQP5 in 2-week air-liquid interface cultures treated for 6 h with lipopolysaccharide (LPS) (10 µg/mL). The levels of AQP5 transcript decreased after exposure to LPS, whereas the levels of AQP3 and AQP4 transcripts were unchanged. Bars indicate means ± standard error of the mean, n = 5 for each group. (b) Dose-dependent decrease in AQP5 messenger RNA (mRNA) after LPS treatment. Cells were incubated in media supplemented with LPS for 6 h. *Significantly different from the control group, P < 0.05. (□) Control, (inline image) LPS.

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AQP5 protein expression in NHBE cells is reduced by LPS treatment

As the levels of AQP5, but not AQP3 or AQP4, transcript decreased after LPS treatment, the effects of LPS on AQP3, AQP4 and AQP5 protein expression were investigated. NHBE cells that had been incubated in media supplemented with 10 µg/mL of LPS for 24 h were used for Western blotting. The AQP3 and AQP4 protein levels remained unchanged (Fig. 4). In addition, the AQP5 protein levels in NHBE cells at 1 and 2 weeks were significantly decreased after LPS treatment (Figs 5,6).

image

Figure 4. Levels of aquaporin (AQP) 3 and AQP4 proteins in 2-week air-liquid interface cultures treated for 24 h with lipopolysaccharide (LPS) (10 µg/mL). AQP3 and AQP4 protein levels in normal human bronchial epithelial cells remained unchanged on day 14 after confluence. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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image

Figure 5. Levels of aquaporin (AQP) 5 protein in 1-week air-liquid interface cultures treated for 24 h with lipopolysaccharide (LPS) (10 µg/mL). The AQP5 protein level in normal human bronchial epithelial cells decreased on day 7 after confluence. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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image

Figure 6. Effects of inhibitors on the lipopolysaccharide (LPS)-induced decrease in aquaporin (AQP) 5 transcript and protein levels in 2-week air-liquid interface cultures. After pretreatment with PD-98059, ML3404 or SP600125 for 30 min, normal human bronchial epithelial cells were incubated in media supplemented with LPS for 6 h for real-time polymerase chain reaction analysis or for 24 h for Western blot analysis. Bars indicate means ± standard error of the mean (n = 8 for each group), expressed as fold changes compared with the control cells. *Significantly different compared with the control cells, P < 0.05. **Significantly different compared with the LPS-treated control cells, P < 0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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Effects of inhibitors on AQP5 gene and protein expression after LPS treatment

Pretreatment with ML3404 and SP600125 prevented LPS-induced AQP5 gene and protein expression, which indicated that ML3404 and SP600125 played an important role in AQP5 expression in airway epithelial cells exposed to LPS. Pretreatment with the extracellular signal-regulated kinase 1/2 inhibitor, PD98059, did not inhibit the downregulation of AQP5 transcript and protein (Fig. 6). The concentrations of inhibitors used in this study were not toxic to the cells (data not shown).15 Total cell lysates were probed with phospho-specific antibodies to p38 or JNK. Phosphorylation of p38 and JNK was increased in cells treated with LPS. LPS-induced phosphorylation of p38 was suppressed by ML3404, and LPS-induced phosphorylation of JNK was suppressed by SP600125 (Fig. 7).

image

Figure 7. Cells were pre-incubated with or without ML3404 (5 µmol/L) or SP600125 (10 µmol/L) and then treated with lipopolysaccharide (LPS) (10 µg/mL) or vehicle for 2 h. Total cell lysates were then probed with phospho-specific antibodies to p38 or c-Jun N-terminal kinase (JNK). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; P-JNK, phospho-JNK; P-p38, phospho-p38; T-JNK, total JNK; T-p38, total p38.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Lesions in the epithelium play a major role in the pathogenesis of numerous respiratory diseases. Loss of water through the nasal epithelium and proximal bronchi is accelerated during inspiration of cold or dry air, which is consistent with the presence of AQP in the apical membrane of epithelial cells.1,9 To better understand the patterns of AQP3, AQP4 and AQP5 expressions during differentiation of human primary bronchial epithelial cells and the response of AQP to LPS stimulation, an in vitro model of primary cultures of human bronchial epithelial cells was established.

