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. 2016 Sep 22:7:12823.
doi: 10.1038/ncomms12823.

Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome

Richard S Beard Jr  1 , Xiaoyuan Yang  1 , Jamie E Meegan  1 , Jonathan W Overstreet  1 , Clement G Y Yang  2 , John A Elliott  1 , Jason J Reynolds  1 , Byeong J Cha  1 , Christopher D Pivetti  3 , David A Mitchell  4 , Mack H Wu  2   5 , Robert J Deschenes  4 , Sarah Y Yuan  1   2
Affiliations

Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome

Richard S Beard Jr et al. Nat Commun. .

Abstract

Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCβ1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCβ1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCβ1 palmitoylation in endothelial inflammation.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1. Inhibition of PATs attenuates microvascular leakage during systemic inflammatory response to burn injury.
The palmitoyl acyltransferase inhibitor 2-bromopalmitate (2-BP, 2 mg kg−1) was intravenously administrated 20 min prior to SIRS induction. (a,b) Transvascular flux of FITC-albumin from mesenteric microvessels observed via intravital microscopy 3 h postburn. (a) Representative images (Scale bars, 100 μm). (b) Quantification of transvascular flux (n=number of animals). For each group, values were normalized to its own t=0 min and presented as mean±s.e.m. *P<0.05 versus sham+vehicle, #P<0.05 versus SIRS+vehicle. (c,d) Plasma protein leakage in the lungs measured as tracer Evans Blue extravasation. (c) Representative images showing tracer leakage captured at 700 nm with infrared imaging scanner and pseudo-colored with a heat-map colour scheme. (d) Quantitative extravasation assay (n=6). Values were normalized to vehicle-treated group subjected to sham operation and presented as mean±s.e.m. **P<0.01 versus sham+vehicle, #P<0.05 versus SIRS+vehicle. (e) Microvascular leakage in the lungs measured as interstitial deposition of small molecule tracer sulfo-NHS-biotin. Upper panels: representative transverse lung slices (500 μm) probed for extravasated sulfo-NHS-biotin with IRDye 800-conjugated streptavidin (SA-IRD) and scanned with infrared imaging system (800 nm). Images were pseudo-colored with heat-map colour scheme (blue=low intensity, red=high intensity). Lower panels: representative confocal micrographs of lung sections probed for sulfo-NHS-biotin with Texas Red-streptavidin (SA-TR). Nuclei were stained with DAPI. Scale bars, 100 μm.
Figure 2
Figure 2. Inhibition of PATs reduces inflammatory mediator-induced endothelial cell–cell junction hyperpermeability.
(a) Time- and dose-dependent effects of 2-BP on thrombin (10 U ml−1)-induced reduction in transendothelial electric resistance (TER), an indicator of cell–cell adhesive barrier function. Values at each time point were normalized to their baseline at t=0. Solid line tracing represents the mean resistance, and shadow represents standard error. Embedded graph indicates peak TER values and presented as mean±s.e.m. (n=3 independent experiments). *P<0.05. (b) Representative confocal images of the adherens junction molecule VE-cadherin (green) and nuclei (blue) 30 min after thrombin with or without pretreatment of 2-BP. Arrows point to discontinued VE-cadherin strands or intercellular gaps. Scale bars, 10 μm. (c) Quantification analysis of VE-cadherin junction discontinuity from three independent experiments, presented as mean±s.e.m. *P<0.05. (d) Histamine-induced changes in hydraulic conductivity (Lp) in intact perfused venules without or with 2-BP (given 30 min prior to histamine and continuously perfused until the end of the observation). Lp indicates venular permeability to water which transports mainly via paracellular pathway. n=number of segment measurements; values in parentheses indicate number of independent experiments. *P<0.05 versus baseline, #P<0.05 versus histamine+vehicle. (e) Representative confocal images of VE-cadherin (green) and nuclei (blue) 10 min after histamine in endothelial cells pre-treated with 2-BP. Arrows point to discontinued VE-cadherin strands or intercellular gaps. Scale bars=10 μm. (f) Quantification analysis of VE-cadherin junction discontinuity from three independent experiments. Data are presented as mean±s.e.m. *P<0.05.
Figure 3
Figure 3. PAT inhibition suppresses leucocyte adhesion and ICAM-1 expression on endothelial surface during inflammatory stimulation.
