Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening.

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial kindreds with pulmonary fibrosis. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no previous connection to telomere biology or disease, with five new heterozygous damaging mutations in unrelated cases and none in controls (P = 1.3 × 10(-8)); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more new damaging and missense variants at conserved residues in cases than in controls (P = 1.6 × 10(-6)). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths, and we observed epigenetic inheritance of short telomeres in family members. Together, these genes explain ~7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction.

Pleiomorphic adenoma gene-like 2 expression is associated with the development of lung adenocarcinoma and emphysema.

Previous study of transgenic mice with long-term expression of pleiomorphic adenoma gene-like 2 (PLAGL2), a surfactant protein C (SP-C) transactivator, in type II cells showed the manifestation of centrilobular emphysema in vivo. Since emphysema is an independent risk factor for bronchogenic carcinoma, we hypothesized that the mouse lungs with induced PLAGL2-expression had increased incidences in developing lung adenocarcinoma. To test the hypothesis, mouse lungs were examined for the presence of tumors. Male mice with induced PLAGL2-expression in the lungs were more vulnerable to tumorigenesis than female mice (p<0.05). Epithelial cells expressing pro-SP-C and Clara cell secretory protein (CCSP) at the terminal bronchioles and the bronchoalveolar duct junction (BADJ) were increased in the induced transgenic mice, suggesting a role of PLAGL2 in expanding SP-C expression cells. Co-expression of TTF-1, pro-SP-C and CD133 (a stem-cell marker) in cancer and distal airway epithelial cells indicated that both cells were derived from common progenitors. This result supported a common-cell-origin mechanism for the comorbid diseases - emphysema and lung cancer. Furthermore, a public lung cancer gene expression profiling database was examined to determine the relevance of PLAGL2 expression and lung adenocarcinoma in humans. Patients with high PLAGL2 expression in lung tumors were readily found. Female patients (N=218) with low PLAGL2 expression (the lowest quartile of total patients) at the early-stage of disease had better prognosis in survival. Male patients, on the other hand, had no such correlation. Generally, their survival rate was significantly poorer than of female patients. Taken together, our data suggested a pathological role of PLAGL2 in lung adenocarcinoma development and a preferable prognosis of low PLAGL2 expression in female patients.

PLAGL2 expression-induced lung epithelium damages at bronchiolar alveolar duct junction in emphysema: bNip3- and SP-C-associated cell death/injury activity.

Emphysema and bronchitis are major components of chronic obstructive pulmonary disease (COPD). Pleomorphic adenoma gene like-2 (PLAGL2), a zinc finger DNA-binding protein, is a transcription factor of the surfactant protein C (SP-C) promoter. Using an inducible transgenic mouse model, PLAGL2 and SP-C were ectopically expressed in lung epithelial cells of terminal bronchiole including the bronchoalveolar duct junction (BADJ), where only few cells express both genes under normal conditions. Ectopic PLAGL2 was also expressed in alveolar type II cells of induced mice. The overexpression of PLAGL2 was associated with the development of air space enlargement in the distal airways of adult mice. Defective alveolar septa and degraded airway fragments were found in the lesions of emphysematous lungs, indicating chronic airway destruction. Female mice were particularly sensitive to the effects of PLAGL2 overexpression with more dramatic emphysematous changes compared with male mice. In addition, analysis of the respiratory system mechanics in the mice indicated that the induction of PLAGL2 resulted in a significant increase in respiratory system compliance. Both TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 analyses showed that apoptotic activity was increased in epithelial cells within the emphysematous lesions as well as at the BADJ. Our results indicate that increased cell injury and/or death could be caused directly by the upregulation of PLAGL2 downstream gene, bNip3, a preapoptotic molecule that dimerizes with Bcl-2, or indirectly by the aberrant expression of SP-C-induced endoplasmic reticulum stress in epithelial cells. Finally, increased expression of PLAGL2 in alveolar epithelial cells correlated with the development of emphysema in the lung of COPD patients. In summary, our data from both animal and human studies support a novel pathogenic role of PLAGL2 in pulmonary emphysema, a critical aspect of severe COPD.

Modulation of PLAGL2 transactivation activity by Ubc9 co-activation not SUMOylation.

Pleomorphic adenoma gene like-2 (PLAGL2), a developmentally regulated and stress inducible zinc finger protein can be post-translationally modified by small ubiquitin-like modifier peptide (SUMO-1); and SUMOylation attenuates PLAGL2 activity on the interactive promoter. Since PLAGL2 was a transactivator of the surfactant protein-C (SP-C) promoter, we hypothesized that SUMOylation down-regulated PLAGL2-activated SP-C promoter activity. Unexpectedly, the SUMO-conjugating enzyme Ubc9 enhanced, rather than reduced, PLAGL2 activated promoter activity but did not affect TTF-1 activation of the promoter. Ubc9 mutant (Ubc9-C93S) defective in SUMO-conjugating activity also enhanced PLAGL2-driven promoter activity suggesting that the stimulatory effect of Ubc9 on SP-C promoter activation was independent of its enzymatic function. PLAGL2 mutants without the K250 and/or K269 SUMOylation sites did not further improve PLAGL2 programmed transcription nor did they abolish Ubc9 enhanced promoter activity supporting the SUMOylation-independent mechanism. Chromatin immunoprecipitation (ChIP) assay demonstrated the association of PLAGL2 and Ubc9 with the SP-C promoter in vivo. Taken together, our data suggests that Ubc9 can function as a co-factor of PLAGL2, uncoupling from its enzymatic activity, to mediate PLAGL2 interactive SP-C promoter activity.

