Cathepsin L induces cellular senescence by upregulating CUX1 and p16^INK4a

CUX1 is not a substrate that is cleaved by CTSL in human ECs and VSMCs

Recently, our post-GWAS functional studies demonstrated that CUX1 is an activator of p16^INK4a, regulating p16^INK4a-dependent cellular senescence, and, consistently, an increased expression of the full length CUX1 in late passage (p10) human ECs was observed compared to early passage (p5) human ECs [13]. Since previous reports showed that CTSL can proteolytically cleave CUX1 to produce a truncated, but more active form of CUX1 with an apparent molecular weight of 110 kDa, instead of 200 kDa for the full length CUX1, in human cancer cells [10, 14, 15], we therefore hypothesized that CTSL expression should be lower in p10 human ECs than that in p5 human ECs. To test this, we first investigated the expression of CTSL as well as CUX1 and p16^INK4a in p5 and p10 human ECs. To our surprise, an increased expression of CTSL as well as CUX1 and p16^INK4a in p10 versus p5 human ECs was detected by both Western blots and qPCR analyses and no truncated CUX1 at 110 kDa was detected (Figure 1A, 1B). The same analyses were also performed in human VSMCs and similar results were observed, demonstrating that an increased expression of CTSL, CUX1 and p16^INK4a in late passage (p15) versus early passage (p8) human VSMCs as shown in Figure 1C, 1D. Together, these data demonstrate that CUX1 is not a substrate of CTSL, at least in human ECs and VSMCs, as both CTSL and CUX1 expression are passage-dependent with an increased expression in late versus early passage human ECs and VSMCs. The same data also demonstrate that CTSL as well as CUX1 and p16^INK4a are all activated by transcription as activation can be detected by qPCR analysis.

Figure 1. Correlated expression of CTSL, CUX1 and p16^INK4a in human ECs and VSMCs. (A, B) Western blots and qPCR analysis showing upregulation of CTSL, CUX1 and p16^INK4a in late passage (p10) versus early passage (p5) human ECs. (C, D) Western blots and qPCR analysis confirming upregulation of CTSL, CUX1 and p16^INK4a in late passage (p15) versus early passage (p8) human VSMCs. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments.

CTSL is an upstream activator of CUX1, regulating cellular senescence

Correlation of expression among CTSL, CUX1, and p16^INK4a in late passage human cells suggests a possible functional relevance among these genes. To determine if CTSL is an upstream regulator of CUX1 and p16^INK4a, we performed siRNA-mediated knockdown of CTSL in p8 human ECs. We detected a significant downregulation of both CUX1 and p16^INK4a expression at both protein and mRNA levels in the siRNA-mediated CTSL knockdown ECs as shown in Figure 2A, 2B. These data further demonstrate that CUX1 is not a substrate of CTSL protease activity as, otherwise, siRNA-mediated CTSL knockdown should result in an upregulation of CUX1. The same data also suggest that CUX1 is a downstream effect gene of CTSL as, otherwise, siRNA-mediated CTSL knockdown should not lead to altered expression of CUX1. Consistent with the reduced expression of CUX1 and p16^INK4a, a significant reduction of cellular senescence in the siRNA-mediated CTSL knockdown ECs was observed as measured by decreased SA-β-gal and γ-H2AX staining (Figure 2C) as well as by decreased expression of the senescence-associated secretory phenotype (SASP) genes IL6, IL-1β, and ICAM-1 (Figure 2D). Therefore, our data demonstrate that CTSL is an upstream activator of CUX1 and/or p16^INK4a, and downregulation of CTSL suppresses CUX1 and/or p16^INK4a as well as cellular senescence, presumably via modulating CUX1 and p16^INK4a expression.

Figure 2. Downregulation of CTSL decreases the expression of CUX1 and p16^INK4a, and inhibits cellular senescence in human ECs. (A, B) Western blot and qPCR analyses showing that siRNA-mediated CTSL knockdown (siCTSL) downregulates CUX1 and p16^INK4a expression in human ECs. (C, D) siRNA-mediated CTSL knockdown (siCTSL) suppresses cellular senescence in human ECs as evidenced by SA-β-gal and γ-H2AX staining as well as by the expression of the SASP genes IL6, IL-1β, and ICAM-1. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel C. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).

