Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression.

Overexpression of the Runt-related transcription factor 2 (RUNX2) has been reported in several cancer types, and the C-X-C motif chemokine receptor 4 (CXCR4) has an important role in tumour progression. However, the interplay between CXCR4 and RUNX2 in melanoma cells remains poorly understood. In the present study, we used melanoma cells and a RUNX2 knockout (RUNX2-KO) in vitro model to assess the influence of RUNX2 on CXCR4 protein levels along with its effects on markers associated with cell invasion and autophagy. Osteotropism was assessed using a 3D microfluidic model. Moreover, we assessed the impact of CXCR4 on the cellular levels of key cellular signalling proteins involved in autophagy. We observed that melanoma cells express both RUNX2 and CXCR4. Restored RUNX2 expression in RUNX2 KO cells increased the expression levels of CXCR4 and proteins associated with the metastatic process. The protein markers of autophagy LC3 and beclin were upregulated in response to increased CXCR4 levels. The CXCR4 inhibitor WZ811 reduced osteotropism and activated the mTOR and p70-S6 cell signalling proteins. Our data indicate that the RUNX2 transcription factor promotes the expression of the CXCR4 chemokine receptor on melanoma cells, which in turn promotes autophagy, cell invasiveness, and osteotropism, through the inhibition of the mTOR signalling pathway. Our data suggest that RUNX2 promotes melanoma progression by upregulating CXCR4, and we identify the latter as a key player in melanoma-related osteotropism.

Characterization and functional analysis of cis-acting elements of the human farnesyl diphosphate synthetase (FDPS) gene 5' flanking region.

Farnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids. Genomic comparison of the 5' upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes. The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors.

The Emerging Role of the RBM20 and PTBP1 Ribonucleoproteins in Heart Development and Cardiovascular Diseases.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

A Potential Role of RUNX2- RUNT Domain in Modulating the Expression of Genes Involved in Bone Metastases: An In Vitro Study with Melanoma Cells.

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling.

Functional characterization of the ribonucleoprotein, PTB-binding 1/Raver1 promoter region.

Raver1 is a ribonucleoprotein, evolutionarily conserved in mammals, which acts as a polypyrimidine tract-binding protein (PTB/hnRNPI) co-repressor in regulating alternative splicing events. The mouse homologue has been identified as a dual compartment protein that interacts with PTB within perinucleolar structures, and localizes at microfilament plasma membrane attachment sites in fibroblasts, epithelial and muscle cells. Human Raver1 gene is localized on chromosome 19p13.2 and encodes for an inferred 756 amino acid protein sharing 87% similarity with the mouse orthologue. The human Raver1 gene expression has not been previously investigated. Here we report the mRNA expression profile of human Raver1 gene and the molecular characterization of its promoter region. From the in silico analysis of 1542 bp of the Raver1 5'-flanking region (GC content=61%), no canonical TATA or CAAT boxes can be highlighted, whereas several consensus Sp1 putative binding sequences can be predicted within 1 kb from the transcription start site (TSS) that we determined by 5'-RACE. Functional analyses established a minimal region involved in the regulation of the human Raver1 promoter activity. Mutational analyses and transfection studies indicated that a GGGAGCTCCC sequence at -531 represents a putative cis signal acting as a negative regulator element of the promoter function. Altogether, our results indicate that human Raver1 gene promoter region shares common features of ubiquitously expressed gene with the interacting splicing regulator PTB.

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

Nuclear localization domains in human thyroid transcription factor 2.

Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin alpha. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.

Identification and analysis of the human neural polypyrimidine tract binding protein (nPTB) gene promoter region.

Neural polypyrimidine tract-binding protein nPTB, originally identified as the neuronal counterpart of the hnRNPI/PTB protein, is an RNA binding protein involved into tissue-specific alternative splicing regulation. Here we describe the functional characterization of the promoter sequence of nPTB in HeLa and neuroblastoma cells. By means of genomic sequence analysis we have isolated and cloned a 1587-base pair region upstream the human nPTB coding region. The nPTB proximal promoter, although rich in G+C content and presenting putative binding sites for the transcription factors Sp1, NF-1, NF-kB and Oct-1, lacks a typical TATA box. Luciferase transient expression assays using deletion mutants have identified the proximal promoter region at -125 relative to the transcription start site. Alignment of human, murine and chimpanzee genomic sequences upstream the nPTB exon 1 has provided evidences for the evolutionary conservation of specific transcription factor binding sites.

Identification of nuclear retention domains in the RBM20 protein.

RBM20 is a nuclear protein which regulates alternative splicing of expressed genes that have a key role in cardiac function. By cloning the human and mouse RBM20 cDNA, producing expressing vectors for truncated proteins, and comparing their sub-cellular distribution in transfected cells, we have identified the sequences necessary for RBM20 full nuclear retention. The region overlaps an RNA binding motif and a serine-arginine domain. The sequence is conserved in many species but belongs only to RBM20 orthologs. The RMB20 tissue specificity, together with the properties of its nuclear localization determinant, demonstrates a specific evolutionary selection of post-transcriptional regulation factors.

Transcriptional regulation of the human Raver2 ribonucleoprotein gene.

Raver2 is a putative modulator of the activity of the polypyrimidine-tract binding protein (PTB), one of the most intensively studied splicing repressors. Little is known about Raver2 expression, and all current data is from mice where it shows tissue specificity. In the present study, by comparing Raver2 transcript expression in human and mouse tissues, we found that human Raver2 is ubiquitously expressed in adult tissues. In order to investigate human Raver2 transcription regulation, we identified and characterized a putative promoter region in a 1000bp region upstream of the transcription starting site of the gene. Dual luciferase reporter assays demonstrated that this region had promoter activity conferred by the first 160bp. By mutagenic analyses of putative cis-acting regulatory sequences, we identified an individual site that decreased the promoter activity by up to 40% when mutated. Together, our results suggest that regulation of human Raver2 expression involves TATA-less transcriptional activity.