
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02111-2
10.1016/j.jbc.2024.107610
107610
Research Article
Cyclophilin J limits linear ubiquitin signaling and controls colorectal cancer progression
Sheng Chunjie shengchj@sysucc.org.cn
∗
Yao Chen
Wang Jing
Mao Yizhi
Fu Lingyi
Chen Shuai chenshuai@sysucc.org.cn
∗
State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou, PR China
∗ For correspondence: Chunjie Sheng; Shuai Chen shengchj@sysucc.org.cnchenshuai@sysucc.org.cn
27 7 2024
9 2024
27 7 2024
300 9 10761010 1 2024
4 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Exorbitant sustained inflammation is closely linked to inflammation-associated disorders, including cancer. The initiation of gastrointestinal cancers such as colorectal cancer is frequently accelerated by uncontrollable chronic inflammation which is triggered by excessive activation of nuclear factor kappa-B (NF-κB) signaling. Linear ubiquitin chains play an important role in activating canonical NF-κB pathway. The only known E3 complex, linear ubiquitin chain assembly complex is responsible for the synthesis of linear ubiquitin chains, thus leading to the activation of NF-κB axis and promoting the development of inflammation and inflammation-associated cancers. We report here cyclophilin J (CYPJ) which is a negative regulator of the linear ubiquitin chain assembly complex. The N terminus of CYPJ binds to the second Npl4 zinc finger (NZF) domain of HOIL-1-interacting protein and the ubiquitin-like domain of Shank-associated RH domain-interacting protein to disrupt the interaction between HOIL-1-interacting protein and Shank-associated RH domain-interacting protein and thus restrains linear ubiquitin chain synthesis and NF-κB activation. Cypj-deficient mice are highly susceptible to dextran sulfate sodium-induced colitis and dextran sulfate sodium plus azoxymethane-induced colon cancer. Moreover, CYPJ expression is induced by hypoxia. Patients with high expression of both CYPJ and hypoxia-inducible factor-1α have longer overall survival and progression-free survival. These results implicate CYPJ as an unexpected robust attenuator of inflammation-driven tumorigenesis that exerts its effects by controlling linear ubiquitin chain synthesis in NF-κB signal pathway.

Keywords

CYPJ
LUBAC
linear ubiquitin
NF-κB
CRC
Abbreviations

AOM azoxymethane

CAC colitis-associated cancer

CFP cyan fluorescent protein

co-IP co-immunoprecipitation

CRC colorectal cancer

CYPJ cyclophilin J

DSS dextran sulfate sodium

FBS fetal bovine serum

FL full length

HIF-1α hypoxia inducible factor-1α

HOIL-1L heme-oxidized iron-regulatory protein 2 ubiquitin ligase 1

HOIP HOIL-1-interacting protein

IB immunoblotting

IBD inflammatory bowel disease

IHC immunohistochemistry

IL interleukin

IP immunoprecipitation

LUBAC linear ubiquitin assembly complex

M1 methionine 1

MS mass spectrometry

NF-κB nuclear factor kappa-B

NZF Npl4 zinc finger

PPIase peptidyl prolyl cis/trans isomerase

PPIL3 PPIase-like 3

PUB PNGase/UBA or UBX-containing proteins domain

qRT-PCR quantitative real-time PCR

RBR RING-in-between-RING

ROI regions of interest

SHARPIN Shank-associated RH domain-interacting protein

TCGA The Cancer Genome Atlas

TNF tumor necrosis factor

UBA Ub-associated domain

UBL ubiquitin-like

WB Western blot

YFP yellow fluorescent protein

ZF zinc finger

Reviewed by members of the JBC Editorial Board. Edited by Paul Shapiro
==== Body
pmcThe linear ubiquitin chain assembly complex (LUBAC) is composed of three members: the catalytic subunit HOIL-1-interacting protein (HOIP) and two regulatory subunits, longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase 1 (HOIL-1L) and Shank-associated RH domain-interacting protein (SHARPIN) (1, 2). As the only known 600-kD multimeric E3 ligase, LUBAC is responsible for peptide bond formation between the C-terminal glycine (G76) of one ubiquitin and the α-NH2 group of methionine 1 (M1) of another ubiquitin. In response to pathogen infections, the linear ubiquitin chain (also known as the M1-linked linear ubiquitin chains; we refer to it as M1-Ub for short) is generated by the LUBAC and plays a vital role in triggering inflammatory pathways such as nuclear factor kappa-B (NF-κB) and activated protein-1 (AP-1) (3, 4). The E3 activity of HOIP is indispensable for the activation of the canonical NF-κB pathway through linear ubiquitination of NEMO (IKKγ), and thus, HOIP-deficient cells are sensitive to tumor necrosis factor (TNF) and lymphotoxin-α-mediated apoptosis and necroptosis. Genetic ablation of Hoip or Hoil-1l results in embryonic lethality mainly due to TNF-induced cell death of endothelial cells (5), while mice with Sharpin deficiency develop chronic proliferative dermatitis (referred to as cpdm mice) (6). Generally, the M1-Ub signal contributes to cell survival while some deubiquitinase such as OTULIN or CYLD hydrolyzes linear ubiquitin chains to inhibit host inflammation. Some pathogenic bacteria have evolved to secrete effector proteins that harbor deubiquitinase activity to disrupt host ubiquitination signaling during infection (7, 8).

LUBAC-mediated M1-Ub signaling is essential for fetal hematopoiesis and the immune response. Humans deficient in functional LUBAC or its mutations have severe immune system disorders and are susceptible to pathogen infections (9). In recent years, the role of LUBAC in cancers has attracted an increasing amount of attentions. Several LUBAC substrates, including LUBAC themselves, NEMO, RIPK1 (10), ASC (11), STAT1 (12), ATG13 (13), ALK1 (14), AGO2 (15), PTEN (16), and GPx4 (17), have been revealed to participate in various biological processes such as promoting cell survival or resistant to apoptosis, necroptosis, or ferroptosis. HOIP, HOIL-1L, and SHARPIN are highly expressed in several cancers (18) including colorectal cancer (CRC). Two germ line single-nucleotide polymorphisms of HOIP, namely Q584H and Q662L (18) were found in patients with activated B-cell-like diffuse large B-cell lymphoma and resulted in constitutive NF-κB activation and lymphomagenesis. Inhibition of LUBAC by small molecule inhibitors is a promising therapeutic strategy for B-cell lymphomas (19).

HOIP is a member of the RING-in-between-RING (RBR) class of the E3 ligase family and functions as the catalytic subunit of LUBAC. HOIP is composed of an PNGase/UBA or UBX-containing proteins (PUB) domain, followed by one zinc finger (ZF) domain, two Npl4 zinc finger (NZF) domains called NZF1 and NZF2, a middle atypical Ub-associated (UBA) domain and a C-terminal RBR domain. The RBR domain of HOIP is capable of specifically generating M1-Ub (20, 21, 22), meanwhile, OTULIN and CYLD bind to the PUB domain of HOIP and cleave the linear ubiquitin linkage (23). The C terminus of SHARPIN shares sequence homology with the N terminus of HOIL-1L. The NZF domains of SHARPIN and HOIL-1L exclusively bind M1-Ub, while their ubiquitin-like (UBL) domains interact with the UBA domain of HOIP to generate the LUBAC (Fig. 1A) (24, 25). Some studies also reported that the UBL domain of SHARPIN interacts with the NZF2 domain of HOIP (Fig. 1A) (4). The association of LUBAC components is crucial to the ability of the E3 ligase complex to synthesize M1-Ub, but its regulation remains largely unknown.Figure 1 CYPJ interacts with LUBAC components.A, a schematic diagram showing the relationship among the three subunits of LUBAC complex: HOIP, HOIL-1L, and SHARPIN. B, the intersection of potential proteins interacting with F-CYPJ identified by mass spectrometry in both 293T and HCT116 cells. C, the HA-CYPJ expression plasmid was cotransfected with Flag-HOIP, Flag-HOIL-1L, or Flag-SHARPIN expression plasmids into 293T cells. Lysates were used for IP with 1 μg anti-Flag antibody and an anti-HA antibody was used for the detection of exogenous CYPJ. D, reciprocal coimmunoprecipitation (co-IP) assays determined the association between CYPJ and HOIP. Flag-HOIP and HA-CYPJ expression plasmids were cotransfected into 293T cells, which were then subjected to IP with 1 μg anti-Flag or 1 μg anti-HA antibody and immunoblotting (IB) with the indicated antibodies. E, reciprocal co-IP assays determined the cooperation between CYPJ and SHARPIN. Flag-SHARPIN and HA-CYPJ expression plasmids were cotransfected into 293T cells, which were then subjected to IP with 1 μg anti-Flag or 1 μg anti-HA antibody and IB with the indicated antibodies. F, lysates of 293T cells were subjected to endogenous co-IP assays with the indicated antibodies to detect the binding of CYPJ to HOIP (upper panel) or SHARPIN (lower panel). G, prebleach and postbleach confocal images were achieved from HeLa cells that were cotransfected with the YFP-CYPJ fusion protein (shown in green) and the indicated CFP-HOIP or CFP-SHARPIN fusion proteins (shown in red). FRET efficiency values were calculated based on the mean fluorescence intensity using FRET module. The scale bars represent 10 μm. CFP, cyan fluorescent protein; co-IP, co-immunoprecipitation; CYPJ, cyclophilin J; LUBAC, linear ubiquitin chain assembly complex; HOIL-1L, heme-oxidized iron-regulatory protein 2 ubiquitin ligase 1; HOIP, HOIL-1-interacting protein; IP, immunoprecipitation; SHARPIN, Shank-associated RH domain-interacting protein; YFP, yellow fluorescent protein.

