Transdifferentiation Of Alveolar Epithelial Cells Research Articles

Study on the expression level of zonula occludens 1 and zonula occludens 1 related nucleic acid binding protein in alveolar epithelial cells in hyperoxia induced bronchopulmonary dysplasia newborn rats

Objective Previous studies have found abnormal proliferation and transdifferentiation of alveolar epithelial cells(AECs)in hyperoxic lung injury of neonatal rats.The purpose of this study was to clarify the expression of zonula occludens 1(ZO-1) and ZO-1 related nucleic acid binding protein(ZONAB)in AECs in hyperoxic lung injury model, in order to investigate its effect on the proliferation and transdifferentiation of AECs in the injured lung tissue. Methods Full-term neonatal Wistar rats were randomly divided into two groups within 12 h after birth, model group(inhaled oxygen concentration 85%)and control group(inhaled air). Lung specimens were collected at 7, 14 and 21 days after exposure.The expression of ZONAB in typeⅡalveolar epithelial cells(AECⅡ)was observed by double immunofluorescence staining.At the same time, AEC Ⅱ was isolated from lung tissues of animal models at these time points, and the expression levels of ZO-1, ZONAB protein and mRNA in lung tissues and AECⅡof the two groups were detected by Western blot and Real-Time PCR.In addition, AEC Ⅱ was isolated from lung tissue of normal newborn rats and then divided into model group(85% oxygen concentration)and control group(21% oxygen concentration). After 48 hours of culture in vitro, the expression levels of ZO-1, ZONAB protein and mRNA were detected, and the expression level and location of ZONAB were observed by immunofluorescence staining. Results Double immunofluorescence staining showed that the expression of ZONAB in AECⅡin model group was significantly lower than that in control group.The protein and mRNA expression levels of ZO-1 and ZONAB in AECⅡisolated from lung tissue of model group were both significantly lower than those from control group, starting from 7 d after hyperoxia exposure.AECⅡisolated from lung tissue of normal newborn rats, were then incubated for 48 hours under hyperoxia or normoxia in vitro, the protein and mRNA expression levels of ZO-1 and ZONAB significantly decreased in model group compared with those in control group.The results of immunofluorescence staining showed that the expression of ZONAB was higher in AECⅡof the control group, and ZONAB was mostly located in the junction and nucleus of cells, while the expression of ZONAB in the model group significantly decreased than that in the control group, and the expression sites were clustered in the cytoplasm, with little expression in the junction and nucleus. Conclusion ZO-1, as a tight junction-related protein, is down-regulated in hyperoxic lung injury model.In addition to destroying pulmonary epithelial barrier to mediate pulmonary edema, it also participated in the regulation of proliferation and differentiation of AECs by regulating transcription factor ZONAB, suggesting that this may be another pathway leading to hyperoxic lung injury. Key words: Hyperoxia; Alveolar epithelial cell; Transdifferentiation; Zonula occludens 1; Zonula occludens 1 related nucleic acid binding protein

Transdifferentiation of type II alveolar epithelial cells induces reactivation of dormant tumor cells by enhancing TGF-β1/SNAI2 signaling.

Dormant tumor cells (DTCs) serve a crucial role in the pathogenesis of metastasis and may compromise the efficacy of existing therapeutic modalities aimed at fully eradicating cancer. However, the mechanisms underlying the dormant-to-proliferating switch of DTCs remain largely unknown. The lung is one of the most common sites of metastatic recurrence. The transdifferentiation of alveolar epithelial cellsII (AECIIs) to AECIs is a hallmark of alveolar epithelial stimulation. However, the role of AECII transdifferentiation during the reactivation of DTCs has not been fully elucidated. In the present study, we found that tumor cells promoted the transdifferentiation of AECIIs. Furthermore, the supernatant of the transdifferentiation of AECIIs to AECIs (Super-TDA) promoted the dormant-to-proliferating switch of DTCs via the autocrine effect of TGF-β1 on the 3D BME culture system invitro. Monensin and LY2109761 blocked the dormant-to-proliferating switch of DTCs induced by Super-TDA. Although lipopolysaccharide did not directly stimulate the reactivation of DTCs, it promoted DTC reactivation by increasing the secretion of TGF-β1 in the Super-TDA. We further demonstrated that the upregulation of SNAI2 expression was required for Super-TDA facilitating the DTC dormant-to‑proliferating switch. Taken together, our findings demonstrated that tumor cells may promote AECII transdifferentiation. Furthermore, the transdifferentiation of AECIIs may, in turn, induce the reactivation of 3D-established DTCs by promoting TGF-β1/SNAI2 signaling. Targeting this process may provide novel therapeutic strategies for the inhibition of the dormant‑to-proliferating switch.

Decreased ZONAB expression promotes excessive transdifferentiation of alveolar epithelial cells in hyperoxia-induced bronchopulmonary dysplasia.

