Targeting hepatic ceruloplasmin mitigates nonalcoholic steatohepatitis by modulating bile acid metabolism.

Nonalcoholic steatohepatitis (NASH) is a condition that progresses from nonalcoholic fatty liver disease (NAFLD) and is characterized by hepatic fat accumulation, inflammation, and fibrosis. It has the potential to develop into cirrhosis and liver cancer, and currently no effective pharmacological treatment is available. In this study, we investigate the therapeutic potential of targeting ceruloplasmin (Cp), a copper-containing protein predominantly secreted by hepatocytes, for treating NASH. Our result show that hepatic Cp is remarkedly upregulated in individuals with NASH and the mouse NASH model. Hepatocyte-specific Cp ablation effectively attenuates the onset of dietary-induced NASH by decreasing lipid accumulation, curbing inflammation, mitigating fibrosis, and ameliorating liver damage. By employing transcriptomics and metabolomics approaches, we have discovered that hepatic deletion of Cp brings about remarkable restoration of bile acid (BA) metabolism during NASH. Hepatic deletion of Cp effectively remodels BA metabolism by upregulating Cyp7a1 and Cyp8b1, which subsequently leads to enhanced BA synthesis and notable alterations in BA profiles. In conclusion, our studies elucidate the crucial involvement of Cp in NASH, highlighting its significance as a promising therapeutic target for the treatment of this disease.

Ceruloplasmin regulating fibrosis in orbital fibroblasts provides a novel therapeutic target for Graves' orbitopathy.

PURPOSE: In diagnosing the pathogenesis of Graves' orbitopathy (GO), there is a growing interest in fibrosis generated by orbital fibroblasts (OFs); nevertheless, the involvement of ceruloplasmin (CP) in OFs remains unknown. METHODS: Differentially expressed genes (DEGs) were identified through bioinformatic analysis. OFs were isolated from orbital tissue and identified with immunofluorescent staining. The levels of DEGs were validated in GO tissue samples and TGF-ß-challenged OFs, and CP was selected for the following laboratory investigations. CP overexpression or knockdown was achieved, and cell viability and fibrosis-associated proteins were investigated to assess the cell phenotype and function. Signaling pathways were subsequently investigated to explore the mechanism of CP function in OFs. RESULTS: CP and cathepsin C (CTSC) are two overlapped DEGs in GSE58331 and GSE105149. OFs were isolated and identified through fibrotic biomarkers. CP and CTSC were downregulated in GO tissue samples and TGF-ß-challenged OFs. CP overexpression or knockdown was achieved in OFs by transducing a CP overexpression vector or small interfering RNA against CP (si1-CP or si2-CP) and verified using a qRT-PCR. CP overexpression inhibited cell viability and reduced the levels of α-SMA, vimentin, fibronectin, and collagen I, whereas CP knockdown exerted opposite effects on OFs. CP overexpression inhibited the phosphorylation of Smad3, Erk1/2, p38, JNK, and AKT; conversely, CP knockdown exerted opposite effects on the phosphorylation of factors mentioned above. CONCLUSION: CP was downregulated in GO and suppressed the expression of fibrosis-associated proteins in both GO and normal OFs. CP might serve as a promising therapeutic agent in the treatment regimens for GO.

A role for ceruloplasmin in the control of human glioblastoma cell responses to radiation.

BACKGROUND: Glioblastoma (GB) is the most common and most aggressive malignant brain tumor. In understanding its resistance to conventional treatments, iron metabolism and related pathways may represent a novel avenue. As for many cancer cells, GB cell growth is dependent on iron, which is tightly involved in red-ox reactions related to radiotherapy effectiveness. From new observations indicating an impact of RX radiations on the expression of ceruloplasmin (CP), an important regulator of iron metabolism, the aim of the present work was to study the functional effects of constitutive expression of CP within GB lines in response to beam radiation depending on the oxygen status (21% O2 versus 3% O2). METHODS AND RESULTS: After analysis of radiation responses (Hoechst staining, LDH release, Caspase 3 activation) in U251-MG and U87-MG human GB cell lines, described as radiosensitive and radioresistant respectively, the expression of 9 iron partners (TFR1, DMT1, FTH1, FTL, MFRN1, MFRN2, FXN, FPN1, CP) were tested by RTqPCR and western blots at 3 and 8 days following 4 Gy irradiation. Among those, only CP was significantly downregulated, both at transcript and protein levels in the two lines, with however, a weaker effect in the U87-MG, observable at 3% O2. To investigate specific role of CP in GB radioresistance, U251-MG and U87-MG cells were modified genetically to obtain CP depleted and overexpressing cells, respectively. Manipulation of CP expression in GB lines demonstrated impact both on cell survival and on activation of DNA repair/damage machinery (γH2AX); specifically high levels of CP led to increased production of reactive oxygen species, as shown by elevated levels of superoxide anion, SOD1 synthesis and cellular Fe2 + . CONCLUSIONS: Taken together, these in vitro results indicate for the first time that CP plays a positive role in the efficiency of radiotherapy on GB cells.

