Signalling in Endocrine Systems

Introduction

A hormone is a chemical messenger released from an endocrine gland/cell in an organised fashion and is transported through the blood to activate or inactivate a target cell/tissue.
 - They are associated with a binding protein used to transport the hormone to its target and binds it to the cell or tissue.

Without entering the blood a hormone may influence the activity of a neighbouring cell, known as the paracrine mechanism, or it may influence the activity of the same cell from which it is released, known as the autocrine mechanism.

Hormones are derived from amino acids (e.g. Thyroxine, Epinephrine, Norepinephrine, Serotonin); cholesterols (e.g. Cortisol, Testosterone, Progesterone) or phospholipids.

Most protein and peptide hormones (e.g. Oxytocin, Vasopressin, IGFs) require the transcription of a single gene through the alpha and beta subunits of the glycoprotein hormones (e.g. TSH, LH and FSH)

The Neuroendocrine System

The hypothalamus releases neuroendocrine hormones into the median eminence running towards the pituitary gland. From here a hormone is released and sent to the target organ (such as the thyroid gland, liver, adrenal gland, pancreas, kidneys, adipose tissue or genital organs).

- e.g. Thyroid Releasing Hormone (TRH) is released from the hypothalamus and stimulates the release of Thyroid Stimulating Hormone (TSH) from the pituitary gland. This then produces Thyroxine (T4) from the thyroid gland which can be converte to Triiodothyronine (T3) in an organ.

Hypothalamus

The hypothalamus is a group of little nuclei located below the thalamus and behind the optic chiasm. It receives input from other brain regions such as the cerebral cortex, thalamus and brainstem; or from hormones released by the pituitary gland, primary target organs or secondary organs.

The hypothalamic anterior nuclei group is found directly above the optic chiasm and consists of the supraoptic nucleus (secretes vasopressin and neurophysin II); the paraventricular nucleus (releases oxytocin, TRH and CRH) and the suprachiasmatic nucleus (involved in circadian rhythms). The periventricular nucleus is located in the wall of the 3rd ventricle and functions in analgesia.

The hypothalamic medial nuclei group consists of the dorsomedial and ventromedial nuclei which are important in controlling energy balances; plus the arcuate nucleus which releases dopamine to inhibit prolactin release.

Finally, the posterior nuclei group is located just anterior to, or within the mammillary bodies. This consists of the posterior nucleus (increases blood pressure, pupillary dilation, releases vasopressin) and the mammillary nuclei (involved in memory). These nuclei integrate information from brain regions and other hormones in order to regulate their own hormonal output.

Pituitary Gland

• Embryology:
  - The posterior pituitary lobe develops from neural tissue at the base of the 3rd ventricle, and the anterior pituitary lobe develops from the roofplate of the pharynx.
  - The boundary epithelal ectoderm in the roof of the pharynx forms a pocket called Rathke's pouch which envelopes the downgrowth of tissue from the 3rd ventricle.
  

Pituitary Gland

• Terminology:
  - The anterior pituitary lobe is also known as the adrenohypophysis, or pars distalis.
  - The posterior pituitary lobe is known as the neurohypophysis, or pars nervosa.
  - The intermediate lobe is known as the pars intermedia.
  - The pars tuberalis is part of the anterior lobe which wraps the pituitary stalk in a highly vascularised sheath.
  
  
• Circulation:
  - The anterior lobe is supplied by the superior hypophyseal artery through which blood enters the primary plexus of the hypophyseal portal sytem.
  - Blood then travels down the hypothalamo-hypophyseal tract, through hypophyseal portal veins (down the pituitary stalk) to the secondary plexus and it leaves via the anterior hypophyseal veins.
  - The posterior lobe is supplied by the inferior hypophyseal artery from which blood travels to the capillary plexus of the infundibular process. Blood then leaves via the posterior hypophyseal veins.

