Distal Epithelium Research Articles

Identification of Amnionless Gene as an Estrogen/ER Alpha-Regulated Gene in the Mouse Pituitary.

Estrogen plays a key role in regulating gonadotropin secretion from the pituitary through both negative and positive feedback pathways. However, the direct mechanisms of this regulation have yet to be defined. We hypothesize that the positive feedback effect of estrogen is achieved by ERα-mediated transcriptional regulation of cellular components needed for LH vesicle transport and secretion of its components into the bloodstream, thus equipping cells with the secretory machinery required for the LH surge. To identify these ERα-regulated components of LH secretion, total RNA was extracted from the pituitaries of cycling mice in metestrus or proestrus (low and high levels of circulating estrogen, respectively), ovariectomized (OVX) WT mice treated with vehicle or estrogen, and OVX ERαKO mice treated with vehicle or estrogen, and subjected to microarray analysis. Overall, 183 genes were found to be differentially expressed in an estrogen/ERα-dependent manner. In this study, we characterized the expression pattern of a gene (amn) that codes for Amnionless (Amn), a type I transmembrane protein known to be involved in vitamin B12 absorption by distal intestinal epithelia and proximal tubules of the kidney. The amn gene was selected as one gene of interest due to significant effect of estrogen treatment on transcription levels and the novelty of its characterization in the pituitary. The microarray analysis revealed a 30-fold increase of amn transcription in proestrus compared to metestrus, and a fold change of 25 in OVX WT mice with estrogen compared to vehicle treatment. ERαKO mice showed no increase in transcription of amn with estrogen treatment. This pattern of amn expression was confirmed using RT-PCR, showing a 9 fold increase in transcription levels in proestrus versus metestrus, and 16-fold increase in the OVX WT estrogen treated compared to vehicle treated groups. Immunohistochemistry confirmed that protein levels increased concurrently with mRNA levels in proestrus versus metestrus, but not in ERαKO mice. In silico analysis of the amn proximal promoter reveals 8 putative EREs within 2 kilo bases upstream of the start codon, evidence of a possible direct regulation of amn expression by the classical ERα pathway, which will be determined by ChIP assay. Currently, we are determining the functional significance of ERα-regulated amn expression in LH secretion using in vitro models. Both primary pituitary cells and isolated gonadotroph cells are treated with anti-Amnionless antibody, in order to block the functional capacity of the protein, followed by GnRH treatment. This experiment in the primary pituitary cells will allow us to determine if Amn in the pituitary plays a critical role in facilitating LH secretion, and the experiment in isolated gonadotroph cells will clarify if the effect on LH secretion is due to Amn in gonadotrophs themselves, or secondarily due to Amn expression in other cell types of the pituitary. For isolation of gonadotroph cells, we have generated ROSA-GFP CgaCre mice, who express Cre, and thus GFP, specifically in gonadotroph. Using flow cytometry, live gonadotrophs can be segregated from other cell types of the pituitary. Future directions will be aimed at elucidation of the role of Amn in preparing the gonadotroph for LH secretion in relation to the well-known function of Amn in facilitating vitamin B12 absorption. In summary, the gene encoding the Amnionless protein has been identified as an estrogen/ERα-regulated gene in the pituitary. The findings we may make from the study of Amn function in the pituitary will add to the understanding of the molecular mechanisms of estrogen priming of the pituitary in preparation for the LH surge. Supported by a COBRE grant from NIH NCRR: P20 RR15592. (poster)

