Procollagen Lysyl Hydroxylase Research Articles

The role of exogenous glutamine on germination, plant development and transcriptional expression of some stress-related genes in onion under salt stress

ABSTRACT The purpose of this research was to explore the role of glutamine (Gln) on plant germination and growth under salinity [0, 50, 100, 150, 200 mM sodium chloride (NaCl)], and on the activation of some stress-related genes [CuZn-Superoxide Dismutase (SOD), Mn-SOD, L-ascorbate oxidase (AO), DNA Polymerase Delta 1 (POLD)-1, Chaperon (CHAPE) and Heat Shock Protein (HSP)-21], genetic template stability and photosynthetic pigment activation. Under salt stress, 2 mM Gln application reduced the mean germination time (MGT) (4.51 days), increased the coefficient of velocity of germination (CVG) (75.85), germination index (GI) (1.46) and germination percentage (GP) (82%), indicating that it was the best application for germination under stress. Gln promoted the development of aboveground plant organs. 3 mM Gln increased the number of leaves from 5.3 to 13.7 and 4.3 to 6.6 under unstress and stress conditions, respectively. Under salt stress, Gln increased photosynthetic pigments and genomic template stability (GTS) (80%). At 2 mM Gln, total chlorophyll and carotenoid content increased from 1.59 mg · g-1 TA to 2.23 mg · g-1 TA and from 0.28 mg · g-1 TA to 0.37 mg · g-1 TA, respectively. The effect on gene expression levels varied with the concentration. The application of 2 mM Gln, which enhanced germination and vegetative parameters under stress, caused a raise in CuZn-SOD gene expression and a 43% decrease in the transcriptional expression of the HSP-21 gene as an indicator of the functionality of the repair mechanism. The improved growth of Gln-treated plants under stress suggests that Gln is involved in bridging the energy deficit of the plant by acting as an alternative fuel for metabolic activities under stress.

Retraction: Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis.

Retraction: Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis.

Overexpressing PLOD Family Genes Predict Poor Prognosis in Pancreatic Cancer.

BackgroundPancreatic cancer is a common malignant tumor. Multiple studies have shown that procollagen lysyl-hydroxylase (PLOD) family genes were closely related to tumor progression and metastasis in a variety of human cancers. This study aimed to explore the prognosis and biological role of PLOD family genes in pancreatic adenocarcinoma (PAAD).MethodsGEPIA, GEO, HPA, CCLE, Kaplan–Meier plotter, cBioPortal, LinkedOmics, DAVID6.8, STRING, and TIMER were employed to determine the prognostic values and biological function of PLOD family members in PAAD.ResultsThe mRNA and protein expression patterns of PLOD family members were noticeably up-regulated in PAAD compared with normal tissues. PLOD family gene expression was also up-regulated in pancreatic cancer cell lines. PLOD1 was correlated with histological and pathological grades of pancreatic cancer. PLOD2 was related to histological grade. The high expression of PLOD1-2 was correlated with the poor overall survival rate and relapse-free survival rate in patients with PAAD. Additionally, PLODs showed high sensitivity and specificity in distinguishing pancreatic cancer from normal tissues. Through the functional enrichment analysis of PLOD-related genes in PAAD, we found that PLODs were enriched in collagen fiber tissue structure, lysine degradation, and collagen biosynthesis. Pathway analysis confirmed that PLODs regulated the proliferation, migration, and metastasis of pancreatic cancer through the RalGEF-Ral signaling pathway. Furthermore, the level of expression of PLOD1-2 was positively correlated with the activity of tumor-infiltrating immune cells, including CD8+T cells, neutrophils, macrophages, and dendritic cells. The level of expression of PLOD3 was inversely correlated with the level of infiltration of CD8+T cells. PLOD1 and PLOD2 were highly expressed in pancreatic cancer tissues with TP53 and KRAS mutations, respectively. However, the level of expression of PLOD3 in SMAD4 wild-type pancreatic cancer was increased.ConclusionThe findings showed that individual PLOD genes or PLOD family genes could be potential prognostic biomarkers for PAAD.

The sex determination gene doublesex regulates expression and secretion of the basement membrane protein Collagen IV

The highly conserved doublesex (dsx) and doublesex/mab-3 related (Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, DsxM in males and DsxF in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane (BM) protein Collagen IV in the fat body. We find that Dsx isoforms regulate the expression of Collagen IV in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase (dPlod) gene, which is involved in the biosynthesis of Collagen IV, as a direct target of Dsx. We further show that Dsx regulates Collagen IV through dPlod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen IV and remotely establish sexual dimorphism.

