Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability To Inhibit Cyst Enlargement Of ADPKD Cells Compared With Nonsecreted Form Ⅲ
Jun 25, 2024
3.4. Effects of mNGAL on Cyst Formation of 2L3 Cells in 3D Culture
Cell proliferation is abnormally increased in 2L3 mice and leads to cyst formation and enlargement [17]. Based on the previous results that mNGAL inhibited 2L3 cell proliferation in 2D culture (Figure 3), we speculated that mNGAL might further inhibit cyst formation in 2L3 cells. To investigate whether mNGAL could inhibit cyst formation in 2L3 cells, we established a 3D culture platform that provided an appropriate environment with Matrigel for cyst formation (Figure 6). Cells were seeded in 96-well plates (1000 cells/well) on top of preformed Matrigel overnight. After the cells were attached to the gel, more Matrigel was added to the cells to form a sandwich-like pattern (Figure 6A). Cells were maintained in 3D culture at 37 ◦C and 5% CO2 for at least 5 days to form cysts. Cysts in 3D culture grew larger with a prolonged culture time (Figure 6B). To confirm that the spherical constructs observed in 3D culture were cysts with a lumen, we fixed and stained them with DAPI and E-cadherin and actin antibodies to identify cell nuclei, cell-cell adh,esion, and apical membrane, respectively. Different optical z sections of a 2L3 cyst in 3D culture from the top (layer 1) to the middle (layer 5) were observed by confocal microscopy, and the lumen was confirmed within the spherical cyst (Figure 6C, Supplementary material
NEW HERBS FOR ADPKD(POLYCYSTIC KIDNEY DISEASE)
To investigate the effects of mNGAL on cyst formation of 2L3 cells in 3D culture, cells in 3D culture were treated with forskolin from Days 1 to 3 to induce cyst formation. The cells were then treated with or without mNGAL from Days 4 to 8, and cyst diameters were measured every day (Figure 6D). Cysts in 3D culture grew proportionally with the number of culture days, with diameters ranging from 40 to 120 µm (Figure 6E–G), while mNGAL significantly suppressed the cyst diameters (Figure 6F) and area (Figure 6G) until Day 8. However, there was no significant difference in the increase in cyst number between the control and mNGAL-treated groups from Days 4 to 8 (Figure 6H). We further analyzed the number of large cysts (diameter > 100 µm) between the two groups and found that mNGAL significantly reduced the percentage of large cysts compared with the control group on Day 8 (Figure 6I).
3.5. Effects of Transfection with pN + LS and pN − LS on NGAL Expression and Cell Proliferation in 2L3 Cells
To study whether the need for a higher concentration of mNGAL (2 mg/mL) in 3D culture to inhibit cyst growth of 2L3 cells (Figure 6) was due to the lack of posttranslational modification of mNGAL, we designed a construct for overexpression of mNGAL in 2L3 cells. In addition, it has been demonstrated that NGAL can interact with NGAL-R and further induce apoptosis [29]. Therefore, to understand whether NGAL interacted with NGAL-R to induce apoptosis and further inhibit cell viability(Figure 3) and cyst growth (Figure 6), a construct for overexpression of nonsecreted NGAL (without the leader sequence) was also transfected into 2L3 cells.
Mouse NGAL (NP_032517.1) and human NGAL (NP_005555.2) are 200-aa and 198-aa proteins, respectively, and both have a 20-aa signal peptide for secretion located at the N-terminus of the NGAL protein (Figure 7A). Sequence comparison showed 62% identity and 80% positivity between mouse NGAL and human NGAL. Mouse NGAL was shown to have two N-linked glycosylation sites on asparagine (Asn, N) at aa 81 and 85, while human NGAL had one site at aa 85 (Figure 7A), revealing the difference between them in posttranslational modification.
