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. 2023 Jun 27;21(1):155.
doi: 10.1186/s12964-023-01061-z.

Mutated CYP17A1 promotes atherosclerosis and early-onset coronary artery disease

Ting-Ting Wu #  1 , Ying-Ying Zheng #  1 , Xiang Ma  1 , Wen-Juan Xiu  1 , Hai-Tao Yang  1 , Xian-Geng Hou  1 , Yi Yang  1 , You Chen  1 , Yi-Tong Ma  2 , Xiang Xie  3
Affiliations

Mutated CYP17A1 promotes atherosclerosis and early-onset coronary artery disease

Ting-Ting Wu et al. Cell Commun Signal. .

Abstract

Background: Coronary artery disease (CAD) is a multi-factor complex trait and is heritable, especially in early-onset families. However, the genetic factors affecting the susceptibility of early-onset CAD are not fully characterized.

Methods: In the present study, we identified a rare nonsense variant in the CYP17A1 gene from a Chinese Han family with CAD. To validate the effect of this variation on atherosclerosis and early-onset coronary artery disease, we conducted studies on population, cells, and mice.

Results: The mutation precisely congregated with the clinical syndrome in all the affected family members and was absent in unaffected family members and unrelated controls. Similar to the human phenotype, the CYP17A1-deficient mice present the phenotype of metabolic syndrome with hypertension, increased serum glucose concentration, and presentation of central obesity and fatty liver. Furthermore, CYP17A1 knockout mice or CYP17A1 + ApoE double knockout mice developed more atherosclerotic lesions than wild type (WT) with high fat diary. In cell models, CYP17A1 was found to be involved in glucose metabolism by increasing glucose intake and utilization, through activating IGF1/mTOR/HIF1-α signaling way, which was consistent in CYP17A1 knockout mice with impaired glucose tolerance and insulin resistance.

Conclusions: Through our study of cells, mice and humans, we identified CYP17A1 as a key protein participating in the pathophysiology of the atherosclerotic process and the possible mechanism of CYP17A1 C987X mutation induced atherosclerosis and early-onset CAD involving glucose homeostasis regulation was revealed. Video Abstract.