Cyclophilin J (CYPJ), also known as peptidyl prolyl cis/trans isomerase (PPIase)-like 3 (PPIL3), participates in innate immune regulation through disrupting the binding of K63-linked ubiquitin chains to TAB2 and TAB3 and leads to NF-κB attenuation (26). In this study, we found that CYPJ inhibits linear ubiquitin chain synthesis in cells by blocking the interaction between HOIP and SHARPIN. Cypj-deficient mice were highly susceptible to dextran sulfate sodium (DSS)-induced colitis, DSS plus azoxymethane (AOM)-driven CRC. Elevated CYPJ expression was observed in human CRC tissues due to the hypoxic microenvironment, and patients with higher expression of both CYPJ and hypoxia-inducible factor-1α (HIF-1α) have a better prognosis. Our findings identified an unexpected function of CYPJ to protect against inflammation-associated carcinogenesis by mitigating linear ubiquitin chain synthesis.

Results

CYPJ interacts with LUBAC components

To identify the potential proteins which interact with CYPJ, we transfected Flag-CYPJ into 293T or HCT116 cells and immunoprecipitated with a Flag-antibody for mass spectrometry (MS) analysis. LUBAC components, HOIL-1L and SHARPIN were found in the intersection of the two MS results (Figs. 1B and S1, A and B; Table S2). We then performed co-immunoprecipitation (co-IP) assays to verify the binding among CYPJ and three LUBAC components and found that CYPJ binds to SHARPIN and HOIP with higher affinities than HOIL-1L (Figs. 1C and S1C). Thus, we focused on HOIP and SHARPIN. Reciprocal co-IP assays confirmed the cooperation of hemagglutinin (HA)-CYPJ with Flag-HOIP (Figs. 1D and S1D) or Flag-SHARPIN (Figs. 1E and S1E) in 293T cells. Besides, endogenous co-IP assay confirmed the interaction among CYPJ and HOIP/SHARPIN (Fig. 1F) in 293T cells. Furthermore, FRET assays affirmed the direct interaction between the cytoplasmic localization of YFP-CYPJ and CFP-HOIP or CFP-SHARPIN in HeLa cells (Fig. 1G). These data suggest that CYPJ forms a complex with components of LUBAC, especially HOIP and SHARPIN.

CYPJ-N interacts with HOIP-NZF2 and SHARPIN-UBL

To further narrow down the binding regions, we constructed a series of truncations covering CYPJ, HOIP, and SHARPIN (Fig. 2A). We expressed and purified GST-fused full-length (FL) CYPJ as well as three segments named CYPJ-1/2/3 from Escherichia coli and incubated them separately with lysates from Flag-HOIP or Flag-SHARPIN-transfected 293T cells. The GST pull-down results showed that CYPJ-FL, CYPJ-1, and CYPJ-2 bound to HOIP, while only CYPJ-FL and CYPJ-1 bound to SHARPIN (Fig. 2B). In another co-IP assay, we constructed three truncated CYPJ plasmids with sequential deletion of these three fragments and called them CYPJ-Δ1, CYPJ-Δ2, and CYPJ-Δ3 (Fig. 2A). Consistent with the GST pull-down assay results, CYPJ-Δ1 and -Δ2 could not interact with HOIP (Fig. 2C), while only CYPJ-Δ1 could not bind to SHARPIN (Fig. 2D). These results demonstrate that the N terminus fragment 1 of CYPJ binds to both HOIP and SHARPIN.Figure 2 The N terminus of CYPJ interacts with the NZF2 domain of HOIP and the UBL domain of SHARPIN.A, a schematic diagram of CYPJ fragments and the various domains in HOIP and SHARPIN, respectively. Highlights are potential interaction domains. B, GST and GST-fused CYPJ truncations involving FL, CYPJ-1, CYPJ-2, and CYPJ-3 were purified from Escherichia coli. and incubated with lysates of 293T cells transfected with Flag-HOIP or Flag-SHARPIN expression plasmids for the GST pull-down assays. C, Flag-HOIP and GFP-tagged CYPJ fragments were cotransfected into 293T cells to perform a co-IP assay. D, Flag-SHARPIN and GFP-tagged CYPJ fragments were cotransfected into 293T cells to perform a co-IP assay. E, GST and GST-fused HOIP portions containing PUB, 3 × NZFs, UBA, RBR, ZF, NZF1 and NZF2 domains were incubated with 1 μg purified His-CYPJ to perform the GST pull-down assay. F, GST and GST-fused SHARPIN portions containing N-terminal, NZF and UBL domains were incubated with 1 μg purified His-CYPJ to perform GST pull-down assays. G, GFP-CYPJ and Flag-tagged HOIP fragments including FL, ΔZF, ΔNZF1, and ΔNZF2, were cotransfected into 293T cells to perform a co-IP assay. One microgram of anti-Flag antibody was applied to perform IP, whereas anti-GFP antibody was applied to perform IB analysis. H, GFP-CYPJ and Flag-tagged SHARPIN sections including FL, N-terminal, ΔUBL and ΔNZF were cotransfected into 293T cells to perform a co-IP assay. One microgram of anti-Flag antibody was applied to perform IP, whereas anti-GFP antibody was applied to perform IB analysis. co-IP, co-immunoprecipitation; CYPJ, cyclophilin J; FL, full length; IB, immunoblotting; IP, immunoprecipitation; HOIP, HOIL-1-interacting protein; NZF, Npl4 zinc finger; PUB, PNGase/UBA or UBX-containing proteins domain; RBR, RING-in-between-RING; SHARPIN, Shank-associated RH domain-interacting protein; UBA, Ub-associated domain; UBL, ubiquitin-like; ZF, zinc finger.

Next, we aimed to identify the interaction regions in HOIP and SHARPIN. Co-IP assays using HOIP-N and HOIP-C indicated that the N terminus of HOIP has a stronger affinity for CYPJ (Fig. S2A). Then, we further purified GST-fused HOIP-PUB, HOIP-3 × NZFs, HOIP-UBA, and HOIP-RBR together with HOIP-ZF, HOIP-NZF1, and HOIP-NZF2 from E. coli (Fig. 2E). GST pull-down assays showed that HOIP-3 × NZFs and HOIP-NZF2 bind strongly to purified His-CYPJ, while HOIP-ZF and HOIP-NZF1 bind weakly (Fig. 2E). For SHARPIN, a GST pull-down assay showed that its UBL domain binds strongly with purified His-CYPJ, and the NZF domain only has a mild interaction with CYPJ (Fig. 2F). In addition, we performed co-IP experiments to verify the interaction results. We deleted the ZF or NZF domains of HOIP individually (HOIP-ΔZF, HOIP-ΔNZF1, and HOIP-ΔNZF2) or together (HOIP-Δ3 × NZF) (Fig. 2A), and co-IP assays showed that the NZF2 domain of HOIP is the primary domain responsible to interact with CYPJ in 293T cells (Fig. 2G). Another co-IP assay showed that SHARPIN without the UBL domain cannot form a complex with CYPJ in 293T cells (Fig. 2H). Taken together, these results suggest that the N terminus of CYPJ is essential to the interplay with the NZF2 domain of HOIP and the UBL domain of SHARPIN, respectively.

