PCSK6-mediated corin activation is essential for normal blood pressure

Journal name:
Nature Medicine
Volume:
21,
Pages:
1048–1053
Year published:
DOI:
doi:10.1038/nm.3920
Received
Accepted
Published online

Hypertension is the most common cardiovascular disease, afflicting >30% of adults1. The cause of hypertension in most individuals remains unknown2, 3, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure4. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia5, 6, 7, 8, 9. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

At a glance

Figures

  1. Corin activation cleavage.
    Figure 1: Corin activation cleavage.

    (a) Corin domains and variants. WT corin and the R801A and S985A variants are diagrammed. An arrow indicates the corin activation site. Filled and open arrowheads indicate ADAM-mediated shedding and corin autocleavage sites, respectively. A disulfide bond (S-S) connects the propeptide and the protease domain. TM, transmembrane; Fz, frizzled; LDLR, LDL receptor; SR, scavenger receptor. His (H), Asp (D) and Ser (S) indicate catalytic residues. (b) Western blot analysis to assess corin activation in HEK293 cells transiently transfected with the indicated plasmids under reducing and non-reducing conditions. The zymogen form of corin (Corin) and the cleaved and activated form of corin (Corin-p, arrow) were detected using an antibody against a V5 tag in plasmid-encoded corin. (c) Western blot analysis of corin activation cleavage in HEK293 cells stably transfected with WT corin and cultured with the protease inhibitors benzamidine (Benz), GM6001, dec-RVKR-cmk (Dec-RVKR) or ALLM. The arrowhead indicates the corin zymogen band on the cell surface (see Supplementary Fig. 3). (d) Western blot analysis of corin activation in HEK293 cells transfected with plasmids encoding PCSK1–PCSK9 or an empty vector (v). (e–j) Western blot analysis of corin activation (Corin-p versus Corin) in HEK293 (eg) and HL-1 (hj) cells transfected with siRNAs targeting the human PCSK6 (siPCSK6) and mouse Pcsk6 (siPcsk6) genes, respectively. Endogenous PCSK6 levels, normalized to GAPDH levels, were measured. *P < 0.05; **P < 0.01 versus control siRNA (Ctr) by one-way ANOVA (ANOVA) and Tukey's post hoc test. Values are mean ± s.d. (n = 4 per group). In e, non-adjacent lanes from the same western blot were used, as indicated.

  2. Cellular mechanism of PCSK6-mediated corin activation.
    Figure 2: Cellular mechanism of PCSK6-mediated corin activation.

    Western blot analysis of corin activation in cell lysates (left) and surface-labeled proteins (right) in HEK293 cells treated with BFA (a,b) or monensin (c,d). Arrowheads indicate corin zymogen bands on the cell surface. Corin-p indicates the cleaved protease domain fragment. (b,d) Percentages of corin activation were calculated on the basis of densitometry from western blots of cell lysates. Values are mean ± s.d. (n = 3 per group). **P < 0.01 versus control by one-way ANOVA and Tukey's post hoc test. (e) Western blot analysis of PCSK6 in the culture medium and lysate from monensin-treated HEK293 cells. (f) A proposed model, in which PCSK6 and corin traffic separately to the cell surface, where PCSK6 activates corin. Monensin blocks PCSK6 secretion via secretory vesicles. (g,h) Western blot analysis of corin activation (bottom) in transfected HEK293 cells treated with increasing amounts of PCSK6-containing conditioned medium (PCSK6 CM, top). *P < 0.05; **P < 0.01 versus control (n = 3 per group). (i) Western analysis of corin activation with PCSK6 CM with or without immunodepletion using a PCSK6-specific antibody (Ab) or control IgG. Corin-transfected HEK293 cells without PCSK6 CM treatment (cells only) or treated with CM from untransfected HEK293 cells (control CM) were used as controls. **P < 0.01 versus control CM (n = 3 per group). (j) Western blot analysis of corin activation by increasing amounts of purified PCSK6. Activation was tested for WT corin (left) and the cleavage site variant R801A (right). Percentages of corin activation were calculated on the basis of densitometry from western blots of WT corin. **P < 0.01 versus control in corin WT (left) (n = 3 per group). In g and j (left), non-adjacent lanes from the same western blot were used, as indicated. Values are mean ± s.d.

