Clara Cell Differentiation Research Articles

Proteomic and ultrastructural analysis of Clara cell and type II alveolar epithelial cell-type lung cancer cells.

BackgroundCurrently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling.MethodsSix human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed.ResultsNCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers.ConclusionsCytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.

Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs.

To study the significance of signal transducer and activator of transcription (Stat) 3 in lung epithelial development of fetal mice, we examined fetal mouse lungs, focusing on the expression of Clara cell secretory protein (CCSP), Forkhead box protein J1 (Foxj1), calcitonin gene-related peptide (CGRP), phosphorylated Stat3 (Tyr705), and hairy/enhancer of split (Hes) 1, and observed cultured fetal lungs upon treatment with IL-6, a Stat3 activator, or cucurbitacin I, a Stat3 inhibitor. Moreover, the interaction of Stat3 signaling and Hes1 was studied using Hes1 gene-deficient mice. Phosphorylated Stat3 was detected in fetal lungs and, immunohistochemically, phosphorylated Stat3 was found to be co-localized in developing Clara cells, but not in ciliated cells. In the organ culture studies, upon treatment with IL-6, quantitative RT-PCR revealed that CCSP mRNA increased with increasing Stat3 phosphorylation, while cucurbitacin I decreased Hes1, CCSP, Foxj1 and CGRP mRNAs with decreasing Stat3 phosphorylation. In the lungs of Hes1 gene-deficient mice, Stat3 phosphorylation was not markedly different from wild-type mice, the expression of CCSP and CGRP was enhanced, and the treatment of IL-6 or cucurbitacin I induced similar effects on mouse lung epithelial differentiation regardless of Hes1 expression status. Stat3 signaling acts in fetal mouse lung development, and seems to regulate Clara cell differentiation positively. Hes1 could regulate Clara cell differentiation in a manner independent from Stat3 signaling.

Genetic control of upper airway patterning (207.2)

In the trachea and bronchi of the mouse, airway smooth muscle (SM) and cartilage are localized to complementary domains surrounding the airway epithelium. Proper juxtaposition of these tissues produces a balance of elasticity and rigidity that is critical for effective air passage. It is unknown how this tissue complementation is established during development. Here we will present data where we dissected the developmental relationship of these tissues by genetically disrupting either SM formation (through Srf inactivation) or cartilage formation (through Sox9 inactivation), and assessed the impact on the remaining tissue. We found that in both the trachea and main bronchi, loss of one lineage led to an increase in cell number of the remaining lineage. However, only in the main bronchi, but not in the trachea, did the loss of one lineage led to a spatial expansion of the remaining lineage. In addition to SM defects, cartilage‐deficient tracheas displayed epithelial phenotypes, including decreased basal cell number, precocious Clara cell differentiation, and increased secretoglobin expression. Our results demonstrate that the development of upper airway tissues is closely coordinated. A disruption in the formation of one lineage may have widespread consequences on the collective function of these tissues.

E-cadherin Controls Bronchiolar Progenitor Cells and Onset of Preneoplastic Lesions in Mice

Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body.associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways.

RNA-Seq quantification of the human small airway epithelium transcriptome

BackgroundThe small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq).ResultsThe data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype.ConclusionsThese observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.

Signaling via Alk5 controls the ontogeny of lung Clara cells

Clara cells, together with ciliated and pulmonary neuroendocrine cells, make up the epithelium of the bronchioles along the conducting airways. Clara cells are also known as progenitor or stem cells during lung regeneration after injury. The mechanisms of Clara cell differentiation are largely unknown. Transforming growth factor beta (TGFbeta)is a multifunctional molecule with roles in normal development and disease pathogenesis. In this study, we deleted the TGFbeta type I receptor Alk5 in the embryonic lung epithelium using Gata5-Cre mice. Absence of Alk5 blocked Clara cell differentiation but had no effect on ciliated or pulmonary neuroendocrine cells. Hairy/Enhancer of Split-1, which is expressed in Clara cell putative ;progenitors' was found to be a downstream target of Alk5 in vivo and in vitro. Loss of Alk5-mediated signaling also stimulated Pten gene expression and inhibited ERK phosphorylation in vivo. Using lung epithelial cells, we show that Alk5-regulated Hes1 expression is stimulated through Pten and the MEK/ERK and PI3K/AKT pathways. Thus, the signaling pathway by which TGFbeta/ALK5 regulates Clara cell differentiation may entail inhibition of Pten expression, which in turn activates ERK and AKT phosphorylation.

