Nuclear receptorsLecture 6

VDR and THR

• Vitamin D reduces colorectal epithelial cell proliferation and inducesdifferentiation, which may be mediated through the VDR. Thus vitamin D may inversely associated with the risk of colorectal, prostate or breastcancers, but epidemiological data are contradictory

Vitamin D and other diseases

• Experiments in animals suggest that vitamin D may be of use in treating the insulin-dependent diabetes and in suppressing transplant rejection.

• Vitamin D supplementation may inhibit or stop the progression of multiple sclerosis (MS). Most MS patients suffer from vitamin D deficiency.

• MS prevalence increases with decreased exposure to the sun, and diets rich in fish, which is rich in vitamin D, may lower MS severity.

• Studies in Japanese population suggested an association between the less active form of VDR and frequency of MS.

Development of type I diabates

- The greater prevalence of T1D at northerly latitudes than in sunnier climates.

- Vitamin D supplemen-tation in early childhood not only helps preventing rickets, but also leads to a reduction in the risk oftype 1 diabetes

Development of type I diabates

Vitamin D and psoriasis

Treatment of psoriasis with vitamin D3

• In a double-blind, placebo-controlled trial, volunteers aged >70 weregiven a daily tablet of 10 g (400 IU) vitamin D3 for up to 3.5 years toinvestigate whether this reduced the risk of fractures. Serum 25-(OH)-D levels were increased in the treated group. However, there was no difference in fracture incidence. No adverse effects were reported.

• In a trial of 800 IU per day vitamin D3 supplementation in 64 monozygotic twin pairs over two years no adverse effects were observed, nor was any significant difference in bone mass density.

• In a 3 year placebo-controlled study of 17.5 g/day vitamin D3 andcalcium supplementation, bone mass density was significantly increasedin the supplemented group. No adverse effects were reported.

• Short term supplementation of elderly women, with 800 IU/day vitamin D3 and calcium for 8 weeks, has been shown to decrease body sway and the incidence of fractures during a 1 year follow-up period.

Clinical trials with vitamin D supplementation

Late closure of the fontanelle (craniotabes) is the earliest sign of rickets. It is detected in infants less than 12 months of age as round unossified areas in the skull.

Beading of the ribs, termed ‘rachitic rosary’, is an almost consistent sign after 6 months of age. This is caused by the swollen cartilaginous ends of the ribs.

Rickets

The chest may be narrow and rather funnel shaped, described as ‘pigeon chest’, in severe cases this may interfere with breathing.

When the child begins to toddle, putting weight on the legs results in the femur becoming bowed.

Severe vitamin D deficiency reduces the growth rate and causes microcephaly, with reduction in brain growth and the eruption of teeth is delayed.

Rickets

• Increases in vitamin D intake since the recognition of the aetiology of rickets, improved the nutrition, resulting in a dramatic reduction in the incidence of rickets.

• During the Second World War, it had become commonplace in Great Britain for dried milk and infant cereals to be fortified with ergocalciferol (vitamin D2), and to give supplementary concentrated vitamin D to infants. Additionally the cod-liver oil was supplied.

Jedrzej Sniadecki1768-1838

1822: describes themethod for treatment of rickets by exposure to sun

RicketsInsufficient vitamin D delivery – e.g. too lowexposure to sunlight, low-fat diet.

Renal osteodystrophy – kidney damage leading to loss of 1-hydroxylase activity, low 1,25(OH)2D3, impaired intestinal calcium absorption (rickets/osteomalacia).

Hereditary rickets type I – inherited defect of kidney 1-hydroxylase prevents formation of active hormone; treatable with exogenous calcitriol.

Hereditary rickets type II – end organ resistance due to mutation in the VDR receptor DNA or hormone binding domains; DNA binding domain mutations unresponsive to administered calcitriol; ligand binding domain mutations can sometimes respond to very high dose 1,25D3 treatment.

Inherited vitamin-D3 insensitivity type II – mutation in VDR

* Inherited, recessive autosomal disease.

* Insufficient mineralization of new-formed bones and ossified cartilages.

* Symptoms of rickets start to be visible during the first few months of live:- pain of bones- general weakness and weakness of muscles- sometimes convulsions resulting from too low level of calcium

* Inhibited growth, underdevelopment of teeth.

* Many children have alopecia both on the head and on the eyebrows oreyelash.

* High doses of vitamin-D are not effective in children with alopecia. Better results can be obtained using high doses of calcium.

