Safe coordinated trafficking of heme and iron with copper maintain cell homeostasis: modules from the hemopexin system.

Studies with patients, animal models of human disease and hemopexin null mice have shown that the heme-binding protein hemopexin is vital for the protection of a variety of cell types and tissues against heme toxicity. The presence of hemopexin in all biological fluids examined to date indicates wide roles in abrogating heme toxicity in human tissues; and, thus, is clinically relevant. Heme-hemopexin endocytosis leads to coordinated trafficking of heme, iron and copper as heme traffics from endosomes to heme oxygenases (HOs) in the smooth endoplasmic reticulum and to the nucleus. This is safe redox-metal trafficking, without oxidative stress, as iron released from heme catabolism by HOs as well as copper taken up with heme-hemopexin move through the cell. To our knowledge, this coordinated metal trafficking has been described only for the hemopexin system and differs from the cell's response to non-protein bound heme, which can be toxic. We propose that defining how cells respond to heme-hemopexin endocytosis, a natural cytoprotective system, will aid our understanding of how cells adapt as they safely respond to increases in heme, Fe(II) and copper. This is relevant for many genetic hemolytic diseases and conditions, stroke and hemorrhage as well as neurodegeneration. Such analyses will help to define a pattern of events that can be utilized to characterize how dysfunctional redox and transition metal handling is linked to the development of pathology in disease states such as Alzheimer's disease when metal homeostasis is not restored; and potentially provide novel targets and approaches to improve therapies.

Clonality studies in giant cell tumor of bone.

Genetic studies including chromosome analysis, telomere reduction and telomere activity, DNA microsatellites and loss of heterozygosity (LOH) studies have been performed on giant cell tumor (GCT) of bone however whether this primary skeletal neoplasm represents a monoclonal or polyclonal proliferation is unknown. Utilizing a new assay to study the polymorphic human androgen receptor locus (HUMARA), the ratio of maternal inactive X-chromosome to the paternal inactive X (Lyon hypothesis) is determined via a methylation--specific polymerase chain reaction (PCR) technique to detect X-chromosome polymorphisms. Characterization of the genetic tumorigenesis of this unpredictable neoplasm may lend insight into its biological behavior and offer improvements in therapeutic intervention, as new information emerges regarding osteoclastic bone resorption. Seventeen female patients with giant cell tumor of bone had their DNA harvested and their X-chromosome inactivation pattern and polymorphisms determined and compared to control. A polyclonal proliferation pattern was identified in all informative samples studied.

Role for copper in transient oxidation and nuclear translocation of MTF-1, but not of NF-kappa B, by the heme-hemopexin transport system.

Heme-hemopexin (2-10 microM) is used as a model for intravenous heme released in trauma, stroke, and ischemia-reperfusion. A transient increase in cellular protein oxidation occurs during receptor-mediated heme transport from hemopexin which is inhibited by the nonpermeable Cu(I) chelator, bathocuproinedisulfonate. Thus, participation of surface redox process involving Cu(I) generation are proposed to be linked to the induction of the protective proteins heme oxygenase-1 (HO-1) and metallothionein-1 (MT-1) by heme-hemopexin. The region (-153 to -42) in the proximal promoter of the mouse MT-1 gene responds to heme- and CoPP-hemopexin in transient transfection assays and contains metal-responsive elements for MTF-1 and an antioxidant-responsive element (ARE) overlapping a GC-rich E-box to which USF-1 and -2 bind. No decreases in DNA binding of the diamide-oxidation sensitive USF-1 and -2 occur upon exposure of cells to heme-hemopexin. MTF-1 and the ARE-binding proteins are relatively resistant to diamide oxidation and are induced approximately eight- and two-fold, respectively, by heme-hemopexin. BCDS prevents the nuclear translocation of MTF-1 by both heme- and CoPP-hemopexin complexes as well as MT-1 mRNA induction by CoPP-hemopexin. Thus, copper is needed for the surface oxidation events and yet the nuclear translocation of MTF-1 in response to hemopexin occurs via copper, probably Cu(I),-dependent signaling cascades from the hemopexin receptor rather than the oxidation per se.