Hypoxia-induced PLOD1 overexpression contributes to the malignant phenotype of glioblastoma via NF-\u03baB signaling

Procollagen lysyl hydroxylase 1 (PLOD1) is highly expressed in malignant tumors such as esophageal squamous cell carcinoma, gastric cancer, and colorectal cancer. Bioinformatics analysis revealed that PLOD1 is associated with the progression of GBM, particularly the most malignant mesenchymal subtype (MES). Moreover, in the TCGA and CGGA datasets, the mean survival time of patients with high PLOD1 expression was significantly shorter than that of patients with low expression. The clinical samples confirmed this result. Therefore, we aimed to investigate the effect of PLOD1 on the development of mesenchymal GBM in vitro and in vivo and its possible mechanisms. Molecular experiments were conducted on the patient-derived glioma stem cells and found that PLOD1 expressed higher in tumor tissues and cancer cell lines of patients with GBM, especially in the MES. PLOD1 also enhanced tumor viability, proliferation, migration, and promoted MES transition while inhibited apoptosis. Tumor xenograft results also indicated that PLOD1 overexpression significantly promotes malignant behavior of tumors. Mechanistically, bioinformatics analysis further revealed that PLOD1 expression was closely associated with the NF-κB signaling pathway. Besides, we also found that hypoxic environments also enhanced the tumor-promoting effects of PLOD1. In conclusion, overexpression of PLOD1 may be an important factor in the enhanced invasiveness and MES transition of GBM. Thus, PLOD1 is a potential treatment target for mesenchymal GBM or even all GBM.

A hierarchical network of hypoxia-inducible factor and SMAD proteins governs procollagen lysyl hydroxylase 2 induction by hypoxia and transforming growth factor β1

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-β1 (TGF-β1), 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-β1-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-β1-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-β1 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-β1-mediated regulation of PLOD2 expression, a key event in the deposition of collagen into the ECM.

SiMPLOD, a Structure-Integrated Database of Collagen Lysyl Hydroxylase (LH/PLOD) Enzyme Variants.

PLOD genes encode for procollagen lysyl hydroxylase enzymes (LH/PLOD), a family of proteins essential for collagen biosynthesis. Several mutations affect these genes, causing severe disorders, such as Ehlers-Danlos and Bruck syndrome, as well a connective tissue disease with phenotype resembling osteogenesis imperfecta caused by lack of LH3 functions. The recently determined three-dimensional (3D) structures of the full-length human LH3/PLOD3 isoform, together with the structure of a fragment of a viral LH/PLOD homolog, are now allowing molecular mapping of the numerous disease-causing mutations, providing insights often suitable for the interpretation of the resulting disease phenotypes. However, the added value of molecular structure interpretation is affected by the limited accessibility of complex molecular data to scientific communities lacking direct expertise in structural biology. In this work, we present a Structurally-integrated database for Mutations of PLOD genes (SiMPLOD), a publicly-available manually-curated online database with an embedded molecular viewer interface for the visualization and interpretation of LH/PLOD mutations on available molecular models. Each SiMPLOD entry is accompanied by manual annotations extrapolated from literature references and comments about the localization of the amino acid variants on the molecular structure. Additional links to the appropriate online resources for clinically-relevant as well as biochemical data are also provided in a standardized format. The web application is available at http://fornerislab.unipv.it/SiMPLOD. © 2019 American Society for Bone and Mineral Research.

