Participation of the TBP-associated factors (TAFs) in cell differentiation.

The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associated factors (TAFs) during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs' regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.

Compound heterozygous mutations in TBPL2 were identified in an infertile woman with impaired ovarian folliculogenesis.

OBJECTIVE: A 32-year-old female was diagnosed with unexplained primary infertility for 10 years. She had roughly normal basal hormone levels, but her basal follicle-stimulating hormone (FSH) levels were elevated. In addition, the level of anti-Mullerian hormone was within the normal range, and she had undergone two failed oocyte collection attempts. We aimed to investigate the genetic cause of female infertility in patients with impaired ovarian folliculogenesis. METHODS: Genomic DNA was extracted from the peripheral blood of the patient and her family members. Whole-exome sequencing was performed on the patient, and TBPL2 mutations were identified and confirmed by Sanger sequencing. The Exome Aggregation Consortium (ExAC) Browser and Genome Aggregation Database (gnomAD) Browser Beta were used to search the allele frequencies of the variants in the general population. The harmfulness of the mutations was analyzed by SIFT, Mutation Taster, and CADD software. RESULT: One novel mutation, c.802C > T (p. Arg268Ter), and one known variant, c.788 + 3A > G (p. Arg233Ter), in TBPL2 were identified in the infertile family. Compound heterozygous mutations in TBPL2 may be the cause of impaired ovarian folliculogenesis, failure of superovulation, and infertility. CONCLUSIONS: We identified compound heterozygous mutations in TBPL2 that caused impaired ovarian folliculogenesis, failure of superovulation, and infertility in patients. These findings suggest an important role for compound heterozygous mutations in TBPL2 and expand the mutational spectrum of TBPL2, which might provide a new precise diagnostic marker for female infertility.

Gene mutations impede oocyte maturation, fertilization, and early embryonic development.

Reproductive diseases are a long-standing problem and have become more common in the world. Currently, 15% of the world's population suffers from infertility, and half of them are women. Maturation of oocytes, successful fertilization, and high-quality embryos are prerequisites for pregnancy. With the development of assisted reproductive technology and advanced genetic assays, we have found that infertility in many young female patients is caused by mutations in various developmental regulators. These pathogenic factors may result in impediment of oocyte maturation, failure of fertilization or early embryonic development arrest. In this review, we categorize these clinically-identified, mutated genetic factors by their molecular characteristics: nuclear factors (PALT2, TRIP13, WEE2, TBPL2, REC114, MEI1 and CDC20), cytoplasmic factors (TLE6, PADI6, NLRP2/5, FBXO43, MOS and BTG4), a factor unique to primates (TUBB8), cell membrane factor (PANX1), and zona pellucida factors (ZP1-3). We compared discrepancies observed in phenotypes between human and mouse models to provide clues for clinical diagnosis and treatment of related reproductive diseases.

Role of the TATA-box binding protein (TBP) and associated family members in transcription regulation.

The assembly of transcription complexes on eukaryotic promoters involves a series of steps, including chromatin remodeling, recruitment of TATA-binding protein (TBP)-containing complexes, the RNA polymerase II holoenzyme, and additional basal transcription factors. This review describes the transcriptional regulation by TBP and its corresponding homologs that constitute the TBP family and their interactions with promoter DNA. The C-terminal core domain of TBP is highly conserved and contains two structural repeats that fold into a saddle-like structure, essential for the interaction with the TATA-box on DNA. Based on the TBP C-terminal core domain similarity, three TBP-related factors (TRFs) or TBP-like factors (TBPLs) have been discovered in metazoans, TRF1, TBPL1, and TBPL2. TBP is autoregulated, and once bound to DNA, repressors such as Mot1 induce TBP to dissociate, while other factors such as NC2 and the NOT complex convert the active TBP/DNA complex into inactive, negatively regulating TBP. TFIIA antagonizes the TBP repressors but may be effective only in conjunction with the RNA polymerase II holoenzyme recruitment to the promoter by promoter-bound activators. TRF1 has been discovered inDrosophila melanogasterandAnophelesbut found absent in vertebrates and yeast. TBPL1 cannot bind to the TATA-box; instead, TBPL1 prefers binding to TATA-less promoters. However, TBPL1 shows a stronger association with TFIIA than TBP. The TCT core promoter element is present in most ribosomal protein genes inDrosophilaand humans, and TBPL1 is required for the transcription of these genes. TBP directly participates in the DNA repair mechanism, and TBPL1 mediates cell cycle arrest and apoptosis. TBPL2 is closely related to its TBP paralog, showing 95% sequence similarity with the TBP core domain. Like TBP, TBPL2 also binds to the TATA-box and shows interactions with TFIIA, TFIIB, and other basal transcription factors. Despite these advances, much remains to be explored in this family of transcription factors.