Signaling via Alk5 Controls Ontogeny of Lung Clara Cells

AbstractClara cells, together with ciliated and pulmonary neuroendocrine cells, make up the epithelium of the bronchioles along the conducting airways. Clara cells are also known as Progenitor/Stem cells during lung regeneration after injury. The mechanisms of Clara cell differentiation are largely unknown. Transforming growth factor-beta is a multifunctional molecule with roles in normal development and disease pathogenesis. In this study, we deleted the TGF-&x03B2; type I receptor, Alk5 in the embryonic lung epithelium using Gata5-Cre mice. Absence of Alk5 blocked Clara cell differentiation but had no effect on ciliated or pulmonary neuroendocrine cells. Hairy/Enhancer of Split-1, which is expressed in Clara cell putative "progenitors" was found to be a downstream target of Alk5 in vivo and in vitro. Loss of Alk5-mediated signaling also stimulated Pten gene expression and inhibited ERK phosphorylation in vivo. Using lung epithelial cells, we show that Alk5-regulated Hes1 expression is stimulated through Pten, MEK/ERK and PI3K/AKT pathways. Thus, the signaling pathway by which TGF-&x03B2;/ALK5 regulates Clara cell differentiation may entail inhibition of Pten expression, which in turn activates ERK and AKT phosphorylation.

β-Catenin Is Not Necessary for Maintenance or Repair of the Bronchiolar Epithelium

Signaling by Wnt/beta-catenin regulates self-renewal of tissue stem cells in the gut and, when activated in the embryonic bronchiolar epithelium, leads to stem cell expansion. We have used transgenic and cell type-specific knockout strategies to determine roles for beta-catenin-regulated gene expression in normal maintenance and repair of the bronchiolar epithelium. Analysis of TOPGal transgene activity detected beta-catenin signaling in the steady-state and repairing bronchiolar epithelium. However, the broad distribution and phenotype of signaling cells precluded establishment of a clear role for beta-catenin in the normal or repairing state. Necessity of beta-catenin signaling was tested through Cre-mediated deletion of Catnb exons 2-6 in airway epithelial cells. Functional knockout of beta-catenin had no impact on expression of Clara cell differentiation markers, mitotic index, or sensitivity of these cells to the Clara cell-specific toxicant, naphthalene. Repair of the naphthalene-injured airway proceeded with establishment of focal regions of beta-catenin-null epithelium. The size of regenerative epithelial units, mitotic index, and restoration of the ciliated cell population did not vary between wild-type and genetically modified mice. Thus, beta-catenin was not necessary for maintenance or efficient repair of the bronchiolar epithelium.

Spectrum of pulmonary adenocarcinoma with ultrastructural correlation: An autopsy study from northern India

In the present study, we have evaluated the use of electron microscopy in subtyping pulmonary adenocarcinomas, comparing the ultrastructural findings with the diagnosis rendered by light microscopy. The gross and histologic features of 16 autopsy cases of pulmonary adenocarcinoma were analyzed and compared with electron microscopic features. The cytologic phenotypes of these cases of well-differentiated pulmonary adenocarcinoma were determined by electron microscopic examination. More than 200 cells in each case were examined, and the tumors were classified according to the predominant feature noted. Eight cases were of Clara cell origin and one case each of type II pneumocyte and bronchial surface cell type. The remaining 6 cases lacked definite discernible features of differentiation towards any specific cell type, other than presence of small nuclear clefts in occasional nuclei. Tumors with Clara cell differentiation were low cuboidal with apical snouts. Type II pneumocyte tumor failed to reveal any characteristic definable as light microscopic feature. Ultrastructural examination is the only definite means of identification of various cell types in the respiratory epithelium and hence forms an invaluable tool in classification of pulmonary adenocarcinoma.

DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation

DC-LAMP is a molecule expressed in mature dendritic cells, but its mRNA is also found in the lung. This study compares the immunostaining spectrum of PE-10, an antisurfactant protein monoclonal antibody; thyroid transcription factor-1 (TTF-1); and DC-LAMP in normal and neoplastic lung in an attempt to characterize the cell type(s) that express DC-LAMP. Electron microscopy was used to define cell types. DC-LAMP marks pulmonary adenocarcinomas that show Clara cell characteristics by electron microscopy. In contrast, PE-10 labels tumors that have Clara cell and type II pneumocyte differentiation. DC-LAMP staining was lost in solid type adenocarcinomas but persisted in well-differentiated areas. CC-10, an antibody that marks Clara cells, was also positive in tumors that labeled for DC-LAMP. There was no prognostic difference in tumors that reacted with DC-LAMP. DC-LAMP and CC-10 reactivity was also observed in endometrial adenocarcinomas but not in other tumor types.

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.

Induction of Tolerance to Naphthalene in Clara Cells Is Dependent on a Stable Phenotypic Adaptation Favoring Maintenance of the Glutathione Pool

Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a gamma-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of gamma-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium.

JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells.

Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers, SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a -150- to -84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the -150- to -84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated the SPRR1B promoter activity. Conversely, a c-jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH(2)-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.

Acute injury to differentiating Clara cells in neonatal rabbits results in age-related failure of bronchiolar epithelial repair.

Nonciliated bronchiolar (Clara) cells are progenitor cells during lung development. During differentiation, they have a heightened injury susceptibility to environmental toxicants bioactivated by cytochrome P450 monooxygenase. When neonatal rabbits are treated with the P450-mediated cytotoxicant 4-ipomeanol (IPO), abnormal bronchiolar epithelium results. This study establishes the impact of IPO cytotoxicity on 3 stages of rabbit Clara cell differentiation, early (2.5 and 5 days postnatal [DPN]), intermediate (7 and 9 DPN), and late (15 and 21 DPN), and relates the cytotoxicity to the extent of bronchiolar repair. Neonates received a single dose of IPO (5 mg/kg) and were assessed by qualitative pathology 48 hours later for injury or at 4 weeks for repair. IPO injured the 3 stages of Clara cell differentiation to the same degree; epithelium was swollen, exfoliated, and squamated. Epithelial repair differed among the 3 stages. Bronchioles of animals treated during early and intermediate stages had simple squamous and irregularly shaped cuboidal cells. Animals treated during late stages were similar to controls. Thus, differentiating Clara cells are susceptible to injury by the P450-mediated cytotoxicant IPO, but the extent of repair varies based on when the initial injury occurs.

TRANSGENIC MOUSE MODELS FOR LUNG CANCER

Transgenic technology allows the ability to target regulatory genes to the lungs in a cell-specific fashion. Using this technology, we have generated a model to investigate the phenotypic consequences of targeting oncogenes to particular cell types in the lungs and are developing a second model for the regulated expression of oncogenes in the lung. The transgenic model involves the constitutive expression of simian virus 40 large T antigen in the Clara cells of mouse lungs. This model has been used to investigate changes in expression of cell cycle regulatory genes in the Clara cells during the transformation process, as well as the expression of the transcription factors regulating the expression of Clara cell differentiation markers. The second model we are developing investigates the regulated expression of the genes in the lungs. This system is based on the establishment of two types of transgenic lines. The regulator line consists of a chimeric transcriptional factor placed under the control of a lung-specific SPC (surfactant protein C) promoter. This chimeric regulator is composed of a transcription activation domain, the GAL4 DNA-binding domain, and a truncated progesterone receptor that is responsive to RU 486, but not to endogenous progesterone. The second transgenic mouse line contains the silent target transgene under the control of a minimal promoter with upstream activating sequences (UAS) that are recognized by the regulator transgene. Upon breeding these two lines, the resulting bitransgenic mice can then be induced to express the target transgene only with the administration of RU 486. Two generations of regulators have been evaluated on their ability to regulate the expression of a growth hormone reporter gene. This system demonstrated the inducible expression of the reporter genes in the distal airways of the lungs.

