LPA₂ upregulation and LPA₃ downregulation correlate with attenuated cell proliferation and increased senescence in Progerin HEK293 cells. (a) Design of vectors expressing Lamin A and Progerin. Puromycin was applied as the selection marker to establish Progerin HEK293 stable cells. (b) PCR with primer targeting outside of the cryptic splicing site shows alternate 150‐nucleotide splicing of Lamin A mRNA in Progerin HEK293 cells. (c) Western blot with anti‐Lamin A/C (ab108595) and anti‐Progerin (sc‐81611) antibody shows expression of Progerin in Progerin HEK293 cells (d) Western blot with LPA receptor antibodies shows upregulation of LPA₂ and downregulation of LPA₃ in Progerin HEK293 cells. Actin was used as a loading control. (e) Cell number measurements show that, in both Lamin A and Progerin HEK293 cells, activating LPA₂ with 5 μM of GRI decreased the cell proliferation rate, but activating LPA₃ with 100 nM of OMPT increased the cell proliferation rate. (f) Representative images of senescence‐associated β‐gal staining assay and its quantified results. Treating Lamin A and Progerin HEK293 cells with 5 μM of GRI for 4 days increased the percentage of β‐gal‐positive cells in both cell lines. Treating Progerin HEK293 cells with 100 nM OMPT for 4 days reduced the percentage of β‐gal‐positive cells. (g) Knockdown of LPA₂ by siRNA (siLPA₂) reduced β‐gal‐positive Progerin HEK293 cells. Knockdown of LPA₃ by siRNA (siLPA₃) increased β‐gal‐positive cells. (h) Overexpression of LPA₃ decreased β‐gal‐positive cells. (i) Real‐time qPCR showed that treatment of 100 nM OMPT for 24 hr reduced mRNA level of p16 in Progerin HEK293 cells. (j) 100 nM OMPT for 24 hr reduced mRNA level of p21. (k) 100 nM OMPT for 24 hr reduced mRNA level of IL6. GAPDH was used as internal control. ANOVA and Student's t test; *p < .05, **p < .01, ***p < .001

LPA₂ increases, but LPA₃ suppresses ROS buildup in Progerin HEK293 cells. (a) By flow cytometry, CM‐DCFDA staining results show that activating LPA₂ by 5 μM GRI for 24 hr increased ROS levels in both Lamin A and Progerin HEK293 cells. (b) Activating LPA₃ by 100 nM OMPT for 24 hr decreased ROS levels in both Lamin A and Progerin HEK293 cells (c) Knockdown of LPA₂ by shRNA (shLPA₂) reduced ROS levels, but knockdown of LPA₃ by shRNA (shLPA₃) increased ROS levels in Progerin HEK293 cells. (d) Overexpression of LPA₃ reduced ROS levels in Progerin HEK293 cells. (e) After 48 hr of 5 μM GRI and 100 nM OMPT, Western blot results showed that activation of LPA₃, but not LPA₂, induced expression of the antioxidant mediators Nrf2, NQO1, SOD2, and Gpx1 in Progerin HEK293 cells. Actin was used as the loading control. ANOVA and Student's t test; *p < .05, ***p < .001

LPA₃ reduction accelerates cell senescence in HGPS patient fibroblasts. (a) Western blot results show lower LPA₂ and LPA₃ protein levels in HGPS patient fibroblasts (AG11 and AG03) than in normal fibroblasts (AG08). (b) CCK‐8 assay revealed that activating LPA₃ with 100 nM OMPT for 5 and 7 days rescued cell proliferation of HGPS AG11 and AG03 fibroblasts after. However, activating LPA₂ with 5 μM GRI had no effect on cell proliferation. (c) Representative images of senescence‐associated β‐gal staining assay and its quantified results. Treating with 100 nM OMPT for 7 days reduced the percentage of β‐gal‐positive HGPS AG03 and AG11 fibroblasts. However, activating LPA₂ with 5 μM GRI had no effect on cell senescence. (d) Real‐time qPCR showed that treatment of 100 nM OMPT for 24 hr reduced mRNA level of p16 in HGPS patient fibroblasts (AG11 and AG03). (e) 100 nM OMPT for 24 hr reduced mRNA level of p21. (f) 100 nM OMPT for 24 hr reduced mRNA level of IL6. GAPDH was used as internal control. (g) By flow cytometry, DCFDA staining results show that activating LPA₃ by 100 nM OMPT for 48 hr decreased ROS levels in both HGPS AG03 and AG11 fibroblasts. However, activating LPA₂ with 5 μM GRI had no effect on ROS level. (h) Western blot results show that activating LPA₃ by 100 nM OMPT for 48 hr stabilized Nrf2 in HGPS AG11 fibroblasts and enhanced protein levels of NQO1, SOD2, and Gpx1, but not Lamin B1. Actin was used as a loading control. ANOVA and Student's t test; *p < .05, **p < .01, ***p < .001

