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Systems approaches to modeling chronic mucosal inflammation.

Kalita M, Tian B, Gao B, Choudhary S, Wood TG, Carmical JR, Boldogh I, Mitra S, Minna JD, Brasier AR - Biomed Res Int (2013)

Bottom Line: EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF- κ B pathways.Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF- κ B2 is required to reduce the noncanonical pathway coupling interval.Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.

View Article: PubMed Central - PubMed

Affiliation: Sealy Center for Molecular Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA.

ABSTRACT
The respiratory mucosa is a major coordinator of the inflammatory response in chronic airway diseases, including asthma and chronic obstructive pulmonary disease (COPD). Signals produced by the chronic inflammatory process induce epithelial mesenchymal transition (EMT) that dramatically alters the epithelial cell phenotype. The effects of EMT on epigenetic reprogramming and the activation of transcriptional networks are known, its effects on the innate inflammatory response are underexplored. We used a multiplex gene expression profiling platform to investigate the perturbations of the innate pathways induced by TGF β in a primary airway epithelial cell model of EMT. EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF- κ B pathways. Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF- κ B2 is required to reduce the noncanonical pathway coupling interval. Experiments using amantadine confirmed the prediction that TRAF-1 and NF- κ B2/p100 production is mediated by an IRES-dependent mechanism. These data indicate that the epigenetic changes produced by EMT induce dynamic state changes of the innate signaling pathway. Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.

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Effect of EMT on canonical-noncanonical NF-κB pathway coupling. (a) Relative changes in TRAF1, NF-κB2, and TNIP1 mRNA expressed as fold-change measurements in the absence or presence of TGFβ-induced EMT as indicated. Each point is the mean of a duplicate biological experiment, measured with three technical replicates. (b) XChIP experiments of HSAECs in the presence (grey bars) or absence (black bars) of TGFβ-induced EMT. Shown is fold change in the TRAF1 promoter quantified by Q-gPCR relative to unstimulated HSAEC signal in duplicate experiments. (c) Computational simulations of p52 processing as a function of translational delay for TRAF1 and NFκB2. Abbreviations; T.d., translational delay. (A) shows the effect of increasing TRAF1 translational delay on p52 processing time while keeping the translational delay of NFκB2 either at nominal rate (90′) or higher than nominal rate or lower than nominal rate. (B) shows similar effect but for increasing NFκB2 translational delay (x-axis). (C) shows the contour plot of all simulations (D, E) amantadine-treated A549 cells (200 μg/mL) were stimulated with poly I:C and TNIP1/Naf1 (D), and IL8 (E) expression was measured by Q-RT-PCR. Data expressed as fold change as compared to untreated cells after normalizing to internal controls, GAPDH. Data analyzed by a 2-way ANOVA with multiple comparisons. Significantly different from amantadine untreated samples: *P < 0.05 and **P < 0.001. Amantadine-treated cells (light bars) showed higher level of noncanonical pathway inhibition compared to untreated cells (dark bars).
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fig5: Effect of EMT on canonical-noncanonical NF-κB pathway coupling. (a) Relative changes in TRAF1, NF-κB2, and TNIP1 mRNA expressed as fold-change measurements in the absence or presence of TGFβ-induced EMT as indicated. Each point is the mean of a duplicate biological experiment, measured with three technical replicates. (b) XChIP experiments of HSAECs in the presence (grey bars) or absence (black bars) of TGFβ-induced EMT. Shown is fold change in the TRAF1 promoter quantified by Q-gPCR relative to unstimulated HSAEC signal in duplicate experiments. (c) Computational simulations of p52 processing as a function of translational delay for TRAF1 and NFκB2. Abbreviations; T.d., translational delay. (A) shows the effect of increasing TRAF1 translational delay on p52 processing time while keeping the translational delay of NFκB2 either at nominal rate (90′) or higher than nominal rate or lower than nominal rate. (B) shows similar effect but for increasing NFκB2 translational delay (x-axis). (C) shows the contour plot of all simulations (D, E) amantadine-treated A549 cells (200 μg/mL) were stimulated with poly I:C and TNIP1/Naf1 (D), and IL8 (E) expression was measured by Q-RT-PCR. Data expressed as fold change as compared to untreated cells after normalizing to internal controls, GAPDH. Data analyzed by a 2-way ANOVA with multiple comparisons. Significantly different from amantadine untreated samples: *P < 0.05 and **P < 0.001. Amantadine-treated cells (light bars) showed higher level of noncanonical pathway inhibition compared to untreated cells (dark bars).

Mentions: EMT effects on the time-dependent expression of the TNIP1/Naf1 gene, a hallmark of the noncanonical NF-κB pathway [24, 27], were also evident in the hierarchical clustering (Figures 3 and 4). As discussed above, the noncanonical pathway is coupled to the canonical pathway through the expression of TNF receptor associated factor-1 (TRAF-1). TRAF-1 is unique for the TRAF isoforms that complexes with, stabilizes, and activates the NIK·IKKα complex to trigger the noncanonical pathway [24]. Analysis of relative fold change of expression showed that TRAF-1 expression was strongly upregulated within 1 h of TNFα stimulation and peaked at 12 h in EMT-HSAECs versus HSAECs, whereas, TRAF-1 was upregulated by 20-fold at 12 h after TNFα stimulation in EMT-HSEACs versus 3-fold in HSEACs (Figure 5(a)).


Systems approaches to modeling chronic mucosal inflammation.

