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.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

Hypoxia-induced PLOD2 promotes clear cell renal cell carcinoma progression via modulating EGFR-dependent AKT pathway activation.

Clear cell renal cell carcinoma (ccRCC) is a type of kidney cancer that is both common and aggressive, with a rising incidence in recent decades. Hypoxia is a key factor that plays a vital role in the tumorigenesis and metastasis of malignancy. However, the precise mechanisms of hypoxia driving ccRCC progression were not totally uncovered. Our study found that hypoxia level was elevated in ccRCC and might be an independent risk factor of prognosis in ccRCC patients. We identified a key protein PLOD2 was induced under hypoxic conditions and strongly associated with poor prognosis in ccRCC patients. When PLOD2 was depleted, the proliferation and migration of ccRCC cells were reduced in vitro and in vivo, while overexpression of PLOD2 had the opposite effect. Mechanically, the study further revealed that PLOD2 was transcriptionally activated by HIF1A, which binds to a specific promoter region of the PLOD2 gene. PLOD2 was also shown to interact with EGFR, leading to the phosphorylation of the receptor. Furthermore, PLOD2 was responsible for binding to the extracellular domain of EGFR, which ultimately activated the AKT signaling pathway, thus promoting the malignant progression of ccRCC. Treatment with the PLOD2 inhibitor Minoxidil significantly suppressed ccRCC progression by inactivating the EGFR/AKT signaling axis. In summary, the findings of this study shed light on the molecular mechanisms behind PLOD2 expression in ccRCC and suggest that it may serve as a potential predictor and therapeutic target for the clinical prognosis and treatment of ccRCC.

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.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

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.

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.

Clinicopathological significances of PLOD2, epithelial-mesenchymal transition markers, and cancer stem cells in patients with esophageal squamous cell carcinoma.

BACKGROUND: To examine the expression level of procollagen-lysine2-oxoglutarate 5-dioxygenase 2 (PLOD2) in esophageal squamous cell carcinoma (ESCC) and analyze its correlation with clinicopathological parameters, in order to explore the mechanism of PLOD2 in regulating invasion and metastasis of ESCC. METHODS: Immunohistochemistry was used to detect the expression level of PLOD2 in tumor tissues and paired adjacent tissues of 172 patients with ESCC, and the relationship between PLOD2 expression and clinicopathological parameters was analyzed. The deposition of collagen fibers in tumor was detected by Sirius red staining. The correlation between tumor stem cells and epithelial-mesenchymal transition (EMT) markers ZEB1 was analyzed by multivariate logistic regression. RESULTS: The expression level of PLOD2 in tumor tissues of patients with ESCC (70.35%, 121/172) was significantly higher than that in paired adjacent tissues (29.65%, 51/172; P < .01). The positive expression rate of PLOD2 in ESCC was related to T classification, lymph node metastasis, and pathological tumor node metastasis of a tumor. The expression rates of ZEB1, CD44, and CD133 in ESCC were correlated with T classification, lymph node metastasis and pathological tumor node metastasis. Scarlet red staining showed that collagen fiber deposition in ESCC tissues with high expression of PLOD2 was significantly higher than that in tissues with low expression of PLOD2 (P < .01). A positive correlation was observed between the expression of PLOD2 and CD133, PLOD2 and CD44, and PLOD2 and N-cadherin (P < .01). Moreover, a negative correlation was noted between the expression of PLOD2 and E-cadherin (P < .01). The combined expression of PLOD2 and ZEB1 were independent prognostic factors for the total survival time of patients with ESCC. CONCLUSION: PLOD2 is highly expressed in ESCC and is closely related to tumor invasion and metastasis. The mechanism of PLOD2 for promoting invasion and metastasis of ESCC may be related to activation of the EMT signaling pathway to promote EMT and tumor stem cell transformation.

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.

Bruck syndrome in 13 new patients: Identification of five novel FKBP10 and PLOD2 variants and further expansion of the phenotypic spectrum.

Bruck Syndrome (BS) is a very rare disorder characterized by osteogenesis imperfecta (OI) associated with congenital contractures and is caused by mutations in FKBP10 or PLOD2 genes. Herein, we describe 13 patients from 9 unrelated Egyptian families with BS. All patients had white sclerae, recurrent fractures, kyphoscoliosis and osteoporosis with variable degrees of severity. Large joint contractures were seen in 11 patients, one patient had contractures of small interphalangeal joints, and one patient had no contractures. Unusual findings noted in individual patients included microcephaly, dental malocclusion, enamel hypoplasia, unilateral congenital dislocation of knee joint, prominent tailbone, and myopathy. Nine different variants were identified in FKBP10 and PLOD2 including five novel ones. FKBP10 variants were found in six families (67%) while PLOD2 variants were identified in three families (33%). The four families, with two affected sibs each, showed inter- and intrafamilial phenotypic variability. In conclusion, we report five novel variants in FKBP10 and PLOD2 thus, expanding the mutational spectrum of BS. In addition, our results expand the phenotypic spectrum, describe newly associated orodental findings, and further illustrate the phenotypic overlap between OI and Bruck syndrome supporting the suggestion of considering BS as a variant of OI rather than a separate entity.

