Relaxin Enhances the Oncogenic Potential of Human Thyroid Carcinoma Cells
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050876
ISSN1525-2191
AutoresSabine Hombach‐Klonisch, Joanna Białek, Bogusz Trojanowicz, Ekkehard Weber, Hans-Jürgen Holzhausen, Josh D. Silvertown, Alastair J. S. Summerlee, Henning Dralle, Cuong Hoang‐Vu, Thomas Klonisch,
Tópico(s)Pregnancy-related medical research
ResumoThe role of members of the insulin-like superfamily in human thyroid carcinoma is primarily unknown. Here we demonstrate the presence of RLN2 relaxin and relaxin receptor LGR7 in human papillary, follicular, and undifferentiated anaplastic thyroid carcinoma suggesting a specific involvement of relaxin-LGR7 signaling in thyroid carcinoma. Stable transfectants of the LGR7-positive human follicular thyroid carcinoma cell lines FTC-133 and FTC-238 that secrete bioactive proRLN2 revealed this hormone to act as a multifunctional endocrine factor in thyroid carcinoma cells. Although RLN2 did not act as a mitogen, it acted as an autocrine/paracrine factor and significantly increased anchorage-independent growth and thyroid carcinoma cell motility and invasiveness through elastin matrices. Suppression of LGR7 expression by LGR7-siRNA abolished the RLN2-mediated accelerated tumor cell motility. The increased elastinolytic activity correlated with enhanced production and secretion of the lysosomal proteinases cathepsin-D (cath-D) and cath-L forms hereby identified as new RLN2 target molecules in human neoplastic thyrocytes. We found the intracellular distribution of procath-L specifically altered in RLN2 transfectants, providing first evidence for selective actions of relaxin on the powerful elastinolytic cath-L production, storage, and secretion in thyroid carcinoma cells. Thus, relaxin enhances the oncogenic potential and acts as novel endocrine modulator of invasiveness in human thyroid carcinoma cells. The role of members of the insulin-like superfamily in human thyroid carcinoma is primarily unknown. Here we demonstrate the presence of RLN2 relaxin and relaxin receptor LGR7 in human papillary, follicular, and undifferentiated anaplastic thyroid carcinoma suggesting a specific involvement of relaxin-LGR7 signaling in thyroid carcinoma. Stable transfectants of the LGR7-positive human follicular thyroid carcinoma cell lines FTC-133 and FTC-238 that secrete bioactive proRLN2 revealed this hormone to act as a multifunctional endocrine factor in thyroid carcinoma cells. Although RLN2 did not act as a mitogen, it acted as an autocrine/paracrine factor and significantly increased anchorage-independent growth and thyroid carcinoma cell motility and invasiveness through elastin matrices. Suppression of LGR7 expression by LGR7-siRNA abolished the RLN2-mediated accelerated tumor cell motility. The increased elastinolytic activity correlated with enhanced production and secretion of the lysosomal proteinases cathepsin-D (cath-D) and cath-L forms hereby identified as new RLN2 target molecules in human neoplastic thyrocytes. We found the intracellular distribution of procath-L specifically altered in RLN2 transfectants, providing first evidence for selective actions of relaxin on the powerful elastinolytic cath-L production, storage, and secretion in thyroid carcinoma cells. Thus, relaxin enhances the oncogenic potential and acts as novel endocrine modulator of invasiveness in human thyroid carcinoma cells. In recent years, the multifunctional peptide hormone relaxin has been identified as an important endocrine player in the reproductive tract, cardiovascular/neural systems, and oncology.1Ivell R Einspanier A Relaxin peptides are new global players.Trends Endocrinol Metab. 