CYPJ limits LUBAC-induced NF-κB activation through impedes the interaction between HOIP and SHARPIN

The E3 activity of HOIP is activated once associated with HOIL-1L or SHARPIN, and the UBL domain of SHARPIN binds to the the UBA and NZF2 domains of HOIP. Our aforementioned data demonstrated that CYPJ primarily interacts with the UBL domain of SHARPIN (Fig. 2H), which impelled us to hypothesize that CYPJ disturbs the HOIP-SHARPIN complex. We expressed and purified the His-tagged or GST-tagged UBL domain of SHARPIN and the UBA/NZF2 domain of HOIP from E. coli (Fig. S2B) and used them to perform GST pull-down assays in different combinations. The results showed that recombinant His-CYPJ disturbs the interaction between the HOIP-UBA/HOIP-NZF2 and SHARPIN-UBL domain (Fig. 3, A and B). We further ectopically expressed HA-CYPJ in 293T cells or two human CRC cell lines (HCT116 and HCT15) and performed endogenous co-IP with a SHARPIN antibody. Immunoblotting (IB) with a HOIP antibody showed that the overexpressed CYPJ reliably interfered with the interaction between HOIP and SHARPIN (Fig. 3C).Figure 3 CYPJ limits LUBAC-induced NF-κB activation through impedes HOIP-SHARPIN complex.A, GST and GST-SHARPIN-UBL protein agarose was incubated with GFP-HOIP-UBA lysates from transfected 293T cells in the absence/presence of 4 μg His-CYPJ to perform pull-down assays. GFP-HOIP-UBA pull-down with GST-SHARPIN-UBL agarose was measured with an anti-GFP antibody in the IB analysis. B, GST and GST-HOIP-NZF2 proteins were incubated with 1 μg His-SHARPIN-UBL in the absence/presence of 4 μg His-CYPJ to perform pull-down assays. His-SHARPIN-UBL pull-down with GST-HOIP-NZF2 agarose was monitored by IB analysis using an anti-His antibody. C, HA-CYPJ alone was transfected into 293T cells (upper panel), HCT116 (middle panel), and HCT15 cells (lower panel) to estimate whether CYPJ affects the binding between endogenous HOIP and endogenous SHARPIN; this was assessed with a co-IP assay using 1 μg anti-SHARPIN antibody and IB analysis using an anti-HOIP antibody. D, Flag-HOIP, Flag-HOIL-1L, and Flag-SHARPIN plasmids were transfected at a ratio of 2:1:1 with or without cotransfection of HA-CYPJ/CYPA into 293T cells. The endogenous linear ubiquitin signal was detected by an antilinear ubiquitin antibody. E, WT and KO bone marrow-derived macrophages (BMDMs) were isolated from WT and KO mice, and WT CYPJ and two CYPJ-deficiency monoclones includes, KO-1 and KO-2 of 293T cells were prepared for the detection of endogenous linear ubiquitin signals. F, effects of HA-CYPJ on NF-κB reporter induced by cotransfection of HOIP-HOIL-1L or HOIP-SHARPIN in 293T cells. G, Flag-HOIP and Flag-SHARPIN expression plasmids with or without HA-tagged CYPJ plasmid were transfected into 293T cells, and the transcription level of TNF and IL-8 was assessed by qRT-PCR. H, Effects of CYPJ re-expression on the activity of the NF-κB reporter induced by HOIP-HOIL-1L or HOIP-SHARPIN in CRISPR-Cas9-mediated CYPJ-KO-1 monoclonal 293T cells. I, Effects of CYPJ re-expression on the activity of NF-κB reporter induced by HOIP-HOIL-1L or HOIP-SHARPIN in CRISPR-Cas9-mediated CYPJ-KO-2 monoclonal 293T cells. J, GFP-CYPJ and its truncations, covering FL, Δ1, Δ2, and Δ3 of CYPJ, were transfected with or without Flag-HOIP and Flag-SHARPIN into KO-1 (upper panel) or KO-2 (lower panel) monoclonal 293T cells. Twenty-fourhours later, the downstream phosphorylation of IκBα and the expression of total IκBα and the indicated proteins were measured by IB analysis. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001. Error bars represent the standard deviation. co-IP, co-immunoprecipitation; CYPA/J, cyclophilin A/J; FL, full length; HA, hemagglutinin; HOIL-1L, heme-oxidized iron-regulatory protein 2 ubiquitin ligase 1; HOIP, HOIL-1-interacting protein; IB, immunoblotting; LUBAC, linear ubiquitin chain assembly complex; NF-κB, nuclear factor kappa-B; NZF, Npl4 zinc finger;qRT-PCR, quantitative real-time PCR; SHARPIN, Shank-associated RH domain-interacting protein; TNF, tumor necrosis factor; UBA, Ub-associated domain; UBL, ubiquitin-like.

The linear ubiquitin chain has been reported to contribute to the activation of canonical NF-κB signaling, and LUBAC is the unique identifiable E3 that is responsible for the synthesis of linear ubiquitin in cells. Given the fact that CYPJ blocks the relationship between HOIP and SHARPIN, we reasonably speculated that CYPJ may reduce the linear ubiquitin signal. As shown in Fig. S2C, our linear ubiquitin antibody shows high specificity of M1-Ub signal. As expected, CYPJ but not its family member cyclophilin A (CYPA) strongly curbs the M1-Ub signal which is synthesized by the LUBAC complex in 293T and HCT116 cells (Fig. 3D; Fig. S2D). Besides, we detected the endogenous linear ubiquitin chains in the bone-derived macrophages (BMDMs) and found that Cypj-deficiency increased the endogenous M1-Ub signal, similar result was also shown in the 293T cells (Fig. 3E).

Next, we tested and verified whether CYPJ suppressed NF-κB downstream signaling through destroying LUBAC activity. First, we performed a luciferase reporter assay and found that CYPJ decreases both HOIP and HOIL-1L, HOIP and SHARPIN-induced NF-κB activation with relatively stronger inhibition of HOIP and SHARPIN (Figs. 3F and S2E). The catalytically dead mutants of CYPJ/CYPA attenuated HOIP and SHARPIN-induced NF-κB activation at levels similar to that of their WT proteins (Fig. S2F), suggesting that their PPIase activity is dispensable for the suppression. In addition, CYPJ overexpression repressed the transcription of the NF-κB target genes TNF and interleukin 8 (IL-8) induced by HOIP and SHARPIN (Figs. 3G and S2G). Rescuing CYPJ expression in two CYPJ KO monoclonal 293T cells restrains again the activation of HOIP and HOIP-1L or HOIP and SHARPIN-induced NF-κB reporter (Fig. 3, H and I). Subsequently, transfection of CYPJ-FL, CYPJ-Δ2, and CYPJ-Δ3, but not CYPJ-Δ1, greatly reduced HOIP and SHARPIN-induced IκBα phosphorylation in two individual CYPJ KO 293T cell lines (Fig. 3J). Given that the N terminus region 1 of CYPJ mediates its collaboration with both HOIP and SHARPIN (Fig. 2, B–D), these phenomena suggest that this region is also important for the inhibitory function of CYPJ. These results provide a novel mechanism by which CYPJ attenuates linear ubiquitin chain triggered NF-κB activation by impeding the formation of the LUBAC complex.

Given the previous findings that CYPJ limits NF-κB activation through attenuating TAB2/3 mediated K63-linked polyubiquitin signal (26), we compared the interplay among CYPJ and TAB2, TAB3, HOIP, and SHARPIN in a co-IP assay, the result showed that CYPJ binds to TAB2, HOIP, and SHARPIN at similar level (Fig. S3A). Furthermore, CYPJ disrupts the linear ubiquitination of NEMO and RIPK1 (Fig. S3, B and C), hints its biological function other than NF-κB.