The Diverse Transformer (Trf) Protein Family in the Sea Urchin Paracentrotus lividus Acts through a Collaboration between Cellular and Humoral Immune Effector Arms.

Sea urchins are long-living marine invertebrates with a complex innate immune system, which includes expanded families of immune receptors. A central immune gene family in sea urchins encodes the Transformer (Trf) proteins. The Trf family has been studied mainly in the purple sea urchin Strongylocentrotus purpuratus. Here, we explore this protein family in the Mediterranean Sea urchin Paracentrotus lividus. The PlTrf genes and predicted proteins are highly diverse and show a typical Trf size range and structure. Coelomocytes and cell-free coelomic fluid from P. lividus contain different PlTrf protein repertoires with a shared subset, that bind specifically to E. coli. Using FACS, we identified five different P. lividus coelomocyte sub-populations with cell surface PlTrf protein expression. The relative abundance of the PlTrf-positive cells increases sharply following immune challenge with E. coli, but not following challenge with LPS or the sea urchin pathogen, Vibrio penaeicida. Phagocytosis of E. coli by P. lividus phagocytes is mediated through the cell-free coelomic fluid and is inhibited by blocking PlTrf activity with anti-SpTrf antibodies. Together, our results suggest a collaboration between cellular and humoral PlTrf-mediated effector arms in the P. lividus specific immune response to pathogens.

A homozygous missense mutation in TBPL2 is associated with oocyte maturation arrest and degeneration.

The genetic causes in most of patients with oocyte maturation arrest remain largely unknown. In this study, we identified a homozygous missense mutation (c.895T>C; p.C299R) in TBPL2 (TATA box binding protein like 2) in two infertile sisters with oocyte maturation arrest and degeneration from a consanguineous family by whole-exome sequencing. The TBPL2 mutation is rare and pathogenic, and impaired the transcription initiation function of the protein. Our results showed that TBPL2 mutation might be associated with female infertility due to oocyte maturation arrest and degeneration.

Changes in the Expression of TBP-2 in Response to Histone Deacetylase Inhibitor Treatment in Human Endometrial Cells.

Histone deacetylase inhibitors (HDACi) induce apoptosis preferentially in cancer cells by caspase pathway activation and reactive oxygen species (ROS) accumulation. Suberoylanilide hydroxamic acid (SAHA), a HDACi, increases apoptosis via altering intracellular oxidative stress through thioredoxin (TRX) and TRX binding protein-2 (TBP-2). Because ROS accumulation, as well as the redox status determined by TBP-2 and TRX, are suggested as possible mechanisms for endometriosis, we queried whether SAHA induces apoptosis of human endometrial cells via the TRX-TBP-2 system in endometriosis. Eutopic endometrium from participants without endometriosis, and ectopic endometrium from patients with endometriosis, was obtained surgically. Human endometrial stromal cells (HESCs) and Ishikawa cells were treated with SAHA and cell proliferation was assessed using the CCK-8 assay. Real-time PCR and Western blotting were used to quantify TRX and TBP-2 mRNA and protein expression. After inducing oxidative stress, SAHA was applied. Short-interfering TRX (SiTRX) transfection was performed to see the changes after TRX inhibition. The mRNA and protein expression of TBP-2 was increased with SAHA concentrations in HESCs significantly. The mRNA TBP-2 expression was decreased after oxidative stress, upregulated by adding 2.5 µM of SAHA. The TRX/TBP-2 ratio decreased, apoptosis increased significantly, and SiTRX transfection decreased with SAHA. In conclusion, SAHA induces apoptosis by modulating the TRX/TBP-2 system, suggesting its potential as a therapeutic agent for endometriosis.

TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes.

BACKGROUND: Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown. METHODS: We used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes. RESULTS: The mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3-/-) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3-/- hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs. CONCLUSIONS: These results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.

Fetal growth restriction shortens cardiac telomere length, but this is attenuated by exercise in early life.