Pituitary Gland

• Innervation:
  - The anterior pituitary gland is innervated by the arcuate nucleus, paraventricular nucleus and medial preoptic nucleus which release hormones into the primary plexus but these hormones interact with the anterior lobe in the secondary plexus.
  - The posterior pituitary gland is innervated by the paraventricular nucleus and supraoptic nucleus which releases hormones directly into the blood stream.
  

Posterior Pituitary Gland

The posterior pituitary releases vasopressin (ADH) which is involved in the maintenance of physiological concentrations. A higher concentration results in the pressor effect.
- This controls blood volume and plasma osmolality. Pathology of this results in diabetes insipidus or SIADH.

It also releases Oxytocin which has a function in females to contract their uterus during labour and eject milk for lactation. It controls sensory/stretch receptors. Damage to this secretion can result in the failure of the suckling reflex.

Anterior Pituitary Gland

The anterior pituitary contains secretory cell types within two classifications:
Basophilic (take up basic stains) - corticotrophs (15-20%) e.g. ACTH and beta-LPH.
- Gonadotrophs (10%) e.g. LH and FSH
- Thyrotrophs (5%) e.g. TSH.

and Acidophilic (take up acidic stains) - Somatotrophs (50%) e.g. GH.
- Lactotrophs (10-25%) e.g. Prolactin.
- Somatomammotrophs (0.5%) e.g. GH and PRL.

Characteristics of human pituitary hormones:
1) Corticotropin-related hormones (small peptides)
- alpha Melanocyte stimulating hormone (aMSH) has 13 amino acids (1823kDa); Corticotropin (ACTH) has 39 amino acids (4507kDa); Beta Lipotropin (BLPH) has 91 amino acids (9500kDa).

2) Glycoprotein hormones (have dissimilar a/b peptide chains)
- Follicle Stimulating Hormone (FSH) has 89 alpha amino acids and 115 beta amino acids (32000); Luteinising Hormon (LH) has the same as does Thyrotropin (TSH).

Anterior Pituitary Gland

3) Somatomammotropic hormones (single chain with 2-3 disulphide bonds)
- Prolactin (PRL) has 198 amino acids and 23510kDa.
- Growth Hormone (GH) has 191 amino acids and 22650kDa

4) Hypophysiotropic Hormones- Corticotropin-releasing hormone (CRH) has 41 amino acids and stimulates ACTH release, as does vasopressin (AVP) which has 9 amino acids.
- Thyrotropin releasing hormone (TRH) stimulates TSH and PRL release.
- LH releasing hormone (LHRH) has 10 amino acids and stimulates LH and FSH release.
- Growth Hormone Releasing Hormone (GHRH) has 44 amino acids and stimulates GH release.

Vasopressin 1

• Distribution of AVP:
  - Vasopressin is a 9 amino acid structured peptide formed into a looped structure and is also known as Arginine Vasopressin (AVP).
  
  

  - The hypothalamic nuclei which release AVP include:
  The supraoptic nucleus;
  the magnocellular neurones of the paraventricular nucleus that project to the posterior pituitary gland;
  the parvocellular neurones of the paraventricular nucleus;
  and the suprachiasmatic nucleus which projects to other areas of the hypothalamus.

  - AVP has 2 major physiological actions: It induces the contraction or relaxation of certain types of smooth muscle, and it promotes the movement of water and Na+ across responsive epithelial tissues.

Vasopressin 1

• Magnocellular Neurosecretory System:
  - Magnocellular neurosecretory cells are within the supraoptic nucleus and paraventricular nucleus of the hypothalamus.
  - The circumventricular organs include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and the Median Pre-optic Nucleus, which project to the supraoptic nucleus. Vasopressin projects down through the median eminence to the pituitary gland.
  
  
• Vasopressin Gene Regulation:- Vasopressin is transcribed in a forward production, whereas oxytocin is transcribed in reverse: 3 exons give rise to the final product.
  - Insitu hybridisation allows the vasopressin mRNA to be identified and shows cellular localisation whereas a northern blot does not.
  - It is found that AVP mRNA expression is upregulated by salt loading and stress, but is unaffected by oestrogen.
  