2002; 13: 343-348Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 2Silvertown JD Summerlee AJ Klonisch T Relaxin-like peptides in cancer.Int J Cancer. 2003; 107: 513-519Crossref PubMed Scopus (63) Google Scholar The thyroid was once considered to be a relaxin target tissue with relaxin reported to increase thyroid weight, radioactive iodine uptake, and protein-bound iodination in rats.3Plunkett ER Squires BP Richardson SJ The effect of relaxin on thyroid weights in laboratory animals.J Endocrinol. 1960; 21: 241-246Crossref PubMed Scopus (4) Google Scholar, 4Plunkett ER Squires BP Heagy FC Effect of relaxin on thyroid function in the rat.J Endocrinol. 1963; 26: 331-338Crossref PubMed Scopus (8) Google Scholar Likely because of the crude relaxin preparations used at the time, these results could not be confirmed.5Braverman LE Ingbar SH Effects of preparations containing relaxin on thyroid function in the female rat.Endocrinology. 1963; 72: 337-341Crossref PubMed Scopus (4) Google Scholar No further investigations were reported thereafter using highly purified relaxin preparations to validate a potential role of relaxin in thyroid tissues and thyroid cell lines. Some 40 years later, the discovery of the G-protein-coupled relaxin-like receptors LGR7 and LGR8 revealed the presence of transcripts for both LGR7 (relaxin receptor) and LGR8 (INSL3/relaxin receptor) in the thyroid gland.6Adham IM Burkhardt E Benahmed M Engel W Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells.J Biol Chem. 1993; 268: 26668-26672Abstract Full Text PDF PubMed Google Scholar, 7Pusch W Balvers M Ivell R Molecular cloning and expression of the relaxin-like factor in the mouse testis.Endocrinology. 1996; 137: 3009-3013Crossref PubMed Scopus (94) Google Scholar, 8Hsu SY Nakabayashi K Nishi S Kumagai J Kudo M Sherwood OD Hsueh AJW Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (672) Google Scholar Relaxin and the relaxin-like INSL3 have been shown to activate cAMP-dependent signaling pathways by binding to either LGR7 or LGR8.8Hsu SY Nakabayashi K Nishi S Kumagai J Kudo M Sherwood OD Hsueh AJW Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (672) Google Scholar, 9Bartsch O Bartlick B Ivell R Phosphodiesterase 4 inhibition synergized with relaxin signaling to promote decidualization of human endometrial stromal cells.J Clin Endocrinol Metab. 2004; 89: 324-334Crossref PubMed Scopus (55) Google Scholar, 10Bartsch O Bartlick B Ivell R Relaxin signalling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity.Mol Hum Reprod. 2001; 7: 799-809Crossref PubMed Scopus (95) Google Scholar, 11Parsell DA Mak JY Amento EP Unemori EN Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1.J Biol Chem. 1996; 271: 27936-27941Crossref PubMed Scopus (81) Google Scholar, 12Bogatcheva NV Truong A Feng S Engel W Adham IM Agoulnik AI GREAT/LGR8 is the only receptor for insulin-like 3 peptide.Mol Endocrinol. 2003; 17: 2639-2646Crossref PubMed Scopus (153) Google Scholar We recently demonstrated the expression and regulation of INSL3 and LGR8 transcripts in human thyroid carcinoma cell lines, identifying hyper- and neoplastic human thyrocytes as a new source and target of the actions of INSL3 and a novel INSL3 splice variant.8Hsu SY Nakabayashi K Nishi S Kumagai J Kudo M Sherwood OD Hsueh AJW Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (672) Google Scholar, 13Hombach-Klonisch S Hoang-Vu C Kehlen A Hinze R Holzhausen HJ Weber E Fischer B Dralle H Klonisch T INSL-3 is expressed in human hyperplastic and neoplastic thyrocytes.Int J Oncol. 2003; 22: 993-1001PubMed Google Scholar Although still primarily undefined, relaxin appears to have oncogenic potential in various organs and tissues, including the human thyroid.14Tashima LS Mazoujian G Bryant-Greenwood GD Human relaxins in normal, benign and neoplastic breast tissue.J Mol Endocrinol. 