The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

MYB mediates downregulation of the colorectal cancer metastasis suppressor heterogeneous nuclear ribonucleoprotein L-like during epithelial-mesenchymal transition.

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

Mithramycin delivery systems to develop effective therapies in sarcomas.

BACKGROUND: Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. RESULTS: In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. CONCLUSIONS: Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.

[High Expression Level of SP1, CSF1R, and PAK1 Correlates with Sensitivity of Leukemia Cells to the Antibiotic Mithramycin].

Acute myeloid leukemia (AML) is a genetically heterogeneous group of oncological diseases of the hematopoietic system, which are extremely difficult to treat. The development of new targeted drugs (Hylteritinib, Venetoclax) significantly improved the survival of patients, but resistance, as well as cytotoxic anti-leukemia drugs, often occurs. The search for new molecular targets for the development of effective approaches for the treatment of AML is very urgent. In blast cells of patients with AML, mutations, chromosomal rearrangements, and increased expression of a number of non-mutant genes, including transcription factor genes, are detected. The transcription factor Sp 1 binds to GC-rich regions of regulatory regions of various genes and thus controls their expression. Sp1 targets include genes responsible for proliferation, cell cycle regulation, and differentiation. In many malignant diseases, a high level of Sp1 gene expression is associated with an unfavorable prognosis, therefore, Sp1 is considered as a promising therapeutic target for cancer. In this paper, we estimated the expression levels of Sp1 in various malignant tissues. Increased Sp1 expression was detected in samples obtained from patients with AML, acute lymphoblastic leukemia, Ewing sarcoma, ovarian and kidney cancer. It is also shown that Sp1 expression correlates with the expression of genes encoding cytokine receptors and growth factors (CSF1R and IL6R), intracellular kinases (CSK, SYK, PAK1, ILK, JAK2), and transcription factor LMO2. The correlation between expression levels of Sp1 and CSF1R, SYK, Jak2 and LMO2 is also characteristic of transplanted human leukemia cells. We measured expression levels of Sp1, CSF1R, ILK, PAK1 in the cells of three transplantable lines of human leukemia and found increased levels of expression of these genes in Kasumi-1 cells. In addition, we showed that Kasumi-1 cells are most sensitive to Mitramycin, a drug that displaces Sp1 from its targets with DNA. Our data indicate the need to identify AML cells that are most sensitive to inhibition of Sp1 activity in order to assess the possibility of suppressing its activity in vivo.

Inhibition of transcription factor SP1 produces neuroprotective effects through decreasing MAO B activity in MPTP/MPP+ Parkinson's disease models.

Monoamine oxidase B (MAO B) inhibitors, which inhibit dopamine decomposition by antagonizing MAO B activity, are approved and widely used for clinical treatment of Parkinson's disease (PD). Nonetheless, the mechanism of the abnormally increased MAO B activity in PD is still unclear. Previous research showed transcription factor specificity protein 1 (SP1) directly regulates MAO B activity by binding the SP1 binding sequence in MAO B promoter. In our study, we first observed that the SP1 protein level and SP1 binding activity in the MAO B promoter were increased in 1-methyl-4-phenylpyridinium (MPP+ ) neurotoxin-induced SH-SY5Y cells. Inhibition of SP1 by pretreatment with SP1 inhibitor mithramycin A (MMA) attenuated the abnormal increase in SP1 binding activity and the MAO B protein level to basal levels. Then, we investigated the neuroprotective effects of SP1 inhibition. In SH-SY5Y cell models of PD, preincubation with MMA or knockdown by SP1-specific small interfering RNA showed potent protection against MPP+ -induced apoptosis via SP1. In a male C57BL/6 mouse model of PD, MAO B activity and MPP+ concentrations in mouse brain following injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were increased, whereas the elevated MAO B activity was decreased after pre-injection of MMA. Moreover, MMA ameliorated MPTP-induced loss of dopaminergic neurons in the substantia nigra pars compacta and mouse behavioral impairments. Altogether, our study suggests that SP1 is a principal factor regulating increases in MAO B activity, and SP1 inhibition produces neuroprotective effects in PD models through decreases in MAO B activity, which may be a new neuroprotective therapeutic strategy for PD treatment.

Mithramycin A Alleviates Osteoarthritic Cartilage Destruction by Inhibiting HIF-2α Expression.

Osteoarthritis (OA) is the most common and increasing joint disease worldwide. Current treatment for OA is limited to control of symptoms. The purpose of this study was to determine the effect of specificity protein 1 (SP1) inhibitor Mithramycin A (MitA) on chondrocyte catabolism and OA pathogenesis and to explore the underlying molecular mechanisms involving SP1 and other key factors that are critical for OA. Here, we show that MitA markedly inhibited expressions of matrix-degrading enzymes induced by pro-inflammatory cytokine interleukin-1β (IL-1β) in mouse primary chondrocytes. Intra-articular injection of MitA into mouse knee joint alleviated OA cartilage destruction induced by surgical destabilization of the medial meniscus (DMM). However, modulation of SP1 level in chondrocyte and mouse cartilage did not alter catabolic gene expression or cartilage integrity, respectively. Instead, MitA significantly impaired the expression of HIF-2α known to be critical for OA pathogenesis. Such reduction in expression of HIF-2α by MitA was caused by inhibition of NF-κB activation, at least in part. These results suggest that MitA can alleviate OA pathogenesis by suppressing NF-κB-HIF-2α pathway, thus providing insight into therapeutic strategy for OA.

Mithramycin has inhibitory effects on gliostatin and matrix metalloproteinase expression induced by gliostatin in rheumatoid fibroblast-like synoviocytes.

OBJECTIVES: Gliostatin (GLS) has angiogenic and arthritogenic activities and enzymatic activity as thymidine phosphorylase. Aberrant GLS production has been observed in the synovial membranes of patients with rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are involved in joint destruction. Promoters of GLS and some MMP genes contain Sp1 binding sites. We examined the inhibitory effect of the Sp1 inhibitor mithramycin on GLS-induced GLS and MMP expression in cultured fibroblast-like synoviocytes (FLSs). METHODS: Synovial tissue samples were obtained from patients with RA. FLSs pretreated with mithramycin were cultured with GLS. The mRNA expression levels of GLS and MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 were determined using reverse transcription polymerase chain reactions. Protein levels were measured using enzyme immunoassay and gelatin zymography. RESULTS: GLS upregulated the expression of GLS itself and of MMP-1, MMP-3, MMP-9, and MMP-13, an effect significantly reduced by treatment with mithramycin. GLS and mithramycin had no effect on MMP-2 expression. CONCLUSIONS: Mithramycin downregulated the increased expression of GLS and MMP-1, MMP-3, MMP-9, and MMP-13 in FLSs treated with GLS. Because GLS plays a pathological role in RA, blocking GLS stimulation using an agent such as mithramycin may be a novel approach to antirheumatic therapy.

LPS depletes PHLPP levels in macrophages through the inhibition of SP1 dependent transcriptional regulation.

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.

Beneficial effect of low-level laser therapy in acute lung injury after i-I/R is dependent on the secretion of IL-10 and independent of the TLR/MyD88 signaling.

The use of low-level laser for lung inflammation treatment has been evidenced in animal studies as well as clinical trials. The laser action mechanism seems to involve downregulation of neutrophil chemoattractants and transcription factors. Innate immune responses against microorganisms may be mediated by toll-like receptors (TLR). Intestinal ischemia and reperfusion (i-I/R) lead to bacterial product translocation, such as endotoxin, which consequently activates TLRs leading to intestinal and lung inflammation after gut trauma. Thus, the target of this study was to investigate the role of TLR activation in the laser (660 nm, 30 mW, 67.5 J/cm2, 0.375 mW/cm2, 5.4 J, 180 s, and spot size with 0.08 cm2) effect applied in contact with the skin on axillary lymph node in lung inflammation induced by i-I/R through a signaling adaptor protein known as myeloid differentiation factor 88 (MyD88). It is a quantitative, experimental, and laboratory research using the C57Bl/6 and MyD88-/- mice (n = 6 mice for experimental group). Statistical differences were evaluated by ANOVA and the Tukey-Kramer multiple comparisons test to determine differences among groups. In order to understand how the absence of MyD88 can interfere in the laser effect on lung inflammation, MyD88-/- mice were treated or not with laser and subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). In summary, the laser decreased the MPO activity and the lung vascular permeability, thickened the alveolar septa, reduced both the edema and the alveolar hemorrhage, as well as significantly decreased neutrophils infiltration in MyD88-deficient mice as well in wild-type mice. It noted a downregulation in chemokine IL-8 production as well as a cytokine IL-10 upregulation in these animals. The results also evidenced that in absence of IL-10, the laser effect is reversed. Based on these results, we suggest that the beneficial effect of laser in acute lung injury after i-I/R is dependent on the secretion of IL-10 and independent of the TLR/MyD88 signaling.

Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription.

Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT.

Epidermal growth factor signals regulate dihydropyrimidine dehydrogenase expression in EGFR-mutated non-small-cell lung cancer.

BACKGROUND: It has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer (NSCLC). However, the relationship between EGFR mutation status and dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, is unknown. METHODS: We elucidated the crosstalk among the EGFR signal cascade, the DPD gene (DPYD), and DPD protein expression via the transcription factor Sp1 and the effect of EGFR mutation status on the crosstalk. RESULTS: In the PC9 (exon19 E746-A750) study, EGF treatment induced up-regulation of both Sp1 and DPD; gefitinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI), and mithramycin A, a specific Sp-1 inhibitor, suppressed them. Among EGFR-mutated (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) and -non-mutated (H1437, H1299) cell lines, EGF administration increased DPYD mRNA expression only in mutated cells (p < 0.05). Accordingly, gefitinib inhibited DPD protein expression only in PC9 and HCC827 cells, and mithramycin A inhibited it in EGFR-mutated cell lines, but not in wild-type. FU treatment decreased the level of cell viability more in gefitinib-treated EGFR-TKI sensitive cell lines. Further, combination treatment of FU and mithramycin A suppressed cell viability even in a gefitinib resistant cell line. CONCLUSIONS: The EGFR signal cascade regulates DPD expression via Sp1 in EGFR mutant cells. These results might be a step towards new therapies targeting Sp1 and DPD in NSCLC with different EGFR mutant status.