Vasopressin 1

• Vasopressin Gene Regulation:
  - A signal peptide is attached to the mRNA which directs it into the RER for biosynthesis, then it is packaged in the golgi where the signal peptide is cleaved off.
  - The peptide is stored in large dense-core vesicles which travel along the axon to the synapse. Along the way, enzymes break the peptide into 3 separate peptides: Vasopressin, Neurophysin and Glycopeptide.
  - Neurophysin then forms a tetrameric structure which forms a molecular chaparone for vasopressin.
  
  

  - Characteristics of Supraoptic Nucleus neurones:
  Vasopressin and Oxytocin are interspersed in the SON. To measure AVP concentration separate to oxytocin, a fluorescent marker should be used after genetically modifying AVP.

  - Electrophysiological recordings show AVPergic and OXYergic neurones together have slow, irregular recordings. OXYergic on their own are fast and continuous whereas AVPergic on their own are phasic.

Vasopressin 1

• Mechanisms of Osmoregulation:
  1) Magnocellular neurones:
  - Supraoptic nuclei neurones respond to an increase in osmolarity. They also respond to volume reduction and suction (evidence for stretch inactivated cation channels).
  - Suction increases electrical activity. In hypertonic solutions the cell shrinks which opens SICs.
  - Sodium channel subunit mRNA expression is upregulated by salt loading. (a11 subunit).
  - An increased production of Na+ channels makes cells more excitable.
  
  

  2) Circumventricular Organs:
  - i.e. SFO and OVLT are found in the anterior ventricular 3rd ventricle area.
  - When the AV3V region is deleted then osmotic responses are abolished.
  - There is a rise in plasma osmolarity with the absence of water when AV3V has not been deleted. When it has been removed, the animals stop drinking water so osmolality increases and vasopressin is not produced.
  - CVO neurones are osmosensitive, so OVLT, MnPO and SON neurones function as an 'osmoreceptor complex'.

Vasopressin 1

• Mechanisms of Osmoregulation:
  3) Astrocytes:
  - Water isn't able to cross the phospholipid bilayer alone, so there are proteins known as aquaporins present in the membrane.
  
  

  - They have 6 transmembrane domains and an NPA motif which forms a water channel.
  - AQP4 mRNA is expressed in the SON and PVN. (the protein is identified using a Western Blot where antibodies are used. A gold, electron-dense particle tags the protein).
  - AQP4 protein expression is glial-specific. Glial processes contain aquaporins, particularly on the endfoot.

Vasopressin 1

• Response to Hyperosmotic Challenges:
  
  Acute:
  - Causes cellular shrinkage (thus activating SICs) which results in the excitation of the neurone.
  - Also, it causes transcellular water movement through glial aquaporins to both the magnocellular neurone and to CVO efferents projecting to the neurone.
  
  

  Chronic:
  - Causes a depletion of pituitary AVP;
  - An increased amount of AVP mRNA expression and an increase in the production of Na+ channel subunit mRNA.

G-Protein Coupled Receptors

There are 3 types of memrane-bound receptors known as either enzyme receptors (which either have intrinsic enzyme activity or are associated with enzymes); receptor channels (ligand gated) or G protein coupled receptors.

• Signal Transduction:
  - GPCRs are 7 transmembrane spanning proteins showing a serpentine structure (snaking in and out of the membrane).
  - It is the receptor for many types of hormone and neurotransmitters, which all exhibit common structural characteristics.

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E1 - E4 = Extracellular loops which form a binding pocket where the hormone/ligand will sit.
H1 - H7 = Transmembrane helices; C1 - C4 = Cytosolic loops linking the TM domains.

G-Protein Coupled Receptors

There is a wide diversity of ligands for GPCRs, and there are different types of receptors:
- Class A are rhodopsin like;
- Class B are secretin-like;
- Class C are metabotropic glutamate-like;
- Orphan receptors have no ligand.

The best characterised receptor type is Beta-adrenergic receptors which bind the catecholamines adrenaline and noradrenaline.
- It has many different physiological effects: The breakdown of glycogen in the liver; and increased contraction rate of the heart.