Figure 7. Effects of transfection with pN + LS and pN − LS on NGAL expression and cell proliferation in 2L3 cells. (A) Alignment of mouse NGAL (NP_032517.1) and human NGAL (NP_005555.2) proteins. The 20-aa signal peptides are shown in blue. The two reported N-linked glycosylation sites on Asn (N) at aa 81 and 85 of mouse NGAL and one at aa 85 of human NGAL are marked by an asterisk (*). Identities: solid line; positives: paired dots. The sequence information above is from the BLASTP program on the National Center for Biotechnology Information (NCBI) website. (B) Construct a diagram of mNGAL with (pN + LS) or without (pN − LS) the secreted Igκ leader sequence on the pLAS2.1w.PeGFP-I2-Bsd vector. (C) The result of gel electrophoresis confirmed the construct of pN + LS (647 bp) and pN − LS (587 bp) digested by NheI and NotI. (D) Timeline of infection and selection of 2L3 cells with pN + LS and pN − LS, respectively. The 2L3 cells were infected pN + LS or pN − LS in the 6a -well plate on day 1. Selection with blasticidin started on day 2 and the medium was changed every 3 days. After selection for a week, cells were collected to be analyzed by RT–QPCR, western blot, and ELISA. (E) Detection of expression of reporter eGFP to examine the transfection efficiency of pN + LS and pN − LS on Day 2. About 10% of cells were infected with the GFP construct. Scale bar: 50 µm. RT–qPCR results showed that pN + LS and pN − LS both had higher expression of Lcn2 (represented total NGAL level) (F) and c-myc (represented transfected exogenous NGAL level) (G) compared with the control group. (H) There was no significant difference oinSlc22a17 (NGAL-R) between the groups. RQ: relative quantification = 2−∆∆Ct; Gapdh was used as the internal control of each sample. Values were normalized to the control group. (I) Western blot results showed pN + LS and pN − LS had higher expression of NGAL than the control group. (J) ELISA showed that pN + LS and pN − LS cells both secreted significantly higher NGAL than the control group, while the secreted NGAL amount of pN − LS cells was significantly less than pN + LS cells. (K) BrdU incorporation showed that pN + LS and pN − LS cells had a lower cell proliferation ratio than the control group, while pN − LS cells had a higher that than pN + LS cells. Student's t-test was performed to determine the significance between the two groups (n = 3 in RT–qPCR and ELISA, n = 4 in western blot and BrdU incorporation). *p < 0.05; ** p < 0.01; *** p < 0.001 vs. 2L3 of each group; # p < 0.05; ### p < 0.001 vs. pN + LS group; ns: no significant difference.
In this study, we used a murine Igκ-chain leader sequence (LS) [44] rather than the original signal peptide of mNGAL to ensure increased secretion of exogenous mNGAL protein. To overexpress secreted or nonsecreted NGAL, NGAL with or without LS in pLAS2.1w.PeGFP-I2-Bsd vectors, namely, pN + LS and pN − LS, were used and are shown in Figure 7B. Two promoters, pCMV and hPGK, were used to drive the independent expression of two proteins, NGAL-c-myc-His6 and eGFP. To ensure insertion of the construct, NheI, and NotI were used to digest the two plasmids, and the gel electrophoresis results showed a 647-bp band from pN + LS and a 587-bp band from pN − LS (due to the lack of a 60-bp leader sequence) (Figure 7C).
