Rationale: Idiopathic pulmonary fibrosis (IPF) is associated with oxidative stress and depletion ... more Rationale: Idiopathic pulmonary fibrosis (IPF) is associated with oxidative stress and depletion of the antioxidant glutathione (GSH) in epithelial lining fluid (ELF) and tissue. However, differential expression of GSH-metabolizing enzymes in bronchoalveolar lavage fluid (BALF) and tissue during lung fibrosis has not been comprehensively studied. Methods: In a label-free whole-proteome approach, we assessed levels of GSH-metabolizing enzymes in BALF and tissue during the onset and resolution of bleomycin-induced pulmonary fibrosis (day 3 through 56) in C57BL/6N mice. Western Blot analysis, assessment of Gpx activity, ELISA, and immunofluorescent stainings were used for validation and comparison with samples derived from interstitial lung disease patients. Results: GSH peroxidase 3 (Gpx3) was the only significantly increased GSH-metabolizing enzyme in BALF during bleomycin-induced lung fibrosis. This data was validated by Western Blot analysis and assessment of GPX activity in BALF.Immunofluorescent (IF) stainings revealed predominant expressionof Gpx3 by epithelial cells. Finally, BALF of ILD patients also contained high levels of GPX3. In tissue, however, Gpx7 was clearly upregulated and, as determined by IF co-stainings, localized to interstitial cells including myofibroblasts, while expression of Gpx1, the most abundant Gpx in tissue, appeared ubiquitous and remained largely unchanged. Conclusion: We show compartment-specific overexpression of selected Gpx during bleomycin-induced lung fibrosis. Hence, upregulation of Gpx enzymes might contribute to depletion of GSH in ELF and tissue, and thereby enhance the oxidant-antioxidant imbalance in pulmonary fibrosis.
Evidence in the current literature suggests that expression and activity of 11β-hydroxysteroid de... more Evidence in the current literature suggests that expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a key regulatory enzyme in glucocorticoid metabolism, is elevated in the liver and reduced in visceral adipose tissue and skeletal muscle following caloric restriction (CR). In order to investigate the influence of CR on 11β-HSD1 in more detail, we assessed expression and activity of 11β-HSD1 in several tissues in two independent CR and re-feeding animal models. Levels and activity of 11β-HSD1 after CR and re-feeding were measured [mouse liver and pig liver, pig visceral adipose tissue and pig skeletal muscle] using semi-quantitative RT-PCR, Western Blot analysis, and HPLC. After CR, no significant difference on mRNA levels was detected in mouse liver. But 11β-HSD1 mRNA expression was upregulated after subsequent re-feeding. In contrast, 11β-HSD1 protein expression after CR was significantly up-regulated, while no difference was detected after re-feeding. Interestingly, upregulation of protein after CR (1.4-fold) was lower than the increase in enzymatic activity (2.6-fold). Furthermore, while no difference was observed in protein levels after two weeks re-feeding, 11β-HSD1 activity increased 2.5-fold. In pig tissues neither 11β-HSD1 mRNA levels, protein expression or enzyme activity were influenced after CR and re-feeding. Overall, the results demonstrate species-dependent differences in regulation of 11β-HSD1 following CR and suggest the presence of an additional regulation step for 11β-HSD1 activity in mouse liver.
