Recent advances in the rationale design of GPER ligands.

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Two novel GPER agonists induce gene expression changes and growth effects in cancer cells.

Although the action of estrogens has been traditionally explained by the binding to and transactivation of the nuclear estrogen receptor (ER)α and ERß, recently the G protein-coupled receptor GPR30/GPER has been involved in the rapid estrogen signaling. We investigated the ability of two original molecules, which were named GPER-L1 and GPERL2, to bind to and activate the GPER transduction pathway in cancer cells. Competition assays, docking simulations, transfection experiments, real-time PCR, immunoblotting, gene silencing technology and growth assays were performed to ascertain the selective action of GPER-L1 and GPER-L2 in activating the GPER-mediated signaling. Both compounds, which did not show any ability to bind to and activate the classical ERs, were able to bind to GPER and to trigger the rapid activation of the GPER/EGFR/ERK transduction pathway which led to the up-regulation of GPER-target genes. Notably, GPER-L1 and GPER-L2 induced the proliferation of SkBr3 breast and Ishikawa endometrial cancer cells at nM concentrations through GPER, hence providing further evidence on their capability to elicit relevant biological responses mediated by GPER. The identification and characterization of these novel compounds as selective GPER agonists represent a valuable tool to further dissect the pharmacology of this novel estrogen receptor and to better differentiate the specific functions elicited by each estrogen receptor subtype in cancer cells.

Structural comparison of the interaction of tubulin with various ligands affecting microtubule dynamics.

Microtubules (MTs), which are highly dynamic assemblies of the protein tubulin, play important and diverse roles in eukaryotic cells. MT dynamics are regulated during the cell cycle by interacting with a large number of endogenous cellular regulators. In addition, many anti-tumour drugs and natural ligands that interact directly with tubulin are able to either stabilise or destabilise MTs and to disrupt the normal dynamics. Herein, we compare the structures of tubulin when complexed with different ligands in order to analyse: (i) various binding-sites of the protein and different positions of ligands within the microtubule (ii) the diverse effect on the microtubule dynamics. The structures and data given are essential for understanding tubulin-ligand interactions and their influence on the regulation of the microtubule system.

Role of the non-receptor tyrosine kinase fes in cancer.

Non receptor protein tyrosine kinases are targets in the treatment of a number of diseases. This review focuses on the role of Fes tyrosine kinase and on the design of inhibitors of this protein. Fes and its homologously related protein Fer are the only two members of a distinct class of non receptor tyrosine kinases and they seem to play a role in cytoskeletal rearrangements and inside-out signaling associated with receptor-ligand, cell-matrix and cell-cell interactions. The knowledge of the three dimensional structure of this protein, in fact, has informed drug design, while at the same time it has helped to shed some light on the molecular mechanism at the basis of kinase activation and functions.

Akt2-mediated phosphorylation of Pitx2 controls Ccnd1 mRNA decay during muscle cell differentiation.

Paired-like homeodomain 2 (Pitx2), first identified as the gene responsible for the Axenfeld-Rieger syndrome, encodes a protein factor that, controlling cell proliferation in a tissue-specific manner, has a crucial role in morphogenesis. During embryonic development, Pitx2 exerts a role in the expansion of muscle progenitors and is expressed at all stages of myogenic progression. In this study, we show that Pitx2 is phosphorylated by the protein kinase Akt2 and is necessary to ensure proper C2C12 myoblast proliferation and differentiation. Pitx2 associates with a ribonucleoprotein complex that includes the mRNA stabilizing factor HuR and sustains Ccnd1 (also known as Cyclin D1) expression, thereby prolonging its mRNA half-life. When the differentiation program is initiated, phosphorylation by Akt2 impairs the ability of Pitx2 to associate with the Ccnd1 mRNA-stabilizing complex that includes HuR and, as a consequence, Ccnd1 mRNA half-life is shortened. We propose that unphosphorylated Pitx2 is required to favor HuR-mediated Ccnd1 mRNA stabilization, thus sustaining myoblast proliferation. Upon Akt2-phosphorylation, the complex Pitx2/HuR/Ccnd1 mRNA dissociates and Ccnd1 mRNA is destabilized. These events contribute to the switch of C2C12 cells from a proliferating to a differentiating phenotype.

Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody.

Tenascin-C exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. Here, we report a human antibody fragment, TN11, derived from a phage library with high affinity for the spliced repeat C and demonstrate that this repeat is undetectable in normal adult tissues, barely detectable or undetectable in breast, lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade astrocytoma (grade III and glioblastoma), especially around vascular structures and proliferating cells. The antibody appears to have potential for development of a therapeutic agent for patients with high grade astrocytoma.

Expression of the rat homologue of the Drosophila fat tumour suppressor gene.

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the b-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.

The human homeodomain protein OTX2 binds to the human tenascin-C promoter and trans-represses its activity in transfected cells.

