Heta Merikallio

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

BackgroundClaudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.Methods47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.ResultsClaudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.ConclusionClaudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) and DJ1 are prognostic factors in lung cancer

Nuclear factor erythroid-derived 2-like 2 (Nrf2) controls the expression of several enzymes that are protective against oxidative stress. We investigated the expression of nuclear factor erythroid-derived 2-like 2, DJ1 (Nrf2 stabilizer), and sulfiredoxin in a large set of lung carcinomas. The cases were analyzed immunohistochemically with antibodies to nuclear factor erythroid-derived 2-like 2, DJ1, and sulfiredoxin with the results being compared with histologic and clinical data. Significant differences were observed in the expression of DJ1 and sulfiredoxin between various types of lung tumors, while expression of nuclear factor erythroid-derived 2-like 2 was more constant. Patients with tumors with cytoplasmic (P = .033) or nuclear (P = .003) DJ1 positivity exhibited worse survival. Separately in squamous cell carcinomas, there was a tendency toward worse survival with both cytoplasmic (P = .013) and nuclear (P = .071) DJ1 positivity. Patients with a strong nuclear factor erythroid-derived 2-like 2 expression in their tumors had worse survival (P = .006). In the Cox regression analysis, nuclear factor erythroid-derived 2-like 2 was an independent prognostic factor (P = .012) along with the T status (P = .008) and DJ1 cytoplasmic positivity (P = .028). Interestingly, smokers and ex-smokers had significantly more sulfiredoxin expression in their tumors (P < .001); and in patients receiving cytostatic drugs or radiation therapy, sulfiredoxin expression predicted a poor prognosis (P = .038). Nuclear factor erythroid-derived 2-like 2 and its stabilizing protein DJ1 affect the prognosis of patients with lung cancer by inducing an elevated stress response to oxidative damage. There were differences in the expression of sulfiredoxin and DJ1 between different lung tumor types, suggesting that the pathways involved in combating oxidative stress vary in different lung cancer types.

Snail promotes an invasive phenotype in lung carcinoma

BackgroundSnail is a transcriptional factor which is known to influence the epitheliomesenchymal transition (EMT) by regulating adhesion proteins such as E-cadherin and claudins as well as matrix metalloproteases (MMP).MethodsTo evaluate the functional importance of snail, a transciptional factor involved in EMT in lung tumors, we investigated its expression in a large set of lung carcinomas by immunohistochemistry. Expression of snail and effects of snail knockdown was studied in cell lines.ResultsNuclear snail expression was seen in 21% of cases this being strongest in small cell lung carcinomas (SCLC). There was significantly greater snail expression in SCLC compared to squamous cell or adenocarcinoma. Positive snail expression was associated with poor survival in the whole material and separately in squamous cell and adenocarcinomas. In Cox regression analysis, snail expression showed an independent prognostic value in all of these groups. In several cell lines knockdown of snail reduced invasion in both matrigel assay and in the myoma tissue model for invasion. The influence of snail knockdown on claudin expression was cell type specific. Snail knockdown in these cell lines modified the expression of MMP2 and MMP9 but did not influence the activation of these MMPs to any significant degree.ConclusionsThe results show that snail plays an important role in the invasive characteristics of lung carcinoma influencing the survival of the patients. Snail knockdown might thus be one option for targeted molecular therapy in lung cancer. Snail knockdown influenced the expression of claudins individually in a cell-line dependent manner but did not influence MMP expressions or activations to any significant degree.

Zeb1 and twist are more commonly expressed in metastatic than primary lung tumours and show inverse associations with claudins.

Background Zeb1 and twist regulate expression of genes which take part in epitheliomesenchymal transition (EMT). Claudins are tight junctional proteins regulating polarity and paracellular permeability of epithelial cells. Aims and methods To study Zeb1 and twist in 289 primary and 54 metastatic lung tumours and their relation to expression of claudins 1–5 and 7 by immunohistochemistry. Results Metastatic tumours showed a significantly lower expression of zeb1 and twist (33.0% and 38.0% vs 5.0% and 14.5%, p<0.001 for both) and they also had a significantly lower expression of claudin 1 (p<0.001), claudin 4 (p<0.001), claudin 5 (p=0.001) and claudin 7 (p<0.001) than did primary tumours. Primary lung tumours showed a strong inverse association between zeb1 and claudin 1 (p=0.024) and claudin 2 (p=0.003). For twist an inverse association was found with claudin 5 (p=0.007). In metastatic tumours zeb1 was inversely associated with claudin 7 (p=0.050) and twist with claudin 5 (p=0.025). Overexpression of claudin 5 was associated with decreased survival (p=0.017). For zeb1 or twist, no association with survival was observed in primary or metastatic tumours. Conclusions According to protein expression, involvement of zeb1 and twist in EMT in primary lung tumours is relatively infrequent. Carcinomas metastatic to the lung showed a significantly higher expression of these transcriptional factors than primary lung tumours, indicating their probable importance in the metastatic process. Zeb1 and twist were inversely associated with several claudins, indicating a role in their down-regulation.

