NF-κB/RelA signaling in secretoglobin progenitors mediates plasticity and MMP-induced barrier disruption in house dust mite-induced allergic asthma.

The mechanisms how aeroallergens induce sensitization are incompletely understood. The house dust mite (HDM) Dermatophagoides pteronyssius (Der p) is a ubiquitous aeroallergen that represents a major cause of allergic rhinitis and asthma. Herein, we tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure is a rapid activator of NF-κB/RelA in the Secretoglobin (Scgb1a1+) lineage associated with upregulation of NF-κB/RelA-dependent markers of epithelial plasticity. To determine the effect of epithelial NF-κB signaling, NF-κB was depleted in a tamoxifen (TMX)-inducible Scgb1a1-CreERTM mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted [RelA knockdown (KD)] mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of metalloproteinase (MMP)9 that are RelA-dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity, and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type (WT) mice. Loss of functional barrier was associated with MMP disruption of zona occludens (ZO)-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability that allows allergen penetration, sensitization producing allergic asthma (AA) in vivo. We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.NEW & NOTEWORTHY Allergic asthma from house dust mite (HDM) allergy causes substantial morbidity. This study examines the dynamic changes in small airway epithelial cells in a mouse model of HDM exposure. Our findings indicate that NF-κB/RelA signaling mediates matrix metalloproteinase production, disrupting the epithelial barrier resulting in allergic sensitization. Our findings bring new insight into mechanisms for epithelial cell-state change in the allergen response, creating a potential therapeutic pathway for maintaining barrier function in asthma.

Genome-wide analyses in Lyme borreliosis: identification of a genetic variant associated with disease susceptibility and its immunological implications.

BACKGROUND: Genetic variation underly inter-individual variation in host immune responses to infectious diseases, and may affect susceptibility or the course of signs and symptoms. METHODS: We performed genome-wide association studies in a prospective cohort of 1138 patients with physician-confirmed Lyme borreliosis (LB), the most common tick-borne disease in the Northern hemisphere caused by the bacterium Borrelia burgdorferi sensu lato. Genome-wide variants in LB patients-divided into a discovery and validation cohort-were compared to two healthy cohorts. Additionally, ex vivo monocyte-derived cytokine responses of peripheral blood mononuclear cells to several stimuli including Borrelia burgdorferi were performed in both LB patient and healthy control samples, as were stimulation experiments using mechanistic/mammalian target of rapamycin (mTOR) inhibitors. In addition, for LB patients, anti-Borrelia antibody responses were measured. Finally, in a subset of LB patients, gene expression was analysed using RNA-sequencing data from the ex vivo stimulation experiments. RESULTS: We identified a previously unknown genetic variant, rs1061632, that was associated with enhanced LB susceptibility. This polymorphism was an eQTL for KCTD20 and ETV7 genes, and its major risk allele was associated with upregulation of the mTOR pathway and cytokine responses, and lower anti-Borrelia antibody production. In addition, we replicated the recently reported SCGB1D2 locus that was suggested to have a protective effect on B. burgdorferi infection, and associated this locus with higher Borrelia burgdorferi antibody indexes and lower IL-10 responses. CONCLUSIONS: Susceptibility for LB was associated with higher anti-inflammatory responses and reduced anti-Borrelia antibody production, which in turn may negatively impact bacterial clearance. These findings provide important insights into the immunogenetic susceptibility for LB and may guide future studies on development of preventive or therapeutic measures. TRIAL REGISTRATION: The LymeProspect study was registered with the International Clinical Trials Registry Platform (NTR4998, registration date 2015-02-13).

Secretoglobins in the big picture of immunoregulation in airway diseases.

The proteins of the secretoglobin (SCGB) family are expressed by secretory tissues of barrier organs. They are embedded in immunoregulatory and anti-inflammatory processes of airway diseases. This review particularly illustrates the immune regulation of SCGBs by cytokines and their implication in the pathophysiology of airway diseases. The biology of SCGBs is a complex topic of increasing importance, as they are highly abundant in the respiratory tract and can also be detected in malignant tissues and as elements of immune control. In addition, SCGBs react to cytokines, they are embedded in Th1 and Th2 immune responses, and they are expressed in a manner dependent on cell maturation. The big picture of the SCGB family identifies these factors as critical elements of innate immune control at the epithelial barriers and highlights their potential for diagnostic assessment of epithelial activity. Some members of the SCGB family have so far only been superficially examined, but have high potential for translational research.

