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. 2010 Jan;6(1):25-33.
doi: 10.1038/nchembio.275. Epub 2009 Dec 6.

Reduced Histone Deacetylase 7 Activity Restores Function to Misfolded CFTR in Cystic Fibrosis

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Free PMC article

Reduced Histone Deacetylase 7 Activity Restores Function to Misfolded CFTR in Cystic Fibrosis

Darren M Hutt et al. Nat Chem Biol. .
Free PMC article

Abstract

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.

Figures

Fig. 1
Fig. 1. HDAC inhibitor treatment increases ΔF508-CFTR expression and trafficking
a, Immunoblot analysis (upper) and quantitation (lower) of CFTR expression following treatment of CFBE41o- cells with 0.1 μM TSA, 5 μM SAHA, 1 μM Scriptaid and 5 μM MS-275. Data are presented as fold change relative to vehicle treatment (mean ± SEM, n ≥ 3) (inset: levels for vehicle treated control). C/B ratio expressed as a fold change relative to vehicle treatment. b, Chemical structure of HDACi tested in a. c, Immunoblot analysis (upper) of CFTR and histone H3 expression and CFTR quantitation (lower) following treatment of CFBE41o- cells with 5 μM SAHA for the indicated time (inset: band C analysis for the first 4 h). Data shown as a percent of band B in vehicle treatment (mean ± SD, n = 2). d, Pulse chase analysis of ΔF508-CFTR (upper) and wild-type CFTR (lower) following treatment of cells with 5 μM SAHA for 24 h. Data shown as percent of maximum CFTR at t = 0 (mean ± SD, n = 2). e, Surface expression of ΔF508extope in BHK cells following treatment with 0.02% DMSO, 25 μM of correctors C3, C4 (11) and 4-PBA (B) (12), 0.1 μM TSA, 5 μM SAHA, 1 μM Scriptaid or 5 μM MS-275 or combinations with C3. (mean ± SEM, n=3). In all panels, asterisk indicates significant differences (p < 0.05) relative to DMSO treatment as determined by two-tailed t-test.
Fig. 2
Fig. 2. Low dose SAHA treatment increases ΔF508-CFTR stability and trafficking
a, (Left panel) Immunoblot analysis of ΔF508 following chronic application of 1 μM SAHA to CFBE41o- cells for the indicated time. The treatment schema is shown beneath the immunoblot. (Right panel) Quantitation of the different glycoforms and the C/B ratio (inset) are shown. Data shown as fold change relative to vehicle treatment (mean ± SEM, n=4). b, (Upper) Immunoblot analysis of ΔF508 following chronic application of 1 μM SAHA to CFBE41o- cells following treatment for 1, 5 and 10 days and subsequent washout. The lower panels show quantitation of bands B and C. The data shown as a ratio of CFTR glycoform at the indicated time relative to the level of band B in the control condition (mean ± SEM; n = 4). c, Cell surface density of extracellular epitope-tagged ΔF508 stably expressed in CFBE41o- cells (CFBE-L-ΔF508) following pretreatment for 5 days and indicated washout (mean ± SEM, n=8). In all panels, asterisk indicates significant differences (p < 0.05) relative to DMSO treatment as determined by two-tailed t-test.
Fig. 3
Fig. 3. HDAC inhibitor treatment activates ΔF508 channel activity
a, The effect of 0.1 μM TSA, 5 μM SAHA, 1 μM Scriptaid, 5 μM MS-275 (24 h at 37°C) on the fold-change in cAMP-mediated iodide efflux relative to the non-treated CFBE41o- cells (mean ± SEM; n ≥ 3). b, Difference in transepithelial short-circuit currents (ΔIsc) in response to Fsk, Fsk + Gst and CFTRinh-172 following treatment of CFBE41o- monolayers at reduced temperature (30°C) or with 10 μM SAHA for 24 h (mean ± SEM; n > 6). In all panels, asterisk indicates significant differences (p < 0.05) relative to DMSO treatment as determined by two-tailed t-test.
Fig. 4
Fig. 4. Silencing of HDAC7 increases ΔF508-CFTR expression, trafficking and activity