MP85-08 DIRECT REGULATION OF COLLAGEN CROSS-LINKING ENZYMES (LOXL2 AND PLOD2) BY TUMOR-SUPPRESSIVE MICRORNA-26A/B IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2016MP85-08 DIRECT REGULATION OF COLLAGEN CROSS-LINKING ENZYMES (LOXL2 AND PLOD2) BY TUMOR-SUPPRESSIVE MICRORNA-26A/B IN RENAL CELL CARCINOMA Akira Kurozumi, Mayuko Kato, Yusuke Goto, Atsushi Okato, Ryosuke Matsushita, Hideki Enokida, Masayuki Nakagawa, Tomohiko Ichikawa, and Naohiko Seki Akira KurozumiAkira Kurozumi More articles by this author , Mayuko KatoMayuko Kato More articles by this author , Yusuke GotoYusuke Goto More articles by this author , Atsushi OkatoAtsushi Okato More articles by this author , Ryosuke MatsushitaRyosuke Matsushita More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , Masayuki NakagawaMasayuki Nakagawa More articles by this author , Tomohiko IchikawaTomohiko Ichikawa More articles by this author , and Naohiko SekiNaohiko Seki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2274AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our recent studies of microRNA (miRNA) expression signatures of human cancers revealed that microRNA-26a (miR-26a) and microRNA-26b (miR-26b) were significantly reduced in cancer tissues, suggesting these miRNAs act as tumor-suppressors. The aim of this study was to investigate the functional significance of miR-26a and miR-26b in RCC and to identify miR-26a/b-mediated target genes involved in RCC oncogenesis and metastasis. Our present data showed that collagen cross-linking enzymes were direct regulation of these miRNAs. Collagens are major components of the extracellular matrix (ECM) and aberrant stiffening of ECM enhances cell growth, survival, integrin signaling, and focal adhesion formation. METHODS Expression levels of miR-26a/b and their candidate target genes were evaluated in clear cell RCC (ccRCC) clinical specimens (fifteen radical nephrectomy specimens) and RCC cell lines (786-O and A498) by PCR methods. Gain-of-function studies (cell proliferation, migration and invasion assays) were performed using transfection of mature miR-26a/b into RCC cells. Genome-wide gene expression analysis and in silico analysis were applied to investigate molecular targets regulated by miR-26a/b in RCC cells. A luciferase reporter assay was carried out to determine whether 3′UTR of target genes have actual binding sites for miR-26a/b. RESULTS The expression levels of miR-26a/b were significantly reduced in ccRCC clinical specimens and RCC cell lines compared with non-cancerous kidney tissues (P < 0.05). Restoration of miR-26a/b significantly inhibited cancer cell migration and invasion in RCC cell lines, 786-O and A498 (P < 0.01). Collagen cross-linking enzymes, lysyl oxidase-like 2 (LOXL2) and procollagen Lysyl Hydroxylase 2 (PLOD2) were identified as direct target genes of miR-26a/b by genome-wide gene expression analysis and in silico analysis. Silencing of LOXL2 or PLOD2 significantly reduced cancer cell migration and invasion in RCC cells. CONCLUSIONS Downregulation of miR-26a/b cause to overexpression of collagen cross-linking enzymes and promoting ECM stiffening. Overexpression of LOXL2 and PLOD2 might enhance RCC cell metastasis. Elucidation of tumor-suppressive miR-26a/b regulated molecular pathways and targets could provide new information on potential therapeutic strategies in RCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1100 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Akira Kurozumi More articles by this author Mayuko Kato More articles by this author Yusuke Goto More articles by this author Atsushi Okato More articles by this author Ryosuke Matsushita More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Tomohiko Ichikawa More articles by this author Naohiko Seki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Procollagen Lysyl Hydroxylase 2 Expression Is Regulated by an Alternative Downstream Transforming Growth Factor β-1 Activation Mechanism

PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) hydroxylates lysine residues in collagen telopeptides and is essential for collagen pyridinoline cross-link formation. PLOD2 expression and subsequent pyridinoline cross-links are increased in fibrotic pathologies by transforming growth factor β-1 (TGFβ1). In this report we examined the molecular processes underlying TGFβ1-induced PLOD2 expression. We found that binding of the TGFβ1 pathway related transcription factors SMAD3 and SP1-mediated TGFβ1 enhanced PLOD2 expression and could be correlated to an increase of acetylated histone H3 and H4 at the PLOD2 promoter. Interestingly, the classical co-activators of SMAD3 complexes, p300 and CBP, were not responsible for the enhanced H3 and H4 acetylation. Depletion of SMAD3 reduced PLOD2 acetylated H3 and H4, indicating that another as of yet unidentified histone acetyltransferase binds to SMAD3 at PLOD2. Assessing histone methylation marks at the PLOD2 promoter depicted an increase of the active histone mark H3K79me2, a decrease of the repressive H4K20me3 mark, but no role for the generally strong transcription-related modifications: H3K4me3, H3K9me3 and H3K27me3. Collectively, our findings reveal that TGFβ1 induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter other than the currently known TGFβ1 downstream co-activators and epigenetic modifications. This also suggests that additional activation strategies are used downstream of the TGFβ1 pathway, and hence their unraveling could be of great importance to fully understand TGFβ1 activation of genes.

Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis.

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs.

A Rab10-Dependent Mechanism for Polarized Basement Membrane Secretion during Organ Morphogenesis

Basement membranes (BMs) are specialized extracellular matrices that are essential for epithelial structure and morphogenesis. However, little is known about how BM proteins are delivered to the basal cell surface or how this process is regulated during development. Here, we identify a mechanism for polarized BM secretion in the Drosophila follicle cells. BM proteins are synthesized in a basal endoplasmic reticulum (ER) compartment from localized mRNAs and are then exported through Tango1-positive ER exit sites to basal Golgi clusters. Next, Crag targets Rab10 to structures in the basal cytoplasm, where it restricts protein delivery to the basal surface. These events occur during egg chamber elongation, a morphogenetic process that depends on follicle cell planar polarity and BM remodeling. Significantly, Tango1 and Rab10 are also planar polarized at the basal epithelial surface. We propose that the spatial control of BM production along two tissue axes promotes exocytic efficiency, BM remodeling, and organ morphogenesis.