Vitamin D insensitivity

* Biochemical features:

- low level of calcium and phosphates in blood

- increased level of alkaline phosphatase in blood

- increased level of PTH

- high level of calcitriol

(there is a difference comparing to rickets type-I, where level of calcitriol is diminished because of deficiency in 1α-hydroxylase).

Inherited vitamin-D3 insensitivity type II – mutation in DBD of VDR

Vitamin D insensitivity

Thyroid glands

• The thyroid gland is a strongly vascularized organ, located in the neck, in close approximation to the first part of the trachea.

• In humans, it has a "butterfly" shape, with two lateral lobes that are connected by isthmus.

• Most animals have two separate glands on either side of the trachea.

Thyroid glands

• Thyroid epithelial cells - the cells responsible for synthesis of thyroid hormones - are arranged in spheres called thyroid follicles.

• The follicle lumen is filled with a thick colloid, which predominantly contains thyroglobulin.

• Thyroglobulin is a highly glycosylated protein of two subunits, each of 330 kDa. The subunit contains 115 tyrosine residues.

• Thyroid hormones are derivatives of tyrosine bound covalently to iodine. The two principal thyroid hormones are:

• thyroxine (T4 or L-3,5,3',5'-tetraiodothyronine; major form released fromthe gland)

• triiodothyronine (T3 or L-3,5,3'-triiodothyronine).

• Although both T3 and T4 are important for normal growth and development and energy metabolism, T3 is ~10 times more active ligand of THR than T4.

Thyroid hormones

Thyroid hormones are basically two tyrosines linked together with the critical addition of iodine at three or four positions on the aromatic rings.

Physiologic effects of thyroid hormones

Mitochondrial ATP production in rats

heart liver soleus plantaris

control

hyperthyroid

Metabolism:

• Thyroid hormones lead to an increase in basal metabolic rate (BMR).

• One consequence of this activity is increasedheat production (nonshivering thermogenesis).

• Thyroid hormones induce expression of UCP-1.

• Thyroid hormones regulate "big time” processes (development, growth and metabolism).

Processes modulated by THR action

Growth:

• Thyroid hormones are necessary for normal growth of juveniles, as evidenced by the growth-retardation observed in thyroid deficiency.

Development:

• A classical experiment in endocrinology was the demonstration that tadpoles deprived of thyroid hormone failed to undergo metamorphosis into frogs.

Rot-Nikcevic & Wassersug, J Exp Biol 2004.

Rana temporariaXenopus laevis

healthy

lack of thyroid gland

Physiologic effects of thyroid hormones

- Amphibian metamorphosis requires adaptation to oxygen rich environment.

- TH is low during embryonic and early larva development and increases beforemetamorphosis.

- Low TH can adapt salamander to low oxygen aquatic environment.

- ? Analogies in mammalian tisses after ischemia and reperfusion? Possibly yes.

Pantos & Mourousis. Oxid Med Cell Longevity 2013

Physiologic effects of thyroid hormones

- A decline in T3 levels occurs aftermyocardial infarction (AMI) or cardiacsurgery.

- At 6 months, recovery of cardiac function iscorrelated with T3 plasma levels.

- Acute T3 (and not T4) administration afterheart ischemia in rats or mice improved thepostischemic recovery of function.

- TR1 is predominantly expressed in themyocardium and regulates genes related to cell differentiation, survival (through PI3K activation) and contractile function (e.g. TRE-mediated upregulation of connexin-43).

Pantos & Mourousis. Oxid Med Cell Longevity 2013

THR and cardiac regeneration

• Mammalian THRs are encoded by two genes, designated and , bothcan be alternatively spliced, generating different isoforms:

* -1, -2,

* -1 (can bind cyclin D1 and p53) and -2.

• The -2 isoform has a unique C-terminus and does not bind T3 and mayact as inhibitor of other THRs.

Thyroid hormone receptor (THR)

• THRs can bind to a TRE as monomers, as homodimers or as heterodimers with the retinoid X receptor (RXR).

• The heterodimer affords the highest affinity binding, and is thought to represent the major functional form of the receptor.

• The most stable binding occurs on the classical DR4 TRE.

• THRs bind to DNA regardless of whether they are occupied by T3.

• Binding of THR alone to DNA leads to repression of transcription, whereas binding of the thyroid hormone-receptor complex leads to activation of transcription.

Thyroid hormone receptor (THR)

Ligand-free state:

• T3-free receptor, as a heterodimer with RXR, assumes a conformation that promotes interaction with a corepressor molecules.

• Binding of T3 induces a conformational change to bind the coactivatorproteins.

