Effects of recombinant human thyroid-stimulating hormone superagonists on thyroidal uptake of 18F-fluorodeoxyglucose and radioiodide.

BACKGROUND: Superagonist analogs of human thyroid-stimulating hormone (hTSH) may stimulate the uptake of (131)I-iodide and (18)F-fluorodeoxyglucose ((18)F-FDG) in thyroid carcinomas to a greater degree than hTSH. We herein report the potency and efficacy of two hTSH analogs, TR1401 and TR1402, to stimulate radioiodide and (18)F-FDG uptake in FRTL-5 cells and compared the effects of hTSH and TR1401 on radioiodide uptake in the thyroid in vivo in mice. METHODS: The effects of hTSH analogs on intracellular levels of cAMP, uptake of (131)I-iodide, and (18)F-FDG were studied in FRTL-5 cells to determine the stimulatory potency and efficacy of the compounds by calculating half-maximum effective concentration (EC(50)) values and maximal stimulatory effects (E(max)). Biodistribution studies (n = 96) and positron emission tomography/computed tomography imaging studies (single animals) on thyroid (125)I/(124)I-iodide uptake were performed with T3-suppressed CD-1 mice in a dose-dependent manner (3, 10, and 30 µg/animal). RESULTS: The EC(50) values of TR1401 and TR1402 demonstrated a 90-fold or 800-fold higher potency for their capacity to increase intracellular cAMP levels in comparison with hTSH (p < 0.05). Similar results were demonstrated for the stimulation of (18)F-FDG uptake. Bovine TSH, TR1401, and TR1402 were 85%-490% more potent to increase iodide uptake than hTSH (p < 0.05). TR1402 was 30% more efficacious to stimulate iodide uptake than hTSH. The agonist-induced increase in radiotracer uptake was paralleled by increases in NIS and GLUT-1 expression. Ex vivo biodistribution studies showed an increased iodide uptake in the thyroid of TR1401-treated mice at the low dose of 3 µg/animal in comparison with hTSH-treated mice (n = 16, p < 0.05). Positron emission tomography/computed tomography imaging studies confirmed the increased thyroidal iodide uptake in TR1401-treated mice in vivo. CONCLUSIONS: TR1401 and TR1402 have considerably higher potency than hTSH to stimulate thyroidal iodide and (18)F-FDG uptake in vitro. Moreover, in vivo studies indicated that at low but not higher doses, TR1401 induced an enhanced ability for the thyroid to concentrate iodide compared with hTSH. These properties makes TR1401 and TR1402 interesting candidates for use in humans to enhance uptake of radioiodine and (18)F-FDG by metastases and recurrences of thyroid carcinoma.

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration.

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K(d)=48nM and the related sFRP-2 with a K(d)=95nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3ß1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.

Purification and Wnt-inhibitory activities of secreted frizzled-related proteins.

Recombinant expression of secreted Frizzled-related proteins (sFRPs) in mammalian expression systems is a convenient source of these proteins for biological studies. Yields of protein vary; screening of clonal lines for high expression is usually worthwhile. Heparin affinity chromatography is an easy step that provides a major enrichment, particularly for sFRP-1 and sFRP-2. Alternatively, sFRP derivatives tagged with poly-histidine at their carboxyl termini are functional and can be readily isolated by chelating chromatography. Once purified, the proteins are stable indefinitely if stored frozen and they tolerate multiple rounds of freeze-thawing. Pre-incubation of Wnt samples with sFRP protein for 30 min at 37 degrees C is sufficient to inhibit Wnt activity in various assays. The concentration of sFRP required to block Wnt signaling should be determined empirically, as it will vary with the Wnt preparation and cellular context.

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate.

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1(lacZ) knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/beta-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.

Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2.

Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.

Identification of a peptide binding motif for secreted frizzled-related protein-1.

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.

Wnt/Frizzled signaling in Ewing sarcoma.

BACKGROUND: The Ewing sarcoma family of tumors (ESFT) is a set of neuroectodermal malignancies that typically presents in the second decade and has a poor prognosis due to metastatic disease. Wnt signaling has a critical role in the normal development of multiple neuroectodermal tissues and also contributes to the neoplastic properties of tumor cells of neuroectodermal origin. PROCEDURE: We surveyed the expression of Wnts and their receptors in nine ESFT cell lines by RT-PCR analysis. We also tested biological response of ESFT cell lines to exogenous Wnts in beta-catenin stabilization, actin stress fiber formation, and chemotaxis assays. RESULTS: We detected Wnt-10b in all the lines, and most also expressed Wnt-5a, Wnt-11, and Wnt-13. Several Frizzleds (Fz) and the Wnt co-receptors, low density lipoprotein-receptor-like proteins 5 and 6 were also expressed. We observed a marked stimulation of the beta-catenin/canonical Wnt pathway in ESFT cells treated with Wnt-3a. Wnt-3a induced morphologic changes characterized by the formation of long cytoplasmic extensions in ESFT cells. We also observed chemotaxis of ESFT cells in response to Wnt-3a. CONCLUSIONS: These results provide evidence that components of Wnt/Fz pathway are expressed and an intact Wnt/Frizzled signaling pathway exists in ESFT cell lines. Activation of the Wnt pathway in ESFT suggests that Wnt modulates cell motility rather than cell proliferation. Hence, activation of this pathway may influence metastatic potential of ESFT.

Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea.

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.

Regulation of fibronectin and metalloproteinase expression by Wnt signaling in rheumatoid arthritis synoviocytes.

OBJECTIVE: The enhanced release of extracellular matrix proteins by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients is suggestive of joint remodeling. Because Wnt proteins play a critical role in joint development, we investigated whether up-regulated Wnt signaling plays a role in the enhanced synthesis of extracellular matrix proteins. The purpose of the present experiments was to determine the role of Wnt-1-like molecules in the expression of matrix proteins by RA FLS and to ascertain the effects of Wnt antagonists on RA FLS function and survival. METHODS: Transfection with a reporter plasmid (TOPflash) was performed to assess whether Wnt signaling is active in RA FLS. Wnt signaling was up-regulated in normal FLS by transfection with a Wnt-1 expression plasmid and was down-regulated in RA FLS by transfection with dominant-negative lymphoid enhancer factor 1 (LEF-1)/T cell factor 4 (TCF-4) and secreted Frizzled receptor protein 1 (sFRP-1) expression plasmids. Recombinant sFRP-1 and anti-Wnt-1 antibody were also administered to RA FLS to block Wnt signaling. RESULTS: RA FLS had constitutive activation of the canonical Wnt signaling pathway. Transfection of normal FLS with a Wnt-1 expression vector enhanced not only fibronectin, but also pro-matrix metalloproteinase 3 (proMMP-3) expression. In a complementary manner, interference with Wnt signaling using anti-Wnt-1 antibody, the Wnt antagonist sFRP-1, or dominant-negative vectors that inhibited the transcription factors TCF-4/LEF-1 blocked the expression of fibronectin by RA FLS and reduced cell survival. Anti-Wnt-1 antibody and sFRP-1 also blocked the expression of proMMP-3. CONCLUSION: Our results indicate that the canonical Wnt pathway regulates fibronectin and metalloproteinase expression in RA FLS.