Previous studies by our group have confirmed excessive transdifferentiation of alveolar epithelial cells (AECs) in a hyperoxia‑induced bronchopulmonary dysplasia (BPD) model, but the underlying mechanism have remained elusive. The transcription factor zonula occludens 1‑associated nucleic acid binding protein (ZONAB) has the biological functions of inhibition of epithelial cell differentiation and promotion of epithelial cell proliferation. The aim of the present study was to explore the regulatory effect of ZONAB on the transdifferentiation and proliferation of AECs in a model of hyperoxia‑induced lung injury. Newborn Wistar rats were randomly allocated to a model group (inhalation of 85% O2) or a control group (inhalation of normal air), and ZONAB expression in lung tissues was detected at different time‑points. TypeII AECs (AECII) isolated from normal newborn rats were primarily cultured under an atmosphere of 85 or 21% O2, and ZONAB expression in the cells was examined. The primary cells were further transfected with ZONAB plasmid or small interfering (si)RNA and then exposed to hyperoxia, and the indicators for transdifferentiation and proliferation were measured. The present study indicated that ZONAB expression in AECII of the BPD rats was significantly decreased from 7days of exposure to hyperoxia onwards. In the AECII isolated from normal neonatal rats, ZONAB expression in the model group was also reduced compared with that in the control group. After transfection with the plasmid pCMV6‑ZONAB, the expression of aquaporin 5 (typeI alveolar epithelial cell marker) decreased and the expression of surfactant proteinC (AECII marker), proliferating‑cell nuclear antigen and cyclinD1 increased, which was opposite to the effects of ZONAB siRNA. Transfection with pCMV6‑ZONAB also alleviated excessive transdifferentiation and inhibited proliferation of AECII induced by hyperoxia treatment. These results suggest that ZONAB expression in AECII decreases under hyperoxia conditions, which promotes transdifferentiation and inhibits proliferation of AECs. This may, at least in part, be the underlying mechanism of lung epithelial injury in the hyperoxia-induced BPD model.

Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

AimsThe purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. Main methodsReal-time PCR analysis, uptake study of [3H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells. Key findingsThe expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [3H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [3H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [3H]Gly-Sar uptake were observed in type I-like cells. The maximal [3H]Gly-Sar uptake was observed at pH6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [3H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC50 value being 4.3μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [3H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0μM. SignificancePEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.

MiR-375 regulates rat alveolar epithelial cell trans-differentiation by inhibiting Wnt/ -catenin pathway

Alveolar epithelial cell (AEC) trans-differentiation is a process where type II alveolar epithelial cells (AEC II) trans-differentiate into type I alveolar epithelial cells (AEC I) during lung recovery after various injuries, in which AEC I are damaged. This process is critical for lung tissue repair. MicroRNAs are a group of small RNAs that regulate gene expression at the post-transcriptional level. They have the potential to regulate almost every aspect of cell physiology. However, whether AEC trans-differentiation is regulated by microRNAs is completely unknown. In this study, we found that miR-375 was downregulated during AEC trans-differentiation. The overexpression of miR-375 with an adenoviral vector inhibited alveolar epithelial trans-differentiation as indicated by an increase in the AEC II marker, surfactant protein C, and decreases in the AEC I markers, T1α and advanced glycosylation end product-specific receptor. miR-375 also inhibited the Wnt/β-catenin pathway. The constitutively activation of Wnt/β-catenin signaling with a stabilized form of β-catenin blocked the miR-375 effects. Frizzled 8 was identified as a target of miR-375. In summary, our results demonstrate that miR-375 regulates AEC trans-differentiation through the Wnt/β-catenin pathway. This discovery may provide new targets for therapeutic intervention to benefit lung recovery from injuries.

More life for a “terminal” cell

More life for a “terminal” cell

Effects of KGF on Alveolar Epithelial Cell Transdifferentiation Are Mediated by JNK Signaling

Rat alveolar epithelial cells (AEC) in primary culture transdifferentiate from a type II (AT2) toward a type I (AT1) cell-like phenotype, a process that can be both prevented and reversed by keratinocyte growth factor (KGF). Microarray analysis revealed that these effects of KGF are associated with up-regulation of key molecules in the mitogen-activated protein kinase (MAPK) pathway. To further explore the role of three key MAPK (i.e., extracellular signal-related kinase [ERK] 1/2, c-Jun N-terminal kinase [JNK] and p38) in mediating effects of KGF on AEC phenotype, primary rat AEC cultivated in minimal defined serum-free medium (MDSF) were treated with KGF (10 ng/ml) from Day 4 for intervals up to 48 hours. Exposure to KGF activated all three MAPK, JNK, ERK1/2, and p38. Inhibition of JNK, but not of ERK1/2 or p38, abrogated the ability of KGF to maintain the AT2 cell phenotype, as evidenced by loss of expression of lamellar membrane protein (p180) and increased reactivity with the AT1 cell-specific monoclonal antibody VIIIB2 by Day 6 in culture. Overexpression of JNKK2, upstream kinase of JNK, increased activation of endogenous c-Jun in association with increased expression of p180 and abrogation of AQP5, suggesting that activation of c-Jun promotes retention of the AT2 cell phenotype. These results indicate that retention of the AT2 cell phenotype by KGF involves c-Jun and suggest that activation of c-Jun kinase may be an important determinant of maintenance of AT2 cell phenotype.