Kalita M, Tian B, Gao B, Choudhary S, Wood TG, Carmical JR, Boldogh I, Mitra S, Minna JD, Brasier AR - Biomed Res Int (2013)

Effect of EMT on canonical-noncanonical NF-κB pathway coupling. (a) Relative changes in TRAF1, NF-κB2, and TNIP1 mRNA expressed as fold-change measurements in the absence or presence of TGFβ-induced EMT as indicated. Each point is the mean of a duplicate biological experiment, measured with three technical replicates. (b) XChIP experiments of HSAECs in the presence (grey bars) or absence (black bars) of TGFβ-induced EMT. Shown is fold change in the TRAF1 promoter quantified by Q-gPCR relative to unstimulated HSAEC signal in duplicate experiments. (c) Computational simulations of p52 processing as a function of translational delay for TRAF1 and NFκB2. Abbreviations; T.d., translational delay. (A) shows the effect of increasing TRAF1 translational delay on p52 processing time while keeping the translational delay of NFκB2 either at nominal rate (90′) or higher than nominal rate or lower than nominal rate. (B) shows similar effect but for increasing NFκB2 translational delay (x-axis). (C) shows the contour plot of all simulations (D, E) amantadine-treated A549 cells (200 μg/mL) were stimulated with poly I:C and TNIP1/Naf1 (D), and IL8 (E) expression was measured by Q-RT-PCR. Data expressed as fold change as compared to untreated cells after normalizing to internal controls, GAPDH. Data analyzed by a 2-way ANOVA with multiple comparisons. Significantly different from amantadine untreated samples: *P < 0.05 and **P < 0.001. Amantadine-treated cells (light bars) showed higher level of noncanonical pathway inhibition compared to untreated cells (dark bars).
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fig5: Effect of EMT on canonical-noncanonical NF-κB pathway coupling. (a) Relative changes in TRAF1, NF-κB2, and TNIP1 mRNA expressed as fold-change measurements in the absence or presence of TGFβ-induced EMT as indicated. Each point is the mean of a duplicate biological experiment, measured with three technical replicates. (b) XChIP experiments of HSAECs in the presence (grey bars) or absence (black bars) of TGFβ-induced EMT. Shown is fold change in the TRAF1 promoter quantified by Q-gPCR relative to unstimulated HSAEC signal in duplicate experiments. (c) Computational simulations of p52 processing as a function of translational delay for TRAF1 and NFκB2. Abbreviations; T.d., translational delay. (A) shows the effect of increasing TRAF1 translational delay on p52 processing time while keeping the translational delay of NFκB2 either at nominal rate (90′) or higher than nominal rate or lower than nominal rate. (B) shows similar effect but for increasing NFκB2 translational delay (x-axis). (C) shows the contour plot of all simulations (D, E) amantadine-treated A549 cells (200 μg/mL) were stimulated with poly I:C and TNIP1/Naf1 (D), and IL8 (E) expression was measured by Q-RT-PCR. Data expressed as fold change as compared to untreated cells after normalizing to internal controls, GAPDH. Data analyzed by a 2-way ANOVA with multiple comparisons. Significantly different from amantadine untreated samples: *P < 0.05 and **P < 0.001. Amantadine-treated cells (light bars) showed higher level of noncanonical pathway inhibition compared to untreated cells (dark bars).
Mentions: EMT effects on the time-dependent expression of the TNIP1/Naf1 gene, a hallmark of the noncanonical NF-κB pathway [24, 27], were also evident in the hierarchical clustering (Figures 3 and 4). As discussed above, the noncanonical pathway is coupled to the canonical pathway through the expression of TNF receptor associated factor-1 (TRAF-1). TRAF-1 is unique for the TRAF isoforms that complexes with, stabilizes, and activates the NIK·IKKα complex to trigger the noncanonical pathway [24]. Analysis of relative fold change of expression showed that TRAF-1 expression was strongly upregulated within 1 h of TNFα stimulation and peaked at 12 h in EMT-HSAECs versus HSAECs, whereas, TRAF-1 was upregulated by 20-fold at 12 h after TNFα stimulation in EMT-HSEACs versus 3-fold in HSEACs (Figure 5(a)).

Bottom Line: EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF- κ B pathways.Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF- κ B2 is required to reduce the noncanonical pathway coupling interval.Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.

View Article: PubMed Central - PubMed

Affiliation: Sealy Center for Molecular Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555, USA.

ABSTRACT
The respiratory mucosa is a major coordinator of the inflammatory response in chronic airway diseases, including asthma and chronic obstructive pulmonary disease (COPD). Signals produced by the chronic inflammatory process induce epithelial mesenchymal transition (EMT) that dramatically alters the epithelial cell phenotype. The effects of EMT on epigenetic reprogramming and the activation of transcriptional networks are known, its effects on the innate inflammatory response are underexplored. We used a multiplex gene expression profiling platform to investigate the perturbations of the innate pathways induced by TGF β in a primary airway epithelial cell model of EMT. EMT had dramatic effects on the induction of the innate pathway and the coupling interval of the canonical and noncanonical NF- κ B pathways. Simulation experiments demonstrate that rapid, coordinated cap-independent translation of TRAF-1 and NF- κ B2 is required to reduce the noncanonical pathway coupling interval. Experiments using amantadine confirmed the prediction that TRAF-1 and NF- κ B2/p100 production is mediated by an IRES-dependent mechanism. These data indicate that the epigenetic changes produced by EMT induce dynamic state changes of the innate signaling pathway. Further applications of systems approaches will provide understanding of this complex phenotype through deterministic modeling and multidimensional (genomic and proteomic) profiling.

Show MeSH
Related in: MedlinePlus