  3. Analyses in Pcsk6-KO mice.
    Figure 3: Analyses in Pcsk6-KO mice.

    (a) Western blot analysis of corin and PCSK6 in WT, Pcsk6- and Corin-KO mouse hearts. Untransfected (293) and corin-transfected (293/corin) HEK293 cells were used as controls. An arrowhead indicates the corin zymogen band. The anti-corin antibody used in this experiment does not recognize the cleaved protease domain fragment. (b) Levels of corin protein in mouse hearts, normalized to GAPDH levels, were calculated. **P < 0.01 versus WT (n = 3 per group). (c,d) Pro-ANP processing activity of cell membranes from mouse hearts, as indicated by pro-ANP conversion to ANP in western blots. HEK293 cell samples were used as controls. Percentages of pro-ANP conversion were calculated on the basis of densitometry from western blots. *P < 0.05; **P < 0.01 versus Pcsk6-KO and Corin-KO mice (n = 3 per group). (e,f) Western blot analysis of pro-ANP in mouse hearts. Levels of pro-ANP in mouse hearts, normalized to GAPDH levels, were calculated. **P < 0.01 versus WT (n = 3 per group). (g) ELISA analysis of pro-ANP levels in mouse hearts. **P < 0.01 versus WT (n = 5 per group). (h) Plasma natriuretic peptide (NP) activity. **P < 0.01 versus WT (n = 5 per group). In d,fh, the results for Pcsk6- and Corin-KO mice were not significantly different. (i) Systolic blood pressure in mice on normal-salt (0.3% NaCl) and high-salt (4% and 8% NaCl) diets at 1 and 3 weeks (wk). P < 0.05, P < 0.01 versus WT for groups on the same diet; **P < 0.01 versus mice of the same genotype on normal-salt diet by two-way ANOVA and Bonferroni post hoc test. The results for Pcsk6- and Corin-KO mice were not significantly different on either the normal-salt or high-salt diets. Values are mean ± s.d.

  4. PCSK6-mediated activation of human corin variants and analysis of the D282N PCSK6 variant.
    Figure 4: PCSK6-mediated activation of human corin variants and analysis of the D282N PCSK6 variant.

    (a) Schematic indicating the location of the amino acid substitutions in the corin variants examined. (b) Western blot analysis of corin activation in HEK293 cells stably overexpressing PCSK6 (HEK293-PCSK6). WT corin and the activation cleavage site mutant R801A were used as positive and negative controls, respectively. (c) Western blot analysis of corin activation in HEK293-PCSK6 cells expressing the indicated corin variants. (d) DNA sequencing traces detecting a G/A mutation in a hypertensive individual. This mutation causes an Asp282Asn (D282N) substitution in the PCSK6 catalytic domain. (e) The PCSK6 signal peptide (Sp) and the pro, catalytic, P and cysteine (Cys)-rich domains are indicated. (f) Western blot analysis of corin activation in HEK293 cells transfected with plasmids expressing WT PCSK6 and the D282N variant or the vector (Vec). Cropped sections of a western blot from the same experiment were used. (g) Percentages of corin activation (Corin-p versus Corin) were calculated on the basis of densitometry of western blots in f. *P < 0.05; **P < 0.01 versus vector (Vec) by one-way ANOVA and Tukey's post hoc test (n = 3 per group). (h) Western blot analysis of PCSK6 expression in the culture medium (CM) and lysate from HEK293 cells expressing WT PCSK6 or the D292N variant. (i,j) Western analysis of corin activation in HEK293 cells transfected with WT corin incubated with increasing amounts of conditioned medium (CM) containing either WT PCSK6 or the D282N variant. *P < 0.05; **P < 0.01 versus vector (Vec) (n = 3 per group). Values are mean ± s.d.

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Author information

  1. Present addresses: Department of Gynecology and Obstetrics, Jiangxi Provincial People's Hospital, Nanchang, China (S.C.); Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA (P.C.); Biochemistry and Molecular Biology Department, Shanghai Medical School, Fudan University, Shanghai, China (C.Z.).

    • Shenghan Chen,
    • Pengxiu Cao &
    • Chunyi Zhang

Affiliations

  1. Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

    • Shenghan Chen,
    • Pengxiu Cao,
    • Jianhao Peng,
    • Chunyi Zhang,
    • Hao Wang,
    • Elizabeth E Martelli,
    • Sathyamangla V Naga Prasad &
    • Qingyu Wu
  2. Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, China.

    • Ningzheng Dong,
    • Tiantian Zhou,
    • Yue Zhang,
    • Yiqing Zhou &
    • Qingyu Wu
  3. Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China.

    • Ningzheng Dong
  4. Department of Cardiology, the First Affiliated Hospital of Soochow University, Suzhou, China.

    • Junhua Yang
  5. Department of Internal Medicine (Rheumatology), Rush University Medical Center, Chicago, Illinois, USA.

    • Rachel E Miller &
    • Anne-Marie Malfait
  6. Department of Biochemistry, Rush University Medical Center, Chicago, Illinois, USA.

    • Rachel E Miller &
    • Anne-Marie Malfait

Contributions

S.C., P.C., N.D. and Q.W. designed the study. S.C., P.C., J.P., C.Z. and H.W. performed molecular biology, biochemistry, cell biology and mouse model studies. N.D., T.Z., J.Y. and Y. Zhang studied hypertensive patients, collected blood samples, sequenced PCSK6 exons, and made plasmids expressing corin variants. E.E.M. and S.V.N.P. did echocardiographic analysis in mice. R.E.M. and A.-M.M. provided Pcsk6-KO mice. S.C., Y. Zhou and Q.W. wrote the manuscript. All authors critically read and commented on the manuscript.

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The authors declare no competing financial interests.

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