1994; 12: 351-364Crossref PubMed Scopus (62) Google Scholar, 15Silvertown JD Geddes BJ Summerlee AJS Adenovirus-mediated expression of human prorelaxin promotes the invasive potential of canine mammary cancer cells.Endocrinology. 2003; 144: 3683-3691Crossref PubMed Scopus (50) Google Scholar, 16Radestock Y Hoang-Vu C Hombach-Klonisch S Relaxin downregulates the calcium binding protein S100A4 in MDA-MB-231 human breast cancer cells.Ann NY Acad Sci. 2005; 1041: 462-469Crossref PubMed Scopus (8) Google Scholar, 17Klonisch T Mustafa T Bialek J Radestock Y Holzhausen H-J Dralle H Hoang-Vu C Hombach-Klonisch S Human medullary thyroid carcinoma (MTC): a source and potential target for relaxin-like hormones.Ann NY Acad Sci. 2005; 1041: 449-461Crossref PubMed Scopus (12) Google Scholar Relaxin affects proliferation and differentiation of MCF-7 human carcinoma cells in a concentration-dependent manner18Bigazzi M Brandi ML Bani G Sacchi TB Relaxin influences the growth of MCF-7 breast cancer cells. Mitogenic and antimitogenic action depends on peptide concentration.Cancer. 1992; 70: 639-643Crossref PubMed Scopus (54) Google Scholar and can modify the extracellular matrix by affecting the expression of matrix metalloproteinases (MMPs), which potentially contribute to relaxin's role as a migration-promoting peptide in mammary carcinoma cells of different species,15Silvertown JD Geddes BJ Summerlee AJS Adenovirus-mediated expression of human prorelaxin promotes the invasive potential of canine mammary cancer cells.Endocrinology. 2003; 144: 3683-3691Crossref PubMed Scopus (50) Google Scholar including humans.19Binder C Hagemann T Hausen B Schulz M Einspanier A Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteinases.Mol Hum Reprod. 2002; 8: 789-796Crossref PubMed Google Scholar In the present study, we have for the first time revealed novel roles for RLN2 in human thyroid carcinoma cells. We identified neoplastic thyroid tissues as a source of RLN2 and LGR7, implicating an active RLN2-LGR7 signaling system in human thyroid carcinoma. Enhanced metabolic activity, anchorage-independent growth, and increased migratory and elastinolytic activity were among the phenotypes observed with stable transfectants of the human follicular thyroid carcinoma cell lines, FTC-133 and FTC-238, which overexpress and secrete bioactive proRLN2. Finally, relaxin up-regulated the production and secretion of cath-L and cath-D, identifying this insulin-like peptide hormone as a novel modulator of the oncogenic potential of human thyroid carcinoma cells. A total of 59 thyroid tissues, including 10 goiter tissues, 9 Graves' disease tissues, and 14 papillary thyroid carcinomas (PTCs), 12 follicular thyroid carcinomas (FTCs), and 14 undifferentiated thyroid carcinomas (UTCs), were collected from patients at the Department of Surgery, University of Halle, by surgical resection for clinical indications (Table 1). This study was approved by the ethical committee of the Martin Luther University, Faculty of Medicine, and all patients gave written consent. Tissues were fixed in formalin and embedded in paraffin and cryopreserved in liquid nitrogen. The human thyroid papillary carcinoma cell line BC-PAP; the follicular carcinoma cell lines FTC-133, FTC-236, FTC-238; and the anaplastic carcinoma cell lines Hth74, C643, UTC-8305, and UTC-8505 were propagated in Dulbecco's minimal essential medium (DMEM)/Ham's F12 (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS) in a 5% CO2 atmosphere at 37°C. Medium was changed every second day, and cells were routinely passaged every 3 to 5 