characterization of new molecular targets involved in iodide ... channel mediating iodide efflux in...

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  • AAllmmaa MMaatteerr SSttuuddiioorruumm –– UUnniivveerrssiittàà ddii BBoollooggnnaa

    DOTTORATO DI RICERCA IN

    ________BIOCHIMICA________

    Ciclo __XXIV___

    Settore Concorsuale di afferenza: 05/E1

    Settore Scientifico disciplinare: BIO/10

    Characterization of new molecular targets

    involved in iodide flux in the thyroid gland: the

    anoctamins

    Presentata da: Dott.sa Carmela Iosco 0000376278

    Coordinatore Dottorato Relatore Correlatore

    Chiar.mo Prof. Chiar.mo Prof. Prof.sa

    Giorgio Lenaz Giovanni Romeo Kerry Rhoden

    Esame finale anno 2012

  • 1

    INDEX

    ABSTRACT

    1. INTRODUCTION .................................................................................................... 5

    IODIDE TRANSPORT 11

    MALIGNANT TUMOURS OF THYROID GLAND 25

    CHLORIDE TRANSPORT 29

    FLUORESCENCE BIOSENSORS 41

    2. AIM ......................................................................................................................... 43

    3. MATERIALS AND METHODS ......................................................................... 45

    CELL CULTURE 45

    MOLECULAR ANALYSIS 52

    IN VIVO FUNCTIONAL EXPERIMENTS 76

    4. RESULTS .............................................................................................................. 96

    MOLECULAR CHARACTERIZATION OF TMEM16A IN THYROID GLAND 96

    FUNCTIONAL CHARACTERIZATION OF TMEM16A IN THYROID GLAND 100

  • 2

    ONGOING EXPERIMENTS 135

    5. DISCUSSION ...................................................................................................... 140

    6. CONCLUSIONS ................................................................................................. 151

    7. PERSPECTIVES ................................................................................................. 155

    BIBLIOGRAPHY .................................................................................................... 157

  • 3

  • 4

    ABSTRACT

    Iodide transport is necessary for the synthesis of thyroid hormones following

    accumulation in the follicular lumen out of thyroid cells, via channels unknown with

    the exception of pendrin.

    According to our hypothesis, TMEM16A could be the main molecular identity of the

    channel mediating iodide efflux in the thyroid gland. TMEM16A is the prior

    candidate for calcium-activated chloride conductance (CaCC). TMEM16A belongs to

    the TMEM16/anoctamin family comprising ten members (TMEM16A-K). Higher

    affinity of TMEM16A for iodide and predicted expression in the thyroid gland

    suggest its mediation of iodide efflux.

    The aim of this project was to identify the role of TMEM16A in iodide transport in

    the thyroid gland, by characterizing its molecular expression and functional

    properties.

    We demonstrated that TMEM16F, H, K transcripts are expressed in FRTL-5 thyroid

    cells, as well as TMEM16A, which is TSH-independent. Tumor tissue from human

    thyroid maintains TMEM16A expression.

    Functional in vivo experiments in FRTL-5, stably expressing YFP-H148Q/I152L

    fluorescent protein as a biosensor, showed that iodide efflux is stimulated by agonists

    of purinergic receptors with an order of potency of ATP>UTP>ADP (compatible with

    an involvement of P2Y purinergic receptors), and by agonists of adrenergic receptors

    (epinephrine, norepinephrine and phenylephrine). Iodide efflux was blocked by α-

    receptor antagonists prazosin and phentolamine, consistent with a role of α1

    adrenergic receptors. Iodide efflux was specifically dependent on calcium mobilized

    from intracellular compartments and induced by the calcium ionophore ionomycin.

    CaCC blockers suppressed ionomycin-/ATP-/epinephrine-stimulated iodide efflux.

    Heterologous expression of TMEM16A in CHO K1 cells induced calcium-activated

    iodide fluxes.

    All these results support the hypothesis of the involvement of TMEM16A in calcium-

    dependent iodide efflux induced by receptor agonists in thyroid cells. TMEM16A

    may represent a new pharmacological target for thyroid cancer therapy, since its

    blockade may enhance the retention of radioiodide by tumour cells enhancing the

    efficacy of radioablative therapy.

  • 5

    CHAPTER 1

    INTRODUCTION

    1.1 THYROID GLAND

    1.1.1 STRUCTURAL AND MORPHOLOGICAL CHARACTERISTICS

    Thyroid gland is an endocrine gland specialized in synthesis, storage and secretion of

    hormones essential for regulation of metabolism and for thermogenesis.

    It is localized at under- hyoid region of neck, on the front surface of trachea. Thyroid

    gland contacts laterally sternocleidomastoid muscle and carotid arteries, rear-recurrent

    laryngeal nerve, trachea and esophagus. Above thyroid gland there is larynx.

    Thyroid gland consists of two side lobes joined in the midline by a thin portion of

    tissue, isthmus. Like all endocrine glands, thyroid is richly vascularised, in particular

    branches of internal carotid artery: two superior thyroid arteries. Each lobe has a

    length of about 4 cm and a width of about 1-2 cm. In adults, normal thyroid gland

    weighs between 15 and 25 grams. Its size, however, may be different according to

    individual, environmental, nutritional factors.

    Secretory epithelial cells produce thyroid peptide hormones T3 (tri-iodothyronine)

    and T4 (tetra-iodothyronine or thyroxin), calcitonin. These cells are classified into two

    types, follicular and parafollicular. Follicular cells (thyrocytes) are organized as a

    single layer of cubic hollow structures called follicles (50-500 uM in diameter),

    representing unity of structure and function of thyroid gland (Fig.1.1). Thyroid gland

    is the only body follicular gland. Thyrocytes synthesize, accumulate and secrete

    thyroid hormones. In fact, it is enclosed in follicular lumen thyroid colloid, a protein

    matrix which acts as a deposit of T3 and T4 thyroid hormones, secreted by thyrocytes

    in response to hormonal stimuli, as the glycoprotein thyroglobulin (TG).

  • 6

    Fig. 1.1 Anatomy and follicular structure of human thyroid gland. (pyroenergen.com)

    Thyroid gland is the only body gland to accumulate hormones outside the cells that

    synthesize them. Due to their active secretion of protein molecules, thyrocytes are

    characterized by a large number of mitochondria and a rough endoplasmic reticulum

    of considerable size.

    Thyrocytes parafollicular cells ("clear" or "C") are among follicular thyrocytes and in

    interfollicular interstitium. Parafollicular cells are not organized into defined

    structures such as follicles, but scattered in small groups. These cells secrete

    calcitonin hormone, which helps body metabolism of calcium, in antagonism with

    parathyroid hormone secreted by dark chief cells of parathyroid glands. The latter are

    in contact with rear side lobes of thyroid gland. Calcitonin inhibits the release of

    minerals from bones causing hypocalcaemia and ipophosphoremia, whereas PTH

    promotes their release causing hypercalcaemia and hyperphosphatemia.

    Hormones T3 and T4 produce rather wide-ranging effects by interacting with several

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