Refining Diagnostic Criteria for High-Grade Dysplasia in Barrett Esophagus

In many respects, the medical community has failed patients with esophageal carcinoma miserably. Even with contemporary treatment, the overall 5-year survival for esophageal cancer in the United States is an abysmal 13%. 1 A number of studies suggest that surveillance is effective in capturing the early lesions for which survival is excellent, but current data indicate that only a small fraction (<5%) of patients in whom gastric cardiac and esophageal carcinomas develop are actually targeted by surveillance programs 2 ; most patients seek care too late in the disease course to benefit from even the most aggressive treatment. Unfortunately, because esophageal cancer is relatively uncommon (there were 16,470 estimated new cases of esophageal cancer in the United States in 2008 compared with 148,419 colon cancers and 215,020 lung cancers), 3 it is not likely, given economic factors, that there will be a push for intensified screening and early detection of esophagus cancers. It is well-known that Barrett esophagus is a precursor lesion for esophageal adenocarcinomas. In the United States, Barrett esophagus has been defined as change of the distal esophageal epithelium of any length that can be recognized as columnar epithelium at endoscopy and that has intestinal metaplasia revealed by biopsy, 4 whereas documentation of goblet cells is not required in the United Kingdom, some of continental Europe (excluding Germany), or Japan. As such, Barrett esophagus is thus often an endoscopic rather than a histologic diagnosis outside the United States. The reasoning for the US definition is the belief that intestinal metaplasia is a marker for a higher likelihood over that of gastric-type mucosa for progression to adenocarcinoma. 5 However, regardless of where the patient resides, most endoscopists biopsy columnar epithelium in the esophagus to assess for neoplasia. Whether definitions will gradually be modified in the United States to transform Barrett esophagus from a histologic diagnosis to an endoscopic one remains a subject of debate (some of which relates to economic issues), but some studies indicate that, in fact, high-grade columnar epithelial dysplasia and early esophageal adenocarcinomas are more likely to be accompanied by gastric cardiac-type mucosa than by metaplastic mucosa with goblet cells. 6 Those of us in the pathology community will be part of any changes that come in the endoscopy community. In the interim, however, we should strive to best serve the lucky minority of at-risk patients who are screened and whose precursor lesions and early cancers are likely to be biopsied. Since the progression from Barrett esophagus to dysplasia to adenocarcinoma is a continuum and presumably the reflection of accumulating genetic events, it is not surprising that pathologists do not always agree in classifying the stages of this progression. This disagreement is present for grading dysplasia 7 and for distinguishing between high-grade

Clara Cell Protein (CC16), a Marker of Lung Epithelial Injury, Is Decreased in Plasma and Pulmonary Edema Fluid From Patients With Acute Lung Injury

Acute lung injury (ALI) and ARDS are common clinical syndromes that are underdiagnosed. Clara cell secretory protein (CC16) is an antiinflammatory protein secreted by the Clara cells of the distal respiratory epithelium that has been proposed as a biomarker of lung epithelial injury. We tested the diagnostic and prognostic utility of CC16 in patients with non-trauma-related ALI/ARDS compared to a control group of patients with acute cardiogenic pulmonary edema (CPE). Plasma and pulmonary edema fluid samples were obtained from medical and surgical patients with ALI/ARDS or CPE requiring intubation for mechanical ventilation. The etiology of pulmonary edema was determined using consensus clinical criteria for ALI/ARDS and CPE and the edema fluid-to-plasma protein ratio. Plasma and edema fluid CC16 levels were measured by sandwich enzyme-linked immunosorbent assay. CC16 levels were log transformed for analysis, and comparisons were made by the Student t test or Chi(2) as appropriate. Compared to patients with CPE (n = 9), patients with ALI/ARDS (n = 23) had lower median CC16 levels in plasma (22 ng/mL [interquartile range (IQR), 9 to 44 ng/mL] vs 55 ng/mL [IQR, 18 to 123 ng/mL], respectively; p = 0.053) and pulmonary edema fluid (1,950 ng/mL [IQR, 1,780 to 4,024 ng/mL] vs 4,835 ng/mL [IQR, 2,006 to 6,350 ng/mL], respectively; p = 0.044). Relative to total pulmonary edema fluid protein concentration, the median CC16 level was significantly lower in patients with ALI/ARDS (45 ng CC16/mg total protein [IQR, 4 to 64 ng CC16/mg total protein] vs 120 ng CC16/mg total protein [IQR, 87 to 257 ng CC16/mg total protein], respectively; p = 0.005). Neither plasma nor edema fluid CC16 levels predicted mortality, the number of days of unassisted ventilation, or ICU length of stay. CC16 is a promising diagnostic biomarker for helping to discriminate ALI from CPE. Larger scale validation is warranted to better characterize the utility of CC16 in the diagnosis of this underrecognized syndrome.