CTSL protease activity is required to activate CUX1 and p16^INK4a as well as cellular senescence

To further demonstrate that CTSL activates cellular senescence by upregulating CUX1 and/or p16^INK4a, we also treated p8 human ECs with 30 μM Z-FY-CHO, a potent and selective inhibitor of CTSL enzymatic activity [26]. Consistent with the results observed in the siRNA-mediated CTSL knockdown human ECs (Figure 2A–2D), a significant reduction of both CUX1 and p16^INK4a expression at both protein and mRNA levels was observed in the Z-FY-CHO-treated human ECs while, as expected, CTSL expression remained unchanged (Figure 3A, 3B). Consequently, a decreased cellular senescence was observed as measured by the reduced SA-β-gal and γ-H2AX staining (Figure 3C) as well as by the decreased expression of the SASP genes IL6, IL-1β, and ICAM-1 (Figure 3D). The same analyses were also performed in human VSMCs, and we verified that Z-FY-CHO inhibits both CUX1 and p16^INK4a expression as well as cellular senescence in human VSMCs as shown in Figure 3E–3H.

Figure 3. Inhibition of CTSL protease activity decreases the expression of CUX1 and p16^INK4a, and inhibits cellular senescence in human ECs and VSMCs. (A, B) Western blot and qPCR analyses showing that inhibition of CTSL protease activity by Z-FY-CHO downregulates CUX1 and p16^INK4a in human ECs. (C, D) inhibition of CTSL protease activity by Z-FY-CHO suppresses cellular senescence in human ECs as evidenced by SA-β-gal and γ-H2AX staining as well as by the expression of the SASP genes IL6, IL-1β, and ICAM-1. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel C. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. (E, F) Western blot and qPCR analyses showing that inhibition of CTSL protease activity by Z-FY-CHO downregulates CUX1 and p16^INK4a in human VSMCs. (G, H) inhibition of CTSL protease activity by Z-FY-CHO suppresses cellular senescence in human VSMCs as evidenced by SA-β-gal and γ-H2AX staining as well as by the expression of the SASP genes IL6, IL-1β, and ICAM-1. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel G. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).

Taken together with the data presented in Figures 1 and 2, these data validate that CTSL is an upstream activator of CUX1 and/or p16^INK4a, and induces cellular senescence, presumably via transcriptionally activating CUX1 and/or p16^INK4a. At the same time, our findings demonstrate that the proteolytic activity of CTSL is required for the transcriptional activation of CUX1 and/or p16^INK4a, which suggests that CTSL is not a direct transcriptional factor of CUX1, and CTSL may activate CUX1 transcription by proteolytically activating a currently unknown transcription factor.

CUX1 is a downstream mediator of CTSL, regulating p16^INK4a and p16^INK4a-dependent cellular senescence

To confirm that CUX1 is the downstream mediator of CTSL, regulating p16^INK4a as well as p16^INK4a-dependent cellular senescence in human ECs, as we previously reported [13], we performed a functional complementation assay. We first overexpressed CTSL in human ECs using a lentiviral expression vector pLVX-CTSL. Overexpression of CTSL was confirmed by Western blot as shown in Figure 4A (Lane 1 and 2). As a result, upregulation of both CUX1 and p16^INK4a was validated at both mRNA and protein levels (Figure 4A, 4B, Lane 1 and 2). Consistent with the increased expression of CUX1 and p16^INK4a, CTSL-overexpressed human ECs showed an increased level of cellular senescence as measured by both the enhanced SA-β-gal and γ-H2AX staining (Figure 4C, Column 1 and 2) as well as by the increased expression of the SASP genes IL6 and IL-1β, and ICAM-1 (Figure 4D, Lane 1 and 2). Next, we performed shRNA-mediated CUX1 knockdown in the CTSL-overexpressing human ECs as previously described [13]. After viral infection, downregulation of CUX1 was first confirmed by both Western blot and qPCR analyses (Figure 4A, 4B, Lane 2 and 3). As a result, downregulation of p16^INK4a was also detected (Figure 4A, 4B, Lane 2 and 3) and, correspondingly, a decreased cellular senescence was observed by the decreased staining of both SA-β-gal and γ-H2AX (Figure 4C, Column 2 and 3) as well as by the decreased expression of the SASP genes IL6 and IL-1β, and ICAM-1 (Figure 4D, Lane 2 and 3). Thus, these data demonstrate that CUX1 is a downstream mediator of CTSL, regulating p16^INK4a and cellular senescence in response to CTSL activation.

Figure 4. CTSL is an upstream regulator of CUX1 and p16^INK4a, inducing replicative senescence. (A, B) Western blot and qPCR analyses showing that overexpression of CTSL (pLVX-CTSL) activates CUX1 and p16^INK4a expression, and that shRNA-mediated CUX1 knockdown (shCUX1) reverses the increased expression of CUX1 and p16^INK4a in CTSL-overexpressing (pLVX-CTSL) human ECs. (C, D) SA-β-gal and γ-H2AX staining and the expression of SASP genes IL6, IL-1β and ICAM-1 verify that overexpression of CTSL (pLVX-CTSL) induces cellular senescence, and that shRNA-mediated CUX1 knockdown (shCUX1) reverses the increased senescence in the CTSL-overexpressing (pLVX-CTSL) human ECs. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel C. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).