CYPJ restricts DSS-induced acute and chronic colitis

NF-κB activation is observed in colonic mucosal tissues of patients with inflammatory bowel diseases (IBD) patients and induces the deterioration of gastrointestinal diseases (27, 28). The inhibitory effect of CYPJ on NF-κB signaling prompted us to evaluate its role in the progression of both acute and chronic colitis in mice. In the acute colitis model, WT and KO mice were initially fed with 3% DSS in their drinking water for 1 week and were then given normal water for another week. All eight KO mice died successively but only one out of nine WT mice died on Day 13 (Fig. 4A). In addition, KO mice exhibited more apparent and severe clinical symptoms, including weight loss, stool consistency, and rectal bleeding, than their WT counterparts (Fig. 4, B–D). Besides, we cut the equal portions of the distal colons into 1-cm2 pieces and cultured them for another 24 h to monitor the secretion of Tnf and Il-1β into the cell supernatant, and the ELISA results showed that tissues from the KO mice secreted significantly increased amounts of cytokines compared to those from the WT mice (Fig. 4E).Figure 4 Cypj-deficient mice are highly susceptible to dextran DSS-induced acute and chronic colitis.A–E, Cypj WT (N = 9) and KO (N = 8) mice were challenged with 3% DSS for 7 days and then fed with chow diet and disease progression was assessed every day. A, the survival curves of the two groups were plotted based over at least 15 days. B-C, weight and weight loss in WT (N = 9) and KO (N = 8) mice were assessed by weighing daily. Because of the increased mortality rate, weight evaluations were suspended on day 8. D, average clinical scores, reflecting weight loss, stool consistency, and bleeding in the stool and rectum, were assessed during the disease progression in WT (N = 9) and KO (N = 8) mice. E, distal colon concentration of Tnf and Il-1β from organ culture supernatants of WT (N = 3) and KO (N = 3) mice were monitored by ELISA. F–L WT (N = 9) and KO (N = 9) mice were administered with three cycles of 2.5% DSS for 5 to 7 days and fed with a chow diet for 14 days to achieve chronic colitis. F–G, weight and weight loss in the WT groups was evaluated throughout the recurring DSS model. H, average clinical scores were assessed throughout recurring colitis progression in both WT and KO groups. I, colon lengths (from cecum to rectum) were evaluated in DSS-induced WT (N = 9) and KO (N = 9) mice as well as WT (N = 3) and KO (N = 3) mice fed a chow diet. J, the distal colon concentrations of Tnf and Il-1β were measured by ELISA from the organ culture supernatants of WT (N = 4) and KO (N = 4) mice, respectively. K, representative micrographs of H&E staining of the distal colons of WT and KO mice. The scale bars represent 100 μm. L, endogenous K63-Ub and M1-Ub signals were detected in distal colons isolated from WT (N = 2) and KO (N = 2) mice. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001. Error bars represent the standard deviation. CYPJ, cyclophilin J; DSS, dextran sulfate sodium; IL, interleukin; M1, methionine 1; TNF, tumor necrosis factor.

Next, we assessed the role of CYPJ in a chronic inflammation model by administering three rounds of 2 to 2.5% DSS treatment. Compared to WT mice, KO mice also exhibited significant weight loss (Fig. 4, F and G) and severe clinical features involving weight loss, stool consistency, and rectal bleeding (Fig. 4H) accompanied by narrowing of the colon (Fig. 4I). Likewise, we cultured dissected colon tissues and found increased secretion of Tnf and Il-1β in the KO group (Fig. 4J). Histopathology analysis by H&E staining revealed a more severe disruption of mucosal structures of the colon, creating a diffuse inflammation pattern in KO mice (Fig. 4K). The M1-Ub signal in colon tissues rose after three cycles of DSS administration, and the KO mice showed comparatively higher M1-Ub expression than the WT mice whereas the change of K63-Ub chains was relatively less obvious (Fig. 4L). Together, these data demonstrated that CYPJ is a negative regulator of inflammation and protects mice from DSS-induced acute and chronic colitis.

CYPJ-deficient mice are highly susceptible to inflammation-driven colon tumorigenesis

Chronic inflammation is a risk factor for CRC. Thus, we next evaluated the role of Cypj in the colitis-associated cancer (CAC), a classical inflammation-induced cancer model using the mutagen AOM combined with three rounds of DSS treatments (Fig. 5A). Compared to WT mice, KO mice showed conspicuously increased weight loss in all the three rounds of DSS treatment (Fig. 5, B and C). Following the completion of the CAC model, we found that all macroscopic polyps were distributed in the distal and middle colons of WT and KO groups and were present in 100% of all mice in the model (Fig. 5D). Representative H&E staining of the distal colon indicated that CYPJ deficiency resulted in obviously severe histopathology features coupled with increased infiltration of immune cells and increased proliferation of cancerous cells in the colon (Fig. 5E). The total number of polyps, the number of polyps with diameters greater than 2 mm, the maximum diameter of macroscopic polyps, and the average clinical score which includes weight loss, stool consistency, and bleeding in the stool and rectum were dramatically increased in KO mice compared to their WT counterparts (Fig. 5F). Besides, compared to the WT mice, KO mice harbored a superior production of inflammatory cytokines of Tnf and Il-1β in the distal colon (Fig. 5G). The KO mice also exhibited a higher linear ubiquitin signal than the control mice in colon tissues (Fig. 5H). In addition, we repeated the experiments in female WT and KO mice and the results were similar to those in male mice which showed that there was no sex specificity in the CAC model (Fig. S4, A–G). Moreover, we assessed whether CYPJ could accelerate the growth and migration of two CRC cell lines with higher CYPJ expression, HCT116 and SW620 (Fig. S5A). CRISPR-Cas9-mediated CYPJ KO did not affect cell proliferation and migration in either cell line (Fig. S5, B–K). These results confirmed that CYPJ is a negative regulator of inflammation-driven tumorigenesis and protects mice from colon cancer initiation.Figure 5 Cypj-deficient mice are highly vulnerable to AOM + DSS-driven CRC tumorigenesis.A, schematic diagram of the inflammation-driven CRC model. WT and KO mice received a single peritoneal injection of a single 10 mg/kg AOM immediately prior to the first 2.5% DSS treatment, which was followed by three rounds of 5 to 7 days DSS and 14 days normal drinking water to recovery. B and C, weight and weight loss of WT (N = 5) and KO (N = 5) mice was evaluated throughout the whole AOM + DSS model. D, representative macroscopic polyps (arrows) were photographed in the distal colons and midcolons removed from the two groups. The scale bar represents 1 cm. E, representative micrographs of H&E staining of the distal colons harvested from WT & KO mice. The scale bars represent 100 μm. F, the number/size of macroscopic polyps, macroscopic polyps with diameters greater than 2 mm, the maximal diameter of macroscopic polyps and the average clinical scores were recorded in the colons of WT (N = 5) and KO (N = 5) counterparts. G, the distal colon concentrations of Tnf and Il-1β from organ culture supernatants of WT (N = 4) and KO (N = 4) mice were measured by ELISA. H, K63-linked ubiquitin signal and M1-Ub signal was detected in distal colons isolated from WT (N = 2) and KO (N = 2) mice. The scale bars represent 100 μm. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001. Error bars represent the standard deviation. AOM, azoxymethane; CRC, colorectal cancer; CYPJ, cyclophilin J; DSS, dextran sulfate sodium; IL, interleukin; M1, methionine 1; TNF, tumor necrosis factor.

Hypoxia induced CYPJ expression in human CRC tissues

To further investigate the clinical relevance of CYPJ in CRC, we analyzed the mRNA abundance of CYPJ in The Cancer Genome Atlas (TCGA) database and found that CYPJ was upregulated in 286 tumor tissues compared to 41 adjacent normal tissues (Fig. 6A). We also analyzed the expression of CYPJ in the TCGA database and found CYPJ is highly expressed in CRC, and the high expression of CYPJ at early stage of tumor is also significantly positively associated with cancer stages and nodal metastasis (Fig. S7A). In addition, we randomly and blindly selected six pairs of CRC and normal tissues and detected the CYPJ level by IB analysis, and the results confirmed higher CYPJ levels in cancer samples than in paired normal tissues (Fig. 6B). Besides, immunohistochemistry (IHC) assays in 111 paired CRC tissues (Fig. 6C) showed that the abundance of CYPJ was apparently higher in tumor tissues than in adjacent normal tissues (Fig. 6D).Figure 6 Hypoxia is a potential factor contributing the high expression of CYPJ.A, the expression profile of CYPJ in various cancer types and normal tissues was identified through the GEPIA website. B, the expression of CYPJ in six paired random CRC cancer and normal tissues was assessed by IB analysis. Relative densitometry of CYPJ in three independent experiments was calculated by Image J software C, the representative expression levels of CYPJ were measured by IHC staining. The scale bars represent 200 μm (10×) or 40 μm (40×). D, Immunohistochemistry (IHC) analysis scores of CYPJ in matched CRC cancer and adjacent normal tissues (N = 111). E, The expression of endogenous HIF-1α, CYPJ, and CYPA in DLD1 and HCT115 cells was evaluated by WB analysis under hypoxia culture (0.4% O2) for 1 h, 2 h and 4 h, respectively (left). Relative densitometry of CYPJ in three independent experiments was calculated by Image J software (right). F–I, Endogenous protein levels of HIF-1α, CYPJ, and CYPA were evaluated by IB analysis under 200 μm CoCl2 treated for indicated times in DLD1 (F), HCT15 (G), HCT116 (H), and SW620 (I) cell lines, respectively. The densitometry in Fig. 6B and E-I was calculated by Image J software. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001. Error bars represent the standard deviation. CRC, colorectal cancer; CYPA/J, cyclophilin A/J; HIF-1α, hypoxia-inducible factor-1α; IB, immunoblotting; WB, Western blot.

