Research ArticleCELL BIOLOGY

Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

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Science Advances  03 Jul 2020:
Vol. 6, no. 27, eaaz8534
DOI: 10.1126/sciadv.aaz8534
  • Fig. 1 Hypoxia-attenuated EC proliferation, CBS expression, and H2S production.

    (A) HUVECs and HAOECs were exposed to normoxia (Con) or hypoxia (1% O2) or treated with 1 mM DMOG for 48 hours. Using the CyQUANT Assay, EC proliferation was evaluated at different time intervals. Normoxic untreated control cells were set to 100%. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing with respective normoxic controls by a one-way analysis of variance (ANOVA). (B) HUVEC and HAOEC were treated with cobalt chloride (CoCl2) at the indicated concentrations for 48 hours and cellular proliferation was assessed using the 5-bromo-2′-deoxyuridine (BrdU) assay. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing with respective nontreated controls by a one-way ANOVA. (C) HUVEC and HAOEC were exposed to either normoxia or 1% O2 for 48 hours. Expression of HIF-1α, PHD2, CBS, CSE, and 3MST was determined by immunoblot. β-Actin was used as a loading control. (D) HUVEC and HAOEC were treated with increasing concentrations of CoCl2 for 48 hours, and the expression of HIF-1α, PHD2, CBS, CSE, 3MST, and β-actin was determined by immunoblotting. (E) HUVEC and HAOEC were exposed to either normoxia (Con) or hypoxia (1% O2) for 48 hours, and H2S levels were determined by methylene blue assay. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing with the respective controls by Student’s t test.

  • Fig. 2 Exogenous hydrogen sulfide supplementation either by chemical donors or CBS overexpression rescued hypoxic stress.

    (A) HUVEC and HAOEC were exposed to normoxia or hypoxia (1% O2) for 48 hours; for the final 2 hours, ECs were exposed to various concentrations (0.1, 0.5, and 1 mM) of an H2S donor, NaHS. Expression of HIF-1α and β-actin was determined by immunoblotting. (B) HUVEC and HAOEC were exposed to normoxia or 1% O2 in the presence or absence of 0.5 mM of another small-molecule slow-release H2S generator, GYY4137 (GYY) for 48 hours, and immunoblotting were performed with the respective indicated antibodies. (C) HUVEC and HAOEC were exposed to normoxia or 1% O2 in the presence or absence of 0.5 mM GYY for 48 hours, and cellular proliferation was assessed using the BrdU assay. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing the specified groups. (D) HUVEC was exposed to normoxia or 1% O2 in the presence or absence of 0.5 mM GYY for 48 hours, and H2S levels were determined by the methylene blue assay. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing with respective untreated normoxic controls by one-way ANOVA. (E) HUVECs were first transfected with either empty vector or the FLAG-CBS construct and then further exposed to normoxia or 1% O2 for 48 hours before determining the expression of HIF-1α, CBS, PHD2, and β-actin by immunoblotting. (F) HUVEC was transfected with either scrambled or VHL siRNA (si-VHL) and was supplemented with or without 0.5 mM GYY for 48 hours, and immunoblotting was performed with the indicated antibodies. SE and LE represent as shorter exposure and longer exposure, respectively, of VHL blots during development. (G) Both si-Con– and si-VHL–transfected HUVEC were supplemented with 0.5 mM GYY for 48 hours, and proliferation was evaluated using the CyQUANT Assay. Data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when compared with the si-Control by a one-way ANOVA. ns, not significant.

  • Fig. 3 Inhibition of CBS stabilized HIF-1α in normoxia by impairing PHD2 activity.

