[Possible Role of Prep 1 Homeodomain Transcription Factor in Cardiac Mesenchymal Stromal Cells].

Homeodomain transcription factors play a significant role in mesenchymal stromal cells (MSCs). Previously, the role of Meis1, Pbx1 and Prep1 proteins from the TALE (Three Amino acid Loop Extension) family in adipocytic and osteogenic differentiation of mouse mesenchymal stromal cells was established. In this work, using ChIP-seq and bioinformatic analysis we investigated the binding pattern of PREP1 with the genomic DNA of human heart MSCs, identified nearby genes, and analyzed their ontology. The target genes of the PREP1 factor in cardiac MSCs have been established. Based on the results, the possible involvement of transcription factor PREP1 in the direct reprogramming of fibroblasts into cardiomyocytes is discussed.

[The Involvement of Cardiomyocyte-Specific Transcription Factors Meis in Adipocyte Differentiation].

Homeodomain transcription factors play a significant role in adipocyte differentiation. The role of Pbx1 and Prep1, proteins of the TALE family (the three amino acid loop extension), was previously established in adipocyte differentiation of mesenchymal stromal cells and 3T3-L1 cell line. In this study, with the use of RNA interference technology we show that another transcription factor from the same family, Meis1, which is a core protein of mature cardiomyocytes, represses adipogenesis to a greater degree than its paralog Meis2. A number of Meis target genes, markers of adipocytes, are identified. This may indicate the transcriptional mechanism of the effect of Meis1 on the adipocyte differentiation of mouse preadipocytes.

Transcriptional control of insulin-sensitive glucose carrier Glut4 expression in adipose tissue cells.

In search for new targets for obesity treatment, we have studied the effect of several transcription factors on the conversion of murine preadipocytes from the 3T3-L1 cell line into adipocytes. We have found that knockdown of Prep1 gene expression affects adipogenic differentiation and results in significant increase in the insulin-sensitive glucose carrier Glut4 gene expression.

Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates.

TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions, and are involved in important pathologies. This review focuses on the PREP, MEIS, and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e., structure, interactors, and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1, and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEIS proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions.

Insulin resistance and adipogenesis: role of transcription and secreted factors.

Insulin stimulates carbohydrate uptake by cells and induces their conversion into lipids as a more efficient form of energy storage. Insulin resistance is associated with a decrease in glucose uptake by muscle and adipose cells and also with a decrease in glycogen synthesis on retention of glucose synthesis by liver cells. Disorders in the insulin signaling cascade on development of insulin resistance can be caused by both changes in functioning of transcriptional factors and in the secretion profile of hormone-like substances. Diacylglycerols and ceramides responsible for activation of some kinases and phosphatases can directly trigger these changes in muscle and liver cells. In adipose tissue, insulin mainly stimulates adipogenesis (adipocyte differentiation) and lipogenesis (lipid accumulation in the cells). Thus, studies on the action mechanisms of factors influencing adipogenesis can be of help for understanding the molecular mechanisms of insulin resistance.

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors.

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.

Oncogenic HoxB7 requires TALE cofactors and is inactivated by a dominant-negative Pbx1 mutant in a cell-specific manner.

The homeobox containing gene HoxB7 is functionally associated with melanoma growth promotion through the direct transactivation of bFGF. Accordingly, the introduction of HoxB7 in the breast cancer line SkBr3 (SkBr3/B7), strongly increases its tumorigenic properties. Here we show that in SkBr3/B7 cells, HoxB7 regulates the expression of TALE Hox cofactors by increasing Pbx2 and Prep1 and decreasing Pbx1. The functional requirement of Hox cofactors in the oncogenic activity of HoxB7 was proven with a dominant-negative Pbx1 mutant, Pbx1NT, which sequesters Prep1 in the cytoplasm. The less aggressive phenotype of the SkBr3/B7/PbxNT cells, evaluated in vitro as well as in vivo, correlated well with increased apoptosis, decreased cycling and up-regulation of p16 and p53. Tumor cell-type specific functional effects of Pbx1NT were observed, possibly related to the presence of different Hox genes in melanoma or breast adenocarcinoma DNA-protein ternary complexes.

Health risk for children from lead and cadmium near a non-ferrous smelter in Bulgaria.