CTSL regulates premature senescence induced by bleomycin and oxidized low density lipoprotein (oxLDL)

Cellular senescence can also be triggered prematurely by various stresses including DNA damage, oxidative stress, oncogenic activation, and metabolic dysregulation [18, 19, 27]. Previously, we demonstrated that both DNA damage and oxidative stress-induced cellular senescence is activated via the CUX1/p16^INK4a signal pathway [13]. To determine if CTSL also participates in DNA damage-induced premature senescence, we first checked the expression of CTSL, CUX1, and p16^INK4a in human ECs treated with 0.5 μg/ml bleomycin, a genotoxic drug known to induce double-stranded DNA breaks [28]. A significant increase of CTSL, CUX1, and p16^INK4a expression was detected at both protein and mRNA levels in the bleomycin-treated human ECs compared to the PBS-treated controls (Figure 5A, 5B, Lane 1 and 2). As expected, an increased expression of CTSL, CUX1, and p16^INK4a in the bleomycin-treated human ECs resulted in an induction of cellular senescence as evident by the increased SA-β-gal and γ-H2AX staining (Figure 5C, Column 1 and 2) as well as the upregulated expression of the SASP genes IL6, and IL-1β, and ICAM-1 (Figure 5D, Lane 1 and 2), suggesting that CTSL is involved in DNA damage-induced cellular senescence. To further demonstrate that CTSL induces DNA damage-induced senescence via activating CUX1 and p16^INK4a, we treated the bleomycin-induced human ECs with Z-FY-CHO. We observed a significant restoration of the expression of CUX1 and p16^INK4a (Figure 5A, 5B, Lane 2 and 3) as well as of cellular senescence (Figure 5C, Column 2 and 3 and Figure 5D, Lane 2 and 3). In addition, we also applied the same functional complementation assay to investigate oxidative stress-induced cellular senescence by treating the 0.1 mg/ml oxLDL-treated human ECs with 30 μM Z-FY-CHO. Similar results were obtained as shown in Figure 6, demonstrating that CTSL is also involved in oxidative stress-induced cellular senescence. Taken together, these data demonstrate that CTSL is an activator of stress-induced cellular senescence.

Figure 5. CTSL is an upstream regulator of CUX1 and p16^INK4a, inducing DNA damage-induced senescence. (A, B) Western blot and qPCR analyses showing that bleomycin (Bleo)-induced activation of CTSL induces CUX1 and p16^INK4a expression, which can be reversed by treating the cells with Z-FY-CHO. (C, D) SA-β-gal and γ-H2AX staining and the expression of the SASP genes IL6, IL-1β, and ICAM-1 verify that bleomycin-induced activation of CTSL induces cellular senescence, which can also be reversed by treating the cells with Z-FY-CHO. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel C. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).

Figure 6. CTSL is an upstream regulator of CUX1 and p16^INK4a, inducing oxLDL-induced senescence. (A, B) Western blot and qPCR analyses show that oxLDL-induced activation of CTSL induces CUX1 and p16^INK4a expression, which can be reversed by treating the cells with Z-FY-CHO. (C–E) SA-β-gal and γ-H2AX staining and the expression of the SASP genes IL6, IL-1β, and ICAM-1 verify that oxLDL-induced activation of CTSL induces cellular senescence, which can also be reversed by treating the cells with Z-FY-CHO. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel D. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).

Cell culture and reagents

Primary human arterial ECs (Cat#: CC-2535) and human arterial VSMCs (Cat#: CC-2571) were purchased from Lonza. Cells were passaged by 1: 4 split ratio when they grow to confluence. All cells are free of mycoplasma. ECs were cultured in basal medium EGM-2, and VSMCs in SmGM-2 supplemented with Bullet Kit (Lonza). All cells were cultured at 37ºC in 5% CO₂ incubator.