The Journal of Steroid Biochemistry and Molecular Biology, 2013
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene HSD11B1) converts glucocorticoid receptor... more 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene HSD11B1) converts glucocorticoid receptor-inert cortisone to receptor-active cortisol. Multiple evidence supports a causal role for 11β-HSD1 in the current obesity epidemic. In obese, HSD11B1 expression is increased in adipose tissue but typically decreased in liver, and the underlying tissue-specific mechanisms are largely unknown. In this context, we investigated a potential role of microRNAs (miRNAs). We used several miRNA target prediction tools to identify possible candidates and a publicly available miRNA expression atlas to further select candidates expressed in hepatocytes. Using a dual luciferase reporter assay, we identified three potential miRNAs, hsa-miR-340, -561 and -579, as potential negative regulators of HSD11B1 expression. Disruption of the corresponding microRNA response elements abolished repression of luciferase activity for hsa-miR-561 and -579, but not for hsa-miR-340. Furthermore, levels of firefly luciferase mRNA were not changed by miR-561 and -579, indicating a mechanism based on translational repression rather than mRNA degradation. Finally, we were able to detect both, miR-561 and -579, in human total liver RNA by reverse-transcription-polymerase chain reaction (RT-PCR). According to the presented results, miR-561 and -579 are likely to be involved in the tissue-specific regulation of HSD11B1 expression. Moreover, literature findings and a pathway enrichment analysis support a potential role of these two miRNAs in glucocorticoid metabolism and signalling and associated diseases.
The airway epithelium is constantly exposed to noxious substances and respiratory disease is amon... more The airway epithelium is constantly exposed to noxious substances and respiratory disease is among the major leading causes of death worldwide. Upon airway injury, restoration of the normal lung architecture would be desirable, but the mechanisms involved in human adult lung regeneration are poorly understood. Notably, most mechanistic studies have been performed in mouse models where exposition to chemicals, most frequently naphthalene (NP) or polidocanol (PL), causes depletion of bronchial epithelial cells followed by subsequent epithelial regeneration. Given the major differences in cellular composition in the upper respiratory epithelium between humans and mice, we have set out to develop a human in vitro model for the analysis of lung injury and regeneration using primary human bronchial epithelial cells (phBECs) cultured at the air-liquid interface. In contrast to the mouse model, NP treatment in human bronchial epithelium in vitro did not induce cell death, let alone specifically deplete club cells. PL treatment, however, led to a dose-dependent loss of epithelial cells with an IC50 of 0.047% PL. Furthermore, loss of differentiated cell types such as goblet, ciliated and club cells were observed post PL treatment followed by subsequent regeneration of the human bronchial epithelium with the appearance of differentiated cell types at the expense of basal cells within two weeks. During regeneration phase, an increase in cell count was observed based on DAPI count from immunofluorescence analysis indicates proliferation. For proof-of-concept, we will next use this human in-vitro model to assess modulation of bronchial epithelial regeneration by Notch signaling.
Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung disease characterized by e... more Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung disease characterized by excessive deposition of collagen. The two IPF therapeutics nintedanib and pirfenidone decelerate disease progression, but the underlying mechanisms are poorly understood. This study comprehensively analyzes the effects of both drugs on collagen synthesis at several regulatory levels. Methods: Primary human IPF fibroblasts were treated with nintedanib (0.01-1.0µM) or pirfenidone (0.1-1.0mM) with or without TGF-s1. Effects on fibrotic marker expression and on collagen chaperones FKBP10 and HSP47 were analyzed by Western Blot and qPCR. Influences on collagen I and III secretion were visualized by Western Blot. Effects of both drugs on collagen I fibril formation were assessed by dynamic light scattering and scanning electron microscopy. Results: Both drugs reduced the expression of collagen I and V, while fibronectin and FKBP10 were only downregulated by nintedanib. Collagen I secretion was reduced by both drugs, while only nintedanib decreased collagen III secretion. Notably, both drugs considerably delayed fibril formation of purified collagen I. Electron microscopy of nintedanib- or pirfenidone-treated fibroblasts revealed less, shorter and frayed collagen fibril bundles. Conclusions: Both drugs act on different regulatory levels in collagen synthesis and processing. In general nintedanib was more effective in downregulating profibrotic gene expression and collagen secretion. Importantly, nintedanib and pirfenidone inhibited collagen I fibril formation and caused reduction and an altered appearance of collagen fibril bundles, which represents a hitherto unknown effect of both drugs.
