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The Role of Glucocorticoid Inducible Transcriptional Repressor PLZF in the Airway Epithelium
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A5812 - The Role of Glucocorticoid Inducible Transcriptional Repressor PLZF in the Airway Epithelium
Author Block: A. Stewart, D. Prodanovic, S. Y. Langenbach, C. Keenan; Pharmacology and Therapeutics, The University of Melbourne, Parkville, Australia.
Introduction: The transcriptional repressor PLZF (promyelocytic leukaemia zinc finger) was first recognized for its role in the pathogenesis of acute promyelocytic leukaemia. Its involvement in control of cell cycle and differentiation, as well as its induction by glucocorticoids (GC) have been reported in different tissues. However, the potential function of PLZF and its induction by GC in the airway epithelium remains to be established. Methods: The localisation of PLZF was investigated in the airway biopsies of healthy and asthmatic (mild, moderate, severe) subjects from Melbourne Epidemiological Study of Childhood Asthma (MESCA) using immunohistochemistry. The localisation of PLZF was also determined in the normal bronchial epithelial cell line BEAS-2B (American Type Culture Collection (ATCC)) using immunofluorescence (IF) and confocal imaging. The specific role of PLZF in the proliferation of bronchial epithelium was determined using BEAS-2B cells and siRNA gene silencing. In vitro studies were used to ascertain whether PLZF is implicated in the GC regulation of several targets, including epithelial sodium channel α subunit (ENaCα) and cyclin dependent kinase inhibitor 1C (CDKN1C). Results: In the airway biopsies, PLZF was localised mostly in the nuclei of basal and ciliated epithelial cells. Although all asthmatic MESCA subjects were on GC treatment, elevated PLZF expression was detected only in the moderate asthma group. PLZF was also localised in the nuclei of BEAS-2B cells using IF. Dexamethasone (Dex) (100nM) strongly induced PLZF mRNA (203±64 fold vehicle, n=4) and protein expression (115±30 fold vehicle, n=4) at 4h and 24h, respectively. PLZF knock down increased cyclin A2 mRNA (PLZF siRNA a: 2.1±0.6 fold non-silencing negative control (NC) siRNA; PLZF siRNA b: 2.2±0.6 fold NC siRNA, n=5) and mitogen-induced BEAS-2B cell number (PLZF siRNA a: 120±12 %NC siRNA; PLZF siRNA b: 120±7 %NC siRNA, n=5). In the absence of PLZF, we found significantly lower Dex-induced ENaCα mRNA levels (PLZF siRNA a: 56±25 %Dex NC siRNA; PLZF siRNA b: 28±10 %Dex NC siRNA, n=4), and notably higher Dex-induced CDKN1C mRNA levels (PLZF siRNA a: 527±263 %Dex NC siRNA; PLZF siRNA b: 375±188 %Dex NC siRNA, n=4). Conclusion: PLZF appears to slow the proliferation of BEAS-2B cells and interferes with GC-induction of selected targets. Our study is the first to provide a foundation for generation of new insights into the roles of PLZF, particularly in mediating selected GC effects in the airway epithelium.
Author Block: A. Stewart, D. Prodanovic, S. Y. Langenbach, C. Keenan; Pharmacology and Therapeutics, The University of Melbourne, Parkville, Australia.
Introduction: The transcriptional repressor PLZF (promyelocytic leukaemia zinc finger) was first recognized for its role in the pathogenesis of acute promyelocytic leukaemia. Its involvement in control of cell cycle and differentiation, as well as its induction by glucocorticoids (GC) have been reported in different tissues. However, the potential function of PLZF and its induction by GC in the airway epithelium remains to be established. Methods: The localisation of PLZF was investigated in the airway biopsies of healthy and asthmatic (mild, moderate, severe) subjects from Melbourne Epidemiological Study of Childhood Asthma (MESCA) using immunohistochemistry. The localisation of PLZF was also determined in the normal bronchial epithelial cell line BEAS-2B (American Type Culture Collection (ATCC)) using immunofluorescence (IF) and confocal imaging. The specific role of PLZF in the proliferation of bronchial epithelium was determined using BEAS-2B cells and siRNA gene silencing. In vitro studies were used to ascertain whether PLZF is implicated in the GC regulation of several targets, including epithelial sodium channel α subunit (ENaCα) and cyclin dependent kinase inhibitor 1C (CDKN1C). Results: In the airway biopsies, PLZF was localised mostly in the nuclei of basal and ciliated epithelial cells. Although all asthmatic MESCA subjects were on GC treatment, elevated PLZF expression was detected only in the moderate asthma group. PLZF was also localised in the nuclei of BEAS-2B cells using IF. Dexamethasone (Dex) (100nM) strongly induced PLZF mRNA (203±64 fold vehicle, n=4) and protein expression (115±30 fold vehicle, n=4) at 4h and 24h, respectively. PLZF knock down increased cyclin A2 mRNA (PLZF siRNA a: 2.1±0.6 fold non-silencing negative control (NC) siRNA; PLZF siRNA b: 2.2±0.6 fold NC siRNA, n=5) and mitogen-induced BEAS-2B cell number (PLZF siRNA a: 120±12 %NC siRNA; PLZF siRNA b: 120±7 %NC siRNA, n=5). In the absence of PLZF, we found significantly lower Dex-induced ENaCα mRNA levels (PLZF siRNA a: 56±25 %Dex NC siRNA; PLZF siRNA b: 28±10 %Dex NC siRNA, n=4), and notably higher Dex-induced CDKN1C mRNA levels (PLZF siRNA a: 527±263 %Dex NC siRNA; PLZF siRNA b: 375±188 %Dex NC siRNA, n=4). Conclusion: PLZF appears to slow the proliferation of BEAS-2B cells and interferes with GC-induction of selected targets. Our study is the first to provide a foundation for generation of new insights into the roles of PLZF, particularly in mediating selected GC effects in the airway epithelium.
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