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Research paper thumbnail of Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

PLOS ONE, Jun 8, 2023

Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3,... more Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1β (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B-and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B-and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokineinduced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.

Research paper thumbnail of NF‐κB and Glucocorticoid Receptor Synergize to Enable Pro‐inflammatory Signaling via Toll‐like Receptor 2 in Pulmonary Epithelial Cells

The FASEB Journal, May 1, 2021

Research paper thumbnail of Insights into Glucocorticoid Resistance: Synergy Between Glucocorticoids and Inflammatory Cytokines on TLR2 Expression

Research paper thumbnail of Role of Nuclear Factor-κB in the Regulation of IκB-kinase-ε and Interferon Regulatory Factor-1 Gene Expression in Human Pulmonary Epithelial Cells

B60. ASTHMA AND ALLERGIC RESPONSES: CELLS, TISSUES, AND ANIMAL MODELS

Research paper thumbnail of Role of Nuclear Factor-κB (NF-κB) in the Regulation of IκB Kinase ε (IKKε) Expression in Human Pulmonary Epithelial Cells

TP111. TP111 ASTHMA AND ALLERGIC RESPONSES: CELLULAR AND MOLECULAR PATHWAYS, 2021

Research paper thumbnail of Crosstalk Between the Glucocorticoid Receptor, NF-κB and P38 MAPK in Pulmonary Epithelial Cells Enables Increased Inflammatory Signaling Due to Enhanced Toll-Like Receptor 2 Expression

A65. EPITHELIAL CELL BIOLOGY: THE PLOT THICKENS, 2020

Research paper thumbnail of Glucocorticoids and Inflammatory Cytokines Synergize to Maintain TLR2 Expression in Airway Epithelial Cells

Research paper thumbnail of Interplay between nuclear factor-κB, p38 MAPK and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells

Journal of Biological Chemistry, 2022

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of... more While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore the synergy that occurs between synthetic glucocorticoids (dexamethasone, budesonide) and pro-inflammatory cytokines (IL1B, TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines, as well as in undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required NF-κB and GR. Likewise, in A549 cells, BEAS-2B cells and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32kb (R1) and ∼7.3kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon response factor-dependent reporter activity, and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Research paper thumbnail of Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Research paper thumbnail of Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

Journal of Biological Chemistry, 2020

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcrip... more Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO)-sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confi...

Research paper thumbnail of Glucocorticoid Receptor Orchestrates Interactions Between Glucocorticoid ‐Responsive and ‐Unresponsive Enhancers to Modulate Gene Expression

Research paper thumbnail of Effect of p53 codon 72 polymorphism on the survival outcome in advanced stage cervical cancer patients in India

Indian Journal of Medical Research, 2016

Research paper thumbnail of Differential regulation of baculoviral repeat containing protein 2 (BIRC2) and 3 (BIRC3) by inflammatory stimuli and glucocorticoids

Research paper thumbnail of Interplay between the Glucocorticoid Receptor and Inflammatory Signaling leads to Enhanced TLR2 Expression in Pulmonary Epithelial Cells

Research paper thumbnail of Somatic Variations in Cervical Cancers in Indian Patients

PloS one, 2016

There are very few reports that describe the mutational landscape of cervical cancer, one of the ... more There are very few reports that describe the mutational landscape of cervical cancer, one of the leading cancers in Indian women. The aim of the present study was to investigate the somatic mutations that occur in cervical cancer. Whole exome sequencing of 10 treatment naïve tumour biopsies with matched blood samples, from a cohort of Indian patients with locally advanced disease, was performed. The data revealed missense mutations across 1282 genes, out of 1831 genes harbouring somatic mutations. These missense mutations (nonsynonymous + stop-gained) when compared with pre-existing mutations in the COSMIC database showed that 272 mutations in 250 genes were already reported although from cancers other than cervical cancer. More than 1000 novel somatic variations were obtained in matched tumour samples. Pathways / genes that are frequently mutated in various other cancers were found to be mutated in cervical cancers. A significant enrichment of somatic mutations in the MAPK pathway ...

