Cammarota, Francesca (2018) Genetic ablation of Hipk2 induces cardiac dysfunction and, in combination with the loss of Hmga1, causes respiratory distress and thyroid dysfunction in mice. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Genetic ablation of Hipk2 induces cardiac dysfunction and, in combination with the loss of Hmga1, causes respiratory distress and thyroid dysfunction in mice
Date: 9 December 2018
Number of Pages: 57
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Dottorato: Medicina molecolare e biotecnologie mediche
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
Avvedimento, Vittorio
Pierantoni, Giovanna MariaUNSPECIFIED
Date: 9 December 2018
Number of Pages: 57
Uncontrolled Keywords: Knock-out model; HIPK2; HMGA proteins
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/04 - Patologia generale
Date Deposited: 09 Jan 2019 13:17
Last Modified: 26 Jun 2020 20:27


The Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine–threonine kinase able to interact with homeobox proteins and other transcription factors. It is a sensor for various extracellular stimuli thus regulating several signal pathways such as apoptosis, embryonic development, DNA-damage response, and cellular proliferation. As consequence, HIPK2 alterations are involved in diseases such as cancer and fibrosis. HIPK2 knock-out (KO) mice have been generated and display a reduction in body size compared to WT mice and severe psychomotor behavioral abnormalities (such as dystonia, impaired coordination, reduced motility, clasping of their posterior limbs when suspended by tails and reduced responses to novelty) that are consistent with cerebellar defects. In this thesis, we have further investigated the in vivo role of HIPK2 analyzing other phenotypic features of the HIPK2-KO mice. Firstly, we investigated the cardiac phenotype consequent to the kinase depletion. Echocardiographic analysis performed on HIPK2- KO mice at 4 and 12 months showed a significant reduction in systolic function in the adulthood, by a left ventricle fractional shortening (LVFS), and an increased in left ventricular (LV) mass/body weight (BW) ratio in HIPK2-KO mice in comparison with wild-type (WT) littermates. Consistently, increased expression levels of cardiac failure hallmarks, such atrial and brain natriuretic peptides, ANP , BNP , and myosin heavy chain, β-MHC were observed. In addition, the histological analysis of the myocardium was performed by WGA staining of cardiac sections at different age (4, 6, 12 and 18 months of age), revealing areas of fibrosis, mild inflammatory infiltrate and sarcoplasmic red deposits. At cellular level, a lot of p62/SQSTM1-positive autophagic vacuoles were observed in sections of hearts from KO mice at 18 months of age. In order to investigate the molecular mechanisms by which HIPK2 depletion causes cardiac failure, H9C2 rat cardiomyoblasts have been stably silenced for HIPK2 gene expression through a doxycycline-inducible system. HIPK2-depleted clones showed a strong upregulation of ANP, BNP and β-MHC markers with respect to control ones, thus representing a good model to study the phenotype observed in KO mice. As HIPK2 protein interacts with the architectural chromatin protein HMGA1 (Pierantoni et al. 2001), Hmga1/Hipk2 double knock-out mice (DKO) have been generated in our laboratory, crossing Hmga1-KO (A1-KO) mice and Hipk2-KO mice, in order to understand the functional role of Hmga1 and Hipk2 complex in vivo. High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasia with a causal role in cancer initiation and progression. HIPK2 and HMGA1 proteins physically interact, and HIPK2 phosphorylates HMGA1 modulating its DNA binding capacity. We observed that the 50% of DKO newborn mice dies within one day of life (P1) and autoptic examination showed that lung phenotype was characterized by collapsed immature sac-like alveoli and unexpanded alveolar spaces. Through molecular analysis, we have demonstrated that HMGA1 and HIPK2 positively regulate surfactant protein expression, since DKO mice show a strongly decreased expression of surfactant genes, both during embryogenesis and at birth. Surfactant proteins are components of lung surfactant which is essential for several lung functions. In addition, autoptic examination of DKO mice at P1 revealed also thyroid abnormalities represented by irregular structures of thyroid follicles devoid of colloid. Immunohistochemistry (IHC) and molecular analysis of thyroid late differentiation markers on DKO thyroid glands showed a reduction of expression levels of Thyroglobulin (Tg), Tireoperoxidase (Tpo) and Thyrotropin receptor (Tshr) DKO mice compared to control mice at P1. The expression of transcription factors required for the expression of the thyroid specific differentiation markers, FOXE1, PAX8 and TTF-1 were strongly reduced in thyroids from DKO at P1 (Gerlini et al. Manuscript in submission). Altogether these data suggest that the lack of both Hmga1 and Hipk2 genes impairs the expression of PAX8 and FOXE1 in thyroid gland and, consequently, of thyroid differentiation markers, indicating that HMGA1/HIPK2 interaction is crucial also for thyroid development.


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