Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing

Abstract

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Fig. 1. THBD is upregulated in senescent cells and aged tissues.

a Schematic depiction of gene expression profiling approach to identify novel senolytic targets. b Top differentially expressed genes between proliferating and erlotinib-induced senescent HBE cells with cellular surface genes highlighted. The x axis represents individual differentially expressed genes, ordered by ranking their expression fold change from large to small. The y axis represents the fold change (erlotnib-induced senescence/proliferating). A total of 331 differentially expressed genes are plotted. c THBD protein expression in IMR90 fibroblasts undergoing replicative (70 passages) and oncogene-induced senescence (IMR90 cells transfected with Tet-ON HRas^G12V and treated with 1 μg/mL Dox for 7 days). d, e Representative co-immunohistochemistry staining and protein expression of THBD (red) and p21 (brown) in young (2 months) and aged (24 months) in murine lung, heart, muscle, and liver tissues. Scale bars, 50 μm.

Fig. 2. THBD signaling is critical for senescent cell survival.

a Quantification of SA-βgal and expression of senescence markers in IMR90 cells stably expressing Tet-ON THBD treated with 1 μg/mL Dox for 9 days. b Expression of apoptotic and senescent markers and cell viability quantification of Tet-ON HRas^G12V stably expressing IMR90 cells infected with control or THBD-targeted shRNA followed by 9 days of Dox (1 μg/mL) treatment using Cell Titer Glo. c Expression of apoptotic and senescent markers and cell viability analysis of senescent Tet-ON HRas^G12V IMR90 cells infected with non-targeting control (NTC) or THBD-targeted shRNA for 10 days. d Quantification of SA-βgal-positive cells and expression of senescence-associated markers in IMR90 cells overexpressing HA-THBD alone or in tandem with uncleavable PAR1 mutant, R46Q for 9 days. e Quantification of SA-βgal-positive cells and expression of senescence-associated markers in IMR90 cells stably expressing Tet-ON THBD treated with Dox (1 μg/mL) alone or with vorapaxar (5 μM) for 9 days. f Cleaved caspase-3 levels and cell viability quantification of Tet-ON HRas^G12V IMR90 cells treated with Dox (1 μg/mL) for 7 days followed by 7 days of vorapaxar (5 μM) treatment. g Schematic representation of findings presented in Fig. 2. THBD is dramatically upregulated in multiple contexts of senescence, promotes senescence, and ensures senescent cell viability through cleaving PAR1’s N-terminus at Arginine 46. THBD depletion or PAR1 inhibition, using vorapaxar, promotes senescent cell death. All data are reported as mean ± SEM of three independent experiments. Unpaired two-tailed Student’s t-test and two-way ANOVA while correcting for multiple comparisons via the Tukey method were performed: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 3. THBD signaling axis is upregulated in senescent cells and aged tissues.

a Schematic representation of THBD-mediated signaling. THBD binds to thrombin and initiates proteolytic cleavage via EPCR-bound PC, resulting in PAR1 activation and intracellular signal transduction through the heterotrimeric G proteins, G_α12/13, G_q, and G_i, and their downstream effector, RhoA. b, c Protein expression (b) and IHC staining (c) of THBD signaling components (PAR1, Protein C, thrombin, EPCR, G_α12/13, RhoA) in young (2 months) and aged (24 months) murine lung, heart, muscle, and liver tissues. Scale bars, 50 μm.

Fig. 4. THBD signaling persists in recycling endocytic vesicles.

a Schematic diagram of two possible fates for the THBD signaling complex following upregulation: stabilization at the cell surface or internalization into endocytic vesicles. b, c Representative co-immunostaining of endogenous THBD (green, b) or PAR1 (green, c) with EEA1, LAMP1, and ubiquitin (red). d Representative EM for THBD in proliferating and palbociclib-induced senescent IMR90 cells. THBD is labeled with electron-dense gold particles. Dashed box regions were magnified and arrows indicate positive THBD labeling. For representative immunofluorescence images, scale bars provided represent 35 μm. For representative EM images, scale bars provided on the larger images and inset images represent 500 nm and 50 nm, respectively.