Cytodifferentiation of atypical adenomatous hyperplasia and bronchioloalveolar lung carcinoma: immunohistochemical and ultrastructural studies.

We used immunohistochemistry and electron microscopy to evaluate the differentiation of cells comprising atypical adenomatous hyperplasia (AAH; n = 26), early bronchioloalveolar lung carcinoma (BAC; n = 11), and overt BAC (n = 16), which are assumed to constitute a continuous spectrum of developmental steps of BAC. Surfactant apoprotein (SAP), a marker for type 2 alveolar cells, was expressed in cells from all the lesions of AAH, early BAC, and overt BAC. However, the proportion of SAP-positive cells decreased and their distribution became more heterogeneous with advancing lesion grade. Urine protein 1, which is identical to the Clara cell-specific 10 kDa protein, was expressed in 70% of overt BAC, whereas only 20% of early BAC showed weak reactivity and none of AAH lesions showed any reactivity at all. Ultrastructurally, type 2 alveolar cell differentiation was predominant among cells from AAH and early BAC. Our results suggest that precursor cells of BAC differentiate predominantly towards type 2 alveolar cells. Cells comprising overt BAC retain this differentiation phenotype, but to a reduced extent. In contrast, concomitantly with progression, cells with Clara cell differentiation emerge and their proportion increases. Such phenotypic changes may reflect metaplasia occurring in tumour cells during the development of BAC.

Pulmonary cytochrome P450 monooxygenase and Clara cell differentiation in mice.

Various studies indicate that cytodifferentiation of Clara cells and development of pulmonary cytochrome P450 (CYP) monooxygenases occur postnatally. The timing of these events is species-specific. Neonatal mice are more susceptible than adult mice are to Clara cell injury by naphthalene, but little is known about the postnatal development of Clara cells and CYP in mice. This study was designed to determine the developmental pattern of Clara cell differentiation and CYP expression in mice. Lungs from mice aged 16 days gestation to 63 days postnatal (DPN) were studied. Clara cell secretory protein (CC10) expression in nonciliated cells was detected earlier in proximal airways than in distal airways, but reached adult levels at 14 DPN in all airway levels. Cilia-associated tubulin expression closely followed the onset of CC10 expression, as did expression of CYP reductase. CYP2B protein expression appeared and differentiated earlier in bronchi than in bronchioles and reached adult levels at 14 and 28 DPN, respectively. CYP2F2 expression appeared earlier in proximal airways, but did not reach adult levels of expression until after 28 DPN. CYP activity, measured by naphthalene metabolism, increased with age and corresponded to CYP2F2 protein expression. We conclude that in the mouse, (1) Clara cell maturation is a postnatal event, (2) Clara cell differentiation is complete at the same age in proximal and distal airways, (3) CYP reductase protein expression occurs at the same time as CC10 expression, but CYP2B and CYP2F2 lag behind, and (4) stereoselective naphthalene monooxygenase activity corresponds with CYP2F2 protein expression.

CD44v6 expression in primary bronchioloalveolar carcinoma and conventional pulmonary adenocarcinoma: Ohori NP, Coppola D, Landreneau RJ. Montefiore-Pathology, University of Pittsburgh, Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582. Mod Pathol 1996;9:507–512