It has been reported that cyclophilin family proteins are induced under stress conditions, such as hypoxia (29), so we explored whether this mechanism mediates the upregulation of CYPJ in colon cancer. We cultured the human colon cell lines DLD1 and HCT15 with low CYPJ expression (Fig. S5A) under hypoxic conditions (1% O2) for 1 h, 2 h, and 4 h, respectively. IB analysis showed that the protein level of CYPJ was significantly increased together with the HIF-1α that was stabilized under low-oxygen conditions as expected (Fig. 6E). CoCl2 treatment, whose effects mimic those of hypoxic culture conditions, also induced CYPJ expression in DLD1, HCT15, HCT116, and SW620 cells (Fig. 6, F–I). Furthermore, we found hypoxia stabilized the protein level of CYPJ through inhibiting its K48-linked ubiquitination (Fig. S6, A–C). These results implied hypoxia is a potential factor that accounts for the high expression of CYPJ in the tumor microenvironment.

To further explore the interaction between HIF-1α and CYPJ during the development of CRC, we performed the IHC staining of HIF-1α in the 111 paired CRC patient samples and found the protein level of HIF-1α was also increased in tumor tissues compared with their adjacent normal mucosal tissues (Fig. 7A). In addition, the IHC scores of HIF-1α and CYPJ showed a positive association based on their staining abundance (Fig. 7B; Pearson R = 0.3126). In 88 CRC samples with prognostic information, Kaplan–Meier survival analysis indicated that high expression of HIF-1α predicts a poor prognosis whereas CYPJ alone is not a prognostic prediction factor (Fig. 7, C and D). Interestingly, among CRC patients with high HIF-1α levels, patients with high expression of CYPJ have preferable overall survival (p ˂ 0.05) and PFS p ˂ 0.05) (Fig. 7, E–G). Moreover, in the TCGA database, we found high expression level of CYPJ correlated with low expression of canonical NF-κB pathway in CRC patients, which is consistent with our study (Fig. S7, B and C).Figure 7 Simultaneously high-expression of HIF-1α and CYPJ predicts a good prognosis in CRC samples.A, the representative expression levels of HIF-1α were measured by IHC staining. The scale bars represent 100 μm (10×) or 20 μm (40×). B, the correlation analysis based on the IHC scores of HIF-1α and CYPJ in matched CRC cancer and adjacent normal tissues (N = 111) was assessed by the Pearson R ratio. C–E, Kaplan–Meier analysis of overall survival and progression-free survival according to the IHC scores of CYPJ and/or HIF-1α in 88 CRC patients. F, the representative high/low expression levels of HIF-1α/CYPJ were measured by IHC staining. The scale bars represent 100 μm or 20 μm. G, statistical form of CRC patients based on the IHC staining abundance of CYPJ/HIF-1α. H, proposed working model of CYPJ in CRC tumorigenesis through attenuating linear ubiquitin chains in NF-κB signal pathway. CRC, colorectal cancer; CYPJ, cyclophilin J; HIF-1α, hypoxia-inducible factor-1α; IHC, immunohistochemistry; NF-κB, nuclear factor kappa-B.

Overall, CYPJ is a hypoxia-inducible tumor suppressor that hinders HOIP/SHARPIN-induced NF-κB activation and plays important roles in restraining tumorigenesis (Fig. 7H). These data also suggest a potential diagnostic and therapeutic value of CYPJ in inflammation and cancer.

Discussion

Cyclophilins are involved in diverse biological processes and were originally identified as intracellular receptors of the immunosuppressive drug cyclosporine A. They belong to the PPIase family and work as chaperones to ensure the correct folding and proper function of proline-rich proteins (30). The earliest discovered and also the most abundant cyclophilin, CYPA, was identified in 1984 and has been studied extensively, but the function of CYPJ remains largely unknown. At present, the PPIase-independent function of cyclophilin also remains to be explored. In our previous study, we reported that infection and inflammation induced CYPJ forms a complex with NZF domain of TAB2/3 through its C terminus, thus blocking K63-linked ubiquitin chain sensing (26). In this study, we further identified that hypoxia induced CYPJ interacts with and inhibits LUBAC through its N terminus to restrain M1-linked linear ubiquitin chain synthesis. Both processes ultimately repressed NF-κB activation. The expressions of TAB2/3 and LUBAC components, as well as the activity of K63- and M1-ubiquitination systems, vary largely according to cell type, physiological/pathological state, or different cell stress stimuli such as inflammation and hypoxia. CYPJ may act on the dominant axis to balance the NF-κB signaling thus avoiding excessive inflammatory responses harmful to the body.

IBD and CRC are the two most common intestinal diseases in the gut, and patients with IBD are at a high-risk of CRC in addition to osteoporosis, venous thromboembolism, and cardiovascular disease (31, 32). Long-term chronic and recurring intestinal inflammation is likely to be carcinogenic (33). Cyclophilins have been implicated in many pathological processes and diseases, including infection, inflammation, and cancers. For instance, CYPA is typically augmented under inflammatory stimuli, including, in ulcerative colitis patients (34). The increase in CYPA in lymphocytes, especially macrophages, is involved in apoptosis, autophagy, and cytokine secretion. CYPA expression is significantly enhanced in CRC tissues compared to normal colon tissues, and the knockdown of CYPA apparently suppresses cell migration and invasion through the attenuation of epithelial-mesenchymal transition (35). Cyclophilin B (CYPB) is also elevated in hepatocellular carcinoma and CRC, and its transcription is induced by activation of transcription factor 6 under hypoxic conditions. Silencing of CYPB results in decreased tumor cell proliferation, migration, and metastasis (36); peptidyl-prolyl isomerase-like 1 (PPIL1) is more highly expressed in colon cancer cells (37) than in adjacent normal colon mucosa cells, and all these findings reveal that cyclophilins can be potential targets for the treatment of CRC and other cancers. However, most of the literature is dependent on clinical observations as well as cancer cell line and xenograft nude mouse studies; thus, additional physiological functions of cyclophilins remain to be ruled out.

CYPJ or PPIL3 was first identified in the human fetal brain (38) and was found to be highly expressed in the liver. Elevated expression of CYPJ is observed in gastric cancer tissue samples and is associated with the progression and prognosis of patients (39). CYPJ also accelerates hepatocellular carcinoma tumor cell growth by regulating the progression of the cell cycle from G1 to S phase via upregulation of cyclin D1, and inhibition of CYPJ with siRNA or its inhibitor cyclosporine A exhibits a therapeutic effect (40, 41). These studies recognized CYPJ as an oncogene that largely relies on its PPIase activity. However, to our knowledge, there is no report on the roles of CYPJ in inflammation-associated colon cancer and primary tumor models in mice. In this study, we identified CYPJ as an extraordinary tumor suppressor that participates in DSS-induced colitis and CAC and mitigates the initiation and progression of CRC in vivo. Interestingly, CYPJ KO does not obviously affect the proliferation and migration of CRC cell lines, including HCT116 and SW620, and PPIase activity is not needed for CYPJ-mediated inhibition of the NF-κB signaling. This phenomenon suggests that CYPJ does not simply affect CRC cell growth or movement in vitro, but it inhibits tumorigenesis which involves complex factors such as the contact of different types of cells, the immune microenvironment, and so on. Further studies are needed to determine at which step CYPJ restrains the development of CRC.