For infection of pN + LS and pN-SP into 2L3 cells, polybrene and lentivirus were added to cells and incubated at 37 ◦C for 24 h, followed by selection with blasticidin (Figure 7D). Detection of GFP expression was performed by fluorescence microscopy after infection (Figure 7E). Infected cells were harvested one week after blasticidin selection for RT–qPCR, western blot, and ELISA to analyze the level of NGAL expression (Figure 7F–J). The RT–qPCR results showed that infection of 2L3 cells with pN + LS and pN − LS led to increasing expression of Lcn2 (Figure 7F) and Lcn2-c-myc (Figure 7G), which represented the levels of total and exogenous NGAL, respectively, while there were no differences in the levels of Slc22a17 (NGAL-R) expression (Figure 7H). The western blot results showed that pN + LS and pN − LS cells had higher levels of intracellular NGAL expression than the control 2L3 cells (Figure 7I). ELISA results for the cell supernatant showed that pN + LS and pN − LS cells both had significantly higher levels of secreted NGAL than the 2L3 cell control group (Figure 7J). In addition, the amount of NGAL secreted by pN − LS cells was significantly less than that secreted by pN + LS cells (Figure 7J), while the two groups had similar NGAL expression levels (Figure 7I). This finding supported our prediction that overexpression of NGAL without a signal peptide (pN − LS) limited the secretion of NGAL despite the presence of a high level of intracellular NGAL. To investigate whether secreted NGAL rather than nonsecreted NGAL inhibited cell proliferation, a BrdU incorporation assay was performed, which showed that pN + LS and pN − LS cells had significantly lower proliferation than the control group (Figure 7K). In addition, pN − LS cells had significantly higher proliferation than pN + LS cells (Figure 7K). These results suggested that NGAL secretion from 2L3 cells was required to inhibit cell proliferation.
3.6. Effects of mental Overexpression Leading to Signaling Pathways in 2L3 Cells
Based on our previous results showing that mNGAL could inhibit the ERK and AKT pathways in 2L3 cells (Figure 4) and further decrease cell proliferation (Figure 3) and cyst enlargement (Figure 6), we used western blotting to analyze whether the ERK and AKT pathways were affected in pN + LS and pN − LS cells (Figure 8). The western blot results showed that the pN + LS and pN − LS groups had a significantly lower ratio of p-ERK (T202/Y204)/ERK than the control group, and the pN − LS group had a higher ratio of p-ERK (T202/Y204)/ERK than the pN + LS group (Figure 8A). The ratio of p-AKT (S473)/AKT was lower in the pN + LS group than in the control group, while pN − LS cells were not significantly different from those in the control and pN + LS groups (Figure 8B). The ratio of p-CREB (S133)/CREB was significantly higher in the pN − LS group than in the pN + LS group, while neither pN + LS nor pN − LS showed a significant difference from the control group (Figure 8C). These results suggested that pN + LS and pN − LS had statistically significant differences in the ratio of p-ERK/ERK (Figure 8A), while the pN + LS group did not show a better effect in AKT (Figure 8B) and CREB (Figure 8C) activation.
According to the previous results that mNGAL application could induce apoptosis (Figure 3) and autophagy in 2L3 cells (Figure 5) and decrease cell proliferation (Figure 3) and cyst enlargement (Figure 6), we used western blotting to investigate the effect on apoptosis and autophagy in pN + LS and pN − LS cells (Figure 9). The western blot results showed that pN + LS cells had a significantly higher ratio of cleaved-/pro-caspase-3 (Figure 9A) and cleaved-/pro-PARP (Figure 9B) than the control group; pN − LS cells showed no significant difference in the ratio of cleaved-/pro-caspase-3 (Figure 9A) and cleaved-/pro-PARP (Figure 9B) compared with the control group, and the ratio of cleaved- /pro-caspase-3 was significantly lower than that in the pN + LS group (Figure 9B). These results demonstrated that overexpression of secreted NGAL rather than nonsecreted NGAL induced apoptosis in 2L3 cells.
A higher ratio of LC3B II/LC3B I (Figure 9C) and a lower ratio of p62/GAPDH (Figure 9D) were identified in pN + LS cells compared with the control group; pN − LS cells had no significant difference in the ratio of LC3B II/LC3B I (Figure 9C) and p62/GAPDH (Figure 9D) compared with the control group, and the ratio of LC3B II/LC3B I was significantly lower than in the pN + LS group (Figure 9C). This result revealed that overexpression of secreted NGAL rather than nonsecreted NGAL induced autophagy in 2L3 cells.