This study showed that culture of NHBE cells on permeable filters in which the membrane polarity of epithelial cells can be established increased the expression of AQP. With a very well-preserved repertoire of the AQP genes expressed by the native tissue, NHBE cells serve as a useful tool for the investigation of gene expression and, hopefully, the functions of AQP in both normal physiology and disease.

Although AQP3, AQP4 and AQP5 are all expressed in cultured NHBE cells, the levels of AQP3 and AQP5 mRNA peaked on day 7 after confluence, and the highest level of AQP4 mRNA was observed on day 14 after confluence, just as in native human bronchial epithelial cells. Recently, two studies on normal human nasal epithelial cells showed that expression of AQP3, AQP4 and AQP5 peaked on day 14.16,17 A possible reason for the difference in AQP expression patterns between this study and previous studies may be the different histological sources of the cultured cells, as the NHBE cells in the present study were from the lower airway, whereas in the previous studies, the cells were obtained from the upper airway.

Our previous study focused on the relationship between AQP5 expression and MUC5AC, which restores the appropriate airway mucus water to mucin ratio in human airway submucosal gland cell line (SPC-A1 cells).15 The present study showed that LPS did not affect AQP3 or AQP4 expression, whereas it reduced AQP5 expression at both mRNA and protein levels. This result is in agreement with previous studies showing that interleukin-13 did not affect AQP3 or AQP4 expression but completely inhibited AQP5 expression at both mRNA and protein levels in human primary nasal epithelial cells.17 Among the three AQP present in the airways, AQP5 is critical for pulmonary physiology. Depletion of AQP5 impairs osmotic equilibration and results in the secretion of reduced volumes of relatively hypertonic fluid, as observed with the secretion of saliva in the upper airways of AQP5 null mice but not AQP3 or AQP4 null mice.11,12 Expression of AQP5 is decreased in the airways of patients with chronic obstructive pulmonary disease.18 In addition, AQP5 RNA interference has been reported to upregulate MUC5AC mucin secretion in SPC-A1 cells.13,19

A number of pathways have been demonstrated to play roles in the regulation of AQP5 gene expression and the propagation of LPS signalling. In this study, PD98059 (an extracellular signal-regulated kinase 1/2 inhibitor), ML3404 (a p38 mitogen-activated protein kinase inhibitor) and SP600125 (a JNK inhibitor) were used to pretreat NHBE cells. ML3404 and SP600125 inhibited the downregulation of both AQP5 mRNA and protein by LPS. These findings indicated that the p38 and JNK pathways may be involved in the expression of AQP5 transcripts in NHBE cells following exposure to LPS. However, another study showed that in murine lung epithelial cells, AQP5 expression was increased in response to hypertonic stress through an extracellular signal-regulated kinase-dependent mechanism.20 AQP5 mRNA and protein expression were decreased in cultured murine lung epithelial cells in response to tumor necrosis factor α (TNF-α), acting through the nuclear translocation of nuclear factor kappa B (NF-κB).21 Discrepancies between these studies and the present study may be related to differences in the types of cells used in the experiments.

In conclusion, this study demonstrated that expression of AQP3, AQP4 and AQP5 was increased when NHBE cells were cultured on permeable filters. AQP5, but not AQP3 or AQP4, gene expression was downregulated by LPS, providing further evidence that AQP5 is important in LPS-mediated changes in mucus composition and modification of cellular permeability and polarization during epithelial repair.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

This project was supported by a Shanghai Leading Academic Discipline Project, Project Number: B115, Grant No.81000012 from the National Science Foundation of China.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Appendix S1 Additional methods.

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RESP_2228_sm_AppS1.doc76KSupporting info item

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