Rationale: Idiopathic pulmonary fibrosis (IPF) is associated with oxidative stress and depletion ... more Rationale: Idiopathic pulmonary fibrosis (IPF) is associated with oxidative stress and depletion of the antioxidant glutathione (GSH) in epithelial lining fluid (ELF) and tissue. However, differential expression of GSH-metabolizing enzymes in bronchoalveolar lavage fluid (BALF) and tissue during lung fibrosis has not been comprehensively studied. Methods: In a label-free whole-proteome approach, we assessed levels of GSH-metabolizing enzymes in BALF and tissue during the onset and resolution of bleomycin-induced pulmonary fibrosis (day 3 through 56) in C57BL/6N mice. Western Blot analysis, assessment of Gpx activity, ELISA, and immunofluorescent stainings were used for validation and comparison with samples derived from interstitial lung disease patients. Results: GSH peroxidase 3 (Gpx3) was the only significantly increased GSH-metabolizing enzyme in BALF during bleomycin-induced lung fibrosis. This data was validated by Western Blot analysis and assessment of GPX activity in BALF.Immunofluorescent (IF) stainings revealed predominant expressionof Gpx3 by epithelial cells. Finally, BALF of ILD patients also contained high levels of GPX3. In tissue, however, Gpx7 was clearly upregulated and, as determined by IF co-stainings, localized to interstitial cells including myofibroblasts, while expression of Gpx1, the most abundant Gpx in tissue, appeared ubiquitous and remained largely unchanged. Conclusion: We show compartment-specific overexpression of selected Gpx during bleomycin-induced lung fibrosis. Hence, upregulation of Gpx enzymes might contribute to depletion of GSH in ELF and tissue, and thereby enhance the oxidant-antioxidant imbalance in pulmonary fibrosis.
Evidence in the current literature suggests that expression and activity of 11β-hydroxysteroid de... more Evidence in the current literature suggests that expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a key regulatory enzyme in glucocorticoid metabolism, is elevated in the liver and reduced in visceral adipose tissue and skeletal muscle following caloric restriction (CR). In order to investigate the influence of CR on 11β-HSD1 in more detail, we assessed expression and activity of 11β-HSD1 in several tissues in two independent CR and re-feeding animal models. Levels and activity of 11β-HSD1 after CR and re-feeding were measured [mouse liver and pig liver, pig visceral adipose tissue and pig skeletal muscle] using semi-quantitative RT-PCR, Western Blot analysis, and HPLC. After CR, no significant difference on mRNA levels was detected in mouse liver. But 11β-HSD1 mRNA expression was upregulated after subsequent re-feeding. In contrast, 11β-HSD1 protein expression after CR was significantly up-regulated, while no difference was detected after re-feeding. Interestingly, upregulation of protein after CR (1.4-fold) was lower than the increase in enzymatic activity (2.6-fold). Furthermore, while no difference was observed in protein levels after two weeks re-feeding, 11β-HSD1 activity increased 2.5-fold. In pig tissues neither 11β-HSD1 mRNA levels, protein expression or enzyme activity were influenced after CR and re-feeding. Overall, the results demonstrate species-dependent differences in regulation of 11β-HSD1 following CR and suggest the presence of an additional regulation step for 11β-HSD1 activity in mouse liver.