days.Table 1List of Thyroid Papillary (PTC), Follicular (FTC), and Dedifferentiated Anaplastic Thyroid Carcinoma Tissues (UTC) Used in This StudyTissueGenderAgePTNMPTC (n = 14)M25T4aN1bM1F51T4N1aM0F56T4N1bM1F14T4N0M0F14T4N1M0M65T3N1MxF71T3N1M1M11T3N1MxM36T2N1MxM63T2aN0M0F27T2N1aMxF59T1N0M0F39T1N0M0F55T1aN0M0FTC (n = 12)F53T4N0M0F60T4NxM1F62T4N1bM1F34T4N0M0F60T4N0M0M60T4NxM2F51T3N1bM0M67T3bN1bM1M43T3N0M0M63T3N0M0F46T3NxM0F54T2NxMxUTC (n = 14)F79T4N2MxF72T4N2MxF76T4N1MxF58T4N1MxF70T4N1aMxM67T4N1MxF87T4N0M1F53T4N0M1F75T4N0M1F68T4N0M1F42T4N0M1M66T4N0M0F69T3NxMxM52T3N0M0M, male; F, female. Those thyroid carcinoma tissues devoid of RLN2 mRNA and immunoreactive protein are indicated in bold. Open table in a new tab M, male; F, female. Those thyroid carcinoma tissues devoid of RLN2 mRNA and immunoreactive protein are indicated in bold. Total RNA was extracted from the human thyroid cell lines and tissues for RT-PCR analysis using the Trizol reagent (Life Technologies, Karlsruhe, Germany) and RNeasy extraction kit (Qiagen, Hilden, Germany), respectively, according to the manufacturers' protocols. The amount of isolated total RNA was determined by spectrophotometry at 260 and 280 nm.20Sambrook J Fritsch EF Maniatis T Maniatis T ed 2. Molecular Cloning—A Laboratory Manual. vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar One μg of total RNA was used for first-strand cDNA synthesis using the Superscript reverse transcriptase kit and 500 ng/ml of oligo d(T) primer (both Life Technologies). Total RNA was also isolated from the human follicular thyroid carcinoma cell lines. For the amplification of RLN2 prorelaxin and the partial coding sequence of human LGR7 relaxin receptor, specific intron-spanning oligonucleotide primers (Table 2) were used to preclude any genomic DNA amplification. Before semiquantitative RT-PCR analysis with the Bio 1D software (LTF, Wasserburg, Germany), cDNA samples were adjusted to equal 18S RNA input. RT-PCR reactions were performed in a 25-μl solution containing 2 μl of cDNA, 2.5 μl of 10× Advantage2 polymerase mix buffer, 10 nmol/L of dNTP, 20 pmol of each primer (Table 2), and 2 U TaqDNA-polymerase (Life Technologies). PCR cycles consisted of an initial denaturation for 5 minutes at 95°C, followed by 35 cycles of denaturation at 95°C for 45 seconds, annealing for 45 seconds (for temperatures see Table 2), elongation for 1.5 minutes at 72°C, and a final extension cycle for 10 minutes at 72°C. PCR products were separated on a 1% low-melting point agarose gel. For sequence analysis, amplicons were purified by Magic column extraction, cloned into the pGEM-T vector (both Promega, Heidelberg, Germany), and sequenced in both directions using the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin-Elmer, Foster City, CA) and T7 or SP6 sequencing primers.Table 2Oligonucleotide Primers Used at Melting Temperatures (TM) Used to Determine the Expression of RLN2, Human LGR7 Relaxin Receptor, and 18S Transcripts in Human Thyroid Carcinoma Cell LinesPrimerPrimer sequencebpTM (°C)Forward RLN25′-CGGACTCATGGGATGGAGGAAG-3′22261Reverse RLN25′-GCTCCTGTGGCAAATTAGCAAC-3′Forward huLGR75′-CCCAATTCTCTATACTCTGACCACAAG-3′24365Reverse huLGR75′-TCATGAATAGGAATTGAGTGTCGTTGATT-3′Forward 18S5′-GTTGTTGGAGCGATTTGTCTGG-3′34461Reverse 18S5′-AGGGCAGGGACTTAATCAACGC-3′ Open table in a new tab For stable transfection of FTC133 and FTC-238 with the pCMV-preproRLN2-IRES-EGFP15Silvertown JD Geddes BJ Summerlee AJS Adenovirus-mediated expression of human prorelaxin promotes the invasive potential of canine mammary cancer cells.Endocrinology. 