BACKGROUND AND AIMS: Fetal and postnatal growth restriction cause a predisposition to cardiovascular disease (CVD) in adulthood. Telomeres are repetitive DNA-protein structures that protect chromosome ends, and the loss of these repeats (a reduction in telomere length) is associated with CVD. As exercise preserves telomere length and cardiovascular health, the aim of this study was to determine the effects of growth restriction and exercise training on cardiac telomere length and telomeric genes. METHODS AND RESULTS: Pregnant Wistar Kyoto rats underwent bilateral uterine vessel ligation to induce uteroplacental insufficiency and fetal growth restriction ("Restricted"). Sham-operated rats had either intact litters ("Control") or their litters reduced to five pups with slowed postnatal growth ("Reduced"). Control, Restricted, and Reduced male rats were assigned to Sedentary, Early exercise (5-9 wk of age), or Late exercise (20-24 wk of age) groups. Hearts were excised at 24 wk of age for telomere length and gene expression measurements by quantitative PCR. Growth restriction shortened cardiac telomere length ( P < 0.001), but this was rescued by early exercise ( P < 0.001). Early and Late exercise increased cardiac weight index ( P < 0.001), but neither this nor telomere length was associated with expression of the telomeric genes Tert, Terc, Trf2, Pnuts, or Sirt1. DISCUSSION AND CONCLUSIONS: Growth restriction shortens cardiac telomere length, reflecting the cardiac pathologies associated with low birth weight. Exercise in early life may offer long-term protective effects on cardiac telomere length, which could help prevent CVD in later life.

Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

Fibroblast growth factor 21 and its novel association with oxidative stress.

Fibroblast growth factor 21 (FGF21) is an endocrine-member of the FGF family. It is synthesized mainly in the liver, but it is also expressed in adipose tissue, skeletal muscle, and many other organs. It has a key role in glucose and lipid metabolism, as well as in energy balance. FGF21 concentration in plasma is increased in patients with obesity, insulin resistance, and metabolic syndrome. Recent findings suggest that such increment protects tissue from an increased oxidative stress environment. Different types of physical stress, such as strenuous exercising, lactation, diabetic nephropathy, cardiovascular disease, and critical illnesses, also increase FGF21 circulating concentration. FGF21 is now considered a stress-responsive hormone in humans. The discovery of an essential response element in the FGF21 gene, for the activating transcription factor 4 (ATF4), involved in the regulation of oxidative stress, and its relation with genes such as NRF2, TBP-2, UCP3, SOD2, ERK, and p38, places FGF21 as a key regulator of the oxidative stress cell response. Its role in chronic diseases and its involvement in the treatment and follow-up of these diseases has been recently the target of new studies. The diminished oxidative stress through FGF21 pathways observed with anti-diabetic therapy is another clue of the new insights of this hormone.

TBP-like protein (TLP) represses myogenesis via inhibition of the myogenin promoter.

TBP-like protein (TLP) is one of the metazoan-restricted transcription factors participating in development and differentiation, though the molecular mechanism by which TLP regulates these processes remains unclear. In this study, we investigated the relationship between TLP and myogenesis of mouse C2C12 myoblasts. We found that TLP gene expression decreases during myogenic differentiation. Overexpression and knockdown of TLP revealed that the levels of muscle-specific myosin heavy chain and the myogenic transcription factor myogenin are downregulated by TLP. TLP inhibits the progression of morphological change from myoblasts to myotubes, thereby suppressing myogenesis. We further show that TLP represses the promoter activity of myogenin. The proximal AT-rich sequence of the myogenin promoter is responsible for TLP-mediated transcriptional repression. The results of this study suggest that TLP inhibits myogenesis through downregulation of the myogenin gene.

TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression.

Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2(-/-) mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.

Activation of a T-box-Otx2-Gsc gene network independent of TBP and TBP-related factors.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors.

Telomere length and telomere repeating factors: Cellular markers for post-traumatic stress disorder-like model.

OBJECTIVE: The aim of the present study was to explore the telomere length of peripheral blood leukocytes from a rat model of post-traumatic stress disorder (PTSD), as well as the expression level of telomere-binding protein in the hippocampal CA1 region. METHODS: The PTSD model was established with 42 adult male Wistar rats. The relative telomere length of the leukocytes was measured by real-time fluorescence quantitative polymerase chain reaction, and the expression levels of telomere repeating factor 1 (TRF1) and telomere repeating factor 2 (TRF2) in the hippocampal CA1 region of the PTSD rat model were determined by immunofluorescence technology. The covariance analysis of repeated measurements by the mixed model approach was used for the telomere length analysis. The comparison of averaged data among groups was performed using least significant difference and analysis of variance. The Student's t test or the Mann-Whitney U test was used for intragroup comparison. The association study among groups was conducted using the Spearman test. RESULTS: The shortening speed of telomere length significantly accelerated in rats after Single Prolonged Stress (SPS) stimulation (P<0.05). The expression levels of TRF1 and TRF2 increased with the progress of PTSD, and the expression peak was shown in day 14, which was significantly different from the control group (P<0.05). CONCLUSION: The shortening speed of the telomere length of peripheral blood leukocytes accelerated in PTSD rats, and the expression levels of TRF1 and TRF2 increased in hippocampus, both of which were closely associated with the pathological progress of the PTSD-like model and unfavorable prognosis.

Tumor suppressor microRNA-18a regulates tumor proliferation and invasion by targeting TBPL1 in colorectal cancer cells.