CD44 is a family of transmembrane glycoproteins involved in cell-to-cell and cell-to-matrix interactions. Of the CD44 isoforms characterized, the v6 variant has been shown to confer metastatic potential in animal models and its expression has been correlated with aggressive behavior in some human malignancies. In an attempt to provide further insight into the heterogeneous appearance and behavior of bronchioloalveolar carcinomas (BACs), the pattern of CD44v6 staining in five mucinous, five nonmucinous, and eight sclerosing stage I BACs were compared with 12 comparably staged conventional pulmonary adenocarcinomas (CPAs). Formalin-fixed, paraffin-embedded sections were stained with monoclonal antibodies to CD44v6 (clone VFF-7). Neoplastic goblet cells, whether growing in a lepidic pattern in mucinous BACs or in an invasive pattern in CPAs, consistently demonstrated low level expression of CD44v6. In contrast, lepidic growth by neoplastic cells of type 2 pneumocyte or Clara cell differentiation demonstrated high levels in five of five cases of nonmucinous BACs, seven of eight cases of sclerosing BACs, and six of nine cases of CPAs. Similar cells of type 2 pneumocyte or Clara cell differentiation within the scarred area of sclerosing BAC demonstrated an intermediate degree of staining with four of eight cases marking with high levels, whereas none of the nine cases of CPAs with invasion by these cells showed high levels in the desmoplastic areas. From these results, two patterns of CD44v6 expression were recognized in BACs and CPAs. First, the expression appeared to be partly dependent on differentiation as cells demonstrating mucinous differentiation, whether in BACs or CPAs, consistently lacked CD44v6 staining. Second, in cells of type pneumocyte or Clara cell differentiation the expression was dependent on the growth pattern and their relationship to the stroma. High level expression was noted in neoplastic cells growing in a lepidic pattern, whereas those demonstrating invasion gradually lost the expression with increasing degrees of stromal reaction. Thus, the expression of CD44v6 in pulmonary BACs and similarly staged CPAs appears to be different from other human malignancies that have a positive correlation with aggressive behavior.

Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice

Clara cells, progenitors for bronchiolar epithelium, are also primary targets for metabolically activated pulmonary cytotoxicants and have an abundance of the cytochrome P-450 monooxygenases required for xenobiotic metabolism. To define the repair pattern after massive Clara cell injury, mice were treated with naphthalene, and lungs evaluated 1-14 days postinjury (DPI). Clara cells of terminal bronchioles were vacuolated and swollen 1 DPI, exfoliated 2 DPI, and resembled controls at 14 DPI. The volume fraction of vacuolated cells was highest 1 and 2 DPI and minimal at 5-7 DPI. The volume fraction of normal nonciliated cells decreased 40% at 1 DPI. Cell proliferation increased within epithelium and interstitium at 1 DPI, was maximal at 2 DPI, and at all other time points was similar to baseline levels. Expression of Clara cell differentiation markers was barely detectable in terminal bronchiolar epithelium at 1 and 2 DPI, clearly detectable at 4 DPI, and gradually returned to control levels at 5-14 DPI. We conclude that bronchiolar epithelial repair after naphthalene injury involves distinct phases of proliferation and differentiation, proliferation of cells that are not differentiated Clara cells, and interaction of multiple cell types including nontarget cells.

Pulmonary cytochrome P-450 monooxygenase system and Clara cell differentiation in rats.

Because a number of studies suggest that the developmental expression of cytochrome P-450s (CYP) in Clara cells is species specific, this study was designed to compare the developmental patterns of the isoform CYP2B and NADPH reductase protein expression and CYP2B activity with the time course of smooth endoplasmic reticulum (SER) formation in Clara cells of rat lung. Pulmonary CYP2B activity measured as pentoxyresorufin O-dealkylation in lung homogenates was not detectable before 7 days postnatal age, but was detectable at adult levels at 50 days postnatal age. In Clara cells, CYP2B and NADPH reductase were detected immunohistochemically at 4 days postnatal age and at adult levels at 10 days postnatal age. The volume density of SER in Clara cells of terminal bronchioles measured morphometrically increased significantly with postnatal age. We conclude that in the rat 1) CYP2B and NADPH reductase distribution and CYP2B activity are age dependent; 2) the increase in Clara cell SER precedes the expression of CYP2B protein; 3) cellular appearance of CYP2B protein precedes CYP activity; and 4) SER appearance and P-450 protein expression do not occur uniformly in differentiating Clara cells, even within the same bronchiole.