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.

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.

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.

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.

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.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

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.

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.

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.

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.

Lysyl hydroxylase 3 is required for normal lens capsule formation and maintenance of lens epithelium integrity and fate.

Lens abnormalities are a major cause of reduced vision and blindness. One mechanism that can lead to reduced lens transparency, i.e. cataract, is abnormal behavior of lens epithelial cells (LECs), the precursors of the transparent lens fiber cells. Here we describe a zebrafish mutation causing the embryonic lens epithelium to generate cellular masses comprising partially differentiated lens fiber cells. We identify the mutant gene as plod3, which encodes for Lysyl hydroxylase 3 (Lh3), an enzyme essential for modification of collagens, including Collagen IV, a main component of the lens capsule. We show that plod3-deficient lenses have abnormal lens epithelium from an early developmental stage, as well as abnormal lens capsules. Subsequently, upregulation of TGFß signaling takes place, which drives the formation of lens epithelial cellular masses. We identify a similar phenotype in Collagen IVα5-deficient embryos, suggesting a key role for the defective lens capsule in the pathogenesis. We propose that plod3 and col4a5 mutant zebrafish can serve as useful models for better understanding the biology of LECs during embryonic development and in formation of lens epithelium-derived cataract.

PLODs are overexpressed in ovarian cancer and are associated with gap junctions via connexin 43.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) play important roles in cancer progression, but their role in ovarian cancer remains elusive. In silico analysis of expression of PLODs in ovarian cancer was performed with reproduction of The Cancer Genome Atlas dataset. PLOD-enriched pathways and related gene(s) were validated by immunohistochemistry (IHC) in 80 ovarian cancer tissue blocks and in vivo xenograft murine models. PLODs (PLOD-1, -2, and -3) were overexpressed in ovarian cancer tissue. Overexpression of individual PLODs showed mutual exclusivity. Each of the three PLODs was differentially expressed between normal and cancer tissue of the ovary. PLOD1 was not prognostic, whereas lower PLOD2 and higher PLOD3 expression were associated with worsened prognosis, respectively. Cases with PLOD overexpression showed enrichment in gap junctions. GJA1 (connexin 43) was significantly overexpressed in cases with PLOD overexpression. IHC in tissue showed the strongest positive correlation between PLOD3 and connexin 43 expression, followed by PLOD2. As per Harmonizome, we selected SKOV3 and CAOV3 cell lines based on constitutive high PLOD1 and PLOD2/PLOD3 expression, respectively for in vitro and in vivo modeling. Only knockdown of PLOD3 was significantly associated with decreased GJA1 expression level in both cell lines. IHC in murine xenograft tumors also showed significantly lower connexin 43 in PLOD3-KD SKOV3 tumors. We conclude that PLODs are generally overexpressed in ovarian cancer and each PLOD may be functionally non-redundant. Association between PLOD3 and gap junctions warrants further investigation.

Involvement of LH3 and GLT25D1 for glucosyl-galactosyl-hydroxylation on non-collagen-like domain of FGL1.

Glucosyl-galactosyl-hydroxylation (GGH) is one type of post-translational modification, which is mainly observed in collagen-like domain-containing proteins. Using LC-MS/MS analysis, we found a GGH-like modification at Lys65 of fibrinogen-like protein 1 (FGL1), although it does not contain a collagen-like domain. To identify the glycosyltransferases responsible for this modification, we established LH3/GLT25D1-knockout FGL1-overexpressing HT1080 cell lines. The result showed that knockout of LH3 or GLT25D1 significantly inhibited the glycosylation. Furthermore, deficiency of GGH by point mutation of the FGL1 protein or knockout of the GGH-related glycosyltransferase reduced FGL1 protein levels. Taken together, these data indicate that Lys65 of FGL1 is glucosyl-galactosyl-hydroxylated by LH3 and GLT25D1. Our results provide novel insights to regulate various FGL1 functions.

Prolyl and lysyl hydroxylases in collagen synthesis.

Collagens are the most abundant proteins in the extracellular matrix. They provide a framework to build organs and tissues and give structural support to make them resistant to mechanical load and forces. Several intra- and extracellular modifications are needed to make functional collagen molecules, intracellular post-translational modifications of proline and lysine residues having key roles in this. In this article, we provide a review on the enzymes responsible for the proline and lysine modifications, that is collagen prolyl 4-hydroxylases, 3-hydroxylases and lysyl hydroxylases, and discuss their biological functions and involvement in diseases.

A Human Pan-Cancer System Analysis of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 3 (PLOD3).