Mode of THR action

Increased by cyclin D1

Thyroid hormone receptor

Reduced by p53

Causes:- Iodine deficiency

- Thyroid disease (e.g. Hashimoto'sdisease, autoimmune disease leading to inflammation).

HShealthy

Hypothyroidism

- Hypothyroidism is a common sideeffect of TKIs.

- Sunitinib is an oral TKI with activityagainst VEGFRs, PDGFRs, and cKit. One-third (14–85%) of patients on sunitinibtreatment develop hypothyroidism.

• Mutations in the ligand-binding domain of THR can lead to generalizedresistance to thyroid hormones.

Mutations in THR

• Clinically, such individuals show a type of hypothyroidism characterized by goiter, but also elevated serum concentrations of T3 and thyroxine and normal or elevated serum concentrations of TSH.

Causes:• Too high secretion of thyroid hormones (less common than hypothyroidism).

• Graves disease, an autoimmune disease in which antibodies bind to and activate the thyroid-stimulating hormone receptor, leading to continuousstimulation of thyroid hormone synthesis.

• Thyroid cancer.

• Hamburger thyroxicosis (rare ).

Exophthalmos

Hyperthyroidism

• Thyrotoxicosis in Minnesota (in 1987) and South Dakota was traced to thyroid-contaminated hamburger. A total of 121 cases were identified.

• The patients complained of sleeplessness, nervousness, headache,fatique, excessive sweating and weight loss.

Hamburger thyroxicosis

• Thyroid hormones are orally active, thusconsumption of thyroid gland tissue can cause thyrotoxicosis.

• Several outbreaks of thyrotoxicosis have been attributed to a practice (banned), where meat in the neck region of slaughtered animals is ground into hamburger.

Hamburger thyroxicosis

Just one contaminated hamburger….

Central nervous system:

• Both decreased and increased concentrations of THs alter the mental state. Too littlethyroid hormone, and the individual tends to feel mentally sluggish, while too much induces anxiety and nervousness.

• Normal levels of THs are essential to the development of the fetal and neonatal brain.

• Congenital thyroid deficiency results in cretinism, which includes dwarfism and mental retardation (an impairment of abstract thought, with preserved vegetative, personal, social functions and memory).

Physiologic effects of thyroid hormones

Thyroid hormone and fetal brain development

• TH have most profound effects on the terminal stages of brain differentiation, including growth of dendrites and axons, and myelination (e.g. direct up regulation of myelin basic protein).

A fetal rat brain produced by in situ hybridization with a probe for the rat thyroid hormone receptor.

Purkinje cells kept for 14 daysTH induced increase in Purkinje cell dendritic branching.

• Human fetuses acquire the ability to synthesize THs at 10 to 12 weeks of gestation.

• There is substantial transfer of maternal THs across the placenta. In the placenta the deiodinases converts T4 to T3.

Thyroid hormone and fetal development

Thyroid deficiency is known to affect fetal development:

* Isolated maternal hypothyroidism:

Typically is not a cause of fetal disease because it usually is associated with infertility. When pregnancy does occur, there is increased risk of intrauterine fetal death and gestational hypertension.

* Subclinical hypothyroidism:

Mild maternal hypothyroidism, diagnosed only retrospectively from banked serum, may lead in children to such effects as slightly lower performance on IQ tests and difficulties with schoolwork.

* Isolated fetal hypothyroidism:

Failure of the fetal thyroid gland. Most children are normal at birth, because maternal thyroid hormones are transported across the placenta. What is absolutely critical is to identify and treat this condition very shortly after birth. If not, the child will become permanently mentally and growth retarded.

few months

thyroid hormone replacement

Isolated fetal hypothyroidism

THR after ligand binding can activate the PI3K pathway via phosphorylation of Akt/PKB (minutes aftet stimulation), leading e.g. to activation of eNOS and nNOS.

- T4 and T3 can bind to the integrin v3, at sites denoted as S1 and S2:* S1 exclusively binds T3 at and leads to PI3K activation.* S2 binds T4, and to a lesser extent T3, and activates the ERK1/2 pathway.

Increased production of TH is associated with progression of ovarian cancer, breastcancer and prostate cancer.

Moeller & Fuhrer. Endocrin-Related Cancer 2013

THR: non-genomic activity

HIF-1

THR and muscle differentiation

Salvatore et al. Nat Rev Endocrinol 2014

deionidase

THR and muscle differentiation

Salvatore et al. Nat Rev Endocrinol 2014

What would be profitable to remember in June:

- Types of rickens

- Effect of vitamin D3 on immune system

- Causes and symptoms of thyroid hormone deficiency

- Splicing forms and mode of action of TR.

Thank you and see you next week...