Recent studies have shown that estrogens promote the growth of lung cancer cells and may potentially be responsible for increased susceptibility to lung cancer in women. These observations raise the possibility of using antiestrogens in treating and preventing lung cancer. However, it is not clear how estrogen receptors (ERs) modulate the growth of non-small cell lung cancer (NSCLC) cells. Our Western blotting and real-time PCR analysis showed that NSCLC cells expressed ERbeta, but not ERalpha. In addition, ERbeta-specific ligands, but not ERalpha-specific ligands, promoted the growth of lung cancer cells. Furthermore, knockdown of ERbeta by short hairpin RNA constructs resulted in loss of estrogen-dependent growth of lung cancer cells. Interestingly, endogenous ERbeta failed to transcriptionally activate estrogen response element (ERE)-luciferase constructs in NSCLC cells, suggesting a lack of genomic function. Upon further investigation, ERbeta was found to be in the cytoplasm in all lung cancer cells and failed to translocate to the nucleus in the presence of estrogen, as observed by biochemical, ArrayScan, and confocal microscopy experiments. Nonetheless, estrogen caused rapid activation of cAMP, Akt, and MAPK signaling pathways in lung cancer cells. Immunohistochemical analysis of lung tumor biopsies showed strong ERbeta staining in the cytoplasm, whereas no staining was observed for ERalpha. In conclusion, our results suggest that that proliferative effects of estrogen in lung cancer cells is mediated primarily, if not exclusively, by the nongenomic action of ERbeta.

Recent studies have demonstrated the presence of estrogen receptor (ER)beta in the mitochondria in various cell types and tissues, but the exact function of this localization remains unclear. In this study, we have examined the function of mitochondrial ERbeta in non-small-cell lung cancer (NSCLC) cells. Down-regulation of ERbeta by short hairpin RNA constructs sensitized NSCLC cells to various apoptosis-inducing agents such as cisplatin, taxol, and etoposide. The increased growth inhibition and induction of apoptosis in ERbeta-knockdown cells was observed irrespective of estrogen treatment, suggesting a ligand-independent role of ERbeta in regulating the intrinsic apoptotic pathway. Further, ERbeta from the mitochondrial fraction physically interacted with the proapoptotic protein Bad, in a ligand-independent manner. Glutathione-S-transferase pull-down assays and molecular modeling studies revealed that the DNA-binding domain and hinge region of ERbeta, and the BH3 domain of Bad were involved in these interactions. Further investigations revealed that ERbeta inhibited Bad function by disrupting Bad-Bcl-X(L) and Bad-Bcl-2 interactions. Reintroduction of ERbeta in the mitochondria of ERbeta knockdown cells reversed their sensitivity to cisplatin. Overall, our results demonstrate a ligand-independent role of ERbeta in regulating apoptosis, revealing a novel function for ERbeta in the mitochondria.

Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models of CDH. We hypothesize that defective parasympathetic innervation may contribute to airway branching abnormalities and, therefore, lung hypoplasia, during lung development in CDH. The murine nitrofen model of CDH was utilized to study the effect of the cholinergic agonist carbachol on embryonic day 11.5 (E11.5) lung explant cultures. Airway branching and contractions were quantified. In a subset of experiments, verapamil was added to inhibit airway contractions. Sox9 immunostaining and 5-bromo-2-deoxyuridine incorporation were used to identify and quantify the number and proliferation of distal airway epithelial progenitor cells. Intra-amniotic injections were used to determine the in vivo effect of carbachol. Airway branching and airway contractions were significantly decreased in nitrofen-treated lungs compared with controls. Carbachol resulted in increased airway contractions and branching in nitrofen-treated lungs. Nitrofen-treated lungs exhibited an increased number of proliferating Sox9-positive distal epithelial progenitor cells, which were decreased and normalized by treatment with carbachol. Verapamil inhibited the carbachol-induced airway contractions in nitrofen-treated lungs but had no effect on the carbachol-induced increase in airway branching, suggesting a direct carbachol effect independent of airway contractions. In vivo treatment of nitrofen-treated embryos via amniotic injection of carbachol at E10.5 resulted in modest increases in lung size and branching at E17.5. These results suggest that defective parasympathetic innervation may contribute to airway branching abnormalities in CDH.

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