Esophageal pH exposure and epithelial cell differentiation

It is proposed that epithelial changes induced by gastroesophageal reflux disease are related to the pH environment of the esophageal lumen. We hypothesized that the various types of esophageal epithelium are associated with specific pH environments that induce their formation. The aim of this study was to compare the luminal pH environment to the histology of the distal esophageal epithelium in patients with gastroesophageal reflux disease. A total of 197 symptomatic patients with increased esophageal acid exposure on 24-hour pH monitoring were grouped according to the histology based on biopsies from the distal esophagus: 17 with squamous epithelium, 126 with cardiac epithelium (CE), and 54 with Barrett's epithelium (BE). All were free of Helicobacter pylori infection and monitored off acid suppression therapy. Acid exposure was expressed as the percent of time the luminal pH was at intervals of 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, and 6-7 over a 24-hour period. Patients with BE spent significantly more time at pH intervals 2-3, 3-4, and 4-5 than those with CE. This pattern switched at pH interval 5-6, where patients with cardiac mucosa spent more time than those with BE. Patients with squamous and CE had similar pH exposure at all intervals. Patients with BE have significantly longer exposure time at the pH interval of 2 to 5 compared to those with cardiac and squamous epithelium. This suggests that the exposure of stem cells to a luminal pH between 2 and 5 may trigger the differentiation of CE into intestinalized CE.

Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had reached 10-15 mg/24 h. Surface expression of proximal Na(+)-coupled transporters and water channels was in part [Na(+)-P(i) cotransporter IIa (NaPi-IIa) and aquaporin-1 (AQP1)] increased by megalin deficiency alone, but unchanged (Na(+)/H(+) exchanger 3) or reduced (NaPi-IIa and AQP1) in GN irrespective of the endocytosis defect. In distal epithelia, significant increases in proteolytic cleavage products of alpha-epithelial Na(+) channel (ENaC) and gamma-ENaC were observed, suggesting enhanced tubular sodium reabsorption. The effects of glomerular proteinuria dominated over those of blunted proximal endocytosis in contributing to ENaC cleavage. Our data indicate that ENaC-mediated sodium entry may be the rate-limiting step in proteinuric sodium retention. Enhanced proteolytic cleavage of ENaC points to a novel mechanism of channel activation which may involve the action of filtered plasma proteases.

Analysis of Adeno-associated Virus Progenitor Cell Transduction in Mouse Lung

Although recombinant adeno-associated virus (rAAV) has been widely used in lung gene therapy approaches, it remains unclear to what extent commonly used AAV serotypes transduce adult progenitors in the lung. In this study, we evaluated the life span and proliferative capacity of rAAV1-, 2-, and 5-transduced airway cells in mouse lung, using a LacZ-CRE reporter transgenic model and Cre-expressing rAAV. In this model, the expression of CRE recombinase led to permanent genetic marking of transduced cells and their descendants with LacZ. To investigate whether the rAAV-transduced cells included airway progenitors, we injured the airways of rAAV-infected mice with Naphthalene, while simultaneously labeling with 5-bromodeoxyuridine (BrdU) to identify slow-cycling progenitor/stem cells that entered the cell cycle and retained label. Both rAAV5 and rAAV1 vectors were capable of transducing a subset of long-lived Clara cells and alveolar type II (ATII) cells that retained nucleotide label and proliferated following lung injury. Importantly, rAAV1 and 5 appeared to preferentially transduce conducting airway epithelial progenitors that had the capacity to clonally expand, both in culture and in vivo following lung injury. These studies suggest that rAAV may be a useful vector for gene targeting of airway stem/progenitor cells.