(a,b) Leucocyte-endothelium interactions were assessed via intravital microscopic analysis of mesenteric microcirculation in rats subjected to SIRS elicited by bacterial lipopolysaccharide (LPS, 10 mg kg−1, IP, 4 h). A group of animals received intravenous 2-BP (2 mg kg−1) immediately after LPS injection. Leucocytes were labelled with acridine orange. (a) Representative images of leucocyte adhesion in postcapillary venules. Scale bars, 30 μm. (b) Quantification of leucocyte slow-rolling flux (number of cells per min), slow-rolling fraction (% of slow rolling cells to total rolling cells), rolling velocity and adhesion. Increased slow-rolling flux or decreased rolling velocity indicates the likelihood of firm adhesion. Data are presented as mean±s.e.m. from 4 rats with ≥10 venules per group. *P<0.05 versus sham+vehicle, #P<0.05 versus SIRS+vehicle. (c,d) The effects of 2-BP on human leucocyte adhesion to human umbilical vein endothelial cells with or without stimulation by IL-1β (100 ng ml−1, 4 h). 2-BP (10 μM) or vehicle was given simultaneously with IL-1β. (c) Representative images of adherent leucocytes (green) on endothelial cells (red) with nuclei stained with DAPI. Embedded images show green channel (leucocytes) alone. Scale bars, 25 μm. (d) Quantification of adherent leucocytes to IL-1β-stimulated ECs. Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus unstimulated+vehicle, #P<0.05 versus IL-1β+vehicle. (e,f) The effects of 2-BP on IL-1β-induced ICAM-1 expression on endothelial surface. Endothelial cells were treated with 100 ng ml−1 IL-1β for 4 h with or without 10 μM 2-BP (given simultaneously with IL-1β). (e) Representative confocal images. Scale bars, 50 μm. (f) Quantification of ICAM-1 surface expression. Results represent mean±s.e.m. from three independent experiments. *P<0.05 versus unstimulated+vehicle, #P<0.05 versus IL-1β+vehicle.
Figure 4
Figure 4. Inflammatory stimuli increase PLCβ1 palmitoylation and its signaling activity.
(a) Increased palmitoylation was detected in endothelial cells after stimulation with SIRS plasma or inflammatory stimuli. Representative images of palmitoylation in cells treated with vehicle, histamine (10 μM, 15 min), thrombin (10 U ml−1, 30 min) or SIRS plasma (20% dilution, 1 h) in the absence or presence of 2-BP (pretreatment at 10 μM). Palmitoylated proteins were metabolically labelled with a palmitic acid analogue ω-alkynyl palmitic acid (Alk-C16). Labelled proteins were visualized by further probing with fluorescent azide via Click chemistry reaction. Palmitoylated proteins are shown in green and nuclei blue. Scale bar, 30 μm. Bar graph shows quantification of total palmitoylation signal intensity. Results are from three independent experiments presented as mean±s.e.m. *P<0.05 versus control+vehicle, #P<0.05 versus stimulus+vehicle. (b,c) The levels of palmitoylated PLCβ1 in endothelial cells were determined using two different methods. (b) Acyl-biotin exchange. Whole cell lysates from thrombin (10 U ml−1) or vehicle treated ECs, with or without 2-BP, were subject to acyl-biotin exchange to isolate palmitoylated proteins in the presence of hydroxylamine (NH2OH). The samples were then used in Western blotting for PLCβ1. Band intensity was quantified and normalized to control. Results represent three independent experiments. *P<0.05 versus vehicle+control, #P<0.05 versus vehicle+thrombin. (c) Pull-down of palmitoylated proteins and immunoblotting for PLCβ1. ECs were incubated with Alk-C16 overnight and then treated with histamine (10 μM) or vehicle. Whole cell lysates were collected, and palmitoylated proteins were pulled down using Azide Agarose Resin via Click chemistry reaction. Palmitoylated protein samples were subject to Western blotting for PLCβ1. Band intensity was quantified and normalized to control group. Bar graph represents four independent experiments. *P<0.05 versus vehicle. (d) IP3 levels in ECs subjected to histamine stimulation (10 μM) with or without 2-BP (10 μM). Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus vehicle, #P<0.05 versus vehicle+histamine.
Figure 5
Figure 5. Endothelial-expressing Zdhhcs and importance of DHHC5/21 in endothelial dysfunction.