Cell-surface events for metallothionein-1 and heme oxygenase-1 regulation by the hemopexin-heme transport system.

A model has been developed for the hemopexin receptor-mediated heme transport system based on iron uptake in yeast. Two steps are required: reduction followed by oxidation by a multi-copper-oxidase. Furthermore, in the hemopexin system, the surface redox events have been linked with gene regulation. The impermeable Cu(I) chelator bathocuproinedisulfonate (BCDS) is shown here to abrogate heme oxygenase-1 (HO-1) mRNA induction by heme-hemopexin. A role for Cu(I) in the regulation of HO-1 and MT-1 (Sung et al., 1999) by hemopexin supports the participation of electron transport processes at the cell surface as does competition by the reductase activator, ferric citrate, which inhibits the induction of MT-1 and HO-1 mRNA by heme-hemopexin. There is a key role for the hemopexin receptor because neither ferric citrate nor iron-transferrin alone regulates MT-1 or HO-1. Cell-surface copper is the first molecule to link the concomitant regulation of HO-1 and MT-1 by the hemopexin receptor. In addition, cytochrome b5 and cytochrome b5 reductase are implicated here in the response of cells to heme-hemopexin. Reduction of one or more electron donors of the reductase and oxidation of the electron acceptor, b5 heme, leads to gene regulation, but only when heme-hemopexin is bound to its receptor. Protein kinase cascades, including JNK, are activated by the hemopexin receptor itself upon ligand binding but are modulated by a Cu(I)-dependent process likely to be heme uptake.

Cellular protection mechanisms against extracellular heme. heme-hemopexin, but not free heme, activates the N-terminal c-jun kinase.

Hemopexin protects cells lacking hemopexin receptors by tightly binding heme abrogating its deleterious effects and preventing nonspecific heme uptake, whereas cells with hemopexin receptors undergo a series of cellular events upon encountering heme-hemopexin. The biochemical responses to heme-hemopexin depend on its extracellular concentration and range from stimulation of cell growth at low levels to cell survival at otherwise toxic levels of heme. High (2-10 microM) but not low (0.01-1 microM) concentrations of heme-hemopexin increase, albeit transiently, the protein carbonyl content of mouse hepatoma (Hepa) cells. This is due to events associated with heme transport since cobalt-protoporphyrin IX-hemopexin, which binds to the receptor and activates signaling pathways without tetrapyrrole transport, does not increase carbonyl content. The N-terminal c-Jun kinase (JNK) is rapidly activated by 2-10 microM heme-hemopexin, yet the increased intracellular heme levels are neither toxic nor apoptotic. After 24 h exposure to 10 microM heme-hemopexin, Hepa cells become refractory to the growth stimulation seen with 0.1-0.75 microM heme-hemopexin but HO-1 remains responsive to induction by heme-hemopexin. Since free heme does not induce JNK, the signaling events, like phosphorylation of c-Jun via activation of JNK as well as the nuclear translocation of NFkappaB, G2/M arrest, and increased expression of p53 and of the cell cycle inhibitor p21(WAF1/CIP1/SDI1) generated by heme-hemopexin appear to be of paramount importance in cellular protection by heme-hemopexin.

Role of heme-hemopexin in human T-lymphocyte proliferation.

Heme-hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 hemopexin receptors per cell (apparent Kd 20 nM), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100-1000 nM heme-hemopexin, consistent with the high affinity of the receptor for hemopexin, and maximal growth is seen in response to 500 nM complex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme-hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme-hemopexin, but not apo-hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme-iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)-hemopexin, which binds to the receptor but is not transported intracellularly [Smith et al., (1993) J. Biol. Chem. 268, 7365], also stimulated cell proliferation in serum-free IMDM but did not "rescue" the cells from desferrioxamine. Furthermore, CoPP-hemopexin effectively competed for the hemopexin receptor with heme-hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme-hemopexin is added to the medium, reaches maximum activity within 5-10 min, and declines to unstimulated levels by 30 min. Heme-hemopexin and CoPP-hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme-hemopexin not only functions as an iron source for T-cells but occupancy of the hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme-hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation.