PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties.

BACKGROUND: Initially discovered for its ability to regenerate ear holes, the Murphy Roth Large (MRL) mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained during adulthood, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that mesenchymal stromal/stem cells (MSC) derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators. METHOD: To address this hypothesis, we compared the secretome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate, Plod2, and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. To assess its contribution to MRL protection against OA, we used an experimental model for osteoarthritis induced by collagenase (CiOA). RESULTS: Among the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2). Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that Plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA. CONCLUSION: Our results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.

METTL3 facilitates renal cell carcinoma progression by PLOD2 m6A-methylation under prolonged hypoxia.

N6-methyladenosine (m6A) is the most prevalent reversible modification in eukaryotic mRNA, and it plays a critical role in tumor progression. The purpose of this study was to investigate the function and regulatory mechanisms of the methyltransferase METTL3 in renal cell carcinoma (RCC). METTL3 expression was upregulated and predicted a poor prognosis in patients with advanced RCC. METTL3 facilitated the proliferation, migration, and invasion of RCC cells, depending on its methylase activity. METTL3 positively regulated the expression of PLOD2, and both genes were triggered under prolonged hypoxia. Mechanistically, hypoxia-induced the binding of HIF-1α to the METTL3 promoter, which enhanced its transcriptional activity. METTL3-mediated m6A modifications of PLOD2 mRNA at 3'UTR region, promoting the translation of PLOD2 protein. Furthermore, silencing METTL3 impaired RCC progression in vitro. In vivo, administration of highly potent and selective METTL3 inhibitor STM2457 showed anti-tumor effects, whereas AAV9-mediated re-transduction of PLOD2 largely abolished the above phenomenon in a subcutaneous mouse model. These findings reveal that hypoxia and HIF-driven METTL3 transcription promote RCC progression by increasing PLOD2 expression in an m6A-dependent manner, suggesting that METTL3 may serve as a novel pharmaceutical intervention for RCC.

SIRT6 deficiency causes ovarian hypoplasia by affecting Plod1-related collagen formation.

SIRT6 is a key member of the mammalian sirtuin family of conserved nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases. Previous studies have shown that SIRT6 can regulate metabolism, DNA damage repair and aging. Ovarian aging process usually share similar mechanisms with general aging, which is characterized by decreases in both numbers of ovarian follicles and the quality of oocytes. It is reported that the expression level of SIRT6 was significantly decreased in the ovaries of aged mice, and the level of SIRT6 was positively correlated with ovarian reserve, indicating that SIRT6 may be potential markers of ovarian aging. However, its biological roles in follicular development are still unclear. Here, we explored the effect of SIRT6 on follicular development and found that ovarian development was interrupted in SIRT6 knockout (KO) mice, leading to disruptions of puberty and the estrus cycle, significant decreases in numbers of secondary and antral follicles, and decreased collagen in the ovarian stroma. Plod1, a lysyl hydroxylase that is vital for collagen crosslinking and deposition, was decreased at both the mRNA and protein levels in SIRT6-deficient ovaries and granulosa cells (GCs). Additionally, we found abnormal estrogen levels in both SIRT6 KO mice and SIRT6 KD GCs, accompanied by decreases in the levels of the estrogen biosynthesis genes Cyp11a1, Cyp19a1, Mgarp, and increases in the levels of TNF-α and NF-κB. These results confirmed the effect of SIRT6 on follicular development and revealed a possible molecular mechanism for SIRT6 involvement in follicular development via effects on estrogen biosynthesis and collagen formation.

Circ-Plod2 destabilizes Mpo mRNA by binding to IGF2BP2 to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

Osteogenic differentiation, proliferation, and/or apoptosis of bone marrow mesenchymal stem cells (BMSCs) are involved in the progression of postmenopausal osteoporosis (PMO). However, circular RNA (circRNA)-mediated changes in the cellular function of BMSCs in PMO are still unclear. This study revealed the excellent ability of circ-Plod2 to promote osteogenic differentiation of BMSCs and its molecular mechanisms. In this study, ovariectomized (OVX) rats and control (Sham) rats were used to simulate PMO. Initially, we found that the expression of circ-Plod2 in OVX BMSCs is down-regulated and the expression of the Mpo gene is up-regulated by sequencing and verification. Further, we confirmed that circ-Plod2 is located in the cytoplasm and belongs to exon-type circRNA. Interestingly, circ-Plod2 promotes Mpo-dependent osteogenic differentiation of BMSCs without affecting proliferation, apoptosis, adipogenic differentiation, or chondrogenic differentiation of BMSCs. Mechanistically, we demonstrated that circ-Plod2 specifically binds IGF2BP2 to form an RNA-protein complex that destabilizes Mpo mRNA. Overexpression of circ-Plod2 in the bone marrow cavity effectively alleviated osteoporosis in OVX rats and inhibited the expression of MPO in BMSCs. Together, this study reveals that circ-Plod2 destabilizes Mpo mRNA by binding to IGF2BP2 to promote osteogenic differentiation of BMSCs to alleviate osteoporosis. The findings of this study may provide biomarkers for the diagnosis of PMO, and may also provide potential strategies for the clinical treatment of PMO.

tDCS Regulates ASBT-3-OxoLCA-PLOD2-PTEN Signaling Pathway to Confer Neuroprotection Following Rat Cerebral Ischemia-Reperfusion Injury.