Characterisation of the Drosophila procollagen lysyl hydroxylase, dPlod

The lysyl hydroxylase (LH) family of enzymes has important roles in the biosynthesis of collagen. In this paper we present the first description of Drosophila LH3 (dPlod), the only lysyl hydroxylase encoded in the fly genome. We have characterised in detail the developmental expression patterns of dPlod RNA and protein during embryogenesis. Consistent with its predicted function as a collagen-modifying enzyme, we find that dPlod is highly expressed in type-IV collagen-producing cells, particularly the haemocytes and fat body. Examination of dPlod subcellular localisation reveals that it is an endoplasmic reticulum resident protein, that partially overlaps with intracellular type-IV collagen. Furthermore, we show that dPlod is required for type-IV collagen secretion from haemocytes and fat body, and thus establish that LH3 enzyme function is conserved across widely separated animal phyla. Our findings, and the new tools we describe, establish the fly as an attractive model in which to study this important collagen biosynthesis enzyme.

The Pitx2c N‐terminal domain is a critical interaction domain required for asymmetric morphogenesis

The paired-like homeodomain transcription factor Pitx2c has an essential role in patterning the left-right axis. However, neither its transcriptional targets nor the molecular mechanisms through which it exerts its patterning function are known. Here we provide evidence that the N-terminal domain of Pitx2c is important for this activity. Overexpression of the Pitx2c N-terminus in ovo randomizes the direction of heart looping, the first morphological asymmetry conserved in vertebrate embryos. In addition, the Pitx2c N-terminal domain blocks the ability of Pitx2c to synergize with Nkx2.5 to transactivate the procollagen lysyl hydroxylase (Plod-1) promoter in transient transfection assays. A five amino acid region containing leucine-41 is required for both of these effects. Our data suggest that the Pitx2c N-terminal domain competes with endogenous Pitx2c for binding to a protein interaction partner that is required for the activation of genes that direct asymmetric morphogenesis along the left-right axis.

Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: A transcriptomic and proteomic study

Hypoxia is a characteristic feature of many human pathologies, including cancer. The sustained proliferation rate of tumor cells leads to alterations of the tumor microenvironment, that progressively becomes more acidic, nutrient-deprived, and hypoxic. The reduced partial pressure of oxygen triggers the onset of an adaptive response, aimed at increasing the local oxygen concentration by several complementary actions. Although directly exposed to the blood stream, endothelial cells lining the vascular lumen in tumors also can be exposed to hypoxia and therefore can contribute to the onset of the adaptive response that leads to tumor angiogenesis. Aiming at getting a detailed insight into the oxygen-dependent regulation of the transcriptional program of vascular endothelial cells and at identifying new relevant markers that may be used as targets for therapeutic intervention in tumor angiogenesis, we have performed a broad-range transcriptomic analysis, using the Affymetrix HG-U133A Gene Chips, of mRNA expression levels in human umbilical cord vein endothelial cells (HUVEC), exposed in vitro to hypoxia for different time periods. The transcriptomic analysis was complemented by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels and alternative splicing for some selected extracellular matrix protein genes, and by a proteomic analysis, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and tandem mass spectrometry for protein separation and identification, of hypoxic and normoxic HUVEC whole-cell lysates and subcellular fractions. Our analysis confirmed previous findings on genes whose expression is regulated by oxygen concentration but also identified new genes (e.g., CXCR4, claudin 3, CD24, tetranectin, Del-1, procollagen lysyl hydroxylase 1 and 2) which are transcriptionally upregulated in hypoxic conditions.

PITX2 Isoform-specific Regulation of Atrial Natriuretic Factor Expression: SYNERGISM AND REPRESSION WITH Nkx2.5

PITX2 and Nkx2.5 are two of the earliest known transcriptional markers of vertebrate heart development. Pitx2-/- mice present with severe cardiac malformations and embryonic lethality, demonstrating a role for PITX2 in heart development. However, little is known about the downstream targets of PITX2 in cardiogenesis. We report here that the atrial natriuretic factor (ANF) promoter is a target of PITX2. PITX2A, PITX2B, and PITX2C isoforms differentially activate the ANF promoter. However, only PITX2C can synergistically activate the ANF promoter in the presence of Nkx2.5. We further demonstrate that the procollagen lysyl hydroxylase (PLOD1) promoter is regulated by Nkx2.5. Mechanistically, PITX2C and Nkx2.5 synergistically regulate ANF and PLOD1 expression through binding to their respective DNA elements. Surprisingly, PITX2A activation of the ANF and PLOD1 promoters is repressed by co-transfection of Nkx2.5 in the C3H10T1/2 embryonic fibroblast cell line. Pitx2a and Pitx2c are endogenously expressed in C3H10T1/2 cells, and these cells express factors that differentially regulate PITX2 isoform activities. We provide a new mechanism for the regulation of heart development by PITX2 isoforms through the regulation of ANF and PLOD1 gene expression and Nkx2.5 transcriptional activity.

PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome.

The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.