Fig 2: SREBP2 represses KISS-1 expression by binding to the KISS-1 promoter.A Half-lives of KISS-1 protein expression in CTRi and SCAPi VSMCs after treatment with 50 μg/ml cycloheximide (CHX) and harvesting at different time points as indicated. B Relative KISS-1 mRNA expression levels in CTRi and SCAPi VSMCs as measured by qRT‒PCR. C Immunofluorescence staining of SREBP2 in CTRi and SCAPi VSMCs. Scale bar = 10 μm. D Immunoblot analysis and quantification of SREBP2 protein expression in the nucleus and cytoplasm of CTRi and SCAPi VSMCs. E A putative SREBP2 binding site was predicted in the promoter region of KISS-1 (422 to 431) by the JASPAR database. F Agarose electrophoresis following ChIP to determine the SREBP2 binding site in the KISS-1 promoter. G The relative luciferase activities of KISS-1 were detected after cotransfection with the pGL3-basic luciferase reporter plasmid vector and different contents of the constructed SREBP2 plasmid for 48 h. H Relative mRNA expression levels of SREBP2, KISS-1, MMP9 and VEGF in CTRi and SREBPi VSMCs as measured by qRT‒PCR. I Secretion concentrations of KISS-1 in culture supernatant as measured by ELISA in VSMCs treated with CTRi and SREBP2i. J Representative images of wound-healing migration assays in VSMCs treated with CTRi and SREBP2i for 0 h and 48 h. K Schematic representation of how SCAP in VSMCs affects embryonic angiogenesis through negative regulation of KISS-1. All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.

Fig 3: Ablation of SCAP in VSMCs inhibits endothelial cell function through intercellular communication.A Heatmap of the related molecules in the VEGF signaling pathway from the placentas of SCAP+/+ and SCAPfl/fl mice. B Secretion concentrations of KISS-1 in culture supernatant as measured by ELISA in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. C Concentrations of VEGF in the culture supernatant as measured by ELISA in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. D Immunoblot analysis of VEGF and ICAM protein expression in HUVECs cocultured with VSMCs of different groups. E Statistical analysis of the relative protein expression levels in (C). F Representative images of the EdU assay of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. G Statistical analysis of the relative cell proliferation rate in (F). H Representative images of the Transwell migration assay of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. I Statistical analysis of the relative cell numbers per field (%) in (C). J Representative images of tube formation assays of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. K Quantitative analysis of number of nodes in (J). L Quantitative analysis of number of Junctions in (J). All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.

Fig 4: Knockdown of KISS-1 expression rescues SCAP-ablated VSMCs from loss of proliferation and migration abilities in vitro.A Representative images of wound-healing migration assays in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i for 0 h and 48 h. B Statistical analysis of the relative VSMC migration rates in (A). C Representative images of the Transwell migration assay in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. D Statistical analysis of the relative cell numbers per field (%) in (C). E Representative images of the EdU assay in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. F Statistical analysis of the relative cell proliferation rate in (E). G Immunoblot analysis and quantification of SCAP, N-SREBP2, KISS-1, GPR54, P-MAPK, MAPK, P-ERK, ERK, P-JNK and JNK protein expression in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.

Fig 5: Kp-10/GPR54 upregulated the expression of Dusp18 and recruited both active Src and its phosphatase Dusp18.a, b IB analysis of lysates from RAW264.7 with indicated dose of Kp-10 treatment for 1 h (a) and quantification results (b). c Quantitative PCR of Dusp18 mRNA in RAW264.7 cells treated with inhibitors of PLC (U73122, 10 µM), PKC (Staurosporine, 0.25 µM), ERK (LY3214996, 20 µM), and P38 (SB203580, 10 µM) for 1 h, and then incubated with indicated dose of Kp-10 for 30 min. d–i Anti-Flag IP of lysates derived from 293 T cells transfected with indicated constructs and treated with PLC inhibitor (U73122, 10 µM), PKC inhibitor (Staurosporine, 0.25 µM), ERK inhibitor (LY3214996, 10 µM), Ca2+/CaMKII inhibitor (KN-93, 10 µM), and Src inhibitor (Saracatinib, 10 µM) for 1 h and then stimulated with 10 nM Kp-10 for 20 min. Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and Src-HA constructs (d) and quantification results (e). Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and DUSP18-HA constructs (f) and quantification results (g). Anti-Flag IP of lysates derived from 293 T cells transfected with Src-HA, and DUSP18-flag constructs (h) and quantification results (i). j, k IB analysis of lysates derived from WT BMMs and Gq/11 KO BMMs with or without Kp-10 treatment for 1 h (j) and quantification of results (k). l Working model of Kp-10 /Gpr54 mediated Src dephosphorylation. Low dose of Kp-10 (0.1, 1 nM) induced the expression of Dusp18 obviously but not by 10 nM Kp-10, which is dependent on Gq/11 signaling. However, both active Src and DUSP18 were recruited by GPR54 through the PR motif in GPR54 CT along with the increase of Kp-10 dose (1, 10 nM). Therefore, phosphorylation of Src was dose dependently suppressed when GPR54 was activated by Kp-10. Data represent means ± SEM. P values were determined by one-way ANOVA analysis (b, c, e, g, i, k). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.