Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.

Activity-dependent, polyneuronal synapse elimination (ADPSE) is a programmed, regressive event in the development of the nervous system and readily studied at the neuromuscular junction, where it is complete 15-20 days after birth. Local excess, or imbalanced, protease activity is one of several possible underlying mechanisms. In this regard, thrombin mediates activity-dependent synapse loss in an in vitro model of ADPSE. To test the involvement of thrombin in vivo, we locally applied the leech thrombin-specific inhibitor, hirudin. We monitored neuromuscular behavior, correlated with acetylcholinesterase and silver nitrate histochemistry at endplates, for changes in the timecourse of in vivo synapse elimination and assayed both thrombin activity and prothrombin expression in developing muscle. Hirudin retarded elimination, without altering motor performance, uniquely at Postnatal Day 5 (P5) and maximally at P9. Reverse transcription-polymerase chain reaction (PCR) showed that neonatal muscle was a source of local prothrombin, with peak expression during the first week after birth. A specific chromogenic assay revealed that local thrombin, activated from muscle-derived prothrombin, peaked during maximal synapse remodeling.

Metal response element-binding transcription factor-1 (MTF-1) is a unique, zinc-inducible transcription factor that binds to metal response elements in the metallothionein promoter and activates transcription in response to metals and oxidative stress. MTF-1 contains six zinc fingers of the Cys 2 -His 2 type. It was previously shown that MTF-1 is reversibly activated to bind DNA in response to changes in zinc status, unlike other zinc finger transcription factors, which do not appear to be reversibly activated by zinc in the cellular environment. Here we show that zinc fingers 2-4 constitute the core DNA-binding domain, whereas fingers 5 and 6 appear to be unnecessary for DNA binding in vitro. Deletion of finger 1 resulted in a protein that bound DNA constitutively in vitro. Furthermore, transfer of MTF-1 finger 1 to a position immediately preceding the three zinc fingers of Sp1 resulted in a chimeric protein that required exogenous zinc to activate DNA binding in vitro, unlike native Sp1, which binds DNA constitutively. Transient transfection experiments demonstrated that intact MTF-1 activated a reporter 2.5-4-fold above basal levels after metal treatment in mouse MTF-1 knockout cells, Drosophila SL2 cells, and yeast. However, the metal response was lost in all three systems when finger 1 was deleted, but was unaffected by deletion of fingers 5 and 6. These data suggest that finger 1 of MTF-1 constitutes a unique metal-sensing domain that, in cooperation with the transactivation domains, produces a zinc-sensing metalloregulatory transcription factor. Metallothioneins (MTs)1 constitute a conserved family of cysteine-rich heavy metal-binding proteins (1). In the mouse, MT-I and MT-II display a wide tissue distribution and have been demonstrated to participate in zinc homeostasis (2), detoxification of cadmium (3, 4), and protection against oxidative stress (5). MT gene transcription is induced dramatically by heavy metals (especially zinc and cadmium) (6). Metal response elements (MREs) are essential for metal induction, and there are multiple copies of the MREs in the proximal promoters of MT genes. MREs were initially shown to mediate the transcriptional response of MT genes to metals (7) and, more recently, to participate in the transcriptional response to oxidative stress (8, 9).MRE-binding transcription factor-1 (MTF-1) was cloned (10, 11) and found to be a six-zinc finger transcription factor in the Cys 2 -His 2 family. Targeted disruption of both MTF-1 alleles in mouse embryonic stem cells demonstrated its essential role for basal as well as heavy metal-induced MT gene expression (12). Unlike other zinc finger transcription factors, which appear to be constitutively active to bind DNA under normal physiological conditions, MTF-1 is reversibly activated to bind DNA in response to changes in zinc availability (9,10,(13)(14)(15)(16). This reversible activation involves direct interaction between zinc and the zinc finger domain of MTF-1. Activation of MTF-1 in response to zinc is reminiscent of copper activat...

Apoptosis, well-established in development and now also in degenerative disease, occurs with regularity in several cell compartments early after controlled contusion spinal cord injury (SCI). Cell death in astrocytic, microglial, and neuronal populations peaks at 3 days, while oligodendroglial apoptosis is found 10-14 days later. In this regard, the executioners of apoptosis, the caspase proteases, are also activated within 3 days of SCI. On the other hand, serine proteases, which have been shown to initiate apoptosis and activate caspases in culture models, have not been extensively studied in regards to nervous system trauma. As part of an ongoing effort to examine the spectrum of genes that are up- and downregulated in the injured rat spinal cord, we synthesized serine protease family specific primers to take advantage of conserved residues in the charge relay system and the codon preferences of these mammalian genes. These primers were then employed in a modified, family-specific differential mRNA display technique. One specific serine protease gene we found that was upregulated after injury was prothrombin. Qualitative and quantitative RT-PCR techniques indicated that this increase occurred early, already evident at 8 h after injury, and reached a maximum level fourfold above baseline at 24 h. Peak expression for prothrombin mRNA occurred prior to peak levels of apoptosis in astrocytic, microglial and neuronal compartments at 72 h. Of additional interest, gene database mining revealed that prothrombin shared approximately 48% similarity with myelencephalon-specific protease (MSP), a neurotoxic serine protease previously found to be increased two- to threefold at 3 days after excitotoxic SCI. Since thrombin induces apoptosis in murine and chick motor and rat hippocampal neurons by activating a member of the novel protease-activated receptor (PAR) gene family known as PAR-1, we also analyzed PAR-1 by similar techniques and found that it, too, was upregulated after SCI with the same kinetics as prothrombin. We confirmed these results with gene array analyses that revealed more than one trypsin subfamily serine protease was activated by SCI. They imply the possibility of using specific, tissue-directed serine protease inhibition at translational or transcriptional levels, and offer a potential paradigm shift in drug discovery for SCI to limit the extent of apoptosis, and consequent functional loss, in the human spinal cord.