LPA₃ is highly internalized and sorted to the lysosome degradation pathway in Progerin cells. (a) Western blot results show that treating Progerin HEK293 cells with 5 μM of MG132 for 6 hr showed no LPA₃ protein restoration. DMSO was used as solvent control (Ctrl). (b) Western blot results show that Progerin HEK293 cells were treated with 5 mM of NH₄Cl or 20 nM of Bafilomycin A1 (BA‐1) for 6 hr to block LPA₃ degradation through the lysosome pathway. ddH₂O was used as solvent control for NH₄Cl (Ctrl in upper panel). DMSO was used as solvent control for BA‐1 (Ctrl in lower panel). (c) Western blot results show that HGPS AG11 fibroblasts were treated with 5 mM of NH₄Cl and 5 μM of MG132 for 6 hr. NH₄Cl treatment rescued LPA₃, but not LPA₁ or LPA₂, in HGPS AG11 fibroblasts. ddH₂O and DMSO were both loaded to control sample as solvent control (Ctrl). (d) Counter‐immunofluorescent staining by LPA₃ and LAMP‐1 shows that LPA₃ localized into the lysosomes in Progerin HEK293 cells. (e) Western blot results show that NH₄Cl treatment rescued LPA₃ in HGPS AG03 fibroblasts. ddH₂O and DMSO were both loaded to control as solvent control (Ctrl). (f) Scheme representing the internalization assay of LPA receptors_. (g) Representative images of time‐dependent LPA₃ internalization. Internalization of LPA₃ was higher in Progerin HEK293 cells. (h) Integrated density of intracellular fluorescence was quantified to indicate internalized LPA₃. ANOVA and Student's t test; **p < .01, ***p < .001

LPA₃ deficiency in zebrafish leads to premature aging phenotypes. (a) Kaplan–Meier survival analysis demonstrated a shorter lifespan in Lpa₃^−/− zebrafish than in corresponding WT siblings (p value = .011, N > 8). (b) Histological analysis shows hematopoietic stem cells loss in kidney marrow of 6‐month‐old Lpa₃^−/− zebrafish. (c) Sperm loss in the testis. (d) Bile duct hyperplasia. (e) Islet hyperplasia in the pancreas. (f) By senescence‐associated β‐gal staining assay, Lpa₃^−/− larvae show a positive β‐gal signal. (g) Representative images of zebrafish swimming tracks obtained and analyzed by locomotor. Locomotor results show that Lpa₃^−/− larvae had lower mobility than WT upon drastic light onset and offset. (h) CM‐DCFDA staining showed increased ROS levels in 5 dpf Lpa₃^−/− larvae. (i) Real‐time qPCR showed enhanced p16 and p21, whereas reduced Gpx1a, SOD2, and NQO1 of 5 dpf Lpa₃^−/− larvae. β‐Actin was used as internal control. ANOVA and Student's t test; *p < .05, ***p < .001

LPA₃ protects fibroblasts against cell senescence caused by oxidative stress. (a) CCK‐8 assay showed that knockdown of LPA receptors alone had no effect on cell proliferation. (b) CCK‐8 revealed that treatment with 10 μM and 50 μM H₂O₂ for 3 days significantly decreased cell proliferation in knockdown of LPA₃, but not of LPA₁ or LPA₂. CCK‐8 reads were normalized to a sham group. (c) Representative images of senescence‐associated β‐gal staining assay. Treatment with 10 μM and 50 μM H₂O₂ for 3 days significantly increased percentages of β‐gal + cells in knockdown of LPA₃, but not of LPA₁ or LPA₂. Quantification was shown in (d). (e) Western blot showed that 100 μM of H₂O₂ treatment for 6 hr increased protein level of Nrf2, NQO1, SOD2, and Gpx1, whereas LPA₃ knockdown abolished the effects. (f) Graphical abstract of this study. ANOVA and Student's t test; *p < .05, **p < .01, ***p < .001