PLAGL2 translocation and SP-C promoter activity--a cellular response of lung cells to hypoxia.

Cobalt is a transition metal which can substitute for iron in the oxygen-sensitive protein and mimic hypoxia. Cobalt was known to be associated with the development of lung disease. In this study, when lung cells were exposed to hypoxia-induced by CoCl(2) at a sub-lethal concentration (100 microM), their thyroid transcription factor-1 (TTF-1) expression was greatly reduced. Under this condition, SP-B promoter activity was down-regulated, but SP-C promoter remained active. Therefore, we hypothesized that other factor(s) besides TTF-1 might contribute to the modulation of SP-C promoter in hypoxic lung cells. Pleomorphic adenoma gene like-2 (PLAGL2), a previously identified TTF-1-independent activator of the SP-C promoter, was not down-regulated, nor increased, within those cells. Its cellular location was redistributed from the cytoplasm to the nucleus. Chromatin immunoprecipitation (ChIP) and quantitative RT-PCR analyses demonstrated that nuclear PLAGL2 occupied and transactivated the endogenous SP-C promoter in lung cells. Thereby, through relocating and accumulating of PLAGL2 inside the nucleus, PLAGL2 interacted with its target genes for various cellular functions. These results further suggest that PLAGL2 is an oxidative stress responding regulator in lung cells.

The TTF-1/TAP26 complex differentially modulates surfactant protein-B (SP-B) and -C (SP-C) promoters in lung cells.

Surfactant protein-B (SP-B) and -C (SP-C) are small hydrophobic surfactant proteins that maintain surface tension in alveoli. Both SP-B and SP-C are regulated by a key factor, thyroid transcription factor-1 (TTF-1), in lung cells. Previously, we identified a 26-kDa, TTF-1-associated protein (TAP26) that was shown to interact with TTF-1 and enhance TTF-1-transactivated SP-B promoter activity. In this study, we hypothesized that TAP26 could also serve as a co-activator of the SP-C promoter. Using the chromatin immunoprecipitation assay (ChIP), we demonstrated that TAP26 was not only a component of the SP-B promoter, but was also a component of the SP-C promoter complex in lung cells. TAP26 could synergistically stimulate TTF-1-activated SP-B and SP-C promoter activities in H441 cells (a lung adenocarcinoma cell). However, in MLE12 cells (a murine lung type II cell), only SP-B, but not SP-C, promoter activity was improved by TAP26 in a concentration-dependent manner. This result indicated that the TTF-1/TAP26 complex-activated SP-C promoter activity was already optimized in MLE12 cells and that the response of the SP-C promoter to the complex was different from that of the SP-B promoter. Via promoter mutation analysis, adjacent TTF-1 binding sites within the proximal promoter region of SP-C were found to be essential for TTF-1/TAP26-enhanced SP-C promoter activity. Thus, a dimerized complex structure was needed for advanced promoter activity. This result also provided a molecular mechanism by which both the SP-B and SP-C promoters could be differentially regulated by the same complex.

Pleiomorphic adenoma gene-like-2, a zinc finger protein, transactivates the surfactant protein-C promoter.

Expression of surfactant protein (SP)-C occurs principally in type II pneumocytes located in the distal lung alveolae. SP-C expression is thought to be primarily regulated by thyroid transcription factor (TTF)-1 and its associated proteins interacting with a previously defined promoter region between -197 and -158 in mice. We screened a human lung cDNA library using a modified yeast one-hybrid system and identified pleiomorphic adenoma gene-like (PLAGL)-2, a ubiquitously expressed zinc finger protein, as a transfactor of the SP-C promoter. The PLAGL2 DNA-binding site was located in the SP-C promoter proximal region close to the TTF-1 sites. This site was demonstrated to be functional by use of electrophoresis mobility shift assay, mutagenesis analysis, and transfection studies. PLAGL2 bound to DNA via its N-terminus zinc fingers and activated the SP-C promoter in a TTF-1-independent manner. Both human and mouse SP-C promoters, but not the SP-B promoter, could be activated by PLAGL2 in transfected human embryonic kidney-293 (HEK293) cells as well as in murine type II (MLE12) cells. The expression of PLAGL2 in isolated human embryonic lung type II cells and its transactivation activity on the SP-C promoter suggest that PLAGL2 may modulate SP-C expression during lung development.