Humans exhibit a rich intestinal microbiome that contain high levels of bacteria capable of producing 3-oxo-lithocholic acid (3-oxoLCA) and other secondary bile acids (BAs). The molecular mechanism mediating the role of 3-oxoLCA in cerebral ischemia-reperfusion (I/R) injury remains unclear. We investigated the role of 3-oxoLCA in a rat cerebral I/R injury model. We found that the concentrations of 3-oxoLCA within the cerebrospinal fluid were increased following I/R. In the in vitro oxygen-glucose deprivation (OGD) model, the levels of intraneuronal 3-oxoLCA was elevated following OGD insult. We showed that the increase of membrane ASBT (apical sodium-dependent bile acid transporter) contributed to OGD-induced elevation of intraneuronal 3-oxoLCA. Increasing intraneuronal 3-oxoLCA promoted ischemia-induced neuronal death, whereas reducing 3-oxoLCA levels were neuroprotective. Our results revealed that PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenases 2) functioned upstream of PTEN (the phosphatase and tensin homolog deleted on chromosome 10) and downstream of 3-oxoLCA to promote OGD-induced neuronal injury. We further demonstrated that direct-current stimulation (DCS) decreased the levels of intraneuronal 3-oxoLCA and membrane ASBT in OGD-insulted neurons, while bilateral transcranial DCS (tDCS) reduced brain infarct volume following I/R by inhibiting ASBT. Together, these data suggest that increased expression of ASBT promotes neuronal death via 3-oxoLCA-PLOD2-PTEN signaling pathway. Importantly, bilateral tDCS suppresses ischemia-induced increase of ASBT, thereby conferring neuroprotection after cerebral I/R injury.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

The PLOD2/succinate axis regulates the epithelial-mesenchymal plasticity and cancer cell stemness.

Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in cancer progression is not completely understood. Using stable isotope-resolved metabolomics analysis, we showed that the epithelial mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate levels. The treatment with cell-permeable succinate induced mesenchymal phenotypes in mammary epithelial cells and enhanced cancer cell stemness. Chromatin immunoprecipitation and sequence analysis showed that elevated cytoplasmic succinate levels were sufficient to reduce global 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional repression of EMT-related genes. We showed that expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated with elevation of cytoplasmic succinate during the EMT process. Silencing of PLOD2 expression in breast cancer cells reduced succinate levels and inhibited cancer cell mesenchymal phenotypes and stemness, which was accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced cells, suggesting that PLOD2 promotes cancer progression at least partially through succinate. These results reveal the previously unidentified function of succinate in enhancing cancer cell plasticity and stemness.

PLOD2 promotes colorectal cancer progression by stabilizing USP15 to activate the AKT/mTOR signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) has been reported as an oncogenic gene, affecting various malignant tumors, including endometrial carcinoma, osteosarcoma, and gastric cancer. These effects are mostly due to the enhanced deposition of collagen precursors. However, more studies need to be conducted on how its lysyl hydroxylase function affects cancers like colorectal carcinoma (CRC). Our present results showed that PLOD2 expression was elevated in CRC, and its higher expression was associated with poorer survival. Overexpression of PLOD2 also facilitated CRC proliferation, invasion, and metastasis in vitro and in vivo. In addition, PLOD2 interacted with USP15 by stabilizing it in the cytoplasm and then activated the phosphorylation of AKT/mTOR, thereby promoting CRC progression. Meanwhile, minoxidil was demonstrated to downregulate the expression of PLOD2 and suppress USP15, and the phosphorylation of AKT/mTOR. Our study reveals that PLOD2 plays an oncogenic role in colorectal carcinoma, upregulating USP15 and subsequently activating the AKT/mTOR pathway.

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis.

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.

General Characteristics and Promotion Properties of Circular PLOD2 in Patients with Glioma.

