Cigarette smoke induces epidermal growth factor receptor-dependent redistribution of apical MUC1 and junctional beta-catenin in polarized human airway epithelial cells.

Cigarette smoke (CS) accounts for nearly 90% of lung cancer deaths worldwide; however, an incomplete understanding of how CS initiates preneoplastic changes in the normal airway hinders early diagnosis. Short-term exposure to CS causes aberrant activation of epidermal growth factor receptor (EGFR) and canonical Wnt/beta-catenin signaling pathways in human bronchial epithelial (HBE) cells. We hypothesize that this response is elicited through the disruption of spatially segregated cell membrane proteins in the polarized airway epithelium. Using an in vitro model of highly differentiated HBE cells, we observed membrane characteristics consistent with the native airway, including the presence of a membrane mucin, MUC1, at the apical cell pole, beta-catenin at the apical-lateral membrane, and EGFR at the basolateral membrane. Following exposure to smoke, intercellular spaces enlarge and cilia disappear. This histopathology is accompanied by molecular events that include perinuclear trafficking of basolateral EGFR, EGFR phosphorylation, pEGFR-mediated phosphorylation of MUC1's cytoplasmic tail (CT), loss of E-cadherin/beta-catenin complexes at the adherens junctions (AJs), intracellular formation and nuclear shuffling of beta-catenin/MUC1-CT complexes, and, ultimately, up-regulation and nuclear localization of Wnt nuclear effector, Lef-1. In the presence of EGFR inhibitor, AG1478, CS-induced histopathology and molecular events were inhibited. These data point to EGFR as a portal through which CS mediates its damaging effects on AJ-mediated cell polarity and activation of canonical Wnt/beta-catenin signaling.

Interleukin-1 as a phenotypic immunomodulator in keratinizing squamous metaplasia of the ocular surface in Sjögren's syndrome.

Chronic inflammation of the ocular surface in Sjögren's syndrome (SS) is associated with a vision-threatening, phenotypic change of the ocular surface, which converts from a nonkeratinized, stratified squamous epithelium to a nonsecretory, keratinized epithelium. This pathological process is known as squamous metaplasia. Based on a significant correlation between ocular surface interleukin (IL)-1beta expression and squamous metaplasia in patients with SS, we investigated the role of IL-1 in the pathogenesis of squamous metaplasia in an animal model that mimics the clinical characteristics of SS. Using autoimmune-regulator (aire)-deficient mice, we assessed lacrimal gland and ocular surface immunopathology by quantifying the infiltration of major histocompatibility complex class II(+) (I-A(d+)) dendritic cells and CD4(+) T cells. We examined squamous metaplasia using a biomarker of keratinization, small proline-rich protein 1B. We used lissamine green staining as a readout for ocular surface epitheliopathy and Alcian blue/periodic acid-Schiff histochemical analysis to characterize goblet cell muco-glycoconjugates. Within 8 weeks, the eyes of aire-deficient mice were pathologically keratinized with significant epithelial damage and altered mucin glycosylation. Although knockdown of IL-1 receptor 1 did not attenuate lymphocytic infiltration of the lacrimal gland or eye, it significantly reduced ocular surface keratinization, epitheliopathy, and muco-glycoconjugate acidification. These data demonstrate a phenotypic modulation role for IL-1 in the pathogenesis of squamous metaplasia and suggest that IL-1 receptor 1-targeted therapies may be beneficial for treating ocular surface disease associated with SS.

Immune profile of squamous metaplasia development in autoimmune regulator-deficient dry eye.