Investigation of the Effect of Rhubarb stalks extracts on Mice Exposed to Oxidative Stress.

Normal blood lipid levels have a crucial role in lowering cardiovascular mortality. This study was designed to investigate the effect of aqueous rhubarb extract on serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde, glutathione, and ceruloplasmin levels, as well as glutathione and malondialdehyde levels in the liver, kidney, and heart tissue in mice exposed to oxidative stress. 40 Balb/c mice were randomly allocated into 8 groups (n=5). Group 1: The control group was left eating feed and water without treatment for (15) days. Group 2: A group exposed to oxidative stress by giving hydrogen peroxide at a rate of (0.5%) with drinking water for 15 days. Group 3: A group exposed to oxidative stress induced by hydrogen peroxide at a rate of (0.5%) for 15 days with injecting on the seventh day, daily for a week, with insulin subcutaneously (15) units/kg. Group (4-8): the Groups were exposed to the oxidative stress induced by hydrogen peroxide (0.5%) for 15 days with injecting on the seventh day into the peritoneal cavity with both the cold aqueous and nonprotein extract, the extract of flavonoids at a dose of 400, 400, 0.4, 8.8, 1.96 mg/kg body weight, respectively. All animals were anesthetized on the last day of the experiment, blood samples were obtained for biochemical testing, and tissue samples from the livers were collected for research. The results revealed that the cold crude aqueous, non-proteinous extracts, flavonoids, proteinous precipitate, and proteinous compound caused a significant decrease (P<0.05) in serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde levels in kidney, liver, and heart. The recorded data showed a significant increase (P<0.05) in serum glutathione and ceruloplasmin in serum and glutathione levels in liver, kidney, and heart tissues in male mice exposed to oxidative stress. The results showed that all Rhubarb extracts have an antioxidant effect in mice exposed to oxidative stress.

Relationship between toxicity of cryoprotectants, osmotic and oxidative stresses in awassi ram sperm.

BACKGROUND: The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated. OBJECTIVE: To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm. MATERIALS AND METHODS: Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode's lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2-propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4 degree C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined. RESULTS: Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels. CONCLUSION: Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress. doi.org/10.54680/fr22210110612.

Therapeutic potential of iron modulating drugs in a mouse model of multiple system atrophy.

Multiple System Atrophy (MSA) is a rare neurodegenerative synucleinopathy which leads to severe disability followed by death within 6-9 years of symptom onset. There is compelling evidence suggesting that biological trace metals like iron and copper play an important role in synucleinopathies like Parkinson's disease and removing excess brain iron using chelators could slow down the disease progression. In human MSA, there is evidence of increased iron in affected brain regions, but role of iron and therapeutic efficacy of iron-lowering drugs in pre-clinical models of MSA have not been studied. We studied age-related changes in iron metabolism in different brain regions of the PLP-αsyn mice and tested whether iron-lowering drugs could alleviate disease phenotype in aged PLP-αsyn mice. Iron content, iron-ferritin association, ferritin protein levels and copper-ceruloplasmin association were measured in prefrontal cortex, putamen, substantia nigra and cerebellum of 3, 8, and 20-month-old PLP-αsyn and age-matched non-transgenic mice. Moreover, 12-month-old PLP-αsyn mice were administered deferiprone or ceruloplasmin or vehicle for 2 months. At the end of treatment period, motor testing and stereological analyses were performed. We found iron accumulation and perturbed iron-ferritin interaction in substantia nigra, putamen and cerebellum of aged PLP-αsyn mice. Furthermore, we found significant reduction in ceruloplasmin-bound copper in substantia nigra and cerebellum of the PLP-αsyn mice. Both deferiprone and ceruloplasmin prevented decline in motor performance in aged PLP-αsyn mice and were associated with higher neuronal survival and reduced density of α-synuclein aggregates in substantia nigra. This is the first study to report brain iron accumulation in a mouse model of MSA. Our results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction and provide evidence that targeting iron in MSA could be a viable therapeutic option.

Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase.

Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called "halogenating cycle". Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

[Clinical manifestations of asthma during combination therapy using ceruloplasmin].

AIM: To estimate the time course of clinical changes in patients with asthma during combination therapy using ceruloplasmin (CP). MATERIALS AND METHODS: A total of 92 asthmatic patients were examined. Their medical history data were collected; external lung function testing and clinical, laboratory, and instrumental examinations, involving the determination of the indicators of lipid peroxidation (LPO) (malonic dialdehyde (MDA), methemoglobin, carboxyhemoglobin) and the antioxidant system (superoxide dismutase (COD), sulfhydryl groups), were performed in all the patients over time. According to the therapy used, the patients were divided into 2 groups matched for gender, age, and clinical manifestations of the disease. A study group consisted of 45 patients who took CP in addition to conventional therapy. A comparison group included 47 patients receiving standard therapy. RESULTS: During the combination therapy using CP, the asthmatic patients showed a reduction in the elevated concentrations of MDA, methemoglobin, and carboxyhemoglobin and increases in the activity of COD and in the levels of sulfhydryl groups, which was followed by a considerable clinical improvement. During the conventional therapy, the indicators of LPO remained high and those of the antioxidant system did low, suggesting permanent oxidative stress. CONCLUSION: CP incorporation into the combination therapy of asthmatic patients contributes to elimination of prooxidant-antioxidant imbalance, which is followed by a marked positive clinical effect.

[CERULOPLASMIN CORRECTION OF DYSLIPIDEMIA CAUSED BY CHRONIC PHYSICAL LOAD IN EXPERIMENT AT SUBMAXIMAL POWER.]

Chronic submaximal physical load of aerobic-anaerobic charater leads to pro-atherogenic dyslipidemia, as manifested by increased levels of triglycerides, total cholesterol, and very-low-density lipoproteins. Ceruloplasmin (total dose 60 mg/kg body weight, triple intraperitoneal introduction on the 1st, 3rd, and 5th day of experiment) produced a partial correction of dyslipidemia, manifested by restoration of the normal levels of triglycerides, total cholesterol, and lipoproteins of very low, low, and high density. At the same time, ceruloplasmin administration did not influence the level of phospholipids.

[Reparative regeneration of connective tissue structures of mammals under antioxidant therapy conditions].

The influence of administration of the antioxidant complexes consisting of nonenzymatic antioxidants (alpha-tocopherol acetate preparation) and enzymatic antioxidants (ceruloplasmin) has been studied in rabbits with experimental arthritis. The introduction of alpha-tocopherol acetate (at a daily dose of 4 mg) improved metabolic processes in the organism (decreased in the rate of erythrocyte precipitation, total leukocytes and their stub and segmental forms; increased in erythrocyte count; reduced the glycosaminoglycan content as determined from uronic acid and hexose level; decreased ceruloplasmin activity and malonic dialdehyde level ion blood serum, all at p < 0.05), thus favoring reduction in the total activity of the inflammatory process as judged from hematological and biochemical data. Intra-articular introduction of ceruloplasmin (1.5 mg/kg, once per week) positively influenced the state of joint structures in damaged knee joints of the animals: decreased the activity of ceruloplasmin (from 5.28 ± 0.06 to 3.94 ± 0.01 AU), and malonic dialdehyde level (0.18 ± 0.02 to 0.08 ± 0.01 µM) in the articular fluid (all at p < 0.05). These effects are probably related to the elimination of inefficiency of the antioxidant system in the synovial medium, thus preventing inflammatory destruction of articular tissues, hindering the development of pannus, and assisting the activation of reparative regeneration of connective tissue structures.

Families with Wilson's disease in subsequent generations: clinical and genetic analysis.

INTRODUCTION: Wilson's disease is an inherited autosomal recessive disorder of copper metabolism. The prevalence of Wilson's disease in most populations is approximately 1 in 30,000. The risk for offspring is 0.5%. The aim of this study was to establish the frequency of disease among offspring of a cohort of Wilson's disease patients. MATERIALS AND METHODS: In February 2014, our registry included 760 cases of diagnosed Wilson's disease. We selected families in which Wilson's disease was diagnosed in the proband's offspring. RESULTS: Between 1957 and 2014, 1,050 relatives of affected members were screened. Wilson's disease in subsequent generations was observed in nine non-consanguineous families, with 12 affected offspring from nine probands. CONCLUSION: We detected a higher (4.08%) than expected (0.5%) frequency of Wilson's disease among proband offspring, which is in accordance with a recent genetic study in the United Kingdom that suggested a higher WD prevalence in the European population.

[The effects of ceruloplasmin in PI3K/PTEN cell signaling pathway change induced by silica].

OBJECTIVE: To investigate the roles of ceruloplasmin (Cp) in PI3K/PTEN cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica. METHODS: HELFs transfected with pGenesil1.1 plasmid and pGenesil1.1 with PTEN shRNA (PT) plasmid were successfully established. 100 µg/ml silica and different concentrations of Cp (10, 20, 30 µg/ml) were used in this experiment and Cp were treated cells after exposed to silica for 1h. Three different cell lines (including HELFs, PT and cells were transfected with p85 dominant negative mutant plasmid (DN-p85)) were divided into control groups, silica groups and silica+different concentrations of Cp groups. MTT assay was used to detect the effects of Cp on silica-induced cell proliferation after inhibiting PTEN and p85. When suppressing the expression of PTEN and p85, western blot assay was performed to detect the levels of p85, p110, AKT308, AKT473 and ERK, JNK and their phosphorylated levels. RESULTS: After inhibition of PTEN, the high levels of p85 induced by 100 µg/ml silica with 30 µg/ml Cp were markedly decreased (P<0.05). When suppressing p85, the increased cell proliferation was not observed. And the high levels of AKT308, AKT473, ERK and phosphorylated JNK and ERK stimulated by 100 µg/ml silica with 30 µg/ml Cp were decrease (P < 0.05). CONCLUSION: Cp could further strengthened silica-induced cell proliferation by PI3K/AKT/MAPK cell signaling pathway, of which the level of p85 was regulated by PTEN.

Myeloperoxidase influences the complement regulatory function of modified C-reactive protein.

In patients with active anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV), there are high levels of circulating C-reactive protein (CRP), which can inhibit the alternative complement pathway by binding factor H and triggering the classical complement pathway by binding C1q. However, the alternative, not the classical, complement pathway has been proven to play an important role in AAV. We found that both purified myeloperoxidase (MPO) and MPO released from ANCA-stimulated neutrophils could bind modified CRP (mCRP), but not pentameric CRP. Furthermore, MPO could block the binding between mCRP and factor H, as well as the binding between mCRP and C1q. Binding with mCRP did not influence the enzymatic activity of MPO. Binding with mCRP also did not influence the binding between MPO and its physical inhibitor, ceruloplasmin, as well as the binding between MPO and MPO-ANCA. The results indicated that MPO might be a complement regulator and inhibit the negative regulatory effect of CRP on the alternative complement pathway.

Anti-inflammatory activity of lycopene isolated from Chlorella marina on type II collagen induced arthritis in Sprague Dawley rats.

The role of commercially available lycopene (all-trans) from tomato in controlling arthritis has been reported. Even though many reports are available that the cis form of lycopene is more biologically active, no report seems to be available on lycopene (cis and trans) isolated from an easily available and culturable sources. In the present study, the anti-arthritic effect of lycopene (cis and trans) from the algae Chlorella marina (AL) has been compared with lycopene (all-trans) from tomato (TL) and indomethacin (Indo). Arthritis (CIA) was developed in male Sprague dawley rats by collagen and the following parameters were studied. The activities of inflammatory marker enzymes like cyclooxygenase (COX), lipoxygenase (LOX) and myeloperoxidase (MPO) were found to be decreased on treatment with AL when compared to TL and Indo. Changes in Erythrocyte sedimentation rate (ESR), white blood cell (WBC) count, red blood cells (RBC) count, hemoglobin (Hb), C-reactive protein (CRP), rheumatoid factor (RF), and ceruloplasmin levels observed in the blood of arthritic animals were brought back to normal by AL when compared to TL and Indo. Histopathology of paw and joint tissues showed marked reduction in edema on supplementation of AL. Thus these results indicate the potential beneficiary effect of algal lycopene on collagen induced arthritis in rats when compared to TL and even to the commonly used anti-inflammatory drug indomethacin. Therefore lycopene from C. marina would be recommended as a better natural source with increased activity and without side effects in the treatment of anti-inflammatory diseases.