Fig. 5. THBD stabilizes the THBD signaling complex in endocytic vesicles by suppressing proteasome- and lysosome-associated degradation processes.

a Time course of THBD signaling component upregulation in IMR90 cells stably expressing Tet-ON HRas^G12V treated with 1 μg/mL Dox for 9 days. b Protein expression of THBD signaling components in IMR90 cell stably expressing Tet-ON HRas treated with Dox for 4 days followed by infection with NTC or THBD-targeted shRNAs for 6 days. c GO enrichment of upregulated genes in senescent Tet-ON HRas^G12V IMR90 cells infected with THBD-targeted shRNA compared with NTC. d Expression of THBD signaling proteins in Tet-ON HRas^G12V IMR90 cells treated with 1 μg/mL Dox for 4 days followed by infection with either an NTC or two THBD-targeted shRNAs and MG132 (1 μM) or chloroquine (5 μM) treatment for 24 h, as indicated.

Fig. 6. THBD suppresses NEDD4L-mediated THBD signaling complex degradation.

a Top 28 upregulated proteosome-associated genes from GO analysis presented in Fig. 5c. b NEDD4L protein and mRNA expression in Tet-ON HRas^G12V IMR90 cells treated with Dox for 7 days. c Protein and mRNA expression of THBD signaling components and NEDD4L in Tet-ON HRas^G12V IMR90 cells treated with 1 μg/mL Dox for the indicated days. d NEDD4L protein and mRNA expression in IMR90 cells stably expressing GFP and HA-THBD for 7 days. e Immunoblot of ubiquitin and NEDD4L co-immunoprecipitated with anti-myc or IgG from lysates of H239T cells transfected with the indicated plasmids. f Expression of THBD signaling receptors and cleaved caspase-3 in Tet-ON HRas^G12V IMR90 cells treated with Dox for 4 days followed by HA-NEDD4L overexpression. g Expression of THBD signaling receptors and cleaved caspase-3 in Tet-ON HRas^G12V IMR90 cells treated with Dox for 4 days followed by infection with THBD- and/or NEDD4-targeted shRNAs, as indicated. h THBD stabilizes the THBD signaling complex in recycling endosomes by suppressing NEDD4L expression and NEDD4L-mediated ubiquitination of the complex for lysosomal and proteasomal degradation. Data are reported as mean ± SEM of three independent experiments. Unpaired two-tailed Student’s t-test were performed: *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 7. THBD signaling antagonism attenuates liver fibrosis through senescent cell clearance.

a Representative THBD staining in healthy (n = 10) and cirrhotic (n = 10) human livers. b Representative THBD staining in MCDE (n = 8), CCL₄ (n = 5), BDL (n = 6) fibrotic murine models. c Representative co-immunohistochemistry staining of THBD (red) and p21 (brown) in murine fibrotic models from b. Magnified images and arrows signify colocalization between THBD and p21 in hepatocytes. d Protein expression of THBD signaling components in hepatocytes isolated from mice on a standard chow (n = 4) or MCDE (n = 4) diet. e Schematic diagram of the vorapaxar regimen and representative IHC staining of SA-βgal and fibrotic markers (Sirius Red, α-SMA, F4/80) following vorapaxar treatment in murine fibrotic models. f Representative SA-βgal, fibrotic markers (Sirius Red, α-SMA), and H&E staining of fibrotic livers from CCL₄ mice treated with 100 mg/kg ABT-263. g Schematic depiction showing that THBD ensures senescent cell survival by activating and stabilizing a proteolytic feed-forward signaling pathway in early endosomes. THBD signaling inhibition results in destabilization and subsequent lysosomal/proteasomal degradation of the multi-protein complex, senescent cell death, and restoration of tissue homeostasis during liver fibrosis. Scale bars, 50 μm.