2003; 144: 3683-3691Crossref PubMed Scopus (50) Google Scholar or the pCMV-IRES-EGFP vector (Clontech, Heidelberg, Germany) we used the Lipofectamine 2000 kit according to the manufacturer's protocol (Life Technologies). Both plasmids had been purified using the DNA Midi isolation kit (Qiagen). Stably transfected clones with the pCMV-preproRLN2-IRES-EGFP (FTC-133, 12 clones; FTC-238, 1 clone) and pCMV-IRES-EGFP vector (FTC-133, 9 clones; FTC-238, 3 clones), respectively, were selected, maintained in medium containing with 800 μg/ml geneticin (Life Technologies), and displayed bright EGFP production in fluorescence microscopy (Zeiss, Jena, Germany). Of those, three FTC-133- and one FTC-238-pCMV-preproRLN2-IRES-EGFP transfectants expressing relaxin transcripts (FTC-133-RLN2 and FTC-238-RLN2) and two clones each of FTC-133 and FTC-238 containing the pCMV-IRES-EGFP vector (FTC-133-EGFP and FTC-238-EGFP) were used in this study. In all assays performed, similar responses were obtained for all RLN2 transfectants when compared with EGFP controls. A medium change was performed every 2 to 3 days and transfectants were passaged every 3 to 5 days using accutase (PAA Laboratories GmbH, Linz, Austria). Before experimentation, cells were seeded at the desired cell densities either in 25-cm2 flasks or in 6-, 24-, or 96-well plates on the day of experimentation (Techno Plastic Products AG, Trasadingen, Switzerland). The day before transfection FTC-133 cells were seeded at 104 cells/well on a six-well plate. Cells were washed three times in phosphate-buffered saline (PBS) before transfection with nonsilencing- or LGR7-siRNA (Qiagen). The nonsilencing siRNA (siNC) conjugated to Alexa Fluor 555 with no sequence similarity to human gene sequences was used as a control (1027099: 5′-AATTCTCCGAACGTGTCACGT-3′). The following LGR7-siRNA target sequences were used: a single LGR7-siRNA at a final concentration of 300 nmol/L 5′-CTGCAGTTACCTGCTTTGGAA-3′ (exon 15) and a combination of two human LGR7-siRNAs at a concentration of 50 nmol/L each 5′-GCTCCAGACCTTGGCAAAGAC-3′ (exon 4) and 5′-TACTAGATAGGAATTGAGTCTC GTTGATT-3′ (exon 20) were found to be optimal. The Lipofectamine 2000 kit (Life Technologies) was used for transfecting the siRNA constructs. After 24 hours the transfection medium was replaced with normal medium. The level of LGR7 mRNA suppression was highest at 72 hours after transfection. Total RNA was isolated and the level of LGR7 mRNA was determined by semiquantitative RT-PCR as described above. All siRNA experiments for RT-PCR, cAMP, and motility assays were performed in triplicates of three independent samples (see below). Transfectants were seeded at 5 × 104 cells/well of a six-well plate and cultured in 1 ml of culture medium for 24 hours in a humidified CO2 incubator. Secreted relaxin in conditioned medium (200 μl) was determined by ELISA according to the manufacturer's instructions (Immunodiagnostic, Bensheim, Germany). The color reaction was determined at 450 nm with an ELISA reader (SLT Labinstruments GmbH, Crailsheim, Germany). Intracellular cAMP levels were determined in FTC-133-RLN2, clone 10, at 1 × 104 cells/well cultured for 24 hours in 96-well plates. Cells were preincubated with 3-isobutyl-1-methyl-xanthine (IBMX; Sigma, Munich, Germany) at 1 mmol/L for 2 hours at 37°C in a water-saturated CO2-incubator. The medium was replaced with 1 ml of 1) fresh medium containing 10 μmol/L forskolin as a positive control (Merck Biosciences, Bad Soden, Germany); 2) supernatants from one FTC-133-EGFP clone as negative control; 3) supernatants from FTC-133-RLN2 clones 4, 10, 11. Supernatants (200 μl) had been collected from six-well plate cultures of FTC-133-EGFP and FTC-133-RLN2, clones (8 × 104 cells/well) after 24 hours of culture and centrifuged at 3000 × g for 30 minutes to remove remaining cells. FTC-133-RLN2 clone 