Role of Secretoglobin+ (club cell) NFκB/RelA-TGFß signaling in aero-allergen-induced epithelial plasticity and subepithelial myofibroblast transdifferentiation.

Repetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFßR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFß1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA+Epcam+ population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFß signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin+ cells results in inhibition of the mucosal TGFß response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin-derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFß1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway.

Secretory carcinoma of the skin with lymph node metastases and recurrence in both lungs: A case report.

Secretory carcinoma of the skin is an extremely rare adnexal tumor, histopathologically identical to homologous lesions in the salivary glands and breast tissue. Although this tumor was previously reported as indolent, we report a case of secretory carcinoma of the skin with metastases and recurrence. The patient, a 31-year-old women, had a subcutaneous mass in the right axilla. The resected specimen contained a circumscribed mass, with proliferating tumor cells that exhibited prominent nucleoli. They exhibited glandular and papillary growth patterns and there were amphophilic secretions in the glands. Immunohistochemically, the tumor cells were positive for mammaglobin and S100. The tumor was surrounded by sweat glands and there was no mammary glandular tissue, suggesting that it was derived from axillary sweat glands. Accordingly, we made a diagnosis of secretory carcinoma of the skin. Four years after the operation, there were metastases in both lungs. The resected specimen revealed a tumor identical to that of the original skin tumor. Next-generation sequencing-based multiplex gene assay performed on the metastatic tissue revealed an ETV6-NTRK3 fusion gene. This is a rare case report of secretory carcinoma of the skin with lymph node metastases and recurrence in both lungs.

[Measurement of PODXL and SCGB1D2 for detecting intraductal papillary mucinous neoplasm].

We determined whether PODXL and SCGB1D2 expressions in whole blood could be useful as diagnostic biomarkers to determine the presence of intraductal papillary mucinous neoplasm (IPMN), as compared to serum CA19-9. A discovery-stage clinical study was performed on 12 patients with IPMN, including 6 intraductal papillary mucinous adenoma (IPMA) patients and 6 intraductal papillary mucinous carcinoma (IPMC) patients who had undergone treatment at the Department of Surgery at Kochi Health Sciences Center and the Department of Gastroenterology and Hepatology at Kochi Medical School Hospital from April 2015 to January 2016;13 controls who did not have pancreatic disease were also enrolled. Serum PODXL and SCGB1D2 levels were measured using ELISA. We found that the area under the receiver-operating characteristic curve (AUC) for IPMN (IPMA+IPMC) diagnosis in IPMN patients and control individuals was 0.89 (95% CI:0.76-1) for PODXL, 0.50 (95% CI:0.25-0.74) for SCGB1D2, and 0.81 (95% CI:0.62-1) for CA19-9. Multivariable logistic regression analysis showed that PODXL was independently able to distinguish IPMN patients from controls. PODXL may be a novel, non-invasive diagnostic biomarker for the detection of IPMN. The AUC for distinguishing IPMC patients from IPMA patients was 0.78 (95% CI:0.47-1) for PODXL, 0.83 (95% CI:0.58-1) for SCGB1D2, and 0.58 (95% CI:0.22-0.95) for CA19-9. Although it was quantitatively demonstrated that the detection of PODXL and SCGB1D2 in the serum may provide a novel, non-invasive approach for distinguishing IPMC patients from IPMA patients, the present findings are preliminary until more elaborate studies are able to clarify whether PODXL and SCGB1D2 are useful as diagnostic markers for IPMC detection.

Differentially expressed Secretoglobin 1C1 gene in nasal polyposis.

