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We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region. Ectopic expression of SP1 enhanced both transcript and protein levels of PHLPP while knockdown of SP1 or pharmacological inhibition of SP1 DNA binding by mithramycin reduced PHLPP expression. Moreover, over-expression of SP1 co-activators CBP/p300 augmented SP1 driven PHLPP promoter activity. Of note, LPS treatment depleted SP1 and CBP protein levels due to which recruitment of SP1 to PHLPP promoter was reduced. Further, we found that re-introduction of SP1 restored promoter activity and transcript levels of PHLPP in LPS stimulated cells. Collectively, our data revealed the molecular mechanism underlying the regulation of PHLPP expression during LPS induced macrophage inflammatory response.

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. In this study, we cloned and identified the proximal 269 bp promoter of survivin gene, which exhibited strong promoter activity in HeLa cells. The TATA-less, GC-rich promoter contains 7 putative binding sites for Sp1, two of which (one at position −148 to −153, the other at position −127 to −140) are essential in regulating basal survivin promoter activity. Not only Sp1 but also Sp3 can activate the survivin promoter, which were proven by EMSA, blocking Sp1 or Sp3 using RNAi or mithramycin treatment of HeLa cells, and overexpression of Sp1 or Sp3. Our results collectively suggest that Sp1 cooperates with Sp3 to regulate survivin promoter activity.

The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation. In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression. By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation. Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively. Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity. In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members. In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression. This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A. Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays. Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

Mithramycin is an mRNA synthesis inhibitor that has been used to decrease bone resorption in patients with humoral hypercalcemia and Paget's disease. During studies on the mechanism of action of mithramycin it became clear that the compound has a direct inhibitory effect on osteoclastic bone resorption in the in vitro bone slice assay. At concentrations of 0.1-100 nM mithramycin directly inhibited osteoclastic bone resorption dose-dependently up to 66 +/- 5% at 100 nM (mean +/- SEM, 3 expts.). Another mRNA synthesis inhibitor, actinomycin D (0.1-100 nM) and the protein synthesis inhibitor, cycloheximide (0.1-10 microM), also dose-dependently inhibited osteoclastic bone resorption by 78 +/- 7% at 100 nM and 76 +/- 7% at 10 microM, respectively. Mithramycin and actinomycin D at 100 nM did not affect osteoclast survival on bone slices and were therefore not cytotoxic at the concentrations used. Mithramycin (100 nM) and cycloheximide (10 microM) both slightly decreased osteoclast cytoplasmic spreading. Addition of 100 nM mithramycin 6 hr after osteoclast adhesion to bone slices still inhibited subsequent resorption by 50%, indicating a continued but lesser requirement for mRNA synthesis during bone resorption. These results show that approximately 75% of osteoclasts obtained from neonatal rat long bones are activated by adhesion to mineralized bone surfaces and require mRNA and protein synthesis in order to resorb bone in vitro.

Mithramycin (MTR) is an antitumor compound that inhibits RNA and DNA polymerase action by forming a non covalent complex with double strand DNA, in the presence of divalent cations. We have shown that in the presence of Mg2+, MTR binds to right-handed poly(dG-m5dC) as a dimer in the right-handed screwness conformation but cannot bind to left-handed poly(dG-m5dC).

Summary Mithramycin (MTR, structure shown in Figure 1) [and the related compound Chromomycin A 3 (CHRA 3 )] are antitumor antibiotics which inhibit DNA dependent RNA polymerase activity via reversible interaction with DNA only in the presence of divalent metal ion such as Mg ++ . In order to understand the role of Mg ++ in MTR-DNA interaction, absorbance and CD spectroscopic techniques are employed to study the binding of MTR to Mg ++ . These studies show : i) the drug alone binds to Mg ++ and ii) two different types of drug-Mg ++ complexes are formed at low (Complex I) and high (Complex II) ratios of the concentration of Mg ++ and MTR. We propose that these two complexes would bind to the same DNA with different affinities and rates. This result suggests that the relative concentration of Mg ++ is an important factor to be taken into account to understand the molecular basis of MTR-DNA interaction.

The involvement of Sp1 in the lipopolysaccharide (LPS)-induced transcription of HDC mRNA in the mouse macrophage-like cell line RAW 264 was analyzed. LPS increased the levels of HDC mRNA 4 h after the stimulation in a concentration-dependent manner. Mithramycin A, an inhibitor of the binding of the Sp family to the GC box, reduced the LPS-induced increase in the levels of HDC mRNA at 4 h and HDC protein at 8 h in a concentration-dependent manner. By conducting electrophoretic mobility shift assays, we found that one of the transcription factors binding to the DNA probe containing the GC box sequence of the mouse HDC gene promoter region was Sp1, and that levels of Sp1-DNA probe complexes were increased by stimulation with LPS although the protein levels of Sp1 were not changed. These results suggested that Sp1 is one of the transcription factors that regulate the LPS-induced expression of HDC in RAW 264 cells.

Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor γ (PPARγ) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after treatment of cycloheximide (a protein synthesis inhibitor). TZD could not repress the expression of the resistin gene in the presence of mithramycin A (an Sp1 binding inhibitor). Sp1 binding site of the resistin promoter (−122/−114 bp) was necessary for the repression. Further investigation of the effect of TZDs on the modification of Sp1 showed that the level of O-glycosylation of Sp1 was decreased in this process. These results suggest that PPARγ activation represses the expression of the resistin gene by modulating Sp1 activity.

We show that a histone deacetylase (HDAC) inhibitor apicidin increases the transcriptional activity of cyclin E gene, which results in accumulation of cyclin E mRNA and protein in a time- and dose-dependent manner. Interestingly, apicidin induction of cyclin E gene is found to be mediated by Sp1- rather than E2F-binding sites in the cyclin E promoter, as evidenced by the fact that specific inhibition of Sp1 leads to a decrease in apicidin activation of cyclin E promoter activity and protein expression, but mutation of E2F-binding sites of cyclin E promoter region fails to inhibit the ability of apicidin to activate cyclin E transcription. In addition, this transcriptional activation of cyclin E by apicidin is associated with histone hyperacetylation of cyclin E promoter region containing Sp1-binding sites. Our results demonstrate that regulation of histone modification by an HDAC inhibitor apicidin contributes to induction of cyclin E expression and this effect is Sp1-dependent.

Lysozyme protects us from the ever-present danger of bacterial infection. The expression of lysozyme is, in part, regulated by the Ets factor, myeloid elf-1-like factor (MEF). MEF binds to the ETS site of the lysozyme promoter at −46 to −40 bp. Closer analysis of the promoter using a series of deletion mutants and point mutants indicated that the region around −75 bp is also essential in regulating the activity of lysozyme. The sequences in this region correspond to the Sp1 consensus binding site. Sp1 is known to regulate a variety of house-keeping and tissue-specific genes by itself or with other transcription factors like AP-1 or ETS. We indicate here that Sp1 regulates the lysozyme gene by binding to the GT-core sequences of lysozyme promoter. Treatment with mithramycin A down-regulated the promoter activity and the transfection of anti-sense Sp1 induced a decrease in the endogenous expression of lysozyme.

Aureolic acid group of antibiotics inhibit transcription by reversible binding to DNA in presence of divalent magnesium. We for the first time report binding of the one of such antitumor antibiotic, mithramycin (MTR), to the major protein component of erythrocyte cytoskeleton, spectrin. A reasonably high apparent dissociation constant was estimated to be 1.5 microM. The binding of mithramycin in the absence of any divalent cation to the large cytoskeletal protein led to quenching in the tryptophan fluorescence of the protein. Stern-Volmer quenching of the tryptophan residues by acrylamide revealed conformational change in the MTR-bound spectrin. This preliminary study might be useful in understanding other possible sites of actions after translocation.

Mithramycin is a DNA-binding antibiotic that has been reported to selectively affect c-myc expression [Snyder, R. C. et al., (1991) Biochemistry30, 4290–4297]. We used in vitro transcription to investigate the specificity of mithramycin action. We found that mithramycin inhibited transcription from the human c-myc P1 and P2 promoters, as well as from a minimal adenovirus-2 major late promoter, with equal efficiencies. Mithramycin also inhibited transcription elongation by creating kinetic blockades to the passage of RNA polymerase II. These data suggest that mithramycin may inhibit transcription non-specifically by affecting general processes such as transcription elongation.

The effects of various drugs on the structure of a bent DNA fragment have been investigated by studying DNA mobility in polyacrylamide gels. This DNA fragment has an anomalously slow rate of migration on account of its phased runs of adenines. Nogalamycin and echinomycin increase the gel mobility of kinetoplast DNA suggesting that the bending has been removed. Mithramycin, actinomycin, distamycin and ethidium have either no effect or cause a further reduction in mobility. These results are compared with other, non-bent DNA species which always show a decrease in gel mobility in the presence of DNA binding drugs.

The interaction of mithramycin with metal ions has been studied by absorbance and fluorescence spectroscopy. Magnesium shifts the drug absorbance spectrum to longer wavelengths and displays a weak binding constant (K a = 1mM); no interaction with calcium was detected. The drug requires magnesium for binding to DNA and this is characterised by small additional hypochromic and bathochromic changes. Mithramycin does not bind to DNA in the presence of calcium. With 10mM magnesium the drug binds to DNA with an association constant of 9.2×10 4 M −1 . The inability of calcium to substitute for magnesium has been confirmed by ‘footprinting’ experiments using both DNase I and hydroxyl radicals.