Joni M. Mäki

Inactivation of the Lysyl Oxidase Gene Lox Leads to Aortic Aneurysms, Cardiovascular Dysfunction, and Perinatal Death in Mice

Background—The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. Methods and Results—Murine Lox gene was disrupted by routine methods. Lox−/− mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox−/− aortas were observed, and electron microscopy of the aortic walls of the Lox−/− fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox−/− fetuses. The wall of the aorta in the Lox−/− fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox+/+ aortas. In Lox−/− fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. Conclusions—Lox has an essential role in the development and function of the cardiovascular system. Inactivation of the Lox gene causes structural alterations in the arterial walls, leading to abnormalities in the cardiovascular functions. Alterations in LOX activity may also play a critical role in certain human cardiovascular diseases.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains.

We report the complete cDNA sequence of the human lysyl oxidase-like 4 (LOXL4) gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 756 amino acids long, including a 24-residue signal peptide. The C-terminal region contains a LO domain similar to those of LOX, LOXL, LOXL2 and LOXL3. The N-terminal region has four subregions similar to scavenger receptor cysteine-rich domains that are highly conserved with LOXL2 and LOXL3. The LOXL4 mRNA is approximately 4 kb in size and is expressed in many tissues, the highest levels among the tissues studied being in the skeletal muscle, testis and pancreas. Recombinant LOXL4 expressed in HT-1080 cells was secreted into the culture medium with no evident proteolytic processing.

Cloning and characterization of a fourth human lysyl oxidase isoenzyme.

We report here the complete cDNA sequence and exon-intron organization of the human lysyl oxidase-like (LOXL)3 gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 753 amino acids in length, including a signal peptide of 25 residues. The C-terminal region, residues 529-729, contains a LO domain similar to those in the LOX (the first characterized LO isoenzyme), LOXL and LOXL2 polypeptides. It possesses the putative copper binding sequence, and the lysine and tyrosine residues that form the lysyltyrosyl quinone cofactor. The N-terminal region, which is similar to that in LOXL2 but not those in LOX and LOXL, contains four subregions similar to scavenger receptor cysteine-rich domains and a putative nuclear localization signal. Recombinant LOXL3, expressed in HT-1080 cells, was secreted into the culture medium but was not detected by immunofluorescence staining in nuclei. The LOXL3 mRNA is 3.1 kb in size and is expressed in many tissues, the highest levels among the tissues studied being seen in the placenta, heart, ovary, testis, small intestine and spleen.

Hearts of Hypoxia-inducible Factor Prolyl 4-Hydroxylase-2 Hypomorphic Mice Show Protection against Acute Ischemia-Reperfusion Injury

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2gt/gt) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34–47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2gt/gt hearts. When isolated Hif-p4h-2gt/gt hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2gt/gt hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1α and Hif-2α by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.

LOXL1 and LOXL4 Are Epigenetically Silenced and Can Inhibit Ras/Extracellular Signal-Regulated Kinase Signaling Pathway in Human Bladder Cancer

Promoter hypermethylation is one of the common mechanisms leading to gene silencing in various human cancers. Using a combination of pharmacologic unmasking and microarray techniques, we identified 59 candidate hypermethylated genes, including LOXL1, a lysyl oxidase-like gene, in human bladder cancer cells. We further showed that LOXL1 and LOXL4 are commonly silenced genes in human bladder cancer cells, and this silence is predominantly related to promoter methylation. We also found LOXL1 and LOXL4 gene methylation and loss of expression in primary bladder tumors. In addition, somatic mutations were identified in LOXL4, but not in LOXL1 in bladder cancer. Moreover, reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells leads to a decrease of colony formation ability. Further studies indicated that the overexpression of LOXL1 and LOXL4 could antagonize Ras in activating the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, our current study suggests for the first time that lysyl oxidase-like genes can act as tumor suppressor genes and exert their functions through the inhibition of the Ras/ERK signaling pathway in human bladder cancer.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by ∼2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

The Ras Signaling Inhibitor LOX-PP Interacts with Hsp70 and c-Raf To Reduce Erk Activation and Transformed Phenotype of Breast Cancer Cells

ABSTRACT The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox−/− mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-κB, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.

Activation of cellular chemotactic responses to chemokines coupled with oxidation of plasma membrane proteins by lysyl oxidase

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of cellular LOX activity by preincubation of vascular smooth muscle cells (VSMC) with β-aminopropionitrile (BAPN), the irreversible inhibitor of LOX activity, resulted in the marked suppression of the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. Plasma membranes purified from VSMC not previously exposed to BAPN contained a group of oxidized plasma membrane proteins, including the PDGF receptor, PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to BAPN-free cells, which had been previously exposed to BAPN, restored the profile of oxidized proteins towards that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells was significantly diminished when compared with cells in which oxidation by LOX was prevented by BAPN. The chemotactic responses of LOX knock-out mouse embryonic fibroblasts mirrored those obtained with VSMC treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.