10 cells (1 × 104 cells/well) were incubated with these media/supernatants, all containing 1 mmol/L IBMX, for 1 hour at 37°C in a water-saturated CO2 incubator. Cells were harvested, washed, and lysed with cAMP extraction buffer and cAMP levels were detected with the colorimetric cAMP Biotrak enzyme immunoassay (Amersham, Freiburg, Germany). The reaction was stopped in 1 mol/L H2SO4 and the product was measured at 450 nm in a microplate reader (SLT). A colorimetric BrdU cell proliferation ELISA (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions. Briefly, on the day of assay, 20 μl of BrdU labeling solution were added to each well of a 96-well plate, except for negative controls that received no BrdU. Cells were incubated for 2 hours at 37°C in a water-saturated CO2 incubator. The assay was performed with 0.25, 0.5, and 1 × 104 stable transfectants/100 μl medium. Thereafter, plates were drained, air-dried for 20 minutes, and blocked with 200 μl/well of ELISA blocking reagent (Roche) for 30 minutes at room temperature. After decanting, 100 μl of BrdU antiserum was added to each well and dishes were incubated for 30 minutes at room temperature. Plates were drained, washed, and incubated with 100 μl/well of substrate solution for 10 minutes at room temperature. Finally, 25 μl of 1 mol/L H2SO4 were added to each well, incubated for 1 minute on a shaker at 300 rpm, and absorbance was measured within 5 minutes at 370 nm in an ELISA reader (SLT). The cytotoxicity Easy-for-You assay kit (Biomedica, Wien, Austria) was performed in 96-well plates according to the manufacturer's instructions. Stable transfectants were plated in 200 μl of medium at 0.25, 0.5, and 1 × 104 cells/well and grown overnight in a CO2 incubator. On addition of chromophorous substrate (20 μl) for 2 hours, NADH2-dependent colored formazan salt formation was monitored hourly for 4 hours at 450 nm using an ELISA reader (SLT). Stable transfectants plated in 96-well plates at a density of 0.25, 0.5, and 1 × 104 cells/well in 100 μl of medium were allowed to grow overnight at 37°C in a CO2 incubator. Wells devoid of cells served as blank. Substrate (100 μl) was added to each well (CellTiter-Glo Luminescent; Promega) and incubated on a shaker and on the benchtop for 2 minutes and 10 minutes at room temperature, respectively. Luminescence was measured with a Sirius luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). Two-layered soft agar assays were performed in six-well plates. The bottom layer of agar (1.5 ml per well) consisted of 5 ml of 3% agar (Roth, Karlsruhe, Germany) in sterile water, 3 ml of FCS (Biochrom), 45 μl of geneticin (50 mg/ml, Life Technologies), 300 μl of a 1:1000 dilution of amphotericin B (stock, 250 μg/ml), and 900 μl of a 1:1000 dilution of gentamicin (stock, 10 mg/ml; all from Sigma) added to 30 ml with Ham's F12 medium. Once solidified at room temperature for 10 minutes, 20,000, 50,000, or 100,000 cells of FTC-133-RLN2 and FTC-133-EGFP were mixed into 1 ml of upper agar layer prepared from a stock consisting of 1.6 ml of 3% agar, 10% FCS, 22.5 μl of geneticin (50 mg/ml), 150 μl (1:1000 dilution) of amphotericin B (250 μg/ml), 450 μl (1:1000 dilution) of gentamicin (10 mg/ml) in 15 ml of culture medium. This cell suspension was carefully layered on top of the bottom layer and once this top agar layer had solidified 1 ml of the culture medium was carefully added and changed once a week. After 4 to 6 weeks, cell colonies in the agar were colored overnight at 37°C in a 5% CO2 atmosphere with 200 μl of iodo-tetrazolium chloride in vivo stain (Sigma). Colored cell colonies were examined by bright-field microscopy (Zeiss). Cellular motility and migration were evaluated in 