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.

First reported case of fragile foal syndrome type 1 in the Thoroughbred caused by PLOD1 c.2032G>A.

BACKGROUND: Warmblood Fragile Foal Syndrome Type 1 (WFFS) is an autosomal recessive disorder reported previously only in warmbloods and thought to be caused by a variant in the gene procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1, c.2032G>A, p.Gly678Arg). Given the presentation of this Thoroughbred case, we hypothesised that a similar genetic mechanism caused this phenotype. OBJECTIVES: To describe the pathological and genetic findings on a foal presenting to a veterinary practice in the UK with skin lesions similar to other Ehlers-Danlos Syndromes, including those documented for warmbloods with WFFS. STUDY DESIGN: A single case report describing a genetic investigation. METHODS: A Thoroughbred foal presenting as dystocia was euthanised for multiple skin lesions and developmental abnormalities. DNA extracted from the foal was tested for the PLOD1 variant (c.2032G>A, p.Gly678Arg) using the commercially available assay. To confirm causality and further interrogate potential novel causes of Ehlers-Danlos Syndrome, 1799 functional candidate genes, including PLOD1, were analysed using whole genome sequencing data generated from DNA extracted from the foal's muscle. These data were compared to 34 control samples from at least 11 other breeds. Variants were prioritised for further evaluation based on predicted impact on protein function. RESULTS: Post-mortem evaluation concluded that this foal suffered from a condition of collagen dysplasia. The foal was homozygous for the c.2032G>A PLOD1 variant. Only two other missense variants identified from whole genome sequencing data were also computationally predicted to be deleterious to protein function, (NPHP3 c.1253T>C, p.Leu418Pro, EPDR1 c.154G>C, p.Glu52Gln). Neither of these genes have been linked to similar phenotypes, or Ehlers-Danlos Syndrome in humans or other species and thus further investigation of these variants as the cause of EDS was not warranted. MAIN LIMITATIONS: This study is a single case report in the Thoroughbred with no additional cases from this breed yet identified to replicate this finding. CONCLUSIONS: Given the clinical presentation similar to WFFS, homozygosity for the PLOD1 variant, and absence of another more plausible causal variant from the WGS experiment, we conclude that PLOD1 c.2032G>A is the likely cause of this foal's condition. This is the first documented evidence of fragile foal syndrome caused by the PLOD1 variant in a breed outside of warmbloods, the Thoroughbred. We therefore recommend a change in the name of this disorder to fragile foal syndrome type 1 (FFS) and utilisation of genetic testing in Thoroughbreds to avoid producing affected foals.

Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration.

Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.

PLOD1 acts as a tumor promoter in glioma via activation of the HSF1 signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) is a collagen-related lysyl hydroxylase and its prognostic value in glioma patients was verified. However, its biological function in glioma has yet to be fully investigated. The PLOD1 mRNA status and clinical significance in gliomas were assessed via the GEPIA database. Overexpression or targeted depletion of PLOD1 was carried out in the human glioma cell line U87 and verified by western blotting. CCK8 and colony formation assays were implemented to examine the impact of PLOD1 on the proliferative and colony-forming phenotypes of U87 cells. Luciferase reporter assays and HSF1-specific pharmacologic inhibitors (KRIBB11) were employed to determine the regulatory relationship between PLOD1 and heat shock factor 1 (HSF1). High expression of PLOD1 was observed in tissue samples of glioblastoma multiforme (GBM) and brain lower-grade glioma (LGG). GEPIA overall survival further demonstrated that both GBM and LGG patients with high PLOD1 displayed worse clinical outcomes compared with those with low PLOD1. Overexpression and targeted depletion of PLOD1 enhanced and suppressed U87 cell proliferation and colony formation, respectively. Luciferase reporter assays showed that PLOD1 significantly enhanced the transcriptional activity of HSF1 in HEK293T cells. PLOD1 deficiency in U87 cells inhibited HSF1-induced survivin accumulation, whereas KRIBB11 also blocked the PLOD1-overexpressing induced survivin expression. An inhibitor of HSF1 signaling events abolished the increased clonogenic potential caused by PLOD1 overexpression in U87 cells. High expression of PLOD1 can increase the proliferation and colony formation of U87 cells by activating the HSF1 signaling pathway. This study suggested PLOD1/HSF1 as an effective therapeutic target for gliomas.