Dataset Information

HUVEC endosome LCMS - ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes

ABSTRACT: Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that regulates pro-inflammatory non-canonical NF-B signaling in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-B-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, as well as allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-B and is a biomarker for complement-mediated endothelial signaling in human renal and synovial tissues. Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for complement-mediated pathologies.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Blood Vessel Endothelial Progenitor Cell, Cell Culture

DISEASE(S): Myocardial Ischemia

SUBMITTER: Caodi Fang

LAB HEAD: Dan Jane-wit

PROVIDER: PXD013381 | Pride | 2019-04-09

REPOSITORIES: Pride

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Publications

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

Fang Caodi C Manes Thomas D TD Liu Lufang L Liu Kevin K Qin Lingfeng L Li Guangxin G Tobiasova Zuzana Z Kirkiles-Smith Nancy C NC Patel Manal M Merola Jonathan J Fu Whitney W Liu Rebecca R Xie Catherine C Tietjen Gregory T GT Nigrovic Peter A PA Tellides George G Pober Jordan S JS Jane-Wit Dan D

Nature communications 20190521 1

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN ...[more]

PMID: 31113953

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Project description:Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells (EC). We recently showed that DNA methyltransferase (DNMT)- and histone deacetylase (HDAC) inhibitors directly repress EC growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDACs are involved in regulation of EC gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes downregulated in tumor-conditioned versus quiescent EC, and re-expressed by 5-aza-2â-deoxycytidine and trichostatin A. Among the 81 genes identified, 77% harboured a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant downregulation in tumor-conditioned EC and reactivation by treatment with a combination of 5-aza-2â-deoxycytidine and trichostatin A, as well as by both compounds separately. Silencing of these genes in tumor-conditioned EC correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, however did not involve DNA methylation of promoter CpG islands. For six genes, downregulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1 and quiescin Q6 are negative regulators of EC growth and angiogenesis. In summary, our data identify novel angiogenesis suppressing genes which become silenced in tumor-conditioned EC in association with promoter histone modifications and reactivated by DNMT- and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis. Experiment Overall Design: A commercial pool (a mixture of 32 donors) of HUVEC (Tebu-bio, Heerhugowaard, The Netherlands) was used for DNA microarray experiments. Total RNA was isolated using the RNeasy RNA isolation kit (Qiagen) according to the supplier's protocol. Possible genomic DNA contaminations were removed by on column DNase treatment with the RNase-free DNase set (Qiagen). The purified RNA was quantified using a Nanodrop spectrophotometer, and RNA quality was evaluated using the Agilent 2100 Bioanalyzer. cDNA synthesis was performed using the Agilent Fluorescent Direct Labelkit with direct incorporation of either cyanine 5 (Cy5) or Cy3 âdCTP nucleotides (Perkin Elmer) according to the manufacturerâs instructions. Labeled cDNA was purified using QIAquick PCR purification columns (Qiagen), followed by concentration by vacuum centrifugation. The Agilent human 1A cDNA microarray (Agilent Technologies, Amstelveen, The Netherlands) contained ~15000 cDNA probes. Labeled cDNA was resuspended in hybridisation buffer and hybridised to Agilent human 1A cDNA microarray for 17 h at 65Â°C, according to the Agilent protocols. All hybridisations were replicated with cy dyes switched. Two fluorescent microarray comparisons were performed: (1) A comparison of tumor-conditioned HUVEC and quiescent HUVEC and (2) a comparison of tumor-conditioned HUVEC treated with or without a combination of DAC and TSA. Experiment Overall Design: Microarray data processing and statistical analysis Experiment Overall Design: The image file was processed using Agilent's Feature Extraction software (version A.6.1.1, Agilent Technologies). This Feature Extraction program was used to identify pixels corresponding to fluorescent signal (as opposed to background) and to remove pixels with intensities that met the default criteria for outliers. The different normalisation routines applied (Local Background, Minimum signal (feature or background) & Average of all background areas) resulted in comparable results. For each identified area of signal and each of the two dyes, the basic measure of RNA abundance was taken to be the mean intensity over pixels in the identified signal area. The log ratio of the red to green intensities for each signal area was used for statistical analyses, with all subsequent analyses done using the R statistical software package (version 1.2). We selected fold change 1.5 as a threshold, since the 4 hybridisations increase the likelihood of statistical reliability.

Dataset Information

HUVEC endosome LCMS - ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes

Publications

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

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