Keywords: Atherosclerosis; CYP17A1; Early-onset coronary artery disease; Pathophysiology.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of the C987X mutation (Tyr-to-stop-gain substitution at the amino acid site 329, a new truncation mutant, CYP17A1ΔECD: missing the N-terminal ectodomain) in the CYP17A1 gene from a Chinese Han family with inherited coronary artery disease (CAD). A Pedigree of a Chinese Han family with CAD. Squares and circles indicate males and females, respectively. Roman numerals indicate generations. Arabic numerals indicate individual family members. CYP17A1 genotype (half filled for C987X carriers, open for wild-type individuals) are shown below each square or circle. A shaded circle or square indicates that the family member had CAD alive, a slash on circle or square indicates that the member was died, and with a shadow and a slash simultaneously indicate that the family member died of CHD. B Genomic structure of human CYP17A1 gene. The del of cytosine in exon 6 causes a stop gain occurrence (indicated by arrow). DNA sequencing data of an unaffected man (III: 3) and an affected man (III: 4) with the heterozygous mutation in CYP17A1. C Glucose levels of the members of Family 1 (A). Data are expressed as mean ± SD. Statistical analyses, unpaired t test. *P < 0.05. D The protein levels of CYP17A1 and truncation mutant. Plasmids encoding Myc-tagged CYP17A1(WT) or CYP17A1(C987X) were transfected into HEK293T cells, and cells were harvested for Immunoblots analysis 48 hours later. β-acting was used as a loading control. E Relative mRNA levels of CYP17A1(WT) or CYP17A1(C987X) transfected at indicated time. F, Densitometric analysis of CYP17A1(WT) or CYP17A1(C987X) proteins shown in (D). Data are presented as mean±SD. Student’s t test; ns., no significant. ****P < 0.0001. n=3 biological replicates
Fig. 2
Fig. 2
The C987X residue is critical for CYP17A1 stability. A HEK 293T cells were transfected with plasmids encoding CYP17A1(WT) or CYP17A1(C987X). After 48 hours, cells were treated with 100 μmol/L cycloheximide (CHX) for the indicated periods. B Densitometric analysis of CYP17A1(WT) and CYP17A1(C987X) proteins shown in (A). The densitometry of CYP17A1(WT) protein at 0 hour is defined as 1. C HEK293T cells were transfected with indicated plasmids and treated with 10 μm MG132 for 30 minutes. Cells were harvested, CYP17A1(WT) or CYP17A1(C987X) was immunoprecipitated by the anti-Myc coupled agarose and probed for the anti-HA antibody. Results shown are representative of two independent experiments. D Alignment of sequences flanking the C987X residue of CYP17A1 in various species. E Subcellular localization of CYP17A1(WT) or CYP17A1(C987X). HEK 293T cells were transfected with a plasmid encoding Myc-FLAG and stained with anti-FLAG and anti-KDEL antibodies. Scale bar =10 μm. F HEK293T cells were transfected with a plasmid encoding CYP17A1-FLAG and stained with anti-Myc tag and anti-COX IV antibodies, followed by microscopic analysis of CYP17A1 expression using a confocal microscope
Fig. 3
Fig. 3
The expression profiles of key enzymes and regulators of glycometabolism in response to CYP17A1 over-expression. HEK293T cell or Huh 7 cells were transiently transfected with plasmids encoding CYP17A1(WT) or CYP17A1(C987X). After 48h following transfection, cell lysates were prepared and subjected to western blot analysis. β-actin expression was used as a loading control. A The effect of CYP17A1 and C987X mutation on key enzymes of glucose transport and aerobic oxidation in HEK293T or Huh7 cells. B The effect of CYP17A1 on regulators of glycolysis. C Schematic model of the CYP17A1-mediated glycometabolism
Fig. 4
Fig. 4
Glucose and insulin homeostasis in CYP17A1-/- mice. A postprandial blood glucose (PBG) levels of wild-type (n=10) and CYP17A1-/- mice (n=22). B fasting blood glucose (FBG) levels of wild-type (n=35) and CYP17A1-/- mice (n=75). Data are expressed as median with standard deviation. Nonparametric test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; C FBG levels of CYP17A1-/- mice at different months. One-way ANOVA. ns. indicates no significance. Glucose tolerance tests were performed in 6-mo-old (D), 12-mo-old (G) wild-type and CYP17A1-/- mice. Bar graphs denote calculated area under the curve for plasma glucose per hour of glucose administration,6-mo-old (E), 12-mo-old (H). Insulin tolerance tests were performed in 6-mo-old (F), 12-mo-old (I) wild-type and CYP17A1-/- mice. Insulin tolerance test data are presented as % of time at 0 plasma glucose levels
Fig. 5
Fig. 5
CYP17A1 whole-body knockout mice develop more aortic atherosclerotic plaques. A Representative images of Oil red stained atherosclerotic aortic plaques obtained from high fat diary (HFD)mice of different age. Genotype of knockout mice (left to right) ApoE-/-(ApoE konckout), CYP17A1-/-ApoE-/- (ApoE and CYP17A1 double knockout), CYP17A1-/- (CYP17A1 whole-body knockout) mice. Corresponding HFD time :6month (row 1),9month (row 2),12month (row 3). B Relative atherosclerotic aortic plaques area of ApoE-/-(n=6) and CYP17A1-/-ApoE-/- (n=6) mice. The plaques area of ApoE-/- is defined as 100%. Data are presented as mean±SD, unpaired t test, **P<0.01. C Atherosclerotic lesion histology in CYP17A1 knockout mice. Representative photomicrographs of Oil red O staining of cross-sections of aortic root in high fat diary WT, CYP17A1-/-, ApoE-/-and CYP17A1-/-ApoE-/- mice (from left to right) at 6 mo of age (A-D) and 9 mo of age (E-H). D Quantitative analysis of atherosclerotic lesion size in aortic root of CYP17-/- (n=6) mice and wide type(n=6). All histology images had the original magnification of 200μm. Data are presented as mean±SD, unpaired t test, ####P<0.0001. E Representative photomicrographs of Oil red O staining of cross-sections of aortic root vessel at 6-mo-old WT and CYP17A1-/- mice in chow or HFD diary mice. Photomicrographs of 6 and 9 mo-old ApoE-/-and CYP17A1-/-ApoE-/- mice with HFD. All histology images had the original magnification of 100μm. F Schematic model, the possible mechanisms of CYP17A1 C987X leading to CAD
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