The overexpression of the enzymes involved in the degradation of procollagen lysine is correlated with various tumor entities. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) expression was found to be correlated to the progression and migration of cancer cells in gastric, lung and prostate cancer. Here, we analyzed the gene expression, protein expression, and the clinical parameters of survival across 33 cancers based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC), function annotation of the mammalian genome 5 (FANTOM5), Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA) and The Cancer Genome Atlas (TCGA) databases. Genetic alteration, immune infiltration and relevant cellular pathways were analyzed in detail. PLOD3 expression negatively correlated with survival periods and the infiltration level of CD8+ T cells, but positively correlated to the infiltration of cancer associated fibroblasts in diverse cancers. Immunohistochemistry in colon carcinomas, glioblastomas, and soft tissue sarcomas further confirm PLOD 3 expression in human cancer tissue. Moreover, amplification and mutation accounted for the largest proportion in esophageal adenocarcinoma and uterine corpus endometrial carcinoma, respectively; the copy number alteration of PLOD3 appeared in all cancers from TCGA; and molecular mechanisms further proved the effect of PLOD3 on tumorigenesis. In particular, PLOD3 expression appears to have a tumor immunological effect, and is related to multiple immune cells. Furthermore, it is also associated with tumor mutation burden and microsatellite instability in various tumors. PLOD3 acts as an inducer of various cancers, and it could be a potential biomarker for prognosis and targeted treatment.

TGFß-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts.

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

A hierarchical network of hypoxia-inducible factor and SMAD proteins governs procollagen lysyl hydroxylase 2 induction by hypoxia and transforming growth factor ß1.

Collagens are extracellular matrix (ECM) proteins that support the structural and biomechanical integrity of many tissues. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) encodes the only lysyl hydroxylase (LH) isoform that specifically hydroxylates lysine residues in collagen telopeptides, a post-translational modification required for the formation of stabilized cross-links. PLOD2 expression is induced by hypoxia and transforming growth factor-ß1 (TGF-ß1), well-known stimuli for the formation of a fibrotic ECM, which can lead to pathological fibrosis underlying several diseases. Here, using human and murine fibroblasts, we studied the molecular determinants underlying hypoxia- and TGF-ß1-induced PLOD2 expression and its impact on collagen biosynthesis. Deletion mapping and mutagenesis analysis identified specific binding sites for hypoxia-inducible factors (HIF) and TGF-ß1-activated SMAD proteins on the human PLOD2 gene promoter that were required for these stimuli to induce PLOD2 expression. Interestingly, our experiments also revealed that HIF signaling plays a preponderant role in the SMAD pathway, as intact HIF sites were absolutely required for TGF-ß1 to exert its effect on SMAD-binding sites. We also found that silencing PLOD2 expression did not alter soluble collagen accumulation in the extracellular medium, but it effectively abolished the deposition into the insoluble collagen matrix. Taken together, our findings reveal the existence of a hierarchical relationship between the HIF and SMAD signaling pathways for hypoxia- and TGF-ß1-mediated regulation of PLOD2 expression, a key event in the deposition of collagen into the ECM.

PLOD2 promotes aerobic glycolysis and cell progression in colorectal cancer by upregulating HK2.

The purpose of this study was to characterize the expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a membrane-bound homodimeric enzyme that specifically hydroxylates lysine in the telopeptide of procollagens, and assess the clinical significance of PLOD2 in colorectal cancer (CRC). Our results show that PLOD2 is highly expressed in CRC tumor tissues and cell lines, both at the mRNA and protein levels. Next, we found that PLOD2 was positively correlated with tumor grade (P = 0.001), T stage (P = 0.001), N stage (P < 0.001), and an advanced TNM stage (P < 0.001). Knockdown of PLOD2 attenuated CRC cell proliferation, migration, and invasiveness, in vitro. Our analysis of the mechanism behind the effects of PLOD2 suggests that PLOD2 affected glycolysis by regulating the expression of hexokinase 2 (HK2). HK2 reverses the inhibitory effects of PLOD2 knockdown in CRC. Furthermore, the data suggest that PLOD2 regulates the expression of HK2 via the STAT3 signaling pathway. Survival analysis revealed that high expression levels of PLOD2 (HR = 3.800, P < 0.001) and HK2 expression (HR = 10.222, P < 0.001) correlated with the overall survival rate. After analyzing their expression and correlation, PLOD2 positively correlated with HK2 (r = 0.590, P < 0.001). Our findings have revealed that PLOD2 is a novel regulatory factor in glucose metabolism, exerted via controlling HK2 expression in CRC cells, suggesting PLOD2 as a promising therapeutic target for CRC treatment.

IFT20 is critical for collagen biosynthesis in craniofacial bone formation.

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.