Jerome A. Roth

Melatonin promotes osteoblast differentiation and bone formation.

Prior studies have demonstrated that the pineal hormone, melatonin, can stimulate chloramphenicol acetyltransferase activity in Drosophila SL-3 cells transfected with a chloramphenicol acetyltransferase reporter construct containing the response element of rat bone sialoprotein (BSP). Based on these findings, studies were performed to determine whether melatonin could similarly modulate the expression of BSP in two cell lines, the MC3T3-E1(MC3T3) pre-osteoblast and rat osteoblast-like osteosarcoma 17/2.8 cell. Initial studies demonstrated that MC3T3 cells grown in the presence of 50 nm melatonin underwent cell differentiation and mineralization by day 12 instead of the 21-day period normally required for cells grown in untreated media. Melatonin increased gene expression of BSP and the other bone marker proteins, including alkaline phosphatase (ALP); osteopontin; secreted protein, acidic and rich in cysteine; and osteocalcin in MC3T3 cells in a concentration-dependent manner. Levels of melatonin as low as 10 nm were capable of stimulating transcription of these genes when cells were grown in the presence of β-glycerophosphate and ascorbic acid. Under these conditions, melatonin induced gene expression of the bone marker proteins; however, this does not occur until the 5th day after seeding the culture dishes. Thereafter, MC3T3 cells responded to melatonin within 2 h of treatment. The fully differentiated rat osteoblast-like osteosarcoma 17/2.8 cells responded rapidly to melatonin and displayed an increase in the expression of BSP, ALP, and osteocalcin genes within 1 h of exposure to the hormone. To determine whether melatonin-induced osteoblast differentiation and bone formation are mediated via the transmembrane receptor, MC3T3 cells were treated in the presence and absence of melatonin with either luzindole, a competitive inhibitor of the binding of melatonin to the transmembrane receptors, or pertussis toxin, an uncoupler of Gi from adenylate cyclase. Both luzindole and pertussis toxin were shown to reduce melatonin-induced expression of BSP and ALP. These results demonstrate, for the first time, that the pineal hormone, melatonin, is capable of promoting osteoblast differentiation and mineralization of matrix in culture and suggest that this hormone may play an essential role in regulating bone growth.

Iron interactions and other biological reactions mediating the physiological and toxic actions of manganese

Chronic exposure to the divalent heavy metals, such as iron, lead, manganese (Mn), and chromium, has been linked to the development of severe, often irreversible neurological disorders and increased vulnerability to developing Parkinsons disease. Although the mechanisms by which these metals elicit or facilitate neuronal cell death are not well defined, neurotoxicity is limited by the extent to which they are transported across the blood-brain barrier and their subsequent uptake within targeted neurons. Once inside the neuron, these heavy metals provoke a series of biochemical and molecular events leading to cell death induced by either apoptosis and/or necrosis. The toxicological properties of Mn have been studied extensively in recent years because of the potential health risk created by increased atmospheric levels owing to the impending use of the gas additive methylcyclopentadienyl manganese tricarbonyl. Individuals exposed to high environmental levels of Mn, which include miners, welders, and those living near ferroalloy processing plants, display a syndrome known as manganism, best characterized by debilitating symptoms resembling those of Parkinsons disease. Mn disposition in vivo is influenced by dietary iron intake and stores within the body since the two metals compete for the same binding protein in serum (transferrin) and subsequent transport systems (divalent metal transporter, DMT1). There appear to be two distinct carrier-mediated transport systems for Mn and ferrous ion: a transferrin-dependent and a transferrin-independent pathway, both of which utilize DMT1 as the transport protein. Accordingly, this commentary focuses on the biochemical and molecular processes responsible for the cytotoxic actions of Mn and the role that cellular transport plays in mediating the physiological as well as the toxicological actions of this metal.

Homeostatic and toxic mechanisms regulating manganese uptake, retention, and elimination

This review attempts to summarize and clarify our basic knowledge as to the various factors that potentially influence the risks imposed from chronic exposure to high atmospheric levels of manganese (Mn). The studies describe the interrelationship of the different systems in the body that regulate Mn homeostasis by characterizing specific, biological components involved in its systemic and cellular uptake and its elimination from the body. A syndrome known as manganism occurs when individuals are exposed chronically to high levels of Mn, consisting of reduced response speed, intellectual deficits, mood changes, and compulsive behaviors in the initial stages of the disorder to more prominent and irreversible extrapyramidal dysfunction resembling Parkinsons disease upon protracted exposure. Mn intoxication is most often associated with occupations in which abnormally high atmospheric concentrations prevail, such as in welding and mining. There are three potentially important routes by which Mn in inspired air can gain access the body to: 1) direct uptake into the CNS via uptake into the olfactory or trigeminal presynaptic nerve endings located in the nasal mucosa and the subsequent retrograde axonal transport directly into the CNS; 2) transport across the pulmonary epithelial lining and its subsequent deposition into lymph or blood; and/or 3) mucocilliary elevator clearance from the lung and the subsequent ingestion of the metal in the gastrointestinal tract. Each of these processes and their overall contribution to the uptake of Mn in the body is discussed in this review as well as a description of the various mechanisms that have been proposed for the transport of Mn across the bloodbrain barrier which include both a transferrin-dependent and a transferrin-independent process that may involve store-operated Ca channels.