Ceruloplasmin alters intracellular iron regulated proteins and pathways: ferritin, transferrin receptor, glutamate and hypoxia-inducible factor-1α.

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp. The purpose of the current study was to determine if cultured retinal pigmented epithelial cells (RPE) also secrete Cp. In addition, the effects of exogenously added Cp on iron regulated proteins and pathways, ferritin, transferrin receptor, glutamate secretion and levels of hypoxia-inducible factor-1α in the nucleus were determined. Like LEC, RPE secrete Cp. Cp was found diffusely distributed within both cultured LEC and RPE, but the cell membranes had more intense staining. Exogenously added Cp caused an increase in ferritin levels in both cell types and increased secretion of glutamate. The Cp-induced increase in glutamate secretion was inhibited by both the aconitase inhibitor oxalomalic acid as well as iron chelators. As predicted by the canonical view of the iron regulatory protein (IRP) as the predominant controller of cellular iron status these results indicate that there is an increase in available iron (called the labile iron pool (LIP)) in the cytoplasm. However, both transferrin receptor (TfR) and nuclear levels of HIF-1α were increased and these results point to a decrease in available iron. Such confounding results have been found in other systems and indicate that there is a much more complex regulation of intracellularly available iron (LIP) and its downstream effects on cell metabolism. Importantly, the Cp increased production and secretion of the neurotransmitter, glutamate, is a substantive finding of clinical relevance because of the neural and retinal degeneration found in aceruloplasminemia patients. This finding and Cp-induced nuclear translocation of the hypoxia-inducible factor-1 (HIF1) subunit HIF-1α adds novel information to the list of critical pathways impacted by Cp.

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide.

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CP's putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.

Ceruloplasmin-induced aggregation of P19 neurons involves a serine protease activity and is accompanied by reelin cleavage.

The cytoarchitectural organization of the nervous system depends partly on extracellular serine proteases, including reelin. This 400K protein, which also exists as the N-terminally-derived 300K and 180K fragments, acts through binding to the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR). Ceruloplasmin (CP), a multifunctional protein found in the circulation and also expressed on glial cells, was shown to bind to, and induce aggregation of neurons newly differentiated from P19 embryonic stem cells. This indicated a potential developmental role of CP in neuronal organization, possibly in relation with reelin and other extracellular serine proteases. Therefore, we analysed the effect of cell-impermeant, large spectrum, serine protease inhibitors on CP-induced neuroaggregation and studied reelin expression. Soybean trypsin inhibitor and aprotinin (SBTI+Apro) inhibited CP neuroaggregative action. Undifferentiated and neurally-differentiating cultures secreted the 400K reelin. The 180K fragment was present during and after differentiation whereas the 300K species was barely detectable. However, CP stimulated generation of the 300K in the differentiated neuronal cultures, and SBTI+Apro abolished this CP effect. Time course profiles and function-blocking antibody indicated that neuroaggregation does not depend on the generation of the 300K fragment or on reelin action. CP neuroaggregative action thus involves a pericellular serine protease, different from reelin. On the other hand, the CP stimulation of reelin cleavage is in line with a possible role of CP in nervous system development. Since P19 cells express ApoER2 and VLDLR, they can help understanding relationships existing between CP, reelin and intervening protease(s).

Ferritin ferroxidase activity: a potent inhibitor of osteogenesis.

Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor alpha-1 (CBF-alpha1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-alpha1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin.

Effect of ceruloplasmin on the number and resistance of erythrocytes during acute physical exercise.

Acute physical exercise was followed by a decrease in the osmotic resistance of erythrocytes, shortening of the time-to-onset of erythrocyte hemolysis, and increase in the sorption capacity of the cell membrane. Administration of ceruloplasmin 24 h before physical exercise normalizes membrane resistance in red blood cells.