Determination of cell uptake pathways for tumor inhibitor lysyl oxidase propeptide.
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ABSTRACT:
SUBMITTER: Ozdener GB
PROVIDER: S-EPMC4549800 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.
| S-EPMC2847845 | biostudies-literature
Project description:Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-alpha-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.
| S-EPMC2211633 | biostudies-literature
Project description:The lysyl oxidase family of proteins is primarily known for its critical role in catalyzing extracellular oxidative deamination of hydroxylysine and lysine residues in collagens, and lysine residues in elastin required for connective tissue structure and function. Lysyl oxidases have additional important biological functions in health and disease. While the enzyme domains are highly conserved, the propeptide regions are less uniform, and have biological activity, some of which are independent of their respective enzymes. This review summarizes what has been published regarding the functions of the propeptide regions of this family of proteins in the context of extracellular matrix biosynthesis, fibrosis and cancer biology. Although much has been learned, there is a need for greater attention to structure/function relationships and mechanisms to more fully understand these multifunctional proteins.
| S-EPMC5842187 | biostudies-literature
Project description:Lysyl oxidase (LOX) is an copper dependent amine oxidase enzyme involved in cross linking of collagens and elastins. Lysyl oxidase propeptide (LOX-PP) is 18 kDa region cleaved during maturation of LOX and its anti-tumorigenic role were studied in various cancers. The effect of LOX-PP overexpression in retinoblastoma cancer cells (Y79) using transient transfection of LOX-PP gene accessed for global gene deregulations. The analysis resulted in RB cancer cell death through deregulation of retinoblastoma in cancer, cell cycle, apoptosis, focal adhesion-PI3K-AKT signaling and DNA repair mechanism pathway. Further validations were done using RT-PCR and western blot analysis. Our results provide evidence that inducing apoptosis through AKT-NFκB signaling.
2019-09-01 | GSE119413 | GEO
Project description:Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.
| S-EPMC3858906 | biostudies-literature
Project description:The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcd(Sid) mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcd(Sid) mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-?B RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.
| S-EPMC4457472 | biostudies-literature
Project description:Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine residues in collagen and elastin, key components of connective tissue. LOX is synthesized as an inactive 50 kD pre-proenzyme, and secreted to the extracellular matrix where it is cleaved into an active 32 kD LOX, and an 18kD free propeptide (LOX-PP), purportedly an inhibitor of fibroblast growth factor-2 (FGF-2) signaling. Given that adipocytes are distributed inside the connective tissue, it is likely that LOX-PP has an important regulatory role in adipogenesis, which has not been studied. Using NIH 3T3-L1 cells, we observed that FGF-2 inhibited adipogenesis, and LOX-PP promoted adipogenesis of 3T3-L1 cells in the presence of FGF-2; the expression of peroxisome proliferator-activated receptor (PPAR) ? and CCAAT-enhancer binding protein (C/EBP) ?, two markers of adipogenesis, were enhanced in the presence of LOX-PP. We further observed that LOX-PP down-regulated AKT and ERK1/2, two proliferative signaling proteins down-stream of FGF-2 signaling. Similarly, inhibition of FGF-2 receptor signaling by canofin, a competitive inhibitor of FGF-2 receptor, promoted adipogenesis albeit less effective compared to LOX-PP. To further explore whether LOX-PP promoted adipogenesis through inhibition of FGF-2 signaling, site directed mutagenesis of LOX-PP, resulting in an Arg158 to Gln158 mutation which abolishes the inhibitory activity of LOX-PP to FGF-2 receptor, attenuated the adipogenic promoting properties of LOX-PP. In summary, for the first time, our data show that LOX-PP enhances adipogenesis at least partially through inhibition of FGF-2 receptor signaling. Our data suggest that LOX-PP may serve as a bona fide therapeutic target for regulating adipogenesis and adipose tissue development.