Previous studies indicated that PPIase superfamily members such as CYPA (34, 42), CYPB (36, 43) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) (44) are highly expressed in different cancer types and are related to HIF-1α under tumor-induced hypoxic conditions. In our study, we found that the abundance of CYPJ is elevated in CRC patient tissues and linked to the stability of HIF-1α in the tumor microenvironment. A positive correlation of CYPJ and HIF-1α in CRC tissues was observed. The prognosis of patients with high expression of both CYPJ and HIF-1α was much better than that of patients with high HIF-1α coupled with low CYPJ expression. Furthermore, higher expression of CYPJ indicated the downregulation of NF-κB signaling in tumor microenvironment which coincided with the better prognosis in CRC patients.

Taken together, these results suggest that CYPJ is a hypoxia-inducible inhibitor of inflammation that acts as a protective factor in colorectal tumorigenesis through attenuating canonical NF-κB signaling and shed light on the development of antiinflammation and antitumor therapeutics.

Experimental procedures

Plasmids

Plasmid encoding HOIP was provided by Mude Shi (Sun Yat-sen University Cancer Center, Guangzhou, China). The HOIP-associated truncations involve HOIP-PUB (1–294aa), HOIP-3 × NZFs (295–438aa), HOIP-ZF (295–329aa), HOIP-NZF1 (330–379aa), HOIP-NZF2 (380–439aa), HOIP-UBA (480–636aa), and HOIP-RBR (693–1072aa) were kindly gifted by Prof. Felix Randow (University of Cambridge, Department of Medicine, United Kingdom) and then subcloned into pGEX-4T-1 vector (GE HealthCare). In addition, HOIP-N (1–438aa), HOIP-C (480–1072aa), HOIP-UBA (480–636aa), HOIP-RBR (693–1072aa), HOIP-ΔZF (Δ295–329aa), HOIP-ΔNZF1 (Δ330–379aa), HOIP-ΔNZF2 (Δ380–439aa), and HOIP-3 × NZFs (Δ295–438aa) were cloned into pcDNA3.0/Flag vector. Plasmid encoding SHARPIN was optimized and synthesized by Synbio Technologies Inc. The SHARPIN-related plasmids including SHARPIN-N (1–232aa), SHARPIN-UBL (233–304aa), and SHARPIN-NZF (350–374aa) were constructed into pGEX-4T-1 and full-length-SHARPIN (1–387aa), SHARPIN-ΔUBL (Δ233–304aa), and SHARPIN- ΔNZF (Δ350–374aa) were cloned into pcDNA3.0/Flag vector. Plasmid encoding HOIL-1L was generated by PCR from human complementary DNA library. CYPJ and its truncations, CYPJ-mut (R44A&F49A), and CYPA-mut (R55A&F60A) were described before (26).

Antibodies and reagents

The rabbit polyclonal antibodies CYPJ (C-term, AP17078b) and CYPJ (C terminus, ab251870) were purchased from Abgent and Abcam. The mouse linear ubiquitin antibody (MABS451) was bought from Millipore. The rabbit linear ubiquitin antibody (clone 1E3, ZRB2114) and mouse antibody Flag (F3165) were ordered from Sigma-Aldrich. The antibodies against HOIP (# 99633), P-IκBα (Ser 32/36, #9246), and IκBα (#4814) were from Cell Signaling Technology. The mouse antibodies specific for Myc (RM1003V), HA (RM1004V), GAPDH (RM2002V), His (RM1001V), and β-Tubulin (RM2003V) were purchased from New Progress Biotechnology. The rabbit anti-SHARPIN (14626-1-AP), anti-HIF-1α (20960-1-AP), anti-GFP (50430-2-AP), mouse anti-GFP (66002-1-Ig) and anti-β-tubulin (66009-1-Ig) were ordered from Proteintech. Secondary antibodies were purchased from The Jackson Laboratory. The cell lysis buffer (10 × ) (#8903) was also bought from Cell Signaling Technology. Protein G PLUS-Agarose immunoprecipitation reagent (SC-2002) was from Santa Cruz Biotechnology. Glutathione Sepharose 4B (17-0756-01) was from GE HealthCare. The 4′,6-diamidino-2-phenylindole staining kit (KGA215) was ordered from KeyGen Biotechnology. Protein Marker (DB180-10) was bought from MIKX. Protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from TargetMol. The dual-luciferase reporter assay system (E1910) was purchased from Promega.

Cell lines

293T and HeLa cells were ordered from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Gibco, C11995500BT) added 10% fetal bovine serum (FBS) (Gibco, 10270-106) in a 37 °C incubator with 5% CO2. Colorectal cancer cells DLD1, HCT15, and HCT116 were purchased from American Type Culture Collection. DLD1 was cultured in RPMI 1640 (Gibco, C11875500BT), HCT15 was cultured in Dulbecco's modified Eagle's medium and HCT116 was cultured in McCoy’s 5A (KeyGEN, KGM4892N-500) supplied with 10% FBS. BMDMs were isolated from the tibia and femur and induced by 20 ng/ml M-CSF for 5 to 7 days. The conditional medium of BMDMs is RPMI 1640 containing 10% FBS, 100U/ml penicillin, 100 μg/ml streptomycin, 1% GlutaMAX, and 20 ng/ml M-CSF. No cell lines used were found in the database of commonly misidentified cell lines that is mentioned by ICLAC and NCBI Biosample. All the human cell lines applied in this study have been confirmed by short tandem repeat identification. Indicated plasmids were transfected into 293T cells with lipo293 (Beyotime, C0521) or colorectal cancer cells with lipo8000 (Beyotime; C0533). To induce hypoxia, cells were transferred to a specially designed chamber containing 1% O2 and cultured in 5% CO2 incubator at 37 °C for indicated times. Mycoplasma contamination was checked by PCR analysis (Myco-F: 5′-GGG AGC AAA CAG GAT TAG ATA CCC T-3′; Myco-R: 5′-GCA CCA TCT GTC ACT CTG TTA ACC TC-3′) and eliminated by Plasmocin treatment (ant-mpt).

Mass spectrometry

To find potential proteins interaction with CYPJ, Flag-CYPJ was transfected in 293T cells and immunoprecipitated with a Flag antibody and then sent to perform LC-MS/MS in Wininnovate Bio-Tech. In brief, the IP fractions were digested in-gel with trypsin and analyzed on the Q Exactive mass spectrometer (Thermo Fisher Scientific). The tandem mass spectra were searched against UniProt human protein database.

Dual-luciferase reporter assay

Cells were transfected with plasmids encoding NF-κB luciferase reporter together with the Renilla luciferase reporter pRL-TK as well as other indicated plasmids. Twenty-four hours later, 293T cells were harvested and the renilla and firefly luciferase activities were successively measured by a dual-luciferase reporter assay system according to the manufacturer’s guidelines.

293T CYPJ-KO cells

Subsequently, 293T KO cells were generated by CRISPR-Cas9 system according to Zhang Lab. The small guide RNA sequences (http://zlab.bio/guide-design-resources) specific for CYPJ were designed as follows: sg-CYPJ-1F 5′-CAC CGA GGA CAC CCA AAA CAT GTG-3′; sg-CYPJ-1R 5′-AAA CCA CAT GTT TTG GGT GTC CTC-3′; sg-CYPJ-2F 5′-CAC CGT GAC ACT GCA TAC AGA TGT-3′; sg-CYPJ-2R 5′-AAA CAC ATC TGT ATG CAG TGT CAC-3′. The small guide RNA sequences were cloned into lentiCRISPR v2 (Addgene, #52961) and positive KO pool cells were screened by 2 μg/ml puromycin for 2 to 4 days. Then 30 to 50 single cells were seeded into one 96-well plate following identified by sequencing and Western blot analysis.

Quantitative real-time PCR

Total RNA was extracted with TRIzol (Magen, R4801) or (Vazyme, R701-02) according to standard protocol. Complementary DNA was synthesized using the HiScript II Q RT SuperMix (Vazyme, R223-01). SYBR Green Mix (GenStar, A301-10) was used for quantitative real-time PCR (qRT-PCR) assays, and the reaction was performed in a CFX96 thermocycler (Bio-Rad). The Cq values were normalized to the control gene GAPDH or 18S in each individual sample and 2-ΔΔCt values were used to evaluate relative expression changes. The specific primer sequences used for qRT-PCR analysis are listed in table S1.

Immunoprecipitation and Western blot

For immunoprecipitation (IP) or co-IP, cells were washed with PBS and then lysed with 1 × lysis buffer. The supernatants were collected and then incubated with 1 μg antibodies for 1 to 2 h followed by adding 20 to 30 μl protein G agarose at 4 °C overnight. The second day, the agaroses were washed three times with fresh cold IP buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 1% Nonidet P40) or 1 × lysis buffer followed by Western blot (WB) analysis.