3.7. Effects of Overexpression of mNGAL on Cyst Formation of 2L3 Cells in 3D Culture
Based on the previous results that mNGAL inhibited 2L3 cell proliferation and cyst enlargement in 2D and 3D culture, respectively (Figures 3 and 6), we suspected that overexpression of secreted NGAL might further inhibit cyst enlargement of 2L3 cells through the inhibition of cell proliferation (Figure 7K).
To investigate the effects of NGAL overexpression in 2L3 cells on cyst formation, the 2L3 control, pN + LS, and pN − LS cells were treated with forskolin on Days 1–3 to induce cyst formation. The cyst diameters, area, and numbers were measured daily after forskolin induction (Figure 10A). Cysts of the 2L3 control, pN + LS, and pN − LS cells in 3D culture grew in a manner dependent on the culture days (Figure 10B) and showed obvious differences in cyst size from Days 4 to 8. The pN + LS group had significantly smaller cyst diameters (Figure 10C) and areas (Figure 10D) than the control group from Days 4 to 8, while the pN − LS group had significantly smaller cyst diameters than the control group on Days 6 and 8 (Figure 10C). There was no significant difference in cyst area between the control and pN − LS groups. In addition, the pN − LS group had significantly larger cyst diameters and areas than the pN + LS group on Day 8 (Figure 10C, D). Although there was no significant difference in the rate of cyst number increases among the control, pN + LS, and pN − LS cells (Figure 10E), the pN + LS and pN − LS groups both had significantly lower percentages of large (diameter > 100 µm) cysts than the control group (Figure 10F) from Days 4 to 8. These results showed that pN + LS and pN − LS both inhibited cyst enlargement, while the inhibitory effect of pN − LS was weaker than that of pN + LS. This finding revealed that overexpression of secreted mNGAL had a superior ability to inhibit cyst enlargement of 2L3 cells in 3D culture than nonsecreted mNGAL.
-A test was performed to determine the significance between groups (n = 3). * p < 0.05; *** p < 0.001 vs. control group; # p < 0.05 vs. pN + LS group
caspase-3 (A) and cleaved-/pro-PARP (B) than the control group, which represented the induction of apoptosis by secreted mNGAL. pN − LS cells had no significant difference in the ratio of cleaved-/pro-caspase-3 and cleaved-/pro-PARP compared with the control group. pN + LS cells had a higherratio of LC3B II/LC3B I (C) and the lower ratio of p62 (D) compared with the control group. pN −LS cells had no significant difference in the ratio of LC3B II/LC3B I and p62 compared with the control group. Tubulin, Vinculin, and GAPDH were used as the internal controls of each sample. Values were normalized to the control group. Student's t-test was performed to determine the significance between groups (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group; # p < 0.05; ## p < 0.01 vs.pN + LS group.
Figure 9. Effects of overexpression of mNGAL protein on apoptosis and autophagy in 2L3 cells. Western blot results showed that pN + LS cells had a significantly higher ratio of cleaved-/pro-caspase-3(A) and cleaved-/pro-PARP (B) than the control group, which represented the induction of apoptosis by secreted mNGAL. pN −LS cells had no significant difference in the ratio of cleaved-/pro-caspase-3 and cleaved-/pro-PARP compared with the control group. pN + LS cells had a higher ratio of LC3BII/LC3B I (C) and the lower ratio of p62 (D) compared with the control group. pN − LS cells had no significant difference in rthe atio of LC3B II/LC3B I and p62 compared with the control group. Tubulin, Vinculin, and GAPDH were used as the internal controls of each sample. Values were normalized to the control group. Student's t-test was performed to determine the significance between groups
(n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group; # p < 0.05; ## p < 0.01 vs. pN + LS group
4–8. Cyst number on Day 4 of each group was used as the control to calculate the cyst number ratio. (F) Percentage of large-size of cysts in the 3D culture of the control group, pN + LS, and pN − LS cells during Days 4–8. Student's t-test was performed to determine the significance between groups (n = 3). * p < 0.05; ** p < 0.01 vs. control group; # p < 0.05; ## p < 0.01 vs. pN + LS group.