(a) Representative images from qualitative PCR analyses of 24 known murine Zdhhcs in primary MLMVECs that confirmed the expression of 11 Zdhhcs. cDNA template of each gene was used as a positive control. (b) Knockdown efficiency of the 11 Zdhhcs expressed in endothelial cells verified by RT-PCR, *P<0.05 versus control siRNA. (c,d) The effects of individual Zdhhc knockdown on thrombin-induced endothelial barrier dysfunction indicated by TER. (c) Representative TER tracings of Zdhhc5 and Zdhhc21 knockdown in response to thrombin (10 U ml−1). Solid line tracing represents the mean resistance, and shadow represents±s.e.m. n=number of measurement; value in parentheses indicates number of independent experiments. (d) Quantification of peak TER reduction upon thrombin treatment in each Zdhhc knockdown group. Values were normalized to TER with control siRNA. Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA. (e,f) The effects of individual Zdhhc knockdown on IL-1β-induced ICAM-1 expression on EC surface. MLMVECs in a 96-well plate were stimulated with IL-1β (100 ng ml−1, 4 h). ICAM-1 surface expression was determined by on-cell western assay using Odyssey CLx Infrared Imaging System. (e) Representative images. CellTag staining was used for cell number normalization. Arrow indicates decreased ICAM-1 expression. (f) Quantification of ICAM-1 expression in Zdhhc knockdown ECs upon IL-1β stimulation. Results represent mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA.
Figure 6
Figure 6. Zdhhc21dep/dep mice are resistant to SIRS-induced lung injury and mortality.
(a,b) Representative images of lung tissues obtained from mice 24 h after thermal injury-induced (a) or LPS-induced SIRS (b) (five mice in each group). Scale bar, 50 μm. (c) Lung histology injury scores in thermal injury-induced SIRS. Results represent mean±s.e.m. from five mice. **P<0.01. (d) Survival rates in mice subjected to thermal injury (n=15 mice). (e) Lung histology injury scores in LPS-induced SIRS. Results represent mean±s.e.m. from five mice. **P<0.01, ***P<0.001. (f) Survival rates in mice subjected to LPS challenge (n=10 mice).
Figure 7
Figure 7. DHHC21 deficiency attenuates leucocyte-endothelial adhesion during inflammation.
(a) Representative images of intravital microscopic analyses of microvessels in mouse ears during burn or LPS challenge. Scale bars, 50 μm. Solid arrowheads point to adherent leucocytes; open arrowheads point to slow-rolling leucocytes. (b) Quantification of leucocyte slow-rolling flux, slow-rolling fraction, rolling velocity and adhesion in Zdhhc21+/+and Zdhhc21dep/dep mice 4 h postburn or LPS. Results represent mean±s.e.m. from six mice. *P<0.05, **P<0.01, ***P<0.001. (c,d) Comparative analysis of the leucocyte adhesion in endothelial versus leucocytic Zdhhc21dep/dep using a chimeric approach. Leucocytes were isolated from either Zdhhc21+/+ or Zdhhc21dep/dep and then applied to endothelial cells from either Zdhhc21+/+ or Zdhhc21dep/dep and stimulated with IL-1β (100 ng ml−1, 4 h). (c) Representative confocal images of adherent leucocytes. Embedded images show leucocyte channel alone. Nuclei were stained with DAPI. Scale bars, 100 μm. (d) Quantification of adherent leucocytes shows that endothelial cells from Zdhhc21dep/dep displayed marked resistance to leukocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Results present mean±s.e.m. from three independent experiments. ***P<0.001 versus unstimulated. #P<0.05 versus IL-1β+Zdhhc21+/+ EC+Zdhhc21+/+ leukocyte.
Figure 8
Figure 8. DHHC21 deficiency prevents inflammation-mediated vascular barrier dysfunction.
(a,b) Following LPS or thermal injury, Zdhhc21dep/dep mice displayed decreased plasma protein extravasation in the lungs. (a) Images of four replicates used to qualitatively demonstrate SIRS-induced Evans Blue extravasation in whole left lung lobes (signal intensity low=blue, high=red). (b) Quantitative results of Evans Blue extravasation assays. Values were normalized to Zdhhc21+/+mice receiving sham operation. Results represent mean±s.e.m. from four independent experiments. *P<0.05 versus Zdhhc21+/++sham, #P<0.05 versus Zdhhc21+/++SIRS. (c,d) Histamine-induced transvascular flux of FITC-albumin from mesenteric microvessels. (c) Representative images at 0, 5, 10 and 15 min after histamine application. Scale bars, 100 μm. Arrowheads indicate the observed microvessel. (d) Quantification of plasma transvascular flux. For each genotype, value was normalized to its own t=0 min. Results represent mean±s.e.m. from six mice. *P<0.05 versus Zdhhc21+/++histamine. (e) The effects of Zdhhc21 knockdown or loss-of-function on endothelial permeability to FITC-albumin with or without 2-BP (100 μM). Thrombin (10 U ml−1) was applied to MLMVEC monolayers and their permeability was calculated based on albumin transendothelial diffusion rate. Bar graph represents mean±s.e.m. at n≥3. *P<0.05 versus Zdhhc21+/+ EC+vehicle, #P<0.05 versus Zdhhc21+/+ EC+thrombin. (f) Dynamic changes of endothelial barrier resistance to thrombin (10 U ml−1) in MLMVECs isolated from Zdhhc21+/+ and Zdhhc21dep/dep mice. TER value of each time point is normalized to baseline. Solid line tracing represents the mean resistance, and shadow represents standard error. Embedded graph shows maximum TER changes. Bar graph represents mean±s.e.m. *P<0.05 versus Zdhhc21+/+ EC+vehicle , #P<0.05 versus Zdhhc21+/+ EC+thrombin. (g) Blunted TER response to thrombin (10 U ml−1) was partially rescued in cells overexpressing wild-type DHHC21. Zdhhc21 (DDK tagged) or empty vector was overexpressed in Zdhhc21dep/dep ECs. Results represent mean resistance±s.e.m. The efficiency of Zdhhc21 overexpression was verified by Western blotting using anti-DDK tag and anti-DHHC21 antibody (Embedded graph).