    (A) HUVEC and HAOEC were transfected with either scrambled (si-Con) or si-CBS for 48 hours. Similarly, HUVEC and HAOEC were incubated with or without AOAA, a chemical inhibitor of CBS, for 48 hours. NT, vehicle treated control. Expression of HIF-1α, CBS, and PHD2 was determined by immunoblotting. β-Actin was used as a loading control. (B) Both si-Control and si-CBS–transfected (for 48 hours) HUVEC and HAOEC were supplemented with 0.5 mM GYY, and immunoblotting was performed with the indicated antibodies. (C) Tube formation assay was performed in si-Control and si-CBS–transfected (48 hours) HUVECs supplemented with or without 0.5 mM GYY on growth-factor reduced Matrigel (2 mg/ml). The images were acquired 4 hours after plating HUVECs on Matrigel in complete endothelial basal medium. Each scale bar represents 200 micron. (D) Both HUVEC and HAOEC cells were transfected with si-Control or si-CBS and incubated with or without 0.5 mM GYY for 48 hours. Cell lysates from each treatment condition were incubated with recombinant biotinylated HIF-1α peptide, and the peptide was then captured using streptavidin-conjugated magnetic beads. Next, these peptide-coated beads were incubated with in vitro translated [35S] pVHL and finally quantified by SDS-PAGE and autoradiography as a measurement PHD2 activity. (E) In vitro prolyl hydroxylation of the purified HIF-1α–ODD protein at 0 and 15 min using lysates from HUVEC cells harboring either si-Control or si-CBS and incubated for 48 hours with or without GYY. Hydroxylation of HIF-1αODD was determined using the hydroxyprolyl-specific antibody at proline-564 (HIF-1α–OHODD), and total HIF-1α was detected (HIF-1αODD). DMOG-treated lysates were used as a negative control, and α-tubulin was used as a loading control. (F) HUVEC cells were treated with either si-Control or si-CBS for 48 hours and both hydroxylated and total HIF-1α were detected from 0 to 120 min after proteasomal blockade with 1 μM MG132. Pico refers to regular chemiluminescence reagents that work in the picomolar range, we detected weak hydroxylated HIF-1α bands, and however, with the highest sensitive femtomolar reagents (Femto) and prolonged exposure more prominent bands could be detected. (G) mRNA expression levels of various HIF-1α target genes in si-Con and si-CBS-transfected (48 hours) HUVECs were determined by quantitative polymerase chain reaction analysis. 18S ribosomal RNA (rRNA) was used as an internal control. si-Con mRNA levels were set to 1 and data represent mean ± SD of three independent experiments performed in triplicate. *P < .05 when comparisons were made by Student’s t test.

  • Fig. 4 Inhibition of CBS-stabilized HIF-1α in zebrafish embryos caused abnormal development of ISVs.

    (A) Zebrafish embryos were injected with either control MO (ConMO) or cbsb MO (cbsb MO), and embryos assessed at 24 or 48 hpf or zebrafish embryos were injected with control and cbsb CRISPR and assessed at 48 hpf. The protein expression of HIF-1α and CBS in the embryo lysates was evaluated by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) ConMO or cbsMO-injected zebrafish embryos were treated with or without 0.5 mM GYY4137 (GYY) at 6 hpf, and at 48 hpf, immunoblotting was performed with the indicated antibodies. (C) Control and cbsb CRISPR fish embryos were treated with or without 0.5 mM GYY4137 (GYY) at 6 hpf, and at 48 hpf, immunoblotting was performed with the indicated antibodies. (D to F) Bright-field whole mount images (D to F) and fluorescent images of the vasculature (D′ to F′) are shown at 52 hpf for embryos from either Tg[flk:EGFP (enhanced green fluorescent protein); CASPER] line (control) or the Tg(flk:EGFP; cbsb CRISPR, CASPER) line (cbsb CRISPR) were treated or not with GYY4137 (0.5 mM) or dimethyl sulfoxide at 6 hpf and were imaged. The box in (D″) indicates the region of enhancement in images (D″) to (F″). The bent axis is marked by the yellow arrow in (E), and disrupted ISVs are marked by the white asterisk in (E″). Each scale bar represents 0.25mm. (G and H) Quantification for axis and ISVs, respectively. Data are the means ± SD of three independent experiments. *P < 0.05 when comparing normal axis/normal ISVs of cbsb CRISPR with all other groups and #P < 0.05 when comparing abnormal axis/abnormal ISVs of cbsb CRISPR versus other groups. The embryo orientation in all the figure panels, left is anterior (A) and right is posterior (P), while top is dorsal (D) and bottom is ventral (V). n = 99 embryos for the control group, n = 52 embryos for the cbsb CRISPR group, and n = 52 embryos for the cbsb CRISPR + GYY group, which are total number of embryos from three independent experiments.

