Thyrotropin (TSH)-Induced Production of Vascular Endothelial Growth Factor in Thyroid Cancer Cells in Vitro: Evaluation of TSH Signal Transduction and of Angiogenesis-Stimulating Growth Factors
Sebastian Hoffmann,
Lorenz C. Hofbauer,
Vera Scharrenbach,
Anette Wunderlich,
Iyad Hassan,
Susanne Lingelbach and
Andreas Zielke
Department of Surgery (S.H., V.S., A.W., I.H., S.L., A.Z.), and Division of Gastroenterology and Endocrinology, Department of Medicine (L.C.H.), Philipps University, D-35032 Marburg, Germany
Address all correspondence and requests for reprints to: Dr. Sebastian Hoffmann, Baldingerstrasse, 35043 Marburg, Germany. E-mail: hoffmans{at}mailer.uni-marburg.de.
Thyroid tumor growth requires angiogenesis, and vascular endothelialgrowth factor (VEGF) has been shown to be the most importantendothelial mitogen. TSH is the major thyrotropic hormone, butits impact to modulate VEGF production has not yet been studied.Several other growth factors have also been shown to affectthyroid cancer cell growth and function in vitro. Therefore,the aim of the current study was to 1) establish the effectof TSH on VEGF as well as 2) evaluate the TSH signal transductionof this effect, and 3) screen other growth factors for the abilityto modulate VEGF in thyroid cancer cell lines. HTC, a follicularcancer cell line lacking endogenous TSH receptor (TSHr), itsreceptor positive variant (HTC TSHr), and a cell line of Huerthlecell origin (XTC) were used. After stimulation with growth factorsin vitro [TSH; epidermal growth factor (EGF), IGF, placentagrowth factor, TGF-, TGF-ß1, fibroblast growth factor,platelet-derived growth factor, and hepatocyte growth factor]cells were analyzed for VEGF gene expression by Northern blottingand for VEGF protein by enzyme immunoassay. TSHr signal transductionwas evaluated by analyzing the effect of stimulators (choleratoxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate)and inhibitors (2',5'-dideoxyadenosine and staurosporine) onVEGF protein levels under basal and TSH-stimulated conditions.TSH increased VEGF mRNA and protein in a dose-dependent mannerin HTC TSHr and XTC cells by up to 40%. The effects of TSH weremediated by protein kinase C (PKC), rather than protein kinaseA (PKA), stimulation, because inhibition of PKC by staurosporineresulted in a decrease in VEGF production of up to 65%, whereasinhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine)resulted in only a minor decrease. TSH was not the most powerfulstimulator of VEGF production. TGF-ß1 and EGF were1.5- to 2-fold more potent. Placenta growth factor and TGF-did not induce VEGF production in TSHr-positive HTC cells, whereasthey did induce VEGF production in TSHr-negative HTC cells.In thyroid cancer cell lines, TSH induces VEGF production involvingthe PKC, rather than the PKA, pathway. However, EGF and TGF-ßincrease the capacity of thyroid cancer cells to provide VEGFmore effectively than TSH. In the absence of a functioning TSHr,additional growth factors, such as TGF-, increase capacity forVEGF stimulation.
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, TGF-ß1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-ß1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-