Housekeeping promoter 5'pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression.

In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25-100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5'UTRs and 5'flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.

Mithramycin A Alleviates Cognitive Deficits and Reduces Neuropathology in a Transgenic Mouse Model of Alzheimer's Disease.

Increasing evidence has shown that specificity protein 1 (Sp1) is abnormally increased in the brains of subjects with Alzheimer's disease (AD) and transgenic AD models. However, whether the Sp1 activation plays a critical role in the AD pathogenesis and selective inhibition of Sp1 activation may have a disease-modifying effect on the AD-like phenotypes remain elusive. In this study, we reported that Sp1 mRNA and protein expression were markedly increased in the brain of APPswe/PS1dE9 transgenic mice, whereas chronic administration of mithramycin A (MTM), a selective Sp1 inhibitor, potently inhibited Sp1 activation in the APPswe/PS1dE9 mice down to the levels of wild-type mice. Specifically, we found that MTM treatment resulted in a significant improvement of learning and memory deficits, a dramatic reduction in cerebral Aß levels and plaque burden, a profound reduction in tau hyperphosphorylation, and a marked increase in synaptic marker in the APPswe/PS1dE9 mice. In addition, MTM treatment was powerfully effective in inhibiting amyloid precursor protein (APP) processing via suppressing APP, beta-site APP cleaving enzyme 1 (BACE1), and presenilin-1 (PS1) mRNA and protein expression to preclude Aß production in the APPswe/PS1dE9 mice. Furthermore, MTM treatment strongly inhibited phosphorylated CDK5 and GSK3ß signal pathways to reduce tau hyperphosphorylation in the APPswe/PS1dE9 mice. Collectively, our findings provide evidence that Sp1 activation may contribute to the AD pathogenesis and may serve as a novel therapeutic target in the treatment of AD. The present study highlights that selective Sp1 inhibitors may be considered as disease-modifying therapeutic agents for AD.

Identification and characterization of natural microbial products that alter the free d-aspartate content of mammalian cells.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.

sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter -56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes.

Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure.

Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells.

Mesenchymal condensation-dependent accumulation of collagen VI stabilizes organ-specific cell fates during embryonic tooth formation.

BACKGROUND: Mechanical compression of cells during mesenchymal condensation triggers cells to undergo odontogenic differentiation during tooth organ formation in the embryo. However, the mechanism by which cell compaction is stabilized over time to ensure correct organ-specific cell fate switching remains unknown. RESULTS: Here, we show that mesenchymal cell compaction induces accumulation of collagen VI in the extracellular matrix (ECM), which physically stabilizes compressed mesenchymal cell shapes and ensures efficient organ-specific cell fate switching during tooth organ development. Mechanical induction of collagen VI deposition is mediated by signaling through the actin-p38MAPK-SP1 pathway, and the ECM scaffold is stabilized by lysyl oxidase in the condensing mesenchyme. Moreover, perturbation of synthesis or cross-linking of collagen VI alters the size of the condensation in vivo. CONCLUSIONS: These findings suggest that the odontogenic differentiation process that is induced by cell compaction during mesenchymal condensation is stabilized and sustained through mechanically regulated production of collagen VI within the mesenchymal ECM.

Increased expression of colonic Wnt9A through Sp1-mediated transcriptional effects involving arylsulfatase B, chondroitin 4-sulfate, and galectin-3.

In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 µg/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a ß-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

Involvement of Sp1 in butyric acid-induced HIV-1 gene expression.

BACKGROUND/AIMS: The ability of human immunodeficiency virus-1(HIV-1) to establish latent infection and its re-activation is considered critical for progression of HIV-1 infection. We previously reported that a bacterial metabolite butyric acid, acting as a potent inhibitor of histone deacetylases (HDACs), could lead to induction of HIV-1 transcription; however, the molecular mechanism remains unclear. The aim of this study was to investigate the effect of butyric acid on HIV-1 gene expression. METHODS: Butyric acid-mediated HIV-1 gene expression was determined by luciferase assay and Chromatin immunoprecipitation assay. Western blot analysis and ELISA were used for the detection of HIV-1. RESULTS: We found that Sp1 binding sites within the HIV-1 promoter are primarily involved in butyric acid-mediated HIV-1 activation. In fact, Sp1 knockdown by small interfering RNA and the Sp1 inhibitor mithramycin A abolished the effect of butyric acid. We also observed that cAMP response element-binding-binding protein (CBP) was required for butyric acid-induced HIV-1 activation. CONCLUSIONS: These results suggest that butyric acid stimulates HIV-1 promoter through inhibition of the Sp1-associated HDAC activity and recruitment of CBP to the HIV-1 LTR. Our findings suggest that Sp1 should be considered as one of therapeutic targets in anti-viral therapy against HIV-1 infection aggravated by butyric acid-producing bacteria.

Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions.

Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in ß-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.