Nasal polyps (NP) are the most common pathological change that occurs in the nasal mucosa and is characterized by mucosal inflammation. Although its etiology and pathogenesis have not been clearly explained, its pathophysiology is arranged by the balance between pro-inflammatory and anti-inflammatory cytokines. The Secretoglobin 1C1 gene synthesizes odor molecule binding proteins (OBPs) in the nasal mucosa and regulates some cytokines. The Secretoglobin 1C1 gene expression could be disrupted by polymorphisms that may act as a possible cause of a disruption in the regulation of the promotor of the gene. Therefore, the main aim of this study was to determine the effects of Secretoglobin 1C1 gene promotor polymorphisms on the gene expression in NP.  In this study, to determine the relationship between the Secretoglobin 1C1 gene promotor polymorphisms and the gene expression, the levels of 48 subjects were sequenced (24 patients with NP and 22 controls without sinonasal disease). The levels' expression of Secretoglobin 1C1 in the subjects' nasal mucosa was also detected using RT-PCR.  In this study, the level of Secretoglobin 1C1's expression increased in NP (P= 0.003). Three polymorphisms were detected in the Secretoglobin 1C1 gene's promotor. The rs113795008 and rs2280540 variations were significantly high in NP (P= 0.005, P= 0.045). The the rs113795008 homozygous mutant type genotype (G/G) was associated with a high mRNA expression level of Secretoglobin 1C1 in NP (P= 0.009). A correlation was found between a high level of Secretoglobin 1C1 expression and its promotor polymorphism, which thus might increase and/or contribute to the susceptibility of developing NP in the Turkish population. These findings suggested that promotor variations in the function of the Secretoglobin 1C1 gene can alter the gene expression biology in NP.

Association Between Secretoglobin Family 3A Member 2 (SCGB3A2) Gene Polymorphisms and Asthma in a Korean Population.

BACKGROUND Secretoglobin family 3A member 2 (SCGB3A2) plays an important role in secreting lung surfactant protein, which is a downstream target of thyroid transcription factor. MATERIAL AND METHODS We investigated whether single-nucleotide polymorphisms (SNPs) of SCGB3A2 gene contribute to susceptibility to asthma. To explore this possible association, 2 promoter SNPs (rs6882292, 659 G/A and rs1368408, -112 G/A) and missense SNP (rs151333009, stop codon) were tested in SCGB3A2 gene in 101 asthma patients and 377 healthy control subjects. SNPStats was used to obtain odds ratio (OR), 95% confidence intervals (CI), and P value adjusted for age and sex as covariables. Logistic regression method in each model (dominant, recessive, and log-additive) was applied to analyze genetic data. RESULTS rs151333009 SNP showed a monomorphic genotype. Two promoter SNPs (rs6882292, -659 G/A and rs1368408, -112 G/A) showed significant association with asthma (rs6882292, OR=2.66, 95% CI=1.42-5.01, p=0.0033 in dominant model, OR=2.45, 95% CI=1.33-4.54, p=0.0055 in log-additive model; rs1368408, OR=1.59, 95% CI=1.02-2.49, p=0.041 in dominant model, OR=3.02, 95% CI=1.15-7.90, p=0.03 in recessive model, OR=1.63, 95% CI=1.63, 95% CI=1.12-2.37, p=0.012 in log-additive model). CONCLUSIONS These results suggest that the promoter SNPs (rs6882292 and rs1368408) of SCGB3A2 gene may contribute to susceptibility to asthma in a Korean population.

Secretoglobin Superfamily Protein SCGB3A2 Alleviates House Dust Mite-Induced Allergic Airway Inflammation in Mice.