Proteolytic activation of the epithelial sodium channel (ENaC) in health and disease

ber of the ENaC/degenerin family of non-voltage gated ion channels. ENaC is localized in the apical membrane of epithelial cells, and is the rate limiting step for sodium absorption in a number of epithelial tissues like the aldosterone-sensitive distal nephron (ASDN), respiratory epithelia, distal colon, sweat and salivary ducts. ENaC is probably a heterotrimer with three well characterized subunits (αβγ). In humans an additional δ-subunit (δ-hENaC) exists but little is known about its function.

Vascular endothelial growth factor expression in the urethral epithelium of castrated adult female rats treated with tamoxifen

Objective. The aim of this study was to evaluate the effects of tamoxifen on vascular endothelial growth factor (VEGF) expression in the urethral epithelium of castrated rats.Materials and methods. Thirty-six adult, castrated, female Wistar-Hannover rats were randomly divided into two groups: group I (n = 16, control), receiving only propylene glycol, and group II (n = 20, tamoxifen), treated with 250 μg/day of tamoxifen for 30 consecutive days by gavage. On the 31st day, the animals were sacrificed and the urethras were immediately removed, separated into the proximal and distal segments and processed for VEGF immunohistochemistry. The data were analysed using Student's t-test (p < 0.05).Results. The mean percentage of VEGF expression in the epithelium of the proximal urethra of the animals in groups I and II was 64.47±3.70 and 74.69±3.03, respectively (p < 0.03), whereas the mean percentage of VEGF expression in the distal urethral epithelium of the animals in groups I and II was 53.49±4.64 and 68.57±3.67, respectively (p < 0.01).Conclusions. Our results indicate that, at the dose and during the time of treatment used, tamoxifen increased VEGF expression in the urethral epithelium of castrated rats.

Apolipoprotein A-I, A-II, C-II, and H expression in the developing lung and sex difference in surfactant lipids

A sex difference in surfactant lipids is associated with a higher incidence of respiratory distress syndrome for males in cases of preterm birth. In animal models, the sex difference in surfactant lipids was shown to be androgen receptor-dependent. This report examines expression of apolipoprotein (apo)A-I, apoA-II, apoC-II, apoE, apoH, and lipoprotein lipase (LPL) by quantitative real-time PCR in pools of male and female fetal lung tissues from various mouse litters from gestation day (GD) 15.5 to 18.5, and in various adult tissues. Although the expression profiles of ApoA-I, ApoA-II, ApoC-II, and ApoH are complex, these genes are co-regulated and they all present a sex difference (P=0.0896, 0.0896, 0.0195, and 0.0607 respectively) with higher expression for females for several litters. Pulmonary expression of apoA-I, apoA-II, and apoH were specific to the developing lung. ApoE and LPL mRNAs showed a significant increase from GD 17.5 to 18.5. An increase in apoA-I-, apoA-II-, apoC-II-, and apoH-mRNA accumulation was observed from GD 16.5 to 17.5 in correlation with the emergence of mature type II pneumonocytes. These four apolipoprotein genes are co-regulated with type 2 and 5 17beta-hydroxysteroid dehydrogenases, which are respectively involved in inactivation and synthesis of androgens. Finally, apoC-II was detected by immunohistochemistry in epithelial cells of the distal epithelium. Positive signals looking like secretory granules were located near the basal membrane. Our results are compatible with a role for apolipoproteins in lipid metabolism and transport in the developing lung in association with the sex difference in surfactant lipid synthesis.

GOLPH2 expression in renal cell cancer

BackgroundRenal cell carcinomas (RCC) are among the most common and most lethal genitourinary malignancies. GOLPH2 (golgi phosphoprotein 2, GOLM1) has recently been proposed as a biomarker for hepatocellular and prostate cancer. In this study we analysed the expression patterns and the prognostic and diagnostic value of GOLPH2 in RCC.MethodsGOLPH2 protein expression was analysed by immunohistochemistry in 104 clinically well characterized RCC cases in comparison with matched normal kidney tissue and in association with clinico-pathological parameters. Statistical analyses including Kaplan Meier analyses were performed with SPSS version 15.0.ResultsGOLPH2 was highly expressed in normal renal tubules and in almost half of RCC with a statistically significant predominance in the papillary and chromophobe histological subtypes. No other associations with clinico-pathological parameters were detectable. The Kaplan-Meier curves showed a weak trend for unfavourable prognosis of tumours with high GOLPH2 expression, but failed significance.ConclusionGOLPH2 protein is expressed in normal renal tissue (especially in distal tubular epithelia) and is down-regulated in the majority of clear cell RCC. In papillary and chromophobe RCC GOLPH2 expression is consistently present. In contrast to its diagnostic value in hepatocellular and prostatic carcinomas, a prognostic or diagnostic value of GOLPH2 in RCC appears to be unlikely.

Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration

Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet ( smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.

Respiratory syncytial virus (RSV) attachment and nonstructural proteins modify the type I interferon response associated with suppressor of cytokine signaling (SOCS) proteins and IFN-stimulated gene-15 (ISG15)

Respiratory syncytial virus (RSV) is a major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. Factors attributing to this problem are associated with an incomplete understanding of the mechanisms by which RSV modulates the host cell response to infection. In the present study, we investigate suppressor of cytokine signaling (SOCS)-1 and SOCS3 expression associated with the type I IFN and IFN-stimulated gene (ISG)-15 response following infection of mouse lung epithelial (MLE-15) cells with RSV or RSV mutant viruses lacking the G gene, or NS1 and NS2 gene deletions. Studies in MLE-15 cells are important as this cell line represents the distal bronchiolar and alveolar epithelium of mice, the most common animal model used to evaluate the host cell response to RSV infection, and exhibit morphologic characteristics of alveolar type II cells, a primary cell type targeted during RSV infection. These results show an important role for SOCS1 regulation of the antiviral host response to RSV infection, and demonstrate a novel role for RSV G protein manipulation of SOCS3 and modulation of ISG15 and IFNβ mRNA expression.

Claudin-7 and claudin-8: immunohistochemical markers for the differential diagnosis of chromophobe renal cell carcinoma and renal oncocytoma

Claudin-7 and claudin-8 code for tight junction proteins expressed in distal nephron epithelium. In a recent oligonucleotide microarray study, we identified claudin-7 and claudin-8 as candidate markers to distinguish chromophobe renal cell carcinoma from other renal tumors, including oncocytoma. Distinction of these lesions can be difficult by light microscopy but is clinically important because chromophobe renal cell carcinoma has malignant biological potential, whereas renal oncocytoma is benign. Claudin-7 and claudin-8 expression was studied by immunohistochemistry in 11 chromophobe renal cell carcinomas and 17 oncocytomas using formalin-fixed paraffin-embedded tissue sections of tumor with adjacent nonneoplastic kidney. Steam antigen retrieval was performed before immunohistochemistry. Specificity was verified by negative control reactions without primary antibody and appropriate membranous staining patterns in positive control tissues (colon carcinoma and adjacent nonneoplastic kidney). Claudin-7 protein was expressed in a membranous pattern in 10 of 11 chromophobe renal cell carcinomas and 4 of 17 oncocytomas (P < .01). Claudin-8 was expressed in multiple patterns: In oncocytoma, 11 of 17 cases showed cytoplasmic, 4 of 17 membranous, and 2 of 17 negative reactions. In chromophobe renal cell carcinoma, 0 of 11 cases showed cytoplasmic, 3 of 11 membranous, and 8 of 11 negative reactions (P < .01). The immunohistochemical pattern of membranous claudin-7 and negative claudin-8 was seen in 7 of 11 chromophobe renal cell carcinomas and 1 of 17 oncocytomas (63% sensitivity, 84% specificity, 88% positive predictive value for chromophobe renal cell carcinoma). Negative claudin-7 and cytoplasmic claudin-8 were observed in 10 of 17 oncocytomas and 0 of 11 chromophobe renal cell carcinomas (59% sensitivity, 100% specificity and positive predictive value for oncocytoma). The distal nephron proteins claudin-7 and claudin-8 have potential use as immunohistochemical biomarkers in the differential diagnosis of chromophobe renal cell carcinoma and oncocytoma. Expression of claudin-7 and claudin-8 may reflect the relationship of chromophobe renal cell carcinoma and oncocytoma to intercalated cells of the cortical collecting duct. It may be necessary to identify additional biomarkers to include with claudin-7 and claudin-8 in a larger immunohistochemical panel to improve diagnostic sensitivity and specificity.