The Journal of Steroid Biochemistry and Molecular Biology, 2013
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene HSD11B1) converts glucocorticoid receptor... more 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene HSD11B1) converts glucocorticoid receptor-inert cortisone to receptor-active cortisol. Multiple evidence supports a causal role for 11β-HSD1 in the current obesity epidemic. In obese, HSD11B1 expression is increased in adipose tissue but typically decreased in liver, and the underlying tissue-specific mechanisms are largely unknown. In this context, we investigated a potential role of microRNAs (miRNAs). We used several miRNA target prediction tools to identify possible candidates and a publicly available miRNA expression atlas to further select candidates expressed in hepatocytes. Using a dual luciferase reporter assay, we identified three potential miRNAs, hsa-miR-340, -561 and -579, as potential negative regulators of HSD11B1 expression. Disruption of the corresponding microRNA response elements abolished repression of luciferase activity for hsa-miR-561 and -579, but not for hsa-miR-340. Furthermore, levels of firefly luciferase mRNA were not changed by miR-561 and -579, indicating a mechanism based on translational repression rather than mRNA degradation. Finally, we were able to detect both, miR-561 and -579, in human total liver RNA by reverse-transcription-polymerase chain reaction (RT-PCR). According to the presented results, miR-561 and -579 are likely to be involved in the tissue-specific regulation of HSD11B1 expression. Moreover, literature findings and a pathway enrichment analysis support a potential role of these two miRNAs in glucocorticoid metabolism and signalling and associated diseases.
The airway epithelium is constantly exposed to noxious substances and respiratory disease is amon... more The airway epithelium is constantly exposed to noxious substances and respiratory disease is among the major leading causes of death worldwide. Upon airway injury, restoration of the normal lung architecture would be desirable, but the mechanisms involved in human adult lung regeneration are poorly understood. Notably, most mechanistic studies have been performed in mouse models where exposition to chemicals, most frequently naphthalene (NP) or polidocanol (PL), causes depletion of bronchial epithelial cells followed by subsequent epithelial regeneration. Given the major differences in cellular composition in the upper respiratory epithelium between humans and mice, we have set out to develop a human in vitro model for the analysis of lung injury and regeneration using primary human bronchial epithelial cells (phBECs) cultured at the air-liquid interface. In contrast to the mouse model, NP treatment in human bronchial epithelium in vitro did not induce cell death, let alone specifically deplete club cells. PL treatment, however, led to a dose-dependent loss of epithelial cells with an IC50 of 0.047% PL. Furthermore, loss of differentiated cell types such as goblet, ciliated and club cells were observed post PL treatment followed by subsequent regeneration of the human bronchial epithelium with the appearance of differentiated cell types at the expense of basal cells within two weeks. During regeneration phase, an increase in cell count was observed based on DAPI count from immunofluorescence analysis indicates proliferation. For proof-of-concept, we will next use this human in-vitro model to assess modulation of bronchial epithelial regeneration by Notch signaling.
Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung disease characterized by e... more Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung disease characterized by excessive deposition of collagen. The two IPF therapeutics nintedanib and pirfenidone decelerate disease progression, but the underlying mechanisms are poorly understood. This study comprehensively analyzes the effects of both drugs on collagen synthesis at several regulatory levels. Methods: Primary human IPF fibroblasts were treated with nintedanib (0.01-1.0µM) or pirfenidone (0.1-1.0mM) with or without TGF-s1. Effects on fibrotic marker expression and on collagen chaperones FKBP10 and HSP47 were analyzed by Western Blot and qPCR. Influences on collagen I and III secretion were visualized by Western Blot. Effects of both drugs on collagen I fibril formation were assessed by dynamic light scattering and scanning electron microscopy. Results: Both drugs reduced the expression of collagen I and V, while fibronectin and FKBP10 were only downregulated by nintedanib. Collagen I secretion was reduced by both drugs, while only nintedanib decreased collagen III secretion. Notably, both drugs considerably delayed fibril formation of purified collagen I. Electron microscopy of nintedanib- or pirfenidone-treated fibroblasts revealed less, shorter and frayed collagen fibril bundles. Conclusions: Both drugs act on different regulatory levels in collagen synthesis and processing. In general nintedanib was more effective in downregulating profibrotic gene expression and collagen secretion. Importantly, nintedanib and pirfenidone inhibited collagen I fibril formation and caused reduction and an altered appearance of collagen fibril bundles, which represents a hitherto unknown effect of both drugs.
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Papers by Claudia Staab-Weijnitz