Nadya I. Tarasova

Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism

Wnt4 and β-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of β-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of β-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of β-catenins helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of β-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca(2+)-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca(2+) and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca(2+)-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.

Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA

Background: Unphosphorylated STAT3 (U-STAT3) regulates gene expression, but the mechanisms of its DNA binding are not fully understood. Results: U-STAT3 binds to the same γ-activated sequence (GAS) DNA-binding site as phosphorylated STAT3. It also binds to AT-rich DNA structures. Conclusion: U-STAT3 regulates gene expression by binding to GAS and influencing chromatin organization. Significance: Our data provide an explanation of mechanisms of U-STAT3 binding to DNA. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ (gamma)-activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to the GAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer.

GTP-Dependent K-Ras Dimerization

Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.

STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region

Background: K-Ras4B modulates downstream signaling at different lipid microdomains. Results: K-Ras4B farnesyl group spontaneously inserts into the disordered lipid microdomains, but phosphorylation prohibits the farnesyl membrane insertion. Conclusion: The farnesyl may determine K-Ras4B function in different membrane microdomain environments. Significance: Figuring out K-Ras4B localization at different membrane microdomains is important for a more complete understanding Ras-effector interactions mediating signaling pathways. K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.

Ligand-induced Internalization of Cholecystokinin Receptors DEMONSTRATION OF THE IMPORTANCE OF THE CARBOXYL TERMINUS FOR LIGAND-INDUCED INTERNALIZATION OF THE RAT CHOLECYSTOKININ TYPE B RECEPTOR BUT NOT THE TYPE A RECEPTOR

Internalization of a variety of different heptahelical G protein-coupled receptors has been shown to be influenced by a number of different structural determinants of the receptors, including the carboxyl terminus. To investigate the role of the carboxyl terminus of cholecystokinin (CCK) receptors in receptor internalization, the rat wild type (WT) CCK-A receptor (WT CCKAR) and the rat WT CCK-B receptor (WT CCKBR) were truncated after amino acid residue 399 (CCKAR Tr399) and 408 (CCKBR Tr408), thereby deleting the carboxyl-terminal 45 and 44 residues, respectively. All WT and mutant CCK receptors were stably expressed in NIH/3T3 cells. Internalization of the CCKAR Tr399 was not significantly different from the WT CCKAR. In contrast, internalization of the CCKBR Tr408 was decreased to 26% compared with the WT CCKBR internalization of 92%. The mutation of all 10 serine and threonine residues (as potential phosphorylation sites) in the carboxyl terminus of the CCKBR to alanines (mutant CCKBR ΔS/T) could account for the majority of this effect (39% internalization). All mutant receptors displayed similar ligand binding characteristics, G protein coupling, and signal transduction as their respective WT receptors, indicating that the carboxyl termini are not necessary for these processes. Thus, internalization of the CCKBR, unlike that of the CCKAR, depends on the carboxyl terminus of the receptor. These results suggest that, despite the high degree of homology between CCKAR and CCKBR, the structural determinants that mediate the interaction with the endocytic pathway reside in different regions of the receptors.

Small Molecule Toxins Targeting Tumor Receptors

Targeting toxic therapeutics to tumors through receptors over expressed on the surface of cancer cells can reduce systemic toxicity and increase the effectiveness of the targeted compounds. Small molecule targeted therapeutics have a number of advantages over toxic immunoconjugates including better tumor penetration, lack of neutralizing host immune response and superior flexibility in selection of drug components with optimal specificity, potency and stability in circulation. Three major components of the targeted drug, the toxic warhead, tumor-specific ligand and the linker can influence the properties of each other and thus have to be optimized for each system. All receptor-targeted drugs are delivered inside the cells through endocytosis and undergo processing liberating the toxins in endosomes and lysosomes. Common delivery route defines a number of general requirements for each drug component. The review addresses currently known possible receptor targets and their ligands along with toxins that have been used and that have a potential to be successfully applied in tumor targeting. Linkers that are stable in circulation, but efficiently cleaved in lysosomes constitute an essential component of receptor-targeted drugs and are evaluated in greater detail.

VIP-ellipticine derivatives inhibit the growth of breast cancer cells

The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC(50) values of 1 and 0.2 microM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED(50) values of 1 and 0.1 microM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC(1) receptor agonists which inhibit MCF-7 cellular viability.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Significance α1-Adrenergic receptors are important for the regulation of vascular function and are targeted clinically for blood pressure control. Here, we provide evidence that α1A/B-adrenergic receptors (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on the cell surface of vascular smooth muscle cells. We show that disruption of α1A/B-AR:CXCR4 heteromeric complexes inhibits α1-AR–mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α1-AR agonist phenylephrine to increase blood pressure. These findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of G protein-coupled receptor heteromerization with important functional implications. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulating blood pressure. Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca2+ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca2+ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.

Endocytosis of gastrin in cancer cells expressing gastrin/CCK-B receptor

Abstract.Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4–7 min) and internalization of fluorescent ligand upon binding at room temperature and 37° C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37° C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.