In an interdisciplinary approach the exposure to lead and cadmium of the population living in the vicinity of a Bulgarian non-ferrous metallurgical plant was studied to determine the routes of exposure. Two exposed villages situated 4 and 6 km SW and W, respectively,--in the plume of the plant's emissions--and a village for comparison situated 14 km SE of the plant were selected for the study. The blood of children aged 3-13 years from the exposed villages (n = 111) and the village for comparison (n = 18) was analyzed for lead (PbB) and cadmium (CdB) as well as for free erythrocyte protoporphyrin (FEP) and haematological parameters. A personal questionnaire concerning nutrition habits and life-style was filled in by interviewers of the children's parents. The cadmium levels in blood were within the variation range stated for Bulgaria. The exposed children had a mean CdB of 0.38 +/- 0.18 microgram/l (range 0.10-0.90 microgram/l) and the comparison group 0.31 +/- 0.35 microgram/l (0.06-1.42 micrograms/l). The mean PbB value measured in the exposed children was 240 +/- 96 micrograms/l (77-631 micrograms/l), while the mean value of PbB for the reference group was 149 +/- 57 micrograms/l (63-285 micrograms/l). Individual PbB levels > 150 and > 200 micrograms/l were found in 85% and 62%, respectively, of the exposed children and FEP > 80 micrograms/dl erythrocytes was detected in 21%. There was a concentration-response and a concentration-effect relationship (r = 0.51, D > 0.001) between PbB and FEP. According to the questionnaire only 10.3% of the interviewed families in the two exposed villages buy all food from the market, the others produce a significant part of their food themselves. The PbB levels of children who consume certain foods of local production (cabbage, cereals, poultry, and beef) were significantly higher compared to the PbB values of children whose families purchase these foods from the market. No significant relationship between lead in children's blood and the use of tin and ceramic utensils, drinking and irrigation water, hygiene habits, family tobacco smoking habits, playgrounds and traffic intensity were revealed. There was a significantly higher incidence of prematurely born babies in the exposed population. The proven relationship between lead intake via the food chain and existing significant health risks is the basis for risk communication. Measures aimed at healthier nutrition and life-style intend to contribute to decreasing the risk of the population living in the smelter region.

Cooperative interactions between PBX, PREP, and HOX proteins modulate the activity of the alpha 2(V) collagen (COL5A2) promoter.

Cell type-specific expression of the human alpha2(V) collagen (COL5A2) gene depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relative to the transcription start site. The present study focused on the characterization of the FPB-bound complex. DNA binding assays and cell transfection experiments revealed that the bipartite core sequence of FPB (5'-ATCAATCA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOXB1 result in binding of the heterodimers to FPB in vitro. Similarly, overexpression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB-driven transcription. In contrast to the composition of the FPB complex purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously introduced into COL5A2-negative cells cannot stimulate FPB-driven transcription unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions among PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V.

Cloning of a human gene closely related to the genes coding for the c-myc single-strand binding proteins.

Southwestern screening of human fibroblast cDNAs with an upstream element of the alpha2(I) collagen promoter (Box 5A) has led to the identification of a novel gene product (RBMS3). RBMS3 contains two pairs of RNA binding motifs and is very closely related to the structure of the c-myc gene single-strand binding proteins (MSSPs). MSSPs are believed to regulate DNA replication, transcription, apopotosis and cell cycle progression by interacting with the C-MYC protein. Consonant with this postulate, RBMS3 binds in vitro to the minus strand of Box 5A and transactivates transcription in the chimeric GAL4 hybrid system. However, the RBMS3 protein mostly localizes to the cytoplasm of transfected cells, in addition to binding strongly in vitro to synthetic poly-U and poly-A oligoribonucleotides. Finally, overexpression in transfected fibroblasts of RBMS3 with and without a nuclear localization signal has no effect on Box 5A-driven transcription. The results thus exclude RBMS3 involvement in the transcriptional regulation of COL1A2 and strongly suggest a cytoplasmic function of this new member of the MSSP family. As part of the initial characterization of RBMS3 we have also established that the gene resides on human chromosome 3p23-p24 and is widely expressed in the embryo and in the adult organism.

Tumor necrosis factor alpha inhibits type I collagen synthesis through repressive CCAAT/enhancer-binding proteins.

Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits ECM accumulation by stimulating the expression of matrix proteolytic enzymes and by downregulating the deposition of structural macromolecules such as type I collagen. Stimulation of ECM degradation has been linked to prolonged activation of jun gene expression by the cytokine. Here we demonstrate that TNF-alpha inhibits transcription of the gene coding for the alpha2 chain of type I collagen [alpha2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-alpha-responsive element. This conclusion was based on the concomitant identification of C/EBPbeta and C/EBPdelta as TNF-alpha-induced factors by biochemical purification and expression library screening. It was further supported by the ability of the C/EBP-specific dominant-negative (DN) protein to block TNF-alpha inhibition of alpha2(I) collagen but not TNF-alpha stimulation of the MMP-13 protease. The DN protein also blocked TNF-alpha downregulation of the gene coding for the alpha1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-alpha-induced signaling pathway that controls ECM formation and remodeling.