24-well Transwell chambers (Costar, Bodenheim, Germany). The upper and lower culture compartments were separated by polycarbonate filters with 8-μm pore size. For migration assays, the upper site of the filters (upper chamber) was coated with 50 μg/ml of human elastin (Sigma) before seeding cells. To investigate the effect of recombinant human RLN2 (generously provided by Dr. Laura Parry, University of Melbourne, Melbourne, Australia) on the motility of untransfected thyroid carcinoma cells, FTC-133 or FTC-238 were plated at 1 × 104 cells/well in Ham's F12 medium with or without 10% FCS and incubated for 24 hours in a 5% CO2 atmosphere at 37°C in the absence or presence of human RLN2 at 100 ng/ml or 500 ng/ml added to the wells (lower chamber). To investigate a similar autocrine/paracrine effect of relaxin secreted by the transfectants, RLN2 and EGFP clones were seeded onto the filter at 1 × 104 cells/well and placed in the well harboring 5 × 104 of either RLN2 or EGFP transfectants (lower chamber). Control experiments were performed to determine the specificity of RLN2-mediated signaling on the motility of FTC-133 and FTC-238 tumor cells seeded on the filter: 1) recombinant RLN2 (500 ng/ml) was heat-inactivated for 10 minutes at 90°C before incubation with the cells; 2) supernatant collected from FTC-133-RLN2, clone 10, grown to confluency in a six-well was diluted 1:1, 1:2, and 1:4 with normal culture medium (v/v) before incubation with FTC-133 on the filter; 3) FTC-133 and FTC-238 were transfected with specific LGR7 siRNA constructs and motility was determined in the presence or absence of recombinant RLN2 at 100 or 500 ng/ml). After a 16-hour incubation period, cells remaining on top of the filter were wiped off with cotton swabs and those transfectants that had traversed the membrane pores to the lower surface of the membrane were washed with chilled PBS, incubated for 5 minutes in 1:1 PBS/methanol (Merck, Darmstadt, Germany) and 15 minutes in methanol before staining with 0.1% toluidine blue (Merck) in 2.5% sodium carbonate (Roth). Migrated cells were counted by light microscopy (Zeiss) in four separate high-power fields per filter. Human paraffin-embedded serial 3-μm thyroid tissue sections (Table 2) were dewaxed and rehydrated in PBS plus 0.1% Tween 20 (PBST). Sections were treated with proteinase K (30 μg/ml; both Sigma) for 30 minutes at 37°C and 3% H2O2 in methanol for 25 minutes at room temperature to inactivate endogenous peroxidase activity. Sections were washed in PBST and nonspecific binding sites were blocked with 10% goat serum in PBST (blocking buffer) for 1 hour at room temperature. Detection of immunoreactive RLN2 in the human thyroid tissue sections was performed with two previously characterized rabbit polyclonal antibodies against RLN2. One polyclonal antiserum against RLN2 was from Immunodiagnostik (Bensheim, Germany)21Dschietzig T Richter C Bartsch C Laule M Armbruster FP Baumann G Stangl K The pregnancy hormone relaxin is a player in human heart failure.FASEB J. 2001; 15: 2187-2195Crossref PubMed Scopus (181) Google Scholar and was diluted 1:300 in blocking buffer overnight at 4°C. Serial sections were also immunostained with the R6 rabbit anti-porcine relaxin antiserum (generously provided by Prof. Bernhard Steinetz, Nelson Institute of Environmental Medicine, New York University Medical Center, Tuxedo, NY) at a dilution of 1:800 in blocking buffer at 4°C overnight. Nonimmune rabbit serum (1:300) served as control. As a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit was used at a dilution of 1:500 for 1 hour at room temperature. After washing in PBST, detection was performed with filtered diaminobenzidine solution for 10 