Tobias N. Cassel

Regulation of Postnatal Lung Development and Homeostasis by Estrogen Receptor β

ABSTRACT Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor α (ERα) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ERβ is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERβ gene (ERβ−/−) revealed decreased numbers of alveoli in adult female ERβ−/− mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ERβ−/− mice, and direct transcriptional regulation of these genes by ERβ was demonstrated. This suggests that estrogens act via ERβ in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ERβ as a previously unknown regulator of postnatal lung development and homeostasis.

Transcription-Associated Recombination Is Dependent on Replication in Mammalian Cells

ABSTRACT Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

Decreased serum and bronchoalveolar lavage levels of Clara cell secretory protein (CC16) is associated with bronchiolitis obliterans syndrome and airway neutrophilia in lung transplant recipients.

Background. The major hinderance for long-term survival after lung transplantation is chronic rejection in the form of bronchiolitis obliterans syndrome (BOS). BOS is a fibrosing process in the small airways causing irreversible airway obstruction. BOS is associated with increased oxidative burden and activation of inflammatory and growth-stimulating mediators. The Clara cell secretory protein (CCSP or CC16) is a secreted differentiation marker for the bronchiolar epithelium with both antioxidative and antiinflammatory/immmunomodulatory properties. We asked whether this molecule could have a role in the development of BOS. Methods. Serum and bronchoalveolar lavage (BAL) fluid samples were collected from 22 consecutive lung transplant recipients, the majority (19) was followed for 2 years. Six patients developed BOS. CCSP in serum was measured in 162 samples from 19 patients with an ELISA method, and CCSP in 191 BAL samples from 22 patients with quantitative Western blot. Results. CCSP in both serum and BAL was significantly lower in BOS compared with acute rejection or no rejection. After the first postoperative month, serum and BAL CCSP levels were consistently lower in the patients who developed BOS than in those who did not. The percentage of neutrophils in BAL correlated negatively with CCSP in BAL. Conclusions. Levels of CCSP in serum and BAL is lowered in BOS. Serum CCSP could have a potential as an early marker for BOS. The correlation between decreased CCSP and increased neutrophils in BAL suggests a loss of local airway defense capacity in BOS.

Regulation of the Clara cell secretory protein/uteroglobin promoter in lung.

Abstract: Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the conducting airway epithelium of the lung in a differentiation‐dependent manner. The proximal promoter region of the rodent CCSP/UG gene directs Clara cell specificity. Previously, it was shown that the forkhead transcription factors HNF‐3α and β and the homeodomain factor TTF‐1 are important transcription factors acting through this region, suggesting that they contribute to cell specificity of the CCSP/UG gene. Members of the C/EBP family of transcription factors can also interact with elements of the proximal rat and mouse CCSP/UG promoters. The onset of C/EBPα expression in Clara cells correlates with the strong increase of CCSP/UG expression. Thus, C/EBPalpha; may play a crucial role for differentiation‐dependent CCSP/UG expression. Transfection studies demonstrate that C/EBPα and TTF‐1 can synergistically activate the murine CCSP/UG promoter. Altogether, these results suggest that C/EBPalpha;, TTF‐1, and HNF‐3 determine the Clara cell‐specific, differentiation‐dependent expression of the CCSP/UG gene in murine lung. The relative importance of these three transcription factors, however, differs in rabbits and humans.

Glucocorticoids regulate the CCSP and CYP2B1 promoters via C/EBPβ and δ in lung cells

Glucocorticoids have several important roles in the lung and play a key role in lung development and maturation. However, the specific molecular mechanisms of glucocorticoid action in lung are unclear. In this study, we have investigated two glucocorticoid-regulated genes expressed in the lung epithelium, the secretory protein CCSP, and the P450-enzyme CYP2B1. In transient transfections of lung epithelial cells, glucocorticoids increased expression from the CCSP and CYP2B1 promoters and we demonstrated that induction was dependent on the integrity of C/EBP-binding sites in both promoters. Electrophoretic mobility shift assays revealed increased DNA-binding of C/EBPβ and C/EBPδ after glucocorticoid treatment, which was not correlated to altered protein levels. The results of this study indicate a previously unknown role for C/EBP transcription factors in glucocorticoid signaling in the lung epithelium.

Different regulation of the LXRα promoter activity by isoforms of CCAAT/enhancer-binding proteins

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRalpha promoter was thus studied to investigate if LXRalpha gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXRalpha gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRalpha promoter. C/EBPalpha upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPbeta and C/EBPdelta had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPalpha and C/EBPbeta downregulated expression of the reporter gene while C/EBPdelta induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRalpha promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRbeta is constitutively expressed during the entire differentiation process while LXRalpha is induced upon addition of differentiation mix.