BACKGROUND: Secretoglobin (SCGB) 3A2, a novel, lung-enriched, cytokine-like, secreted protein of small molecular weight, was demonstrated to exhibit various biological functions including anti-inflammatory, antifibrotic and growth-factor activities. Anti-inflammatory activity was uncovered using the ovalbumin-induced allergic airway inflammation model. However, further validation of this activity using knockout mice in a different allergic inflammation model is necessary in order to establish the antiallergic inflammatory role for this protein. METHODS: Scgb3a2-null (Scgb3a2-/-) mice were subjected to nasal inhalation of Dermatophagoides pteronyssinus extract for 5 days/week for 5 consecutive weeks; control mice received nasal inhalation of saline as a comparator. Airway inflammation was assessed by histological analysis, the number of inflammatory cells and various Th2-type cytokine levels in the lungs and bronchoalveolar lavage fluids by qRT-PCR and ELISA, respectively. RESULTS: Exacerbated inflammation was found in the airway of Scgb3a2-/- mice subjected to house dust mite (HDM)-induced allergic airway inflammation compared with saline-treated control groups. All the inflammation end points were increased in the Scgb3a2-/- mice. The Ccr4 and Ccl17 mRNA levels were higher in HDM-treated lungs of Scgb3a2-/- mice than wild-type mice or saline-treated Scgb3a2-/- mice, whereas no changes were observed for Ccr3 and Ccl11 mRNA levels. CONCLUSIONS: These results demonstrate that SCGB3A2 has an anti-inflammatory activity in the HDM-induced allergic airway inflammation model, in which SCGB3A2 may modulate the CCR4-CCL17 pathway. SCGB3A2 may provide a useful tool to treat allergic airway inflammation, and further studies on the levels and function of SCGB3A2 in asthmatic patients are warranted.

Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer.

HOXB7 encodes a transcription factor that is overexpressed in a number of cancers and encompasses many oncogenic functions. Previous results have shown it to promote cell proliferation, angiogenesis, epithelial-mesenchymal transition, DNA repair and cell survival. Because of its role in many cancers and tumorigenic processes, HOXB7 has been suggested to be a potential drug target. However, HOXB7 binding sites on chromatin and its targets are poorly known. The aim of our study was to identify HOXB7 binding sites on breast cancer cell chromatin and to delineate direct target genes located nearby these binding sites. We found 1,504 HOXB7 chromatin binding sites in BT-474 breast cancer cell line that overexpresses HOXB7. Seventeen selected binding sites were validated by ChIP-qPCR in several breast cancer cell lines. Furthermore, we analyzed expression of a large number of genes located nearby HOXB7 binding sites and found several new direct targets, such as CTNND2 and SCGB1D2. Identification of HOXB7 chromatin binding sites and target genes is essential to understand better the role of HOXB7 in breast cancer and mechanisms by which it regulates tumorigenic processes.

Mammary analog secretory carcinoma, low-grade salivary duct carcinoma, and mimickers: a comparative study.

Mammary analog secretory carcinoma (MASC) is a recently recognized low-grade salivary carcinoma characterized by a specific ETV6 rearrangement. We describe 14 new MASCs and examine their immunophenotypic and genetic profiles in the context of look-alikes, namely, low-and high-grade salivary duct carcinoma and acinic cell carcinoma. ETV6 rearrangement, and robust expression of mammaglobin and S100, were demonstrated in 11/11, 14/14, and 12/14 MASCs, respectively. All low-grade salivary duct carcinomas coexpressed S100/mammaglobin (6/6); none harbored ETV6 rearrangements (0/5). Given that S100/mammaglobin coexpression and absence of zymogen granules are features of both MASC and low-grade salivary duct carcinoma, these two are best distinguished histologically. The former is predominantly an extraductal neoplasm with bubbly pink cytoplasm, whereas the latter is a distinct intraductal micropapillary and cribriform process. Querying ETV6 gene status may be necessary for difficult cases. No acinic cell carcinoma expressed mammaglobin (0/13) or harbored an ETV6 rearrangement (0/7); only 1/13 acinic cell carcinomas weakly expressed S100. DOG1 expression was limited or absent among all tumor types, except acinic cell carcinoma which expressed DOG1 diffusely in a canalicular pattern. Therefore, histology and immunohistochemistry (mammaglobin, S100, DOG1) suffices in distinguishing acinic cell carcinoma from both MASC and low-grade salivary duct carcinoma. HER2 (ERBB2) amplification was detected in only 1/10 acinic cell carcinomas, but none of the MASCs or low-grade salivary duct carcinomas tested. High-grade salivary duct carcinomas frequently expressed mammaglobin (11/18) and harbored HER2 amplifications (13/15); none harbored ETV6 rearrangements (0/12). High-grade salivary duct carcinomas can easily be distinguished from these other entities by histology and HER2 amplification.