  • Fig. 5 PHD2 is persulfidated by CBS and persulfidation of PHD2 at zinc finger domain maintains its activation.

    (A) Modified biotin switch assay for detecting persulfidation of PHD2 in untreated and NaHS-treated (1 mM) HUVEC and HAOECs. Dithiothreitol (DTT) treatment (1 mM) for 30 min reversed PHD2 persulfidation. (B) Modified biotin switch assay to determine the persulfidation status of PHD2 in si-Control and si-CBS–treated HUVEC and HAOECs after 48 hours transfections. (C) HUVEC and HAOEC were transfected with either scrambled (si-Control) or two PHD2 siRNA (si-PHD2_08 and si-PHD2_09) for 48 hours. Expression of PHD2 was determined by immunoblotting. β-Actin was used as a loading control. (D) HUVECs were first transfected with either si-Control or si-PHD2 for 24 hours and further transfected with HA–empty vector (EV) or cDNAs encoding HA-PHD2 [wild type (WT), C21S (M1), C33S (M2), C127S (M3), C201S (M4), or C208S (M5)] for another 36 hours, and mRNA expression levels of PHD2 gene was determined. 18S rRNA was used as an internal control. si-Control–pretreated and EV-transfected mRNA levels were set to 1, and data are the means ± SD of three independent experiments performed in triplicate. *P < 0.05 when comparing between EV-treated groups in both si-Control or si-PHD2–pretreated group. (E) HUVECs were first transfected with si-PHD2 for 24 hours and further transfected with HA–empty vector (EV) or cDNAs encoding HA-PHD2 [WT, C21S (M1), C33S (M2), C127S (M3), C201S (M4), or C208S (M5)] for another 36 hours, and lysates from each treatment conditions were incubated with recombinant biotinylated HIF-1α peptide and further captured using streptavidin-conjugated magnetic beads. Next, these peptide-coated beads were incubated with in vitro–translated [35S] pVHL and finally quantified by SDS-PAGE and autoradiography as a measurement PHD2 activity. (F) HUVEC cells were transfected with either si-Control, si-PHD2, or si-PHD2 along with reexpression of WT or mutant PHD2 constructs. Cellular lysates served as a source of PHD2 and were incubated with GST or GST–HIF-1α prebound to GSH-agarose for 2 hours at 4°C. Respective agarose pull-down were then subjected to immunoblotting for PHD2 and HIF-1α, while cellular lysates served as input control for PHD2 and β-actin.

  • Fig. 6 Schema of cystathione β-synthase–mediated activation of HIF and PHD2 through persulfidation.

    PHD2 is targeted by H2S under normoxia to maintain its prolyl hydroxylation activity to degrade HIF-1α by VHL-mediated ubiquitin-proteasome pathway. Inhibition of CBS depletes endogenous H2S level and stabilizes HIF-1α by inhibiting PHD2 activity in normoxia. Mechanistically, PHD2 is persulfidated by CBS and persulfidation of PHD2 at zinc finger domain augments activation in normoxia.

Supplementary Materials

  • Supplementary Materials

    Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

    Anindya Dey, Shubhangi Prabhudesai, Yushan Zhang, Geeta Rao, Karthikeyan Thirugnanam, Md. Nazir Hossen, Shailendra Kumar Dhar Dwivedi, Ramani Ramchandran, Priyabrata Mukherjee, Resham Bhattacharya

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