For the WB assay, protein samples were prepared, normalized, and equal amounts of samples were boiled for 5 min together with 2 × (FD003) or 5 × loading buffer (FD006) and then used to perform SDS-PAGE. The proteins were transferred onto a 0.22 μm polyvinylidene difluoride membrane (Roche), sealed with 5% nonfat milk for 1 to 2 h, following incubated with appropriate primary antibody at 4 °C overnight. The next day, after washing three times with tris-buffered salined with Tween 20, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and then flushed with enhanced chemiluminescence (FD8000) and visualized by ChemiDoc Touch (Bio-Rad).

GST pull-down assay

GST and GST fusion proteins were expressed in E. coli strain BL21 (TransGen, CD901) and purified by Glutathione Sepharose, His-tagged proteins were expressed in E. coli strain BL21 (DE3) (TransGen, CD601) and purified by Ni-NTA His Bind Resin (7sea Biotech, PAN001-001C). Cell lysis of 293T transfected into indicated plasmids or equivalent His-tagged proteins were collected and incubated with GST or GST-fused proteins in 1 × lysis buffer overnight at 4 °C. The following day, GST-fused agaroses were washed at least three times and then analyzed by WB.

FRET microscopy

We used cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) donor/acceptor pairs to examine interactions beSHARPIN at a subcellular/SHARPIN at a subcellular level. Indicated plasmids encoding YFP-CYPJ and CFP-HOIP or CFP-SHARPIN were cotransfected into HeLa cells. Twenty-four hours later, FRET measurements were evaluated using the acceptor photobleaching method in a laser confocal fluorescence microscopy (ZEISS LSM880). After identifying a cotransfected cell, we selected several boxed regions of interest (ROI) within the cell for acceptor photobleaching: the ROIs were subjected to 10 to 12 exposures to ensure >80% acceptor bleaching efficiency. Nonphotobleached ROI in the same cell were used as a negative control. Prebleach and postbleach image set and generated pseudocolored images showing FRET efficiency values were achieved by the ZEISS FRET module. All images were background-subtracted.

Mice

The Cypj-deficient mice were generated by Shanghai Biomodel Organism Science & Technology Development Co, Ltd using CRISPR-Cas9 gene targeting technology and were confirmed by PCR analysis from the genomic DNA isolated from mice tails. All mice initially used were 20g ± 2g with gender and weight matched. Mice were maintained under a specific pathogen free condition with the approval of Sun Yat-sen University and all animal experiments were raised in line with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The serous concentration of Tnf and Il-1β were measured by ELISA Kit from R&D Systems. H&E staining of distal colon tissues and liver tissues were performed by Servicebio Technology.

DSS-induced colitis and AOM + DSS induced colon cancer

DSS (36–50 kDa) (160110) was bought from MP Biomedicals, and AOM (A5486) was ordered from Sigma-Aldrich. The chronic colitis or recurring colitis model is achieved by feeding the WT/KO mice with three cycles of 2 to 2.5% (wt/vol) DSS dissolved in drinking water for 1 week and recovering for another 2 weeks. Alternatively, the period of DSS administration could be shortened to 4 to 5 days according to the degree of weight loss (45).

For inflammation-driven CRC progression, mice were given a single intraperitoneal (i.p.) injection with 10 mg/kg AOM at the first day, immediately followed by three cycles of 2 to 2.5% DSS administration (typically 7 days DSS and 14 days water). Mice were monitored for body weight, disease progression, and each parameter was scored to generate a semiquantitative clinical score (46).

When the DSS or AOM + DSS model ended, the blood, the colon, and macroscopic polyps were collected. For colon organ culture, the washed and equivalent weight of strips were placed into 24-wells plates for 24 h and then the cell-free supernatants were harvested and assayed for indicated cytokines by ELISA. A total of 20 to 40 μg equal amount of distal colon samples were used for SDS-PAGE, and histopathological analysis was assessed by H&E staining (47).

IHC staining and prognostic value analysis

Protein levels of CYPJ and HIF-1α were detected by IHC and performed by Servicebio Biotechnology. The staining intensity and the percentage of positive area of CYPJ and HIF-1α were evaluated by two independent pathologists who were blinded to the clinical parameters. The final scores of CYPJ and HIF-1α were calculated and recorded according to both the intensity and the percentage in cancer tissue and adjacent normal tissue, respectively. The patients were categorized based on the scores of CYPJ and HIF-1α to do overall survival and progression-free survival analysis in GraphPad Prism, and the Log-rank (Mantel-Cox) test was used to compare difference.

Statistical analysis

All in vitro experiments were performed at least three times, and animal models were carried out at least twice. Statistical analyses were generated by the GraphPad Prism software and two-tailed Student’s t test was used in Figure 3, F and G, Figure 4, D, H, and J, Figure 5, F and G, one-way ANOVA analysis was used in Figure 3, H and I, Figure 4, E and I and Figure 6E, two-way ANOVA analysis was used in Figure 4, B and F, Figure 5B. A p value that was less than 0.05 was considered statistically significant for all data, and p value was indicated by asterisks as followed: ∗p ˂ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and n. s. means no significant, respectively.

Ethics approval

All animal experiments were approved by the Animal Welfare and Ethics Committee of the Sun Yat-sen University Cancer Center Institutional Board (L025501202312007). Experiments involving patient specimens were approved by the Ethics Committee of Sun Yat-sen University Cancer Center Institutional Board (G2023-019-01). Written informed consent was obtained from all the patients. The study methodologies conformed to the tenets of the Declaration of Helsinki. The detailed information of CRC patients was mentioned above (48).

Data availability

Raw data that support the findings of this study have been deposited in the Research Data Deposit database (http://www.researchdata.org.cn) with the Approval Number RDDB2024670089. Any reasonable requests for this study are available from the corresponding author.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Supplementary Table 2

Supplementary Tables and Figures

Acknowledgments

We thank Prof. Felix Randow (University of Cambridge) for kindly gifting HOIP-truncated plasmids.

Author contributions

C. S. and S. C. writing–review and editing; C. S. and S. C. investigation; C. S., C. Y., and S. C. resources; C. S. and S. C. writing–original draft; C. S., C. Y., and J. W. formal analysis; C. S. and S. C. funding acquisition; C. S. data curation; C. S., J. W., Y. M., and L. F. methodology. C. S. and J. W. software; S. C. supervision; C.S. and S. C. project administration; C. S. and S. C. conceptualization.

Funding and additional information

This work was supported by grants from the 10.13039/100014717 National Natural Science Foundation of China (81902845 , 82273045 ), Guangzhou Basic and Applied Basic Research Foundation (SL2023A04J01981 ) and 10.13039/501100021171 Guangdong Basic and Applied Basic Research Foundation (2022A1515011930 ).
==== Refs
References