Homeodomain-containing proteins mediate many transcriptional processes in eukaryotes during development. Recently, mammalian homeodomain proteins involved in the anterior head formation have been discovered, but their effect on gene transcription has never been investigated. Here we report on the ability of the human homeodomain protein OTX2 to bind with high affinity to a target sequence present in the promoter of the gene encoding the human extracellular matrix protein tenascin-C and to repress its transcriptional activity in transiently transfected cells.

The first untranslated exon of the human tenascin-C gene plays a regulatory role in gene transcription.

The transcription of the human tenascin-C (TN-C) gene is directed by a single promoter. Here we demonstrate, in transiently transfected cells, that two distinct regions of the untranslated 179 bp-long exon 1 play antagonistic roles in transcriptional regulation: bases from 1 to 20 strongly increase the transcription of the reporter gene CAT directed by the human TN-C gene promoter, while bases from 79 to 179 significantly reduce this activation.

The glucagon gene is transcribed in beta-like pancreatic cells.

In this report we demonstrate that approximately 1.1 kb of the rat glucagon gene promoter upstream of the transcriptional start site specifically directs the transcription of the reporter gene chloramphenicol acetyl transferase (CAT) (p[-1.1]GLU-CAT) in insulinoma beta-TC1 cells. On the contrary, the 350 bp closest to the transcription start site (p[-0.35]GLU-CAT) are ineffective in beta-TC1 cells. Both constructs are transcriptionally active in InR1-G9 glucagonoma cells. While protein kinase A and protein kinase C activators, acting through independent pathways, strongly increase both the transcription of p[-1.1]GLU-CAT and the accumulation of glucagon transcript in beta-TC1 cells, they are weaker activators in InR1-G9 cells. Our experiments suggest that some positive transcription control elements, necessary for the glucagon gene transcription in insulinoma beta-TC1 cells, are localized in the -350/-1100 region of the glucagon gene. Furthermore, our data indicate that glucagon gene transcription can be strongly activated through the protein kinase A pathway in some specific cellular contexts.

Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences.

This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to -2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene.

Cell-cycle dependent alternative splicing of the tenascin primary transcript.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin.

We have obtained and characterized 11 monoclonal antibodies (mAbs) specific for different domains of human tenascin (TN). Five of these mAbs reacted with epitopes contained in the TN area that undergoes alternative splicing and are thus able to recognize specific TN isoforms. These mAbs are a useful tool to study the expression and distribution of TN and its different isoforms in normal and pathological tissues.

Steady-state levels of different tenascin mRNAs in various normal human tissues.

Northern blot analysis of TN mRNA from different human tissues shows two major bands of about 6 and 8 kb which correspond to two different mRNAs generated by alternative splicing of the primary transcript. In liver, pancreas and kidney only the 6 kb TN mRNA was detectable. The highest levels of 8 kb TN mRNA were observed in placenta and skin representing 30% and 52% of total TN mRNA, respectively. In all other tissues tested the 8 kb TN mRNA represented less than 20% of total TN mRNA.

Tenascin and fibronectin distribution in human normal and pathological synovium.

Tenascin is a glycoprotein found mainly in the extracellular matrix of developing and malignant tissues. The distribution of this molecule in normal and pathological synovia from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) was investigated by indirect immunofluorescence utilizing specific monoclonal antibodies. The same technique was used to study total fibronectin (tFn) in synovial tissues as well as ED-A and ED-B containing fibronectin (Fn) isoforms (A-Fn, B-Fn), generated by alternative splicing of pre-mRNA. Tenascin was found in normal synovium just beneath the whole lining cell layer. However, a higher density and spreading pattern of distribution was observed in OA and RA sections. A-Fn and B-Fn isoforms were prominent and widespread throughout the normal synovial lining; in hypercellular synovial lining (in RA and OA samples), A-Fn and B-Fn were also observed spreading in the sublining, as well as tFn.

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.

Procedure for the purification of the fibronectin proteolytic fragments containing the ED-B oncofetal domain.

Fibronectin (FN) is the blend of structurally different molecules (isoforms) whose makeup varies depending on the FN sources. Fibronectin polymorphism is caused by three sequences (called ED-A, ED-B, and IIICS) which may be included or excluded from the FN molecule depending on the alternative splicing patterns of a single primary transcript. The sequence ED-B, which is a complete type III repeat of 91 amino acids, presents some interesting peculiarities: it is the most conserved FN region and, in normal adult tissues, the ED-B-containing FN has an extremely restricted distribution while having a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989, J. Cell Biol. 108, 1139-1148), suggesting that the ED-B sequence may confer to the FN molecules specific biological activities required during ontogenesis and oncogenetic processes. Here we describe a detailed procedure to purify fibronectin fragments containing the ED-B sequence. These purified fragments are useful reagents in the study of the biological function(s) of the ED-B-containing FN molecules.