Placental Growth Factor and Vascular Endothelial Growth Factor Receptor-2 in Human Lung Development

We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of <2 days and 7 with respiratory distress syndrome of >10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression in capillary endothelium in bronchopulmonary dysplasia might be a reflection of the dysregulation of vascular development that is characteristic of bronchopulmonary dysplasia.

Increased COX-2 Expression of Esophageal Squamous Epithelium in the Patients with Reflux Esophagitis

Background/Aims: Reflux-induced injury and oxidative stress result in esophageal inflammation and the potential for progression to intestinal metaplasia and adenocarcinoma. Previous studies have suggested that increased expression of COX-2 is important in the genesis of esophageal adenocarcinoma. The aim of this study was to compare the COX-2 expression of distal esophageal squamous epithelium between the patients with reflux esophagitis and normal controls. Methods: Biopsies of the distal esophageal squamous mucosa were taken 3 cm above the squamocolumnar junction in 19 reflux esophagitis patients and 21 normal controls. The expression of COX-2 was confirmed by immunohistochemistry using monoclonal antibodies. The staining index was obtained by adding up the scores for staining intensity with the staining area, which ranged from 0 to 6. Results: COX-2 expression in the squamous epithelium from paraffin embedded biopsies in patients with reflux esophagitis was significantly increased compared to controls (31.6% vs. 0%, P = 0.038). The mean COX-2 expression score was 2.2 in the reflux esophagitis patients with positive COX-2 expression. Conclusions: It suggests that the changes in COX-2 expression occur before any metaplastic changes in the esophageal squamous epithelium of the patients with reflux esophagitis.

TGF- receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung

Transforming growth factor (TGF)-beta signalling plays important roles in regulating lung development. However, the specific regulatory functions of TGF-beta signalling in developing lung epithelial versus mesenchymal cells are still unknown. By immunostaining, the expression pattern of the TGF-beta type II receptor (TbetaRII) was first determined in the developing mouse lung. The functions of TbetaRII in developing lung were then determined by conditionally knocking out TbetaRII in the lung epithelium of floxed-TbetaRII/surfactant protein C-reverse tetracycline transactivator/TetO-Cre mice versus mesenchyme of floxed-TbetaRII/Dermo1-Cre mice. TbetaRII was expressed only in distal airway epithelium at early gestation (embryonic day (E)11.5), but in both airway epithelium and mesenchyme from mid-gestation (E14.5) to post-natal day 14. Abrogation of TbetaRII in mouse lung epithelium resulted in retardation of post-natal lung alveolarisation, with markedly decreased type I alveolar epithelial cells, while no abnormality in prenatal lung development was observed. In contrast, blockade of TbetaRII in mesoderm-derived tissues, including lung mesenchyme, resulted in mildly abnormal lung branching and reduced cell proliferation after mid-gestation, accompanied by multiple defects in other organs, including diaphragmatic hernia. The primary lung branching defect was verified in embryonic lung explant culture. The novel findings of the present study suggest that transforming growth factor-beta type II receptor-mediated transforming growth factor-beta signalling plays distinct roles in lung epithelium versus mesenchyme to differentially control specific stages of lung development.

