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Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells.

ABSTRACT: We have examined the effects of l-thyroxine (T4) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal growth factor (EGF). T4, at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10-20 min. Activation by T4 of STAT3 was maximal at 30 min (15+/-4-fold enhancement; mean+/-S.E.M.) in 18 experiments. This effect was reproduced by T4-agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T4. In the nuclear fraction of T4-treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin fibroblasts (BG-9). T4 also potentiated the EGF-induced nuclear translocation of activated STAT1alpha and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T4 of STAT3, and the potentiation of EGF by T4, require activities of PKC, PTK and an intact MAPK pathway.

SUBMITTER: Lin HY

PROVIDER: S-EPMC1220069 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:PurposeAngiogenesis is involved in many pathological and physiological processes and is mainly driven by hypoxia. Karyopherin subunit alpha 2 (KPNA2), a member of the nuclear transport protein family, was recently shown to be induced by hypoxia in various types of tumours, so we aimed to investigate the role and mechanism of KPNA2 in angiogenesis under hypoxia.Materials and methodsAfter overexpression or knockdown of KPNA2 in human umbilical vein endothelial cells (HUVEC) by adenovirus vector infection, the tube formation, proliferation and migration of HUVEC under hypoxia were detected by tubule formation assay, 5-ethynyl-2'-deoxyuridine (EdU) staining and Transwell assay, respectively. After overexpression or knockdown of KPNA2 in a murine hindlimb ischemia model by local injection of purified adenovirus vector into the gastrocnemius muscle, blood flow changes were examined with a laser Doppler system. Changes in KPNA2-binding proteins under hypoxia were detected by immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP). The effect of KPNA2 on signal transducer and activator of transcription 3 (STAT3) was detected by Western blotting and quantitative RT‒PCR.ResultsKPNA2 was upregulated in the HUVEC hypoxia model and murine hindlimb ischemia model. Overexpression of KPNA2 increased the proliferation, migration and tube formation of HUVEC under hypoxia, while knockdown of KPNA2 reduced the proliferation, migration and tube formation of HUVEC. Overexpression of KPNA2 promoted the restoration of blood flow in the murine hindlimb ischemia model, while knockout of KPNA2 inhibited the restoration of blood flow in the murine hindlimb ischemia model. Mechanistically, hypoxia promoted the binding of STAT3 to KPNA2. Overexpression of KPNA2 promoted STAT3 phosphorylation and then upregulated vascular endothelial growth factor (VEGF) and angiopoietin 2(ANGPT2), whereas knockdown of KPNA2 inhibited STAT3 phosphorylation and then downregulated VEGF and ANGPT2.ConclusionOur study demonstrates that hypoxia promotes the binding of STAT3 to KPNA2 and KPNA2 promotes angiogenesis under hypoxia by promoting the binding of STAT3 and JAK1 and regulating STAT3 phosphorylation.

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Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells.

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