| S-EPMC5358703 | biostudies-literature
Project description:Angiogenesis is a critical process involved in normal physiology. Pathological angiogenesis is observed in vascular diseases and neoplasia. The propeptide domain of LOX (LOX-PP) has been shown to inhibit tumorigenesis in various cancers. In this study, we explored the role of both overexpressed and recombinant LOX-PP in naïve human umbilical vein endothelial cell with the addition of vascular endothelial growth factor (VEGF). Primarily, we observed a significant reduction in the angiogenesis signaling pathways upon LOX-PP overexpression by proteomic analysis. Further functional analysis showed that the VEGF induced cell proliferation, migration, adhesion and tube formation was inhibited by LOX-PP. Moreover, LOX-PP arrested cells at S-phase, reduced F-actin levels and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK). The anti-angiogenic effect of LOX-PP was further confirmed by the reduction in the vascular network formation in chick chorioallantoic membrane (CAM). These results indicate that inhibition of angiogenesis events is not only achieved by overexpressing LOX-PP but also by addition of rLOX-PP. Taken together our findings discovered the anti-angiogenic role of LOX-PP in endothelial cells which suggests that harnessing this potential can be a promising strategy to inhibit angiogenesis.
| S-EPMC6041307 | biostudies-literature
Project description:BackgroundIonizing radiation (IR) is a mainstay of cancer therapy, but irradiation can at times also lead to stress responses, which counteract IR-induced cytotoxicity. IR also triggers cellular secretion of vascular endothelial growth factor, transforming growth factor ? and matrix metalloproteinases, among others, to promote tumor progression. Lysyl oxidase is known to play an important role in hypoxia-dependent cancer cell dissemination and metastasis. Here, we investigated the effects of IR on the expression and secretion of lysyl oxidase (LOX) from tumor cells.MethodsLOX-secretion along with enzymatic activity was investigated in multiple tumor cell lines in response to irradiation. Transwell migration assays were performed to evaluate invasive capacity of naïve tumor cells in response to IR-induced LOX. In vivo studies for confirming IR-enhanced LOX were performed employing immunohistochemistry of tumor tissues and ex vivo analysis of murine blood serum derived from locally irradiated A549-derived tumor xenografts.ResultsLOX was secreted in a dose dependent way from several tumor cell lines in response to irradiation. IR did not increase LOX-transcription but induced LOX-secretion. LOX-secretion could not be prevented by the microtubule stabilizing agent patupilone. In contrast, hypoxia induced LOX-transcription, and interestingly, hypoxia-dependent LOX-secretion could be counteracted by patupilone. Conditioned media from irradiated tumor cells promoted invasiveness of naïve tumor cells, while conditioned media from irradiated, LOX- siRNA-silenced cells did not stimulate their invasive capacity. Locally applied irradiation to tumor xenografts also increased LOX-secretion in vivo and resulted in enhanced LOX-levels in the murine blood serum.ConclusionsThese results indicate a differential regulation of LOX-expression and secretion in response to IR and hypoxia, and suggest that LOX may contribute towards an IR-induced migratory phenotype in sublethally-irradiated tumor cells and tumor progression.
| S-EPMC4223762 | biostudies-literature
Project description:The propeptide (LOX-PP) domain of the lysyl oxidase proenzyme was shown to inhibit the transformed phenotype of breast, lung and pancreatic cells in culture and the formation of Her2/neu-driven breast cancer in a xenograft model. A single nucleotide polymorphism (SNP, rs1800449) positioned in a highly conserved region of LOX-PP results in an Arg158Gln substitution (humans). This arginine (Arg)→glutamine (Gln) substitution profoundly impaired the ability of LOX-PP to inhibit the invasive phenotype and xenograft tumor formation. To study the effect of the SNP in vivo, here we established a knock in (KI) mouse line (LOX-PPGln mice) expressing an Arg152Gln substitution corresponding to the human Arg158Gln polymorphism. Breast cancer was induced in wild-type (WT) and LOX-PPGln female mice beginning at 6 weeks of age by treatment with 7,12-dimethylbenz(a)anthracene (DMBA) in combination with progesterone. Time course analysis of tumor development demonstrated earlier tumor onset and shorter overall survival in LOX-PPGln versus WT mice. To further compare the tumor burden in WT and LOX-PPGln mice, inguinal mammary glands from both groups of mice were examined for microscopic lesion formation. LOX-PPGln glands contained more lesions (9.6 versus 6.9 lesions/#4 bilateral). In addition, more DMBA-treated LOX-PPGln mice had increased leukocyte infiltrations in their livers and were moribund compared with DMBA-treated WT mice. Thus, these data indicate that the Arg→Gln substitution in LOX-PP could be an important marker associated with a more aggressive cancer phenotype and that this KI model is ideal for further mechanistic studies regarding the tumor suppressor function of LOX-PP.
| S-EPMC6692853 | biostudies-literature