Transforming Growth Factor‐β1 Inhibits CFTR‐dependent cAMP‐stimulated Alveolar Epithelial Fluid Transport: Critical Role of Interleukin‐8

β‐adrenergic agonist‐dependent stimulation of the lung fluid clearance is an important mechanism that protects the lung from alveolar flooding. In this study we hypothesized that critical mediators of acute lung injury (ALI), such as transforming growth factor‐β1 (TGF‐β1) and interleukin‐8 (IL‐8), could directly antagonize the epithelial response to β‐adrenergic agonists. Short circuit current experiments revealed that TGF‐β1 inhibits CFTR‐specific β‐agonist‐stimulated vectorial Cl‐transport across the apical membrane of primary rat and human alveolar epithelial type II (ATII). TGF‐β1 also significantly decreased β‐agonist‐stimulated cAMP production resulting in the inhibition of the CFTR promoter activity and gene expression in ATII cells. We found that this TGF‐β1‐dependent inhibition required IL‐8 and was mediated by desensitization the β‐adrenergic receptor through both TGF‐β1 and IL‐8 signaling pathways. Consistent with the in vitro results, we showed that TGF‐β1 requires IL‐8 to inhibit the β‐agonist‐stimulated fluid transport across the distal airspace epithelium in vivo in rats. In summary, the results show for the first time that TGF‐β1 and IL‐8 inhibit the stimulated vectorial chloride transport across the distal lung epithelium, thus impairing the β‐adrenergic‐dependent protective mechanism against alveolar flooding.NIH P50HL074005 (JFP), ALA Award and T32 GM008440 (JR)

Morphometric analysis of the urethra of castrated female rats treated with tamoxifen

Objectives The aim of this study was to evaluate the effects of tamoxifen on the weight and thickness of the urethral epithelium of castrated female rats. Methods Forty castrated adult female Wistar–Hannover rats were randomly divided into two groups: Group I ( n = 20) in which the animals received only the vehicle (propylene glycol) and Group II ( n = 20) in which the rats received tamoxifen 250 μg/day by gavage. After 30 days of treatment, all animals were sacrificed and the urethra was immediately removed for weighing. Next, the urethra was divided into the proximal and distal segments, which were fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The data were analyzed using the weighted minimum mean-square error method and Student's t-test for two independent samples ( p < 0.05). Results There was a significant increase in the mean weight of the urethra in the rats of Group II compared to the control group, 32.0 ± 2.0 mg and 22.0 ± 1.6 mg, respectively ( p < 0.001). The mean thickness of the distal urethral epithelium of the animals treated with tamoxifen was significantly greater than that of the control group, 42.8 ± 2.0 μm and 36.6 ± 1.5 μm, respectively ( p < 0.001). There was no statistically significant difference between the two groups with respect to the epithelial thickness of the proximal urethra ( p = 0.514). Conclusion Treating castrated adult rats with 250 μg/day of tamoxifen for 30 days may increase the weight of the urethra and the thickness of the distal urethral epithelium.

Importance of microsomal epoxide hydrolase (mEH) in toxicity, metabolism and formation of covalent protein adducts from the volatile air pollutant, naphthalene (NA): Comparison of mEH null and wild type (WT) mice

Human exposure to NA is due to combustion of both environmental and man‐made materials. Toxicity of NA is P450‐dependent, occurring in murine, but not rat, airways. However, the extent to which specific metabolic pathways (to epoxides, quinones and diol epoxides) contribute to cytotoxicity and the formation of covalent protein adducts is unknown. Upon inhalation exposure to NA (4h at 5–20 ppm) male mEH null animals had increased cytotoxicity in the bronchioles, but decreased susceptibility in the trachea and lobar bronchus as compared to WT mice, despite having a normal pattern of P450‐containing CC10 positive Clara cells. Formation rates of NA metabolites (conjugates plus diol) in the trachea were lower in EH null animals, but equivalent in the airways compared to WT. As expected, rates of diol formation were undetected/low in mEH null animals. Protein adducts detected after ip administration of NA were 2–4 fold higher in airway epithelium than elsewhere in the lung, and EH null animals showed significantly higher levels compared to WT. Bound liver adducts showed no differences between mEH and WT animals. These studies suggest that metabolites (1,2‐quinone, diol epoxide) arising via the diol pathway are not critical to NA toxicity or formation of covalent adducts in distal airway epithelium. This work also suggests that there are compensatory changes associated with disruption of mEH. NIEHS 04311, 12720 and 04699.

Targeting the MEK1 Cascade in Lung Epithelium Inhibits Proliferation and Fibrogenesis by Asbestos

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.