GATA-binding protein 3, gross cystic disease fluid protein-15 and mammaglobin have distinct prognostic implications in different invasive breast carcinoma subgroups.

AIMS: To evaluate the prognostic significance of GATA-binding protein 3 (GATA-3), gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin (MGB) in invasive breast carcinomas (IBCs). METHODS AND RESULTS: GATA-3, GCDFP-15 and MGB were expressed in 37.9% (370/976), 26.0% (254/978) and 35.3% (348/986) of this cohort of 1017 IBCs, respectively. GCDFP-15 was an independent favourable prognostic factor in all cases [disease-free survival (DFS), hazard ratio (HR) 0.587, P = 0.049; overall survival (OS), HR 0.512, P = 0.049], as well as in oestrogen receptor (ER)-negative (DFS, HR 0.353, P = 0.012; OS, HR 0.310, P = 0.017) and HER2-positive (DFS, HR 0.279, P = 0.036; OS, HR 0.235, P = 0.050) cases; it also refined the prognostication of molecular apocrine cancers. GATA-3 and MGB did not show any prognostic significance. CONCLUSIONS: The commonly used breast carcinoma biomarkers vary in their prognostic implications. GCDFP-15 independently indicated a favourable prognosis, especially in ER-negative, HER2-positive and molecular apocrine cancers. GATA-3 and MGB were not associated with outcome.

Gene expression abnormalities in histologically normal breast epithelium from patients with luminal type of breast cancer.

The gene expression profile of breast cancer has been described as a great breakthrough on the way to comprehend differences in cancer origin, behavior and therapy. However, gene expression profile in histologically normal epithelium (HNEpi) which could harbor genetic abnormalities predisposing breast tissue to develop malignancy was minor scope for scientists in the past. Thus, we aimed to analyze gene expressions in HNEpi and breast cancer tissue (BCTis) in order to establish its value as potential diagnostic marker for cancer development. We evaluated a panel of disease-specific genes in luminal type (A/B) of breast cancer and tumor surrounding HNEpi by qRT-PCR Array in 32 microdissected samples. There was 20.2 and 2.4% deregulation rate in genes with at least 2-fold or 5-fold over-expression between luminal (A/B) type breast carcinomas and tumor surrounding HNEpi, respectively. The high-grade luminal carcinomas showed higher number of deregulated genes compared to low-grade cases (50.6 vs. 23.8% with at least 2-fold deregulation rate). The main overexpressed genes in HNEpi were KLK5, SCGB1D2, GSN, EGFR and NGFR. The significant differences in gene expression between BCTis and HNEpi samples were revealed for BAG1, C3, CCNA2, CD44, FGF1, FOSL1, ID2, IL6R, NGFB, NGFR, PAPPA, PLAU, SERPINB5, THBS1 and TP53 gene (p < 0.05) and BCL2L2, CTSB, ITGB4, JUN, KIT, KLF5, SCGB1D2, SCGB2A1, SERPINE1 (p < 0.01), and EGFR, GABRP, GSN, MAP2K7 and THBS2 (p < 0.001), and GSN, KLK5 (p < 0.0001). The ontological gene analyses revealed high deregulations in gene group directly associated with breast cancer prognosis and origin.

Transgenically-expressed secretoglobin 3A2 accelerates resolution of bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice. METHODS: A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis. RESULTS: Adult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs. CONCLUSIONS: These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.

Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways.

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.

Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis.

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress.

Searching for mammary analogue [corrected] secretory carcinoma of salivary gland among its mimics.