BACKGROUND: Circular RNAs are closed endogenous RNAs that are involved in the progression of diverse tumors. Even with the most advanced combined treatments, patients with glioblastoma multiforme have a median survival time of <15 months. This study aimed to investigate the roles of circular PLOD2 (circPLOD2) in glioma tumorigenesis and tumor development and to clarify its tumor-promoting effects by bioinformatics analysis and molecular experiments. METHODS: To determine the characteristics of circPLOD2 expression, quantitative real-time polymerase chain reaction was conducted. Stable knockdown of circPLOD2 was implemented for functional assays. Cell Counting Kit-8 and colony formation assays were used to measure cell proliferation. Transwell assays and tube formation assays were used to evaluate cell invasion and angiogenesis abilities, respectively. An intracranial xenograft model was established to determine the function of circPLOD2 in vivo. Further biochemical and Western blot analyses were conducted to evaluate proteins associated with circPLOD2. RESULTS: circPLOD2 was upregulated in glioma tissues and cells. High expression of circPLOD2 was significantly associated with tumor size, World Health Organization grade, and molecular characteristics of glioma. circPLOD2 deregulation affected glioblastoma multiforme cell proliferation, invasion, and angiogenesis. Knockdown of circPLOD2 inhibited tumorigenesis in vivo. Further biochemical analysis showed that circPLOD2 was involved in oncogenic pathways and correlated with the expression of proteins related to proliferation, invasion, and angiogenesis. CONCLUSIONS: Our data indicate that circPLOD2 promotes glioma tumorigenesis and tumor development in vitro and in vivo and that suppressing circPLOD2 could be a novel therapeutic strategy for glioma.

Effect of Dexamethasone on Adhesion of Human Neutrophils and Concomitant Secretion.

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

circRNA PLOD2 promotes tumorigenesis and Warburg effect in colon cancer by the miR-513a-5p/SIX1/LDHA axis.

Increasing evidence has proved that circRNAs might act as potential biomarkers for tumor diagnosis and prognosis. However, the functions and mechanisms of multiple circRNAs in colon cancer remains unclear. Here, we found circPLOD2 was dramatically upregulated in colon cancer tissue and cell lines. In vitro CCK-8, colony formation and transwell assays, and in vivo tumor transplantation assay were performed and explored that circPLOD2 might promote tumor proliferation, migration and invasion in vitro and in vivo. Moreover, based on the analysis of RNA pull-down, RNA immunoprecipitation, luciferase and rescued assays, we confirmed that the interactions between circPLOD2, miR-513a-5p and SIX1. It suggested that circPLOD2 acted as a sponge of miR-513a-5p to regulate the activation of the target gene SIX1. In addition, as a key transcription factor of Warburg effect related genes, SIX1 was proved to enhance the transcriptional expression of LDHA by chromatin immunoprecipitation assay, thereby regulating glycolysis in colon cancer cells. Therefore, we identified that circPLOD2 promoted colon cancer progression through miR-513a-5p/SIX1/LDHA axis, and acted as a new biomarker for colon cancer prognosis and treatment.

Modulation of blood pressure regulatory genes in the Agtrap-Plod1 locus associated with a deletion in Clcn6.

The AGTRAP-PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt-Sensitive (SS) rat background (SS-Clcn6) resulted in lower diastolic blood pressure compared to SS-WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS-Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS-Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS-WT. Nppb mRNA expression in SS-Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors.

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes.

Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

P3H4 and PLOD1 expression associates with poor prognosis in bladder cancer

PurposeThe prolyl 3-hydroxylase family member 4 gene (P3H4) is involved in the development of human cancers. The association of P3H4 with bladder cancer (BC) prognosis is unclear. This study aimed to analyze the association of P3H4 with BC prognosis.MethodsRNA-Seq data were downloaded from The Cancer Genome Atlas project and BC microarray datasets (GSE13507, GSE31684, and GSE32548) were downloaded from the Gene Expression Omnibus database. We analyzed the differences in P3H4 expression levels between BC tumors and non-tumor tissues and between samples with different clinical information. The association of P3H4 and P3H4-related genes with BC prognosis and the possibility of using P3H4 expression as a prognostic biomarker in BC patients were also analyzed. RevMan was used to perform the meta-analysis.ResultsP3H4 was upregulated in BC tissues compared with the adjacent non-tumor tissues (p = 4.06e−08). Univariate Cox regression analysis and meta-analysis showed that high P3H4 expression level contributed to a poor BC prognosis (Hazard ratio, HR = 1.348, 95% CI 1.140–1.594, p = 4.89e−04; meta-analysis: HR = 1.45, 95% CI 1.10–1.91; p = 9.00e−03). Among the genes related to P3H4, the PLOD1 gene was closely associated with P3H4 expression (r = 0.620, p = 2.49e−44). Also, a meta-analysis showed that PLOD1 expression was associated with a poor prognosis in BC patients (HR = 1.77, 95% CI 1.31–2.38; p = 2.00e−04). (AU)

PLOD2 Is a Prognostic Marker in Glioblastoma That Modulates the Immune Microenvironment and Tumor Progression.