• General Mechanisms:
  - The intracellular loop (i.e. C3) connecting helices 5 and 6 binds to signal transducing G proteins. Binding of the ligand causes the movement of helices which alters the conformation of the loop and thus activating the G protein.
  - G proteins bind guanine nucleotides (such as GTP) and use GTPs energy to alter their conformation. They form the link between receptor and effector.
  
  

G-Protein Coupled Receptors

• G-Proteins:
  - G proteins linked to 7 transmembrane receptors are heterotrimeric proteins with 3 subunits termed alpha, beta and gamma.
  - There are many different types of G proteins, the function and specificity of which is determined by:
  The alpha subunit which is unique for each type of G protein. There are structural differences in domains involved in both receptor and effector recognition. A common domain binds GTP.
  e.g/ as - stimulates adenylate cyclase.
  ai - is inhibitory to adenylate cyclase.
  aq - stimulates phospholipase C.
  
  
• Pathology:
  - Cholera is caused by the bacterium Vibrio cholerae which secretes a protein toxin that binds to the gut and is taken up. This modifies (ADP ribosylates) the alpha subunit of Gs to lack the enzyme in an active, GTP-bound state. Causing a persistant rise in cAMP and excessive secretion of Na+ and water.
  

G-Protein Coupled Receptors

• Pathology:- Whooping Cough is caused by Bordetella pertussis which produces a 2 component toxin, one of which inhibits Gi preventing an inhibition of adenylate cyclase. This increases cAMP and increases mucus secretion from the epithelial cells of airways.
  - Ras oncogenes and cancer: Variants of a monomeric G protein are carried by oncogenic viruses. These variants are inefficient at hydrolysing GTP which maintains activity and promotes tumour formation.
  
• G-Protein Activation:- Step 1: At resting state, the G proteins alpha subunit is associated with GDP.
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  - Step 2: The binding of a hormone to the receptor produces a conformational change in the receptor to allow the G protein complex to bind to the receptor at the C3 loop.
  

G-Protein Coupled Receptors

• G-Protein Activation:- Step 3: The GDP bound to the Gs protein is replaced by GTP so the subunits of the protein dissociate.
  
  

  - Step 4: The GSa subunit binds to adenylate cyclase to activate synthesis of cAMP. The hormone then tends to dissociate.

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  - Step 5: Hydrolysis of GTP to GDP causes the GSa subunit to dissociate from adenylate cyclase and binds to the Gb, y subunits regenerating a conformation of Gs that can be activated by an R hormone complex.

G-Protein Coupled Receptors

• Regulation of GPCRs:- A variety of mechanisms control the activity of these receptors, e.g. in constant presence of adrenaline, B-adrenergic receptors will bind the agonist but there will be no signal transduction (desensitization).
  - Receptors can also be down regulated where there is a reduction in the production of the receptor mRNA so the number of receptors decreases.
  
  

  - Phosphorylation modulates activity during desensitization: Sites of the receptor can be phosphorylated by B-adrenergic receptor kinase (BARK) which only phosphorylates agonist-occupied receptors. Following phosphorylation by BARK, a protein called B-arrestin binds to the phosphorylated receptor and blocks interactions with Gs.

G-Protein Coupled Receptors

• Cyclic Nucleotides:- Adenosine 3'5' monophosphate (cAMP) and guanosine 3'5' monophosphate (cGMP) are important second messengers whose intracellular concentration is controlled by the balance between synthesis (by cyclases) and degradation (phosphodiesterase):
  
  

  ATP/GTP -> 3'5' Cyclic AMP/GMP -> AMP/GMP.
  e.g. under the action of adenylyl cyclase, the ATP chain becomes a loop.

  - cAMP is more abundant than cGMP and has many physiological roles including the modulation of muscle contraction, glucose synthesis and glycogenolysis, secretion of hormones and proteins, and the secretion of ions and fluid.
  - cAMP transfers information from the plasma membrane to intracellular sites, and mediates amplification of the initial signal.