Mammary analog secretory carcinoma of salivary gland is a recently described entity with unique morphologic, clinical, and genetic characteristics, including the characteristic t(12;15)(p13;q25) with ETV6-NTRK3 translocation found in secretory carcinomas of the breast. Before their initial description, these salivary gland tumors were generally diagnosed as acinic cell carcinoma or adenocarcinoma. For the purpose of this study, all cases of salivary gland acinic cell carcinoma, cribriform cystadenocarcinoma, and adenocarcinoma, not otherwise specified (NOS), diagnosed over a 10-year period were retrieved from our surgical pathology files. There were a total of 11 cases diagnosed as acinic cell carcinoma, 10 cases of adenocarcinoma, NOS, and 6 cases of cribriform cystadenocarcinoma. All slides were reviewed by two pathologists (AP, CGF) and tumors that show morphologic features of mammary analog secretory carcinoma according to the recent literature were selected. This process narrowed down the initial number to six cases originally diagnosed as acinic cell carcinoma, three cases originally diagnosed as adenocarcinoma, NOS, and one case originally diagnosed as cribriform cystadenocarcinoma. The 10 cases were subjected to immunohistochemistry for S-100, mammaglobin, and ANO1, as well as fluorescence in situ hybridization analysis for t(12;15)(p13;q25) with ETV6-NTRK3 fusion rearrangement. The ETV6-NTRK3 gene rearrangement was detected in three tumors. These three tumors, initially diagnosed as acinic cell carcinomas, stained positive for S-100 and mammaglobin, and negative for ANO1 by immunohistochemistry. Two of the three patients were male (2/3). In summary, mammary analog secretory carcinoma is a newly described diagnostic entity that should be in the differential diagnosis of salivary gland tumors that morphologically mimic other neoplasms, mainly acinic cell carcinomas. They differ from conventional acinic cell tumors immunohistochemically and molecularly. Positivity for mammaglobin and S-100, and negativity for ANO1 are useful screening tools before confirmatory molecular studies.

Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland.

Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPß, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

Differential expression of secretoglobins in normal ovary and in ovarian carcinoma--overexpression of mammaglobin-1 is linked to tumor progression.

Secretoglobins (SCGB), such as mammaglobin 1 (MGB1, SCGB2A2), mammaglobin 2 (MGB2, SCGB2A1) and lipophilin B (LIPB, SCGB1D2), have been related to carcinogenesis. We profiled expression of MGB1, MGB2 and LIPB in human tissues and ovarian carcinoma and explored the impact of SCGB overexpression on cell proliferation. MGB1, MGB2 and LIPB mRNA are expressed at variable levels in most human tissues and we observed significant bilateral correlations between the different secretoglobins. Concerted overexpression of MGB1 and LIPB resulted in significant increase in cell proliferation. In clinical specimens of ovarian carcinoma we measured elevated concentrations of secretoglobin mRNA and for MGB1 this up-regulation was confirmed on the protein level. Overexpression of MGB1 positively correlated with the FIGO stage, the tumor grade and the mitotic index suggesting a patho-physiological role of the protein. Our data indicate that MGB1, MGB2 and LIPB mRNAs are expressed at low levels in human tissues but basal expression is upregulated in ovarian cancer. The in vivo correlation between nuclear MGB1 localization and the mitotic rate in ovarian cancer as well as the increased cell proliferation induced by secretoglobin overexpression in ovarian cancer cell lines suggest a pathophysiological role of these proteins in ovarian cancer.

Secretoglobin superfamily protein SCGB3A2 deficiency potentiates ovalbumin-induced allergic pulmonary inflammation.

Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, which may play a role in lung inflammation, is predominantly expressed in airway epithelial cells. In order to understand the physiological role of SCGB3A2, Scgb3a2(-/-) mice were generated and characterized. Scgb3a2(-/-) mice did not exhibit any overt phenotypes. In ovalbumin- (OVA-) induced airway allergy inflammation model, Scgb3a2(-/-) mice in mixed background showed a decreased OVA-induced airway inflammation, while six times C57BL/6NCr backcrossed congenic Scgb3a2(-/-) mice showed a slight exacerbation of OVA-induced airway inflammation as compared to wild-type littermates. These results indicate that the loss of SCGB3A2 function was influenced by a modifier gene(s) in mixed genetic background and suggest that SCGB3A2 has anti-inflammatory property. The results further suggest the possible use of recombinant human SCGB3A2 as an anti-inflammatory agent.