Divergence of tight and adherens junction factors in alveolar epithelium in pulmonary fibrosis.

It has been proposed that an epithelial injury may be one of the multiple primary events in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to characterize the tight junction and adherens junction proteins in normal human lung, IPF, cryptogenic organizing pneumonia, and asbestosis. We determined the immunohistochemical cell-specific expression of tight junction proteins claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-7, as well as 3 adherens junction proteins, E-cadherin, N-cadherin, and β-catenin. We further analyzed the expression of claudin-1, claudin-3, and claudin-4 and E-cadherin, N-cadherin, and β-catenin at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of both tight junction and adherens junction proteins were elevated in regenerative alveolar epithelium in pulmonary fibrosis as compared with the expression of these proteins in normal alveolar epithelium. In particular, the expression levels of claudins-1 and claudin-3 were clearly elevated in all diseases. Furthermore, the amounts of adherens junction proteins messenger RNAs (mRNAs) were also all increased in pulmonary fibroses in comparison with healthy controls, with N-cadherin showing the greatest increase in mRNA levels in all diseases. However, the amounts of claudin-1, claudin-3, and claudin-4 mRNAs in fibrotic lung were similar to or even lower than those measured in the healthy controls. It is possible that the diminished capacity to produce claudin mRNAs may be one explanation for poor repair capacity of alveolar epithelial cells in IPF.

Impact of smoking on the expression of claudins in lung carcinoma

RATIONALE Tight junctions regulate the paracellular permeability and orientation of cells and claudins are key components of tight junctions. OBJECTIVES To study the influence of cigarette smoke on claudin expression in vitro and in lung cancer patients. METHODS We studied the effect of smoking on claudin expression by exposing a bronchial cell line (BEAS-2B) and two carcinoma cell lines (SK-LU1 and SK-MES1) to tobacco smoke for 48 h and analysed their claudin mRNA expression. The relation between smoked pack years and protein expression of claudins 1-5 and 7 in 344 lung cancer patients was determined by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS In BEAS-2B cells and SK-LU1 cells, an initial increase was followed by a decline in the mRNA expression of several claudins. In SK-MES1 cells, no evident elevation in claudin expression was observed. Intense claudin 1 and 4 positivity was found more often in cancer samples of smokers and ex-smokers compared to non-smokers (p<0.001 and p=0.003, respectively). Heavy smokers with longer than 40 pack-years consumption had more often intense claudin 1 (p=0.011), 4 (p=0.050) or 7 (p=0.058) expression in squamous cell carcinoma compared to non-smokers or smokers with fewer pack-years. Claudin 1 positivity predicted a better survival in adenocarcinoma (p=0.044) and in squamous cell carcinoma (p=0.027) and claudin 4 positivity in adenocarcinoma only (p=0.048). In squamous cell carcinoma, claudin 7 positivity was associated with a better survival (p=0.011). CONCLUSIONS Bronchial BEAS-2B cells and SK-LU1 cells respond to tobacco smoke by changing their claudin mRNA synthesis and resulting tight junction permeability changes may thus contribute to tobacco induced carcinogenesis both during initiation and progression. This concept is strengthened by findings in the clinical tumour material, where tobacco consumption was associated with claudin expression.

Glutathione-S-transferases in lung and sputum specimens, effects of smoking and COPD severity

BackgroundOxidative stress plays a potential role in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Glutathione S-transferases (GSTs) detoxify toxic compounds in tobacco smoke via glutathione-dependent mechanisms. Little is known about the regulation and expression of GSTs in COPD lung and their presence in airway secretions.MethodsGST alpha, pi and mu were investigated by immunohistochemistry in 72 lung tissue specimens and by Western analysis in total lung homogenates and induced sputum supernatants from non-smokers, smokers and patients with variable stages of COPD severity.ResultsGST alpha was expressed mainly in the airway epithelium. The percentage of GST alpha positive epithelial cells was lower in the central airways of patients with very severe (Stage IV) COPD compared to mild/moderate COPD (p = 0.02). GST alpha by Western analysis was higher in the total lung homogenates in mild/moderate COPD compared to cases of very severe disease (p < 0.001). GST pi was present in airway and alveolar epithelium as well as in alveolar macrophages. GST mu was expressed mainly in the epithelium. Both GST alpha and pi were detectable in sputum supernatants especially in patients with COPD.ConclusionThis study indicates the presence of GST alpha and pi especially in the epithelium and sputum supernatants in mild/moderate COPD and low expression of GST alpha in the epithelium in cases of very severe COPD. The presence of GSTs in the airway secretions points to their potential protective role both as intracellular and extracellular mediators in human lung.