Treatment

To test the effect of stress-induced senescence, ECs were treated with 0.5 μg/ml bleomycin (Cayman, Cat#: 13877) for 24 h and with 0.1 mg/ml oxidized Low Density Lipoprotein (oxLDL) (Invitrogen, Cat#: L34357) for 48 h. ECs were also treated with 30 μM of the CTSL inhibitor Z-FY-CHO (Santa Cruz Biotechnology, Cat#: 167498-29-5) for 48 h.

qPCR

Total RNA was isolated with the RNeasy Mini kit (Qiagen, Cat#: 74104). cDNA was synthesized with SuperScript^® III Reverse Transcriptase (Invitrogen, Cat#: 18080044) after the RNA samples were treated with DNase I (Invitrogen, Cat#: AM8170G). All the procedures were performed following the manufacturer’s protocols. qPCR was performed with the StepOne real-time PCR system according to the protocol for the Power SYBR Green PCR Master Mix (Applied Biosystems, Cat#: A46109) and for TaqMan Universal PCR Master Mix (Applied Biosystems, Cat#: 4318157). The following probe/primer mixes for TaqMan PCR were purchased from Applied Biosystems: CUX1 Hs00738851_m1, p16^INK4a Hs02902543_mH, and GAPDH internal control (Hs02786624_g1). Other Primers used, including the primers for SASP genes IL6 and IL-1β, and ICAM-1, are listed in Supplementary Table 1. All the data represent the combination of three independent samples (n = 3), each with two technical repeats.

Western blot

Whole cell proteins were isolated with RIPA buffer (Sigma, Cat#: R0278). Proteins were separated on SDS-PAGE gel and transferred to PVDF membranes. Proteins were detected with protein-specific antibodies. All antibodies were purchased and used as listed in Supplementary Table 2. For control, α-tubulin was used. The data represent three independent biological replicates (n = 3).

Senescence β-galactosidase staining

The Senescence β-Galactosidase Staining Kit (Cell Signaling, Cat#: 9860S) was used to detect senescent cells by following the manufacturer’s instruction (https://www.cellsignal.com/products/cellular-assay-kits/senescence-b-galactosidase-staining-kit/9860). Senescent cells were visualized using an RVL-100-G microscope (Echo Laboratories, San Diego, CA, USA). Images were analyzed using ImageJ software (version 1.52K, NIH). The data represent three independent biological replicates (n = 3).

γ-H2AX staining

γ-H2AX staining was performed as previously described [13]. Cells were seeded on glass coverslips on 24 well plates and fixed in 4% paraformaldehyde. Cell membrane was solubilized in PBS containing 5% FBS and 0.5% Triton X-100. Cells were first incubated with γ-H2AX antibodies in the solubilizing buffer for 1 h and then with Alexa Fluor 488-conjugated secondary antibody. γ-H2AX staining was visualized using an RVL-100-G microscope (Echo Laboratories). Images were analyzed using ImageJ software (version 1.52K, NIH). For control, nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma, Cat#: D9542). The data represent three independent biological replicates (n = 3).

RNAi knockdown

For siRNA transient CTSL knockdown in human ECs, ON-TARGETplus Human CTSL siRNA was purchased from Horizon Discovery. siRNA transfection was performed by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat# 13778150) according to the manufacturer’s protocol (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.99253.file.dat/protocols.par.67200.file.dat/huvec-rnaimax.pdf). 24 h after transfection, cells were harvested for RNA isolation.

Overexpression of CTSL

For overexpression of human CTSL, Cathepsin L expression vector was purchased from Addgene (Cat #: 11250). The full length CTSL cDNA was cloned into pLVX-M-puro vector (Addgene, Cat#: 125839) and confirmed by sequencing. Lentiviruses were generated by transfecting 293T cells with pLVX-CTSL, together with psPAX2 (Addgene, Cat#:12260) and pMD2.G (Addgene, Cat#:12259), according to Addgene’s protocol (https://www.addgene.org/protocols/lentivirus-production/). Generated viruses were infected into human ECs by incubating the virus with 10 μg/ml polybrene for 3 h at 37°C.

BrdU proliferation assay

Human ECs proliferation was determined by BrdU incorporation using the BrdU Cell Proliferation Assay Kit (Cell Signaling, Cat#: 6813). Briefly, human ECs were subcultured (10,000 cells per well) in 96-well plates for 24 h after virus infection. Then 1× BrdU was added to the culture medium for DNA labeling. The labeling medium was removed after 2 h, then cells were fixed, and DNA was denatured by the addition of 100 μl of fixing/denaturing solution for 30 min. The incorporated BrdU was then detected by a mouse anti-BrdU monoclonal antibody and measured using an anti-mouse IgG, horseradish peroxidase-linked antibody following the manufacturers’ instructions. Data for the BrdU proliferation assay represent n = 6 independent biological samples.

Statistical analysis

P-values were calculated using Student’s T-test with 2 tails. Error bars represent the median with S.E. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3). Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the SA-β-gal staining and γ-H2AX staining panel.

Data availability

All data and reagents are available upon reasonable request.