Recent advances in the understanding of microRNA have rendered microRNAs (miRNAs) a compelling novel class of biomarker in cancer biology. However, the specific function of miRNA­18a (miR­18a) in colorectal cancer (CRC) remains unclear. In the present study, the role of miR­18a in the carcinogenesis of CRC was investigated. miR­18a expression was assessed in CRC specimens and cell lines using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The targets of miR­18a were predicted using bioinformatics tools. Luciferase reporter assays were used to confirm the functional association between miR­18a and its target genes. The effect of miR­18a on cell proliferation, invasion and migration was confirmed in vitro by a methylthiazol tetrazolium assay, cell invasion assay, and wound healing assay. Gene and protein expression was examined using RT­qPCR and western blotting, respectively. It was demonstrated that the expression of miR­18a in CRC tissues and cell lines was markedly lower than in normal control tissues and cells, respectively. In addition, miR­18a inhibited cell proliferation, invasion and migration in CRC cells. Moreover, TATA box­binding protein­like protein 1 (TBPL1) was identified as a potential target gene of miR­18a in the bioinformatics analysis and luciferase reporter assays, and miR­18a directly inhibited TBPL1 expression by targeting its 3'­untranslated region. Furthermore, TBPL1 was downregulated and inversely correlated with miR­18a expression in tissues. These findings demonstrate that miR­18a exhibits a protective role in CRC via inhibiting proliferation, invasion and migration of CRC cells by directly targeting the TBPL1 gene.

MiR-133b acts as a tumor suppressor and negatively regulates TBPL1 in colorectal cancer cells.

INTRODUCTION: MicroRNAs have emerged as post-transcriptional regulators that are critically involved in tumorigenesis. This study was designed to explore the effect of miRNA 133b on the proliferation and expression of TBPL1 in colon cancer cells. METHODS: Human colon cancer SW-620 cells and human colon adenocarcinoma HT-29 cells were cultured. MiRNA 133b mimcs, miRNA 133b inhibitors, siRNA for TBPL1 and scrambled control were synthesized and transfected into cells. MiR-133b levels in cells and CRC tumor tissue was measured by real-time PCR. TBPL1 mRNA was detected by RT-PCR. Cell proliferation was studied with MTT assay. Western blotting was applied to detect TBPL1 protein levels. Luciferase assays were conducted using a pGL3-promoter vector cloned with full length of 3'UTR of human TBPL1 or 3'UTR with mutant sequence of miR-133b target site in order to confirm if the putative binding site is responsible for the negative regulation of TBPL1 by miR- 133b. RESULTS: Real time PCR results showed that miRNA 133b was lower in CRC tissue than that in adjacent tissue. After miR-133b transfection, its level was elevated till 48h, accompanied by lower proliferation in both SW-620 and HT-29 cells. According to that listed in http://www.targetscan.org, the 3'-UTR of TBPL1 mRNA (NM_004865) contains one putative binding site of miR-133b. This site was confirmed to be responsible for the negative regulation by miR-133b with luciferase assay. Further, Western blotting and immunohistochemistry both indicated a higher TBPL1 protein expression level in CRC tissue. Finally, a siRNA for TBPL1 transfection obviously slowed down the cell proliferation in both SW-620 and HT-29 cells. CONCLUSION: MiR-133b might act as a tumor suppressor and negatively regulate TBPL1 in CRC.

Interaction between transactivation domain of p53 and middle part of TBP-like protein (TLP) is involved in TLP-stimulated and p53-activated transcription from the p21 upstream promoter.

TBP-like protein (TLP) is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1) in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.

Comparative expression of thioredoxin-1 in uterine leiomyomas and myometrium.

Uterine leiomyomas are benign tumors that develop from smooth muscle cells (SMCs). The reactive oxygen species (ROS) have been shown to be involved in the signaling pathways that stimulate proliferation of a variety of cell types. Thioredoxin-1 (TRX-1) is a redox-regulating protein, which is overexpressed in various tumors. In the present study, we investigated the expressions of TRX-1 and its related molecules in uterine leiomyomas and matched adjacent myometrium. Our results showed the expression of TRX-1 was increased in leiomyomas compared with the matched adjacent myometrium by quantitative RT-PCR and western blotting. FOXO3A expression was increased in leiomyomas compared with myometrium by western blotting. The mRNA levels of hypoxia-inducible factor-1α, cyclooxygenase-2 and cyclin D1 were increased in leiomyomas compared with the adjacent myometrium. The mRNA level of (thioredoxin-1-binding protein) TBP-2 in leiomyomas was not altered when compared with the matched adjacent myometrium. These results suggest that TRX-1 and some of its related molecules are associated with the pathogenesis of uterine leiomyomas. The identification of TRX-1 signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.