Research paper thumbnail of Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

PLOS ONE, Jun 8, 2023

Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3,... more Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1β (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B-and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B-and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokineinduced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.

Research paper thumbnail of NF‐κB and Glucocorticoid Receptor Synergize to Enable Pro‐inflammatory Signaling via Toll‐like Receptor 2 in Pulmonary Epithelial Cells

The FASEB Journal, May 1, 2021

Research paper thumbnail of Insights into Glucocorticoid Resistance: Synergy Between Glucocorticoids and Inflammatory Cytokines on TLR2 Expression

Research paper thumbnail of Role of Nuclear Factor-κB in the Regulation of IκB-kinase-ε and Interferon Regulatory Factor-1 Gene Expression in Human Pulmonary Epithelial Cells

B60. ASTHMA AND ALLERGIC RESPONSES: CELLS, TISSUES, AND ANIMAL MODELS

Research paper thumbnail of Role of Nuclear Factor-κB (NF-κB) in the Regulation of IκB Kinase ε (IKKε) Expression in Human Pulmonary Epithelial Cells

TP111. TP111 ASTHMA AND ALLERGIC RESPONSES: CELLULAR AND MOLECULAR PATHWAYS, 2021

Research paper thumbnail of Crosstalk Between the Glucocorticoid Receptor, NF-κB and P38 MAPK in Pulmonary Epithelial Cells Enables Increased Inflammatory Signaling Due to Enhanced Toll-Like Receptor 2 Expression

A65. EPITHELIAL CELL BIOLOGY: THE PLOT THICKENS, 2020

Research paper thumbnail of Glucocorticoids and Inflammatory Cytokines Synergize to Maintain TLR2 Expression in Airway Epithelial Cells

Research paper thumbnail of Interplay between nuclear factor-κB, p38 MAPK and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells

Journal of Biological Chemistry, 2022

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of... more While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore the synergy that occurs between synthetic glucocorticoids (dexamethasone, budesonide) and pro-inflammatory cytokines (IL1B, TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines, as well as in undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required NF-κB and GR. Likewise, in A549 cells, BEAS-2B cells and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32kb (R1) and ∼7.3kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon response factor-dependent reporter activity, and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Research paper thumbnail of Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Research paper thumbnail of Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

Journal of Biological Chemistry, 2020

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcrip... more Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO)-sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confi...

Research paper thumbnail of Glucocorticoid Receptor Orchestrates Interactions Between Glucocorticoid ‐Responsive and ‐Unresponsive Enhancers to Modulate Gene Expression

Research paper thumbnail of Effect of p53 codon 72 polymorphism on the survival outcome in advanced stage cervical cancer patients in India

Indian Journal of Medical Research, 2016

Research paper thumbnail of Differential regulation of baculoviral repeat containing protein 2 (BIRC2) and 3 (BIRC3) by inflammatory stimuli and glucocorticoids

Research paper thumbnail of Interplay between the Glucocorticoid Receptor and Inflammatory Signaling leads to Enhanced TLR2 Expression in Pulmonary Epithelial Cells

Research paper thumbnail of Somatic Variations in Cervical Cancers in Indian Patients

PloS one, 2016

There are very few reports that describe the mutational landscape of cervical cancer, one of the ... more There are very few reports that describe the mutational landscape of cervical cancer, one of the leading cancers in Indian women. The aim of the present study was to investigate the somatic mutations that occur in cervical cancer. Whole exome sequencing of 10 treatment naïve tumour biopsies with matched blood samples, from a cohort of Indian patients with locally advanced disease, was performed. The data revealed missense mutations across 1282 genes, out of 1831 genes harbouring somatic mutations. These missense mutations (nonsynonymous + stop-gained) when compared with pre-existing mutations in the COSMIC database showed that 272 mutations in 250 genes were already reported although from cancers other than cervical cancer. More than 1000 novel somatic variations were obtained in matched tumour samples. Pathways / genes that are frequently mutated in various other cancers were found to be mutated in cervical cancers. A significant enrichment of somatic mutations in the MAPK pathway ...