Contribution of Sulfate Conjugation, Deamination, and O-Methylation to Metabolism of Dopamine and Norepinephrine in Human Brain

Abstract: The kinetic constants were determined for dopamine (DA) and norepinephrine (NE) metabolism by phenolsulfotransferase (PST), type A and B monoamine oxidase (MAO), and membrane‐bound and soluble catechol‐O‐ methyltransferase (COMT) in frontal lobe preparations of human brain. PST and membrane‐bound COMT were found to have the lowest Km, values for both catecholamines. By means of the appropriate rate equations and the calculated kinetic constants for each enzyme, the activity of each enzymatic pathway was determined at varying concentrations of DA and NE. Results indicate that deamination by MAO is the principal pathway for the enzymatic inactivation of DA whereas NE is largely metabolized by MAO type A and membrane‐bound COMT under the in vitro assay conditions used. At concentrations less than 100 μM, soluble COMT’contributes less than 5% to the total catabolism of either catecholamine. PST can contribute up to 15% of the total DA metabolism and 7% of NE metabolism.

Distinct Cellular Localization of Membrane‐Bound and Soluble Forms of Catechol‐O‐Methyltransferase in Brain

Abstract: The cellular localization of the two forms of catechol‐O‐methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane‐bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane‐bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the Km membrane‐bound catechol‐O‐methyltransferase may be localized predominantly in neurons, whereas the high‐Km soluble enzyme is found in glial cells.

Sulfation of Dopamine and Other Biogenic Amines by Human Brain Phenol Sulfotransferase

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit‐ters in vivo, the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3‐methoxytyramine (3‐MT), tyramine, norepinephrine, 3‐methoxy‐4‐hydroxyphenylethyleneglycol, octopamine, 5‐hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3‐MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the Km value for DA was found to be 5.0 μM, while the Km value for the sulfate‐donor 3′‐phosphoadenosine‐5′‐phosphosulfate was 0.25 μM. The ratio of 3‐O‐ to 4‐O‐DA‐sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3‐O‐ and 4‐O‐esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol‐O‐methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.

Characterization of membrane-bound and soluble catechol-O-methyltransferase from human frontal cortex

Abstract: Catechol‐O‐methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane‐bound form. Attempts to solubilize the membrane‐bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X‐100 was capable of solubilizing membrane‐bound COMT suggested that the membrane‐bound transferase is an integral membrane protein. The membrane‐bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH‐activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl2, both enzymes displaying 65% inhibition at 2.5 mM CaCl2. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed Ki values for the membrane‐bound transferase five‐ to 10‐fold lower than those observed for the soluble transferase. Solubilization of membrane‐bound COMT in Triton X‐100 resulted in an increase in the apparent Km value of the membrane‐bound transferase for dopamine. The increase in Km appeared to be due to apparent competitive inhibition by Triton X‐100 and reached a limiting value of approximately 80 μM. These results confirm that membrane‐bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.

Manganese‐induced rat pheochromocytoma (PC12) cell death is independent of caspase activation

Manganese (Mn) is an essential mineral that at high concentrations can produce an irreversible syndrome resembling Parkinsons disease. To examine the mechanism by which Mn elicits its toxic response, we have selected the rat pheochromocytoma cells (PC12) as our model system because it possesses much of the biochemical machinery associated with dopaminergic neurons. Mn‐induced PC12 cell death is both time and concentration dependent with approximately 50% cell survival at 48 hr in the presence of 0.3 mM Mn. To determine whether oxidative stress contributed to cytotoxicity induced by Mn, lipid peroxidation was assessed in Mn‐treated in PC12 cells. The highly sensitive HPLC assay that measures the lipid peroxide product, 9‐HODE, was used and results of these experiments demonstrate there was no increase in the lipid peroxidation in cells exposed to 0.3 mM Mn for 24 hr. Mn was found to stimulate the activation of the apoptotic marker proteins, p38 and caspase‐3 within the first 24 hr of treatment. The selective inhibitor of caspase‐3, DEVD‐CHO, and the nonselective caspase inhibitor, Z‐VAD‐FMK, however, fail to prevent Mn‐induced PC12 cell death. Studies were performed to determine the role of mitochondria in initiating or supporting Mn cytotoxicity, because Mn has been reported to cause changes in membrane permeability. Mn caused a decrease in ATP levels in PC12 cells in both a time and concentration dependent manner. We hypothesize that both apoptosis and necrosis contribute to PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited. J. Neurosci. Res. 61:162–171, 2000.

Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation.

Mn is a neurotoxin that leads to a syndrome resembling Parkinsons disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytoma (PC12) cells as a model since they possess much of the biochemical machinery associated with dopaminergic neurons. Mn, like nerve growth factor (NGF), can induce neuronal differentiation of PC12 cells but Mn-induced cell differentiation is dependent on its interaction with the cell surface integrin receptors and basement membrane proteins, vitronectin or fibronectin. Similar to NGF, Mn-induced neurite outgrowth is dependent on the phosphorylation and activation of the MAP kinases, ERK1 and 2 (p44/42). Unlike NGF, Mn is also cytotoxic having an IC50 value of approximately 600 microM. Although many apoptotic signals are turned on by Mn, cell death is caused ultimately by disruption of mitochondrial function leading to loss of ATP. RT-PCR and immunoblotting studies suggest that some uptake of Mn into PC12 cells depends on the divalent metal transporter 1 (DMT1). DMT1 exists in two isoforms resulting from alternate splicing of a single gene product with one of the two mRNA species containing an iron response element (IRE) motif downstream from the stop codon. The presence of the IRE provides a binding site for the iron response proteins (IRP1 and 2); binding of either of these proteins could stabilize DMT1 mRNA and would increase expression of the +IRE form of the transporter. Iron and Mn compete for transport into PC12 cells via DMT1, so removal of iron from the culture media enhances Mn toxicity. The two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells.

Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion

DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; +/-IRE). We have compared 1A/+IRE and 2/-IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was approximately 10-fold greater than that of untreated cells, while expression in the untreated cells was approximately 5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at approximately 1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at approximately 5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.