a, (Upper panel) Immunoblot analysis of CFTR following siRNA-mediated HDAC silencing in CFBE41o- cells. (Lower panel) quantitative analysis of total CFTR, glycoforms and C/B ratio following silencing of HDACs 1, 2, 3 and 7. Data are expressed as fold change relative to siScr control (mean ± SEM; n=3). The inset represents the glycoform levels for siScr control samples. b, Analysis of HDAC1 and HDAC7 protein levels following their respective silencing in CFBE41o- cells (mean ± SEM; n=3). c, Pulse-chase of CFTR after HDAC7 silencing in CFBE41o- cells. Quantitation of CFTR band B glycoform levels is presented as the percentage of maximal CFTR seen at t = 0. (mean ± SEM; n = 3). d, The effect of siHDAC1 and siHDAC7 on the fold-change in cAMP-mediated iodide efflux relative to the non-treated CFBE41o- cells (mean ± SEM; n ≥ 3). In all panels, asterisk indicates significant differences (p < 0.05) relative to siScr treatment as determined by two-tailed t-test.
Fig. 5
Fig. 5. SAHA rescues ΔF508-CFTR activity in primary human bronchial epithelial cells
a, ΔIsc in response to Fsk, Fsk + Gst and CFTRinh-172 following treatment of ΔF/ΔF-HBE with 10 μM SAHA or DMSO. Asterisk indicates a significant change (p ≤ 0.05) relative to vehicle treatment. b, ΔIsc as in a, following treatment of ΔF/ΔF-HBE with 1 μM SAHA for the indicated time. Asterisk indicates a significant change (p ≤ 0.05) relative to 2-day treatment. c, ΔIsc as in a, following treatment of ΔF/ΔF-HBE with 1 μM SAHA and washout for the indicated time. Asterisk and # indicate a significant change (p ≤ 0.05) relative to non-treatment (NT),and 8 days without washout (8 day + 0) respectively. Data shown as a percent of 8 day + 0. d, CFTR immunoblot of ΔF/ΔF-HBE lysates following treatment with 1 μM SAHA for the indicated treatment and washout time. e, CFTR band C analysis in non-CF HBE, ΔF/ΔF-HBE and ΔF/ΔF-HBE treated with 0.02% DMSO or 10 μM SAHA. Inset depicts non-CF HBE lysates. Data shown as ratio of band C to actin and the non-CF HBE ratio normalized to 100% (mean ± SEM; n = 3). f, Representative images depicting the effect of 0.1 μM TSA, 5 μM SAHA, 5 μM MS-275 and 1 μM Scriptaid on the localization of ΔF508extope in primary HBE cells (scale bar = 10 μM). In panels a-c data is mean ± SEM, n > 6. In all panels, p values determined by two-tailed t-test between compared data points.
Fig. 6
Fig. 6. Mechanism of HDAC7-mediated ΔF508 multi-target pathway correction
a, Clustered display of the preprocessed expression matrix, showing probeset-scaled expression values (adjusted p-value<0.01) following siHDAC7 treatment of CFBE41o-cells. In all panels, the color gradient goes from blue (low) to orange (high). The reported biological effect on CFTR of indicated proteins is coded as follows: red: negatively affect CFTR stability, trafficking and/or activity; green: positively affect CFTR stability, trafficking and/or activity; black: unknown effects. Bold typeface indicates an increased expression change greater then 2-fold. Functional assignments based on the CF pathways platform are depicted in colored boxes to the left of the gene name with the key code depicted in Supplemental Fig. S7. b, Cartoon depicting potential steps in a pathway of HDAC7-based correction. The diagram illustrates a branched pathway in which the observed alteration in the transcriptional environment (colored arrowhead indicating up- or down-regulation in a) affects the stability, trafficking and/or activity of ΔF508. Bold solid lines represent steps favoring positive effect on correction of function while dotted lines represent steps leading to loss of function. c, Hypothetical model by which the HDAC7-sensitive transcriptional regulation of CFTR interacting genes leads to correction of the ΔF508 trafficking defect.

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