1 Gerlach B. Cordier S.M. Schmukle A.C. Emmerich C.H. Rieser E. Haas T.L. Linear ubiquitination prevents inflammation and regulates immune signalling Nature 471 2011 591 596 21455173
2 Taraborrelli L. Peltzer N. Montinaro A. Kupka S. Rieser E. Hartwig T. LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L Nat. Commun. 9 2018 3910 30254289
3 Teh C.E. Lalaoui N. Jain R. Policheni A.N. Heinlein M. Alvarez-Diaz S. Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis Nat. Commun. 7 2016 1335
4 Iwai K. Fujita H. Sasaki Y. Linear ubiquitin chains: NF-kappaB signalling, cell death and beyond Nat. Rev. Mol. Cell Biol. 15 2014 503 508 25027653
5 Peltzer N. Rieser E. Taraborrelli L. Draber P. Darding M. Pernaute B. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death Cell Rep. 9 2014 153 165 25284787
6 Ang R.L. Chan M. Legarda D. Sundberg J.P. Sun S.C. Gillespie V.L. Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death Proc. Natl. Acad. Sci. U. S. A. 118 2021 2001602118
7 Wan M. Wang X. Huang C. Xu D. Wang Z. Zhou Y. A bacterial effector deubiquitinase specifically hydrolyses linear ubiquitin chains to inhibit host inflammatory signalling Nat. Microbiol. 4 2019 1282 1293 31110362
8 De Jong M.F. Liu Z. Chen D. Alto N.M. Shigella flexneri suppresses NF-kappaB activation by inhibiting linear ubiquitin chain ligation Nat. Microbiol. 1 2016 16084
9 Boisson B. Laplantine E. Prando C. Giliani S. Israelsson E. Xu Z. Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency Nat. Immunol. 13 2012 1178 1186 23104095
10 Spit M. Rieser E. Walczak H. Linear ubiquitination at a glance J. Cell Sci. 132 2019 jcs208512
11 Rodgers M.A. Bowman J.W. Fujita H. Orazio N. Shi M. Liang Q. The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation J. Exp. Med. 211 2014 1333 1347 24958845
12 Zuo Y. Feng Q. Jin L. Huang F. Miao Y. Liu J. Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling Nat. Commun. 11 2020 1146 32123171
13 Chu Y. Kang Y. Yan C. Yang C. Zhang T. Huo H. LUBAC and OTULIN regulate autophagy initiation and maturation by mediating the linear ubiquitination and the stabilization of ATG13 Autophagy 17 2021 1684 1699 32543267
14 Fu Y. Wang H. Dai H. Zhu Q. Cui C.P. Sun X. OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin Mol. Cell 81 2021 3187 3204.e3187 34157307
15 Zhang H. Zhao X. Guo Y. Chen R. He J. Li L. Hypoxia regulates overall mRNA homeostasis by inducing Met (1)-linked linear ubiquitination of AGO2 in cancer cells Nat. Commun. 12 2021 5416 34518544
16 Guo Y. He J. Zhang H. Chen R. Li L. Liu X. Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression Oncogene 41 2022 4877 4892 36192478
17 Dong K. Wei R. Jin T. Zhang M. Shen J. Xiang H. HOIP modulates the stability of GPx4 by linear ubiquitination Proc. Natl. Acad. Sci. U. S. A. 119 2022 e2214227119
18 Yang Y. Schmitz R. Mitala J. Whiting A. Xiao W. Ceribelli M. Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms Cancer Discov. 4 2014 480 493 24491438
19 Jo T. Nishikori M. Kogure Y. Arima H. Sasaki K. Sasaki Y. LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells Blood 136 2020 684 697 32325488
20 Liu J. Wang Y. Gong Y. Fu T. Hu S. Zhou Z. Structural insights into SHARPIN-mediated activation of HOIP for the linear ubiquitin chain assembly Cell Rep. 21 2017 27 36 28978479
21 Elliott P.R. Leske D. Hrdinka M. Bagola K. Fiil B.K. McLaughlin S.H. SPATA2 links CYLD to LUBAC, activates CYLD, and controls LUBAC signaling Mol. Cell 63 2016 990 1005 27591049
22 Stieglitz B. Rana R.R. Koliopoulos M.G. Morris-Davies A.C. Schaeffer V. Christodoulou E. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP Nature 503 2013 422 426 24141947
23 Smit J.J. Monteferrario D. Noordermeer S.M. van Dijk W.J. van der Reijden B.A. Sixma T.K. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension EMBO J. 31 2012 3833 3844 22863777
24 Shimizu S. Fujita H. Sasaki Y. Tsuruyama T. Fukuda K. Iwai K. Differential involvement of the Npl4 zinc finger domains of SHARPIN and HOIL-1L in linear ubiquitin chain assembly complex-mediated cell death protection Mol. Cell Biol. 36 2016 1569 1583 26976635
25 Noad J. von der Malsburg A. Pathe C. Michel M.A. Komander D. Randow F. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-kappaB Nat. Microbiol. 2 2017 17063
26 Sheng C. Yao C. Wang Z. Chen H. Zhao Y. Xu D. Cyclophilin J limits inflammation through the blockage of ubiquitin chain sensing Nat. Commun. 9 2018 4381 30348973
27 Rhodes J.M. Campbell B.J. Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared Trends Mol. Med. 8 2002 10 16 11796261
28 Ibrahim S. Zhu X. Luo X. Feng Y. Wang J. PIK3R3 regulates ZO-1 expression through the NF-kB pathway in inflammatory bowel disease Int. Immunopharmacol 85 2020 106610
29 Andreeva L. Motterlini R. Green C.J. Cyclophilins are induced by hypoxia and heat stress in myogenic cells Biochem. Biophys. Res. Commun. 237 1997 6 9 9266819
30 Wang P. Heitman J. The cyclophilins Genome Biol. 6 2005 226 15998457
31 Chu T.P.C. Moran G.W. Card T.R. The pattern of underlying cause of death in patients with inflammatory bowel disease in england: a record linkage study J. Crohns Colitis 11 2017 578 585 28453767
32 Mooiweer E. Baars J.E. Lutgens M.W. Vleggaar F. van Oijen M. Siersema P.D. Disease severity does not affect the interval between IBD diagnosis and the development of CRC: results from two large, Dutch case series J. Crohns Colitis 6 2012 435 440 22398065
33 Lee T.W. Editorial: risk of cancer development in IBD-inflammation vs immunosuppression Inflamm. Bowel Dis. 26 2020 460 461 31323095
34 Xu Z. Liu R. Huang L. Xu Y. Su M. Chen J. CD147 aggravated inflammatory bowel disease by triggering NF-kappaB-Mediated pyroptosis Biomed. Res. Int. 2020 2020 5341247
35 Yamamoto T. Takakura H. Mitamura K. Taga A. Cyclophilin a knokdown inhibits cell migration and invasion through the suppression of epithelial-mesenchymal transition in colorectal cancer cells Biochem. Biophys. Res. Commun. 526 2020 55 61 32188574
36 Kim Y. Jang M. Lim S. Won H. Yoon K.S. Park J.H. Role of cyclophilin B in tumorigenesis and cisplatin resistance in hepatocellular carcinoma in humans Hepatology 54 2011 1661 1678 21748762
37 Obama K. Kato T. Hasegawa S. Satoh S. Nakamura Y. Furukawa Y. Overexpression of peptidyl-prolyl isomerase-like 1 is associated with the growth of colon cancer cells Clin. Cancer Res. 12 2006 70 76 16397026
38 Zhou Z. Ying K. Dai J. Tang R. Wang W. Huang Y. Molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (PPIL3) from human fetal brain Cytogenet. Cell Genet. 92 2001 231 236 11435694
39 Gong Z. Mu Y. Chen J. Chu H. Lian P. Wang C. Expression and significance of cyclophilin J in primary gastric adenocarcinoma Anticancer Res. 37 2017 4475 4481 28739742
40 Zhao X. Xia C. Wang X. Wang H. Xin M. Yu L. Cyclophilin J PPIase inhibitors derived from 2,3-quinoxaline-6 amine exhibit antitumor activity Front Pharmacol. 9 2018 126 29520233
41 Chen J. Chen S. Wang J. Zhang M. Gong Z. Wei Y. Cyclophilin J is a novel peptidyl-prolyl isomerase and target for repressing the growth of hepatocellular carcinoma PLoS One 10 2015 e0127668
42 Choi K.J. Piao Y.J. Lim M.J. Kim J.H. Ha J. Choe W. Overexpressed cyclophilin A in cancer cells renders resistance to hypoxia- and cisplatin-induced cell death Cancer Res. 67 2007 3654 3662 17440077
43 Jeong K. Kim K. Kim H. Oh Y. Kim S.J. Jo Y. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells Oncol. Lett. 9 2015 2854 2858 26137159
44 Han H.J. Kwon N. Choi M.A. Jung K.O. Piao J.Y. Ngo H.K. Peptidyl prolyl isomerase PIN1 directly binds to and stabilizes hypoxia-inducible factor-1alpha PLoS One 11 2016 e0147038
45 Wirtz S. Popp V. Kindermann M. Gerlach K. Weigmann B. Fichtner-Feigl S. Chemically induced mouse models of acute and chronic intestinal inflammation Nat. Protoc. 12 2017 1295 1309 28569761
46 Neufert C. Becker C. Neurath M.F. An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression Nat. Protoc. 2 2007 1998 2004 17703211
47 Allen I.C. Wilson J.E. Schneider M. Lich J.D. Roberts R.A. Arthur J.C. NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-kappaB signaling Immunity 36 2012 742 754 22503542
48 Shi W. Ye Z. Zhuang L. Li Y. Shuai W. Zuo Z. Olfactomedin 1 negatively regulates NF-kappaB signalling and suppresses the growth and metastasis of colorectal cancer cells J. Pathol. 240 2016 352 365 27555280