Expression of snail, twist, and Zeb1 in malignant mesothelioma.

We examined 56 malignant mesotheliomas, 117 lung adenocarcinomas, and 34 metastatic lung adenocarcinomas with antibodies to transcription factors involved in epitheliomesenchymal transition. The tumors were stained immunohistochemically with antibodies zeb1, twist, and snail. Malignant mesotheliomas exhibited a stronger expression of zeb1 and twist than lung adenocarcinomas (p < 0.001 for both). Metastatic adenocarcinomas displayed a more frequent expression of zeb1 and twist (p < 0.001 for both) than lung adenocarcinomas. Patients with snail positive mesotheliomas experienced a better survival (p = 0.046), whereas in lung adenocarcinomas, this trait predicted worse survival (p = 0.024). Biphasic mesotheliomas had a more frequent expression of snail or zeb1 than epithelioid and sarcomatoid mesotheliomas as a group (p = 0.034 and p = 0.005, respectively). E‐cadherin was more commonly present in epithelioid mesotheliomas (p = 0.002). Cases with snail positivity showed a significantly lower apoptotic index (p = 0.039). Malignant mesotheliomas display strong twist and zeb1 expression, which may be an indication of transdifferentiation between the epithelioid and sarcomatoid cell types with biphasic mesotheliomas representing tumors in active transition. Metastatic adenocarcinomas show a higher expression of zeb1 and twist than primary lung tumors, confirming our previous findings and highlighting their significance in the metastatic process. Snail expression is associated with poor prognosis in lung adenocarcinomas, but an opposite effect was seen in mesothelioma, a fact which may be related to the different cellular origin of epithelial and mesothelial cells.

Gender differences in the T-cell profiles of the airways in COPD patients associated with clinical phenotypes

T lymphocytes are believed to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How T cells are recruited to the lungs and contribute to the inflammatory process is largely unknown. COPD is a heterogeneous disease, and discriminating disease phenotypes based on distinct molecular and cellular pathways may provide new approaches for individualized diagnosis and therapies. Bronchoalveolar lavage (BAL) and blood samples were obtained from 40 never-smokers, 40 smokers with normal lung function, and 38 COPD patients. T-cell chemokine receptor expression was analyzed with flow cytometry, and soluble BAL cytokines and chemokines were measured using a cytokine multiplex assay. Correlations with gender and clinical characteristics including lung imaging were investigated using multivariate modeling. Th1/Tc1- and Th2/Tc2-associated soluble analytes and T-cell chemokine receptors were analyzed as cumulative Th1/Tc1 and Th2/Tc2 immune responses. A higher expression of chemokine receptor CCR5 on CD8+ T cells in BAL and higher percentage of CXCR3+CD8+ T cells in blood was found in female smokers with COPD compared to those without COPD. CCR5 expression on CD4+ and CD8+ T cells was lower in BAL from male smokers with COPD compared to those without COPD. Among female smokers with COPD, Th1/Tc1 immune response was linked to BAL macrophage numbers and goblet cell density, and Th2/Tc2 response was associated with the measures of emphysema on high-resolution computed tomography. The highly gender-dependent T-cell profile in COPD indicates different links between cellular events and clinical manifestations in females compared to males. Our findings may reveal mechanisms of importance for the difference in clinical course in female COPD patients compared to males.

GASC1 expression in lung carcinoma is associated with smoking and prognosis of squamous cell carcinoma.

GASC1 (gene amplified in squamous cell carcinoma 1) encodes a nuclear protein that epigenetically catalyses the lysine demethylation of histones. We investigated the expression of GASC1 in different histological subtypes of lung cancer (n=289). Percentage value of GASC1 immunohistochemical expression was evaluated separately in the nuclei and cytoplasms of epithelial cancer cells. The results were compared with clinicopathologic factors and the smoking history of the patients. In lung tumor cells, 38% of nuclei and 54% of the cytoplasms stained positive for GASC1. Adenocarcinomas expressed more GASC1 nuclear (p=0.00011) and cytoplasmic (p=0.00074) positivity than squamous cell carcinoma. Smokers displayed less nuclear and cytoplasmic GASC1 expression than non-smokers (p=0.028 and p=0.036, respectively). Similarly, patients with more cytoplasmic positive staining had fewer pack years (p=0.043). Nuclear GASC1 expression had an impairing effect on survival when all histological lung cancer types were analysed together (p=0.039) and separately in squamous cell lung carcinoma (p=0.016). The results reveal that GASC1 expression is higher in adenocarcinoma than squamous cell carcinoma. Smoking decreases GASC1 expression in tumor cells, indicating that tobacco smoke may influence the methylation of histone 3 lysine residues in lung cancer. Nonetheless, nuclear GASC1 predicts a poor prognosis, especially in squamous cell carcinoma.