Conditional, genetically encoded, small molecule–regulated inhibition of NFκB signaling in RPE cells

Khiem T. Vu, Fang Zhang, John D. Hulleman

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

PURPOSE. Nuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule. METHODS. We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach. RESULTS. This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled ‘‘on’’ or ‘‘off’’ within the same cell population repeatedly to confer protection at desired time points. CONCLUSIONS. These studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.

Original languageEnglish (US)
Pages (from-to)4126-4137
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume58
Issue number10
DOIs
StatePublished - Aug 1 2017

Fingerprint

Retinal Pigment Epithelium
Tetrahydrofolate Dehydrogenase
Interleukin-1
Trimethoprim
Cytokines
Retinal Diseases
Doxycycline
Complement Activation
Electrophoretic Mobility Shift Assay
Interleukin-6
Cell Survival
Transcription Factors
Up-Regulation
Western Blotting
Enzyme-Linked Immunosorbent Assay
Inflammation
Gene Expression
Polymerase Chain Reaction
Population

Keywords

  • Destabilized domain
  • Inflammation
  • IκBα
  • NFκB
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Conditional, genetically encoded, small molecule–regulated inhibition of NFκB signaling in RPE cells. / Vu, Khiem T.; Zhang, Fang; Hulleman, John D.

In: Investigative Ophthalmology and Visual Science, Vol. 58, No. 10, 01.08.2017, p. 4126-4137.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. Nuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule. METHODS. We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach. RESULTS. This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93{\%}). This strategy is dosable, completely reversible, and can be cycled ‘‘on’’ or ‘‘off’’ within the same cell population repeatedly to confer protection at desired time points. CONCLUSIONS. These studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.",
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AU - Vu, Khiem T.

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AU - Hulleman, John D.

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N2 - PURPOSE. Nuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule. METHODS. We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach. RESULTS. This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled ‘‘on’’ or ‘‘off’’ within the same cell population repeatedly to confer protection at desired time points. CONCLUSIONS. These studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.

AB - PURPOSE. Nuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule. METHODS. We developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach. RESULTS. This destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled ‘‘on’’ or ‘‘off’’ within the same cell population repeatedly to confer protection at desired time points. CONCLUSIONS. These studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.

KW - Destabilized domain

KW - Inflammation

KW - IκBα

KW - NFκB

KW - Retinal pigment epithelium

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