minutes and immunostained sections were counterstained with hematoxylin. Immunoreactive relaxin receptor LGR7 in human thyroid tissues was detected with a previously characterized rabbit polyclonal antiserum against human LGR7 (generously provided by Prof. Aaron Hsueh, Dept. of Obstetrics and Gynecology, Stanford University, Stanford, CA) used at 1:800 in blocking buffer.8Hsu SY Nakabayashi K Nishi S Kumagai J Kudo M Sherwood OD Hsueh AJW Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (672) Google Scholar All immunostained tissue sections (two sections per patient) were evaluated by two independent reviewers blinded to the histological diagnosis. Planimetric measurement of immunostained tissues was performed semiquantitatively using the Axioplan light microscope and the Zeiss KS300 software (Zeiss). The percentage of immunostained tissue areas per total section area (100%) was classified into four groups: 0 to 10% negative; 10 to 40% low; 40 to 80% moderate; ≥80% high. For immunofluorescent detection of cathepsin-D (cath-D), cathepsin-L (cath-L), mannose-6-phosphate receptors (M-6-PR), and the lysosomal marker CD63, transfectants were plated on sterile glass slides at 2 × 104 cells/ml and incubated in a humidified atmosphere at 5% CO2 for 2 to 3 days before fixation of cells with 4% formaldehyde. Sections were incubated overnight at 4°C with previously characterized primary rabbit polyclonal antiserum to human cath-D22Fiebiger E Meraner P Weber E Fang IF Stingl G Ploegh H Maurer D Cytokines regulate proteolysis in major histocompatibility complex class II-dependent antigen presentation by dendritic cells.J Exp Med. 2001; 193: 881-892Crossref PubMed Scopus (152) Google Scholar diluted at 1:500 and mouse monoclonal antibodies (mAbs) specific for human procath-L (2D4) and all three cath-L forms (procath-L, single-chain, and heavy chain of cath-L; 33/1)23Tolosa E Li W Yasuda Y Wienhold W Denzin LK Lautwein A Driessen C Schnorrer P Weber E Stevanovic S Kurek R Melms A Bromme D Cathepsin V is involved in the degradation of invariant chain in human thymus and is overexpressed in myasthenia gravis.J Clin Invest. 2003; 112: 517-526Crossref PubMed Scopus (111) Google Scholar, 24Yasuda Y Li Z Greenbaum D Bogyo M Weber E Bromme D Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages.J Biol Chem. 2004; 279: 36761-36770Crossref PubMed Scopus (157) Google Scholar at 1:100 each, respectively, in blocking solution (2% bovine serum albumin, 0.05% saponin in PBS; Sigma). Cells were washed with PBST and incubated with 10% goat or donkey antiserum for 30 minutes before a 1-hour incubation with the secondary rhodamine-conjugated goat anti-rabbit antiserum (1:400; Dianova, Hamburg, Germany) or rhodamine-conjugated donkey anti-mouse antiserum (1:100; Jackson ImmunoResearch, West Grove, PA), respectively. Immunofluorescent localization of mannose-6-phosphate receptors (M6PR type I+II; Abcam, Cambridge, UK) and lysosomal marker CD63 (Sigma) was performed with mAbs diluted at 1:200 and 1:1000 in blocking buffer (for M6PR containing 0.05% saponin; Sigma), respectively. After blocking nonspecific binding sites with 10% donkey serum, the secondary rhodamine-conjugated donkey anti-mouse antiserum (1:100; Jackson ImmunoResearch) was used for visualization. In all cases, nuclear Hoechst stain (1:100; Sigma) was used, and labeled cells were embedded in Fluoroguard anti-fade reagent (Bio-Rad, Munich, Germany) before fluorescence microscopy or confocal laser-scanning microscopy (KLSM-410; both Zeiss). For the detection of relaxin, cellular proteins of stable RLN2 and EGFP transfectants as well as untransfected FTC-133 and FTC-238 cells we
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