Differential expression of a V-type ATPase C subunit gene, Atp6v1c2, during culture of rat lung type II pneumocytes

The lung alveolar epithelium consists of type I and type II pneumocytes. In vivo, the type II cell is the progenitor cell from which the type I cell originates. When freshly-isolated type II cells are cultured under conventional conditions they rapidly lose their phenotypic properties and attain characteristics of type I cells. Taking advantage of this transdifferentiation, we sought to identify genes that are differentially expressed during culture of rat type II cells. Using suppression subtractive hybridization (SSH), a vacuolar-type H+-ATPase (V-ATPase) C2 subunit gene (Atp6v1c2) was found to be enriched in freshly isolated rat type II cells compared to those cultured for 4 days. Northern blotting and reverse-transcription polymerase chain reaction (RT-PCR) confirmed the differential expression of Atp6v1c2 during in vitro culture of isolated type II cells. Expression ofAtp6v1c2 was significantly reduced early during in vitro culture: almost 90% reduction was observed after 24 h of incubation as determined by real-time PCR. In situ hybridization showed that Atp6v1c2 is expressed in both bronchiolar and alveolar lung epithelial cells, an expression pattern similar to that of surfactant protein B (SP-B). Multi-tissue Northern blotting revealed a unique tissue distribution with Atp6v1c2 expression limited to lung, kidney and testis. The presence and expression of Atp6v1c2 gene transcript isoforms, resulting from alternative splicing, were also investigated. Elucidation of differential expression of Atp6v1c2 in type II cells and further studies of its regulation may provide information useful in understanding the molecular mechanism underlying phenotypic and functional changes during transdifferentiation of alveolar epithelial cells.

Temporal expression of Notch in preterm rat lungs exposed to hyperoxia

To explore the mechanism of Notch in hyperoxia-induced preterm rat lung injury, 2-days-old preterm SD rats were randomized into control and hyperoxia group (FiO2 > or = 0.85). On day 1, 7, 14 and 21, 8 rat pups of each time point were used to assess histopathological changes of lung with HE staining and to evaluate the expression of Notch1 and Notch3 with immunohistochemistry. Notch1, Notch3, Aquaprin5 (AQP5) and surfactant protein C (SP-C) mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lung injury in the hyperoxia group was characterized by retarded lung alveolization and differentiation of alveolar epithelial type II cells (AEC II). Positive staining of Notch1 in hyperoxia group was weaker than controls at every time point (except for day 7), while positive staining of Notch3 was much stronger (P < 0.05, P < 0.01). Notch1, Notch3 mRNA level showed similar change as protein level. AQP5, SP-C mRNA decreased significantly as compared with that of the controls (P < 0.01). We are led to conclude that hyperoxia results in abnormal expression of Notch, which is likely to contribute to the pathogenesis of lung injury through regulating proliferation and transdifferentiation of alveolar epithelial cells.

Identification of three genes of known function expressed by alveolar epithelial type I cells.

To identify genes of known function expressed by type I (AT1) cells, changes in gene expression during transdifferentiation of alveolar epithelial cells (AEC) in primary culture from type II (AT2) to type I-like cell phenotype were evaluated. Total RNA from AEC on Day 0 or Day 8 was hybridized to a rat microarray for screening. Eight upregulated genes on Day 8 were selected for further investigation. Northern analysis confirmed upregulation of three of these genes, PAI-1, P2X4, and P15INK4B. The corresponding proteins were evaluated in cultured AEC and results correlated with expression in AT1 cells. In AEC monolayers, all three proteins increased between Day 1 and Day 8. In mixed populations of freshly isolated rat lung cells, concurrent labeling with the AT1 cell-specific antibody, VIIIB2, localized these proteins to AT1 cells. In whole lung, all three proteins were detected in alveolar epithelium in a location consistent with expression in AT1 cells. Identification of novel AT1 cell genes of known function suggests an active role for AT1 cells in alveolar homeostasis. Furthermore, expression of these gene products in AT1-like cells, in freshly isolated AT1 cells, and AT1 cells in whole lung indicates that AT1-like cells reflect many of the properties of AT1 cells in situ.

Reversible transdifferentiation of alveolar epithelial cells.

Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.