In vivo modulation of glucocorticoid receptor mRNA by inhaled fluticasone propionate in bronchial mucosa and blood lymphocytes in subjects with mild asthma

BACKGROUND In vivo regulation of the glucocorticoid receptor (GR) by glucocorticoids provides a means of modulating sensitivity of targeted cells. OBJECTIVE We sought to determine the in vivo modulation of GR mRNA expression by fluticasone propionate (FP) in subjects with mild asthma. METHODS Ten atopic asthmatic subjects were treated with FP 250 microg twice daily for 4 weeks. Before and after treatment, the patients underwent fiberoptic bronchoscopy with endobronchial biopsy and sampling of venous blood for measurements of GR mRNA levels. A solution hybridization assay was used for quantitative analysis of GR mRNA. In addition, a 24-hour urinary cortisol excretion and an adrenocorticotropic hormone test before and after treatment with FP were performed. RESULTS A high interindividual variation in GR mRNA expression was seen. However, we detected a significant reduction of the GR mRNA levels in the endobronchial biopsy specimens after FP treatment (36.6 +/- 23.1 and 25.0 +/- 10.9 amol GR mRNA/microg RNA, respectively; P <.01). In the peripheral blood lymphocytes an even more striking downregulation of the GR by its cognate ligand was documented (30.3 +/- 26.5 and 8.8 +/- 5 amol GR mRNA/microg RNA, respectively; P <.001), possibly reflecting differences in glucocorticoid sensitivity between tissues. A small but significant reduction of the 24-hour urinary cortisol excretion was observed (233 +/- 109 and 157 +/- 66 nmol/L, respectively; P <.01), whereas the feedback regulation of glucocorticoid synthesis by means of the hypothalamic-pituitary-adrenal axis as assessed by the adrenocorticotropic hormone test remained normal after treatment with FP. CONCLUSION The results in this study confirm the potency of the inhaled corticosteroid FP and provide evidence for a considerable tissue-specific interindividual variation in the expression of the GR.

C/EBPα and TTF-1 Synergistically Transactivate the Clara Cell Secretory Protein Gene

In the lung, the Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the Clara cells of the small airway epithelium in a differentiationdependent manner. Although the physiological function of CCSP is unknown, the differentiation-dependent expression of this protein makes it a good model to study the mechanisms behind pulmonary epithelial gene expression during lung development and differentiation. In the rat developing lung, low levels of CCSP expression are first detected around embryonic day 16. As development proceeds, CCSP expression starts increasing at day 18–19 and further increases until birth.1 Previously, the homeodomain thyroid transcription factor-1 (TTF-1) and the forkhead hepatocyte nuclear factor-3 (HNF-3) have been identified as major regulators of pulmonary CCSP gene expression.2–4 Both transcription factors are expressed in the bronchiolar epithelium and in Clara cells. However, during lung development, the expression of HNF-3 factors and TTF-1 does not temporally reflect the expression pattern of CCSP. Therefore, the increase in CCSP expression levels seen late in lung development cannot be explained by the action of HNF-3 factors or TTF-1 alone. The CCAAT/enhancer binding protein alpha (C/EBPα) is expressed in the lung epithelium and is first detected in the rat lung at embryonic day 18–19,5 correlating to the increase in CCSP expression. Recently, we have characterized a compound C/EBPresponse element in the proximal CCSP promoter.6 C/EBPα as well as C/EBPδ, another C/EBP family member that is expressed in the bronchiolar epithelium, transactivate the CCSP promoter through interaction with the two C/EBP-binding sites that form the compound element.6 The C/EBP-binding sites are situated in close proximity to the previously characterized TTF-1 and HNF-3 binding sites. Together, this prompted us to investigate putative cooperative interactions between these transcription factors in the regulation of CCSP. To address the question of whether C/EBP, TTF-1, and HNF-3 factors interact in the regulation of CCSP, we used Drosophila SL-2 cells. These cells lack many mammalian transcription factors and are thus well suited for studies of cooperative inter-

Pre-vaccination incidence of genital warts in 15-23-year-old women and men attending youth clinics in Stockholm, Sweden.

Abstract Background: Human papillomavirus (HPV) vaccines were introduced to the market in 2006 and 2007. The present pilot study was designed to examine the incidence of genital warts in the population up to 23 y of age in the county of Stockholm before the start of mass HPV vaccination. Methods: Data from the electronic health records of 9 youth clinics in the county of Stockholm were collected retrospectively for the y 2006–2008. Results: In total, 49,985 patients visited the study youth clinics during 2006–2008. Of these, 1817 were denoted genital warts patients. An extrapolation of the study data was done in an attempt to estimate the annual number of genital warts cases in the full Stockholm County population aged 15–23 y. Results showed that there were approximately 1792 genital warts patients in the age group 15–23 y each year in Stockholm County. Female cases represented approximately 62% of all cases in the age group 15–23 y. The peak incidence was at around 20 y of age for females, while males had a more flattened peak incidence around 19–23 y of age. Conclusion: This pilot study demonstrates that, compared to other reported data, genital warts are at least as common in Sweden as in other countries among 15–23 y old females and males.