This study aimed to investigate the role of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) in glioblastoma (GBM) pathophysiology. To this end, PLOD2 protein expression was assessed by immunohistochemistry in two independent cohorts of patients with primary GBM (n1 = 204 and n2 = 203, respectively). Association with the outcome was tested by Kaplan−Meier, log-rank and multivariate Cox regression analysis in patients with confirmed IDH wild-type status. The biological effects and downstream mechanisms of PLOD2 were assessed in stable PLOD2 knock-down GBM cell lines. High levels of PLOD2 significantly associated with (p1 = 0.020; p2< 0.001; log-rank) and predicted (cohort 1: HR = 1.401, CI [95%] = 1.009−1.946, p1 = 0.044; cohort 2: HR = 1.493; CI [95%] = 1.042−2.140, p2 = 0.029; Cox regression) the poor overall survival of GBM patients. PLOD2 knock-down inhibited tumor proliferation, invasion and anchorage-independent growth. MT1-MMP, CD44, CD99, Catenin D1 and MMP2 were downstream of PLOD2 in GBM cells. GBM cells produced soluble factors via PLOD2, which subsequently induced neutrophils to acquire a pro-tumor phenotype characterized by prolonged survival and the release of MMP9. Importantly, GBM patients with synchronous high levels of PLOD2 and neutrophil infiltration had significantly worse overall survival (p < 0.001; log-rank) compared to the other groups of GBM patients. These findings indicate that PLOD2 promotes GBM progression and might be a useful therapeutic target in this type of cancer.

Using the yeast three-hybrid system for the identification of small molecule-protein interactions with the example of ethinylestradiol.

Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo.

P3H4 and PLOD1 expression associates with poor prognosis in bladder cancer.

PURPOSE: The prolyl 3-hydroxylase family member 4 gene (P3H4) is involved in the development of human cancers. The association of P3H4 with bladder cancer (BC) prognosis is unclear. This study aimed to analyze the association of P3H4 with BC prognosis. METHODS: RNA-Seq data were downloaded from The Cancer Genome Atlas project and BC microarray datasets (GSE13507, GSE31684, and GSE32548) were downloaded from the Gene Expression Omnibus database. We analyzed the differences in P3H4 expression levels between BC tumors and non-tumor tissues and between samples with different clinical information. The association of P3H4 and P3H4-related genes with BC prognosis and the possibility of using P3H4 expression as a prognostic biomarker in BC patients were also analyzed. RevMan was used to perform the meta-analysis. RESULTS: P3H4 was upregulated in BC tissues compared with the adjacent non-tumor tissues (p = 4.06e-08). Univariate Cox regression analysis and meta-analysis showed that high P3H4 expression level contributed to a poor BC prognosis (Hazard ratio, HR = 1.348, 95% CI 1.140-1.594, p = 4.89e-04; meta-analysis: HR = 1.45, 95% CI 1.10-1.91; p = 9.00e-03). Among the genes related to P3H4, the PLOD1 gene was closely associated with P3H4 expression (r = 0.620, p = 2.49e-44). Also, a meta-analysis showed that PLOD1 expression was associated with a poor prognosis in BC patients (HR = 1.77, 95% CI 1.31-2.38; p = 2.00e-04). CONCLUSIONS: The P3H4 and PLOD1 genes might be used as reliable prognostic biomarkers for BC.

PLOD3 regulates the expression of YAP1 to affect the progression of non-small cell lung cancer via the PKCδ/CDK1/LIMD1 signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3) is a crucial oncogene in human lung cancer, whereas protein kinase C δ (PKCδ) acts as a tumor suppressor. In this study, we aimed to explore the regulation by PLOD3 on the expression of YAP1 to affect the progression of non-small cell lung cancer (NSCLC) via the PKCδ/CDK1/LIMD1 signaling pathway. We found that PLOD3, CDK1, and YAP1 were highly expressed, while LIMD1 was poorly expressed in NSCLC tissues. Mechanistic investigation demonstrated that silencing PLOD3 promoted the cleavage of PKCδ in a caspase-dependent manner to generate a catalytically active fragment cleaved PKCδ, enhanced phosphorylation levels of CDK1, and LIMD1 but suppressed nuclear translocation of YAP1. Furthermore, functional experimental results suggested that loss of PLOD3 led to increased phosphorylation levels of CDK1 and LIMD1 and downregulated YAP1, thereby suppressing the proliferation, colony formation, cell cycle entry, and resistance to apoptosis of NSCLC cells in vitro and inhibiting tumor growth in vivo. Taken together, these results show that PLOD3 silencing activates the PKCδ/CDK1/LIMD1 signaling pathway to prevent the progression of NSCLC, thus providing novel insight into molecular targets for treating NSCLC.