Figure 9
Figure 9. DHHC21 deficiency reduces inflammation-induced PLCβ1 palmitoylation and signaling activity.
(a) The level of palmitoylated PLCβ1 in Zdhhc21+/+ and Zdhhc21dep/dep ECs with or without thrombin (10 U ml−1) was determined using resin-assisted capture. Palmitoylated proteins in whole cell lysate were isolated by thiol-reactive sepharose resin in the presence of hydroxylamine (NH2OH) and then used for immunoblotting with anti-PLCβ1. Blot is representative of four independent experiments. (b) Membranous and cytosolic fractionation assay showing the subcellular distribution of PLCβ1 and its downstream signal PKC in Zdhhc21+/+ and Zdhhc21dep/dep ECs with or without thrombin (10 U ml−1). Images are representatives of three independent experiments. Gapdh and caveolin-1 were used as internal loading controls (Gapdh for whole cell and cytoplasm fraction, caveolin-1 for plasma membrane fraction). (c) IP3 levels indicative of PLCβ1 signaling activity in Zdhhc21+/+ and Zdhhc21dep/dep ECs subjected to thrombin (10 U ml−1) stimulation. Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus Zdhhc21+/+ EC+control, #P<0.05 versus Zdhhc21+/+ EC+thrombin. (d,e) Intracellular calcium spike indicative of PLCβ1 activation in ECs isolated from Zdhhc21+/+ or Zdhhc21dep/dep. Data represents three independent experiments. (d) Representative images of live EC monolayers labelled with the calcium indicator Fluo-4 before and after thrombin stimulation. (e) Representative tracings of intracellular calcium concentration.
Figure 10
Figure 10. DHHC21-mediated PLCβ1 palmitoylation contributes to barrier dysfunction in inflammation.
Molecular manipulations of DHHC21 or PLCβ1 via knockdown and site-specific mutagenesis indicate the involvement of DHHC21-PLCβ1 palmitoylation in endothelial barrier response. (a) Verification of PLCβ1 siRNA knockdown by Western blotting. β-actin serves as loading control. Results represent mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA. (b) Verification of wild-type PLCβ1 or C17S mutant PLCβ1 (V5 tagged) overexpression in Zdhhc21+/+ or Zdhhc21dep/depECs. PLCβ1 C17S mutant was created by site-specific mutagenesis at Cys17, the highly predicted palmitoylation site. Empty pLX304 vector serves as vector control. β-actin serves as loading control. (c) Dynamic recording of TER responses to thrombin (10 U ml−1) in PLCβ1 knockdown ECs. Solid line tracing represents the mean resistance, and shadow represents±s.e.m. Embedded bar graph shows peak TER changes and presented as mean±s.e.m. from three independent experiments. ***P<0.001. (d) The effect of overexpressing wild-type PLCβ1 on TER responses to thrombin (10 U ml−1) in Zdhhc21+/+ ECs. Solid line tracing represents the mean resistance, and shadow represents±s.e.m. Embedded bar graph shows peak TER value changes. Bar graph represents mean±s.e.m. from three independent experiments. ***P<0.001. (e) The effect of overexpressing C17S mutant PLCβ1 (palmitoylation impaired) on TER responses to thrombin (10 U ml−1) in Zdhhc21+/+ ECs. Solid line tracing represents the mean resistance, and shadow represents s.e.m. Embedded bar graph shows peak TER value changes. Bar graph represents mean±s.e.m. from three independent experiments. n.s.=not significant. (f) The effect of overexpressing wild-type PLCβ1 on TER responses to thrombin (10 U ml−1) in Zdhhc21dep/dep ECs. Solid line tracing represents the mean resistance, and shadow represents s.e.m. Embedded bar graph shows peak TER changes. Bar graph represents mean±s.e.m. from three independent experiments. n.s.=not significant.
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