PURPOSE: Squamous metaplasia of the ocular surface epithelium in severe Sjögren syndrome (SS) dry eye has been implicated to be associated with chronic engagement of immune-mediated inflammation. While the detailed immunopathological mechanism underlying keratinization of the ocular muco-epithelium in this setting remains unclear, mice deficient in the autoimmune regulator gene (Aire) demonstrate SS-like pathological changes in the exocrine organs and ocular surface including squamous metaplasia. Using this murine model, we sought to determine the specific immune events that predict squamous metaplasia of the cornea in Aire deficiency. METHODS: Lissamine green staining, goblet cell density, and corneal small proline-rich protein 1B (SPRR1B) were compared in Aire-sufficient and -deficient mice at 4, 8, and 16 weeks of age. Corneal, limbal and conjunctival infiltration of CD4(+) and CD8(+) T cells as well as CD11c(+) and MHC class II (I-A(d+)) dendritic cells (DCs) were examined at the same time points. Ordinary least squares regression was used to model SPRR1B's relationship with lissamine green staining, goblet cell density, and immune cell infiltration. RESULTS: Lissamine green staining was present in Aire-deficient mice by four weeks of age and increased over time. Compared to Aire-sufficient controls, conjunctival goblet cell density (GCD) decreased and corneal SPRR1B increased in Aire-deficient mice with significant differences noted at both 8 and 16 weeks. Immune-mediated CD4(+) T cell infiltration of the conjunctiva and limbus peaked at eight weeks and then decreased. In contrast, corneal T cell infiltration continued to increase over time, reaching a maximum cell number at 16 weeks. CD11c(+) myeloid-derived DCs were found in the conjunctiva and limbus at all time points. As the mice aged, there was a notable increase in corneal CD11c(+) cell counts. Interestingly, the dynamic of activated MHC class II(+) DCs was nearly identical to that of CD4(+) T cells, peaking first in the limbus at eight weeks with maximum infiltration of the cornea by 16 weeks. Regression analysis showed that squamous metaplasia biomarker, SPRR1B, is strongly related to the lissamine green staining of the ocular surface. Corneal infiltration of activated DCs was most prognostic of corneal SPRR1B expression while the presence of precursor DCs, activated DCs, and CD4(+) T cells in the limbus were also significant predictors of SPRR1B. CONCLUSIONS: Aire-deficient mice represent a useful model to study Sjögren-like autoimmune-mediated ocular surface disease. Results of the current study suggest that squamous cell precursor protein, SPRR1B, provides an important readout to evaluate ocular surface damage and specific events related to immune-mediated inflammation. Results also define an appropriate time frame for interventional studies to develop more effective therapies for keratinizing ocular surface disease.

Small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia in dry eye disease.

PURPOSE: Squamous metaplasia occurs in ocular surface diseases like Sjögren's syndrome (SS). It is a phenotypic change whereby epithelial cells initiate synthesis of squamous cell-specific proteins such as small proline-rich protein 1B (SPRR1B) that result in pathologic keratin formation on the ocular surface. The authors hypothesized that inflammation is a key inducer of pathologic keratinization and that SPRR1B represents an analytical biomarker for the study of the molecular mechanisms. METHODS: Real-time quantitative RT-PCR and immunohistochemistry were used to examine SPRR1B mRNA and protein in two different mouse models of dry eye and patients with SS. Adoptive transfer of mature lymphocytes from mice lacking the autoimmune regulator (aire) gene was performed to examine the role of inflammation as an inducer of squamous metaplasia. SPRR1B expression in response to several cytokines was examined in vitro, whereas the expression of cytokines IL1beta and IFNgamma was quantified in ocular tissues of aire-deficient mice and patients with SS. RESULTS: SPRR1B was increased across the ocular surface of mice with both desiccating stress and autoimmune-mediated, aqueous-deficient dry eye and in patients with SS. Adoptive transfer of CD4(+) T cells from aire-deficient mice to immunodeficient recipients caused advanced ocular surface keratinization. IL1alpha, IL1beta, IL6, IFNgamma, and TNFalpha induced SPRR1B expression in vitro and the local expression of IL1beta and IFNgamma was elevated in ocular tissues of patients with SS and aire-deficient mice. CONCLUSIONS: SPRR1B is a valid biomarker for the study of the molecular mechanisms of squamous metaplasia. There is a definitive link between inflammation and squamous metaplasia in autoimmune-mediated dry eye disease, with IL1beta and IFNgamma likely acting as key participants.

A novel Krüppel related factor consisting of only a KRAB domain is expressed in the murine trigeminal ganglion.

The largest family of zinc-finger (ZnF) transcription factors is that containing the Krüppel-associated box, or KRAB domain. The amino-terminal KRAB domain of these proteins functions as a transcriptional repressor with the downstream ZnF motifs providing DNA-binding specificity. Here we report the identification and characterization of a novel murine Krüppel-related factor (KLF), MIF1, which contains a KRAB domain but lacks a ZnF motif. Western blot analysis identified MIF1-like proteins in the murine trigeminal ganglion (TG) and immunostaining localized these proteins primarily to the cytoplasm of TG neuronal cell bodies. In situ hybridization for Mif1 transcripts confirms the selective expression of Mif1 in TG neurons. Consistent with the non-nuclear localization of MIF1 we could detect no transcriptional repressor activity of the MIF1 protein. However MIF1 appears to be capable of interacting with the co-repressor TIF1beta and exhibits transcription repressor activity when fused to yeast GAL4 binding domain protein. Genomic analysis of Mif1 sequence suggests that the Mif1 transcript may result from splicing of a longer KRAB-ZnF containing transcript.

The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.

The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.

The roadblock light chain binds a novel region of the cytoplasmic Dynein intermediate chain.

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.