Furthermore, NO and S-nitrosylation have two faces in cancer [
18]. Excessive S-nitrosylation induces tumor development, but S-nitrosylation could also facilitate tumor cell death. Similarly, although excessive S-nitrosylation inhibits VEGFD in LUAD, the function of normal S-nitrosylated VEGFD remain unknown. We treated the NCI-H1975 cells with L-NAME. The cell culture fluid tested by the ELISA kit showed that L-NAME promotes the secreted VEGFD (Fig.
7A). On the contrary, the cell culture fluid tested by the ELISA kit showed that GSNO represses the secreted VEGFD (Fig.
7B). Interestingly, NCI-H1975 cell were treated with GSNO at different doses, the cellular protein and secreted protein levels of VEGFD were inconsistent at the same time and dose (Fig. S
4A and B), it indicated that S-nitrosylation may affect secretion of VEGFD. VEGFD is a secreted protein that is cleaved by proteolytic enzymes to form the mature form, which is then secreted outside the cell. However, the previous data indicated that S-nitrosylation suppresses VEGFD at protein level. To avoid the influences of VEGFD transcription levels, cells transfected with VEGFD were treated with Actinomycin D (ACTD) to decrease RNA synthesis. The ELISA results of cell culture fluid indicated that S-nitrosylation could promote the secretion of VEGFD while denitrosylation disrupts the secretion (Fig.
7C and D). Meanwhile, immunofluorescence displayed that the VEGFD
C277S mutant was significantly increased in the cytoplasm (Fig. S
4C). Proteins in the culture medium were concentrated by TCA precipitation and Western blot results showed the reduction of secretion in the VEGFD
C277S mutant (Fig.
7E). We used an ELISA kit to detect secreted VEGFD, the ELISA results of cell culture fluid indicated that the VEGFD
C277S mutant decreased secretion (Fig.
7F). The secretion of VEGFD is significant to angiogenesis and lymphangiogenesis, PROX1, TIE1, and MMP7 are target genes of VEGFD in angiogenesis and lymphangiogenesis. The quantitative RT-PCR results showed that the VEGFD
C277S mutant deactivates the target genes (Fig.
7E). To verify the effect of the VEGFD
C277S mutant on its function, we conducted a tube formation assay by HUVEC cells in vitro. HUVEC cells were transfected with Vector, WT, and C277S mutant, respectively, and tube formation assays showed that the VEGFD
C277S mutant was dysfunctional (Fig. S
4D, E, and F). These results suggest that normal S-nitrosylation is essential for the secretion of VEGFD.
As the mechanism of S-nitrosylation affects secretion, there are multiple proteolytic enzymes, including CTSD, KLK3, Plasmin, Furin, PC5, and PC7 [
42‐
44]. The expression of these proteolytic enzymes is related to the maturation and secretion of VEGFD. To explore the functions of S-nitrosylation on proteolytic enzymes, we treated BASE-2B cells with L-NAME. The quantitative RT-PCR results show that proteolytic enzymes of VEGFD are activated (Fig. S
3A-F). On the contrary, BASE-2B cells were stimulated with GSNO, and the expression of these enzymes was significantly decreased (Fig. S
3G-L). To further validate our results, HEK-293T cells transfected with PAI/PC-5/PC-7 were treated with GSNO (10µM), and the ELISA assay showed that proteolytic-activated VEGFD is inhibited by GSNO (Fig. S
3M-O). These results indicated that NO affects the expression of VEGFD-related proteolytic enzymes. Proteolytic cleavages of VEGFD require two concerted proteolytic cleavages (Fig.
7H) The intracellular cleavage occurs between the central VEGF homology domain (VHD) and the C-terminal propeptide. protein convertases constitutively cleave VEGF-D before secretion. The extracellular cleavage occurs between the N-terminal propeptide and the VHD and can be mediated by different proteases [
45]. We speculate that the VEGFD
C277S mutant cannot be cleaved by proteolysis. There are loads of proteases in the proteolysis of VEGFD C-terminal propeptide [
46]. To further explore the mechanism of S-nitrosylation regulating secretion of VEGFD, we co-transfected HEK-293T cells with VEGFD/VEGFD
C277S and PAI/PC-5/PC-7, the ELISA results suggest that the VEGFD
C277S mutant couldn’t be influenced by PC-7 not Plasmin/PC-5 (Fig.
7I). Western blot results showed the same results (Fig.
7J), indicating that the VEGFD
C277S mutant disrupts PC-7-dependent proteolysis. Plasmin and PC-5 can hydrolyze carboxyl-terminal and N-terminal amino acids and of VEGFD. However, PC-7 is a proteolytic enzyme that only hydrolyzes carboxyl-terminal amino acids of VEGFD [
46]. These results suggest that S-nitrosylation of VEGFD is responsible for PC-7-dependent proteolysis.