  - cGMP was discovered shortly after cAMP but has fewer apparent physiological roles. In contract to adenylyl cyclase, there are both membrane bound and soluble forms of guanylyl cyclase.

G-Protein Coupled Receptors

cGMP has roles as a second messenger in photoreceptor cells, involving the action of cGMP phosphodiesterase:
 - Mediation of atrial natriuretic peptide (ANP) actions. ANP is secreted by heart muscle cells to lower blood pressure.

A novel receptor type was discovered in the 1980s whose extracellular domain binds ANP, a single membrane spanning sequence and a guanylyl cyclase catalytic domain on the cytoplasmic side.
Binding of ANP induces a conformational change that activates guanylyl cyclase.
- This produces cGMP which activates Protein Kinase G to decrease Calcium concentration, thus relaxing myofibrils.

• cAMP Phosphodiesterases:
  - Are distributed in different intracellular compartments and subject to complex regulation. They are involved in the catalysis of hydrolysing cAMP.
  - Cells contain multiple forms of these phosphodiesterases (PDEs) which can be either single polypeptide chains or dimers.
  - Inhibitors of PDEs have been used in cAMP research, e.g. caffeine (1,3,7-trimethylxanthine); IBMX (1-methyl-3-isobutylxanthine) both of which manipulate PDE activity.

G-Protein Coupled Receptors

cAMP phosphodiesterases are activated by cAMP-dependent protein kinases as a feedback mechanism, or by insulin in the liver.
In unstimulated cells, concentration is about 1um which is not sufficient enough to activate Protein Kinases because a large proportion is bound to phosphodiesterase and other proteins.

Stimulation of Adenylyl Cyclase causes an increase in free cAMP which activates protein kinases.

In glycogenolysis, glucose is released from glycogen after activation by adrenaline through a B-adrenergic receptor in liver and muscle cells:
1) Adrenaline activates adenylyl cyclase to convert ATP into cAMP.

2) cAMP activates the cAMP-dependent protein kinase which inactivates glycogen synthase and phosphoprotein phosphatase to inhibit the synthesis of glycogen, while also activating glycogen phosphorylase kinase (using ATP).

3) This Kinase then activates glycogen phosphorylase using ATP, which converts glycogen and nPi to nGlucose and P.

G-Protein Coupled Receptors

• Recent Concepts in GPCR Signalling:
  Heterodimerisation:
  - When a receptor can form a dimer with another GPCR so they can both respond to different hormones.

  Regulators:
  - RGS proteins bind to the G protein and prevent it from reassociating with or activating the receptor.

Actions of Vasopressin

• The Vasopressin (V2) Receptor:- The V2 receptor of a rats kidney was first cloned in 1992 when the gene was located and mRNA expression was observed.
  - The V2 receptor has a classic GPCR structure where the external loop forms a binding pocket and the C3 cytosolic loop interacts with the G protein.
  - In condition of dehydration, the amount of V2 receptor mRNA expression decreases (is down regulated) so the kidney gradually becomes desensitized to the presence of AVP.
  
  
• Renal Action of AVP:
  - In the absence of AVP, there is no water movement through principal cells as the apical membrane is impermeable.
  - When AVP binds, the G protein dissociates to activate adenylyl cyclase. Then subluminal vesicles fuse with the apical membrane to cause the permeability of the membrane to change.
  

Renal Actions of AVP

• Aquaporin 2:- Was discovered and cloned in the 1990s, it is found within the inner medullary collecting duct (IMCD).
  - AQP2 is a water channel and the way to explore its function is to express the protein in a cell type which is impermeable to water, e.g. a frog cell.
  - If they express AQP2 then the cell will increase in size as water enters through the membrane.- The hydropathy profile shows that AQP2 has 6 hydrophobic domains with 2 loops folding into the membrane and 2 NPA motifs. The protein folds around itself.
  - AQP2 mRNA expression is regulated by the hydration state (i.e. AVP). When thirsty, AQP2 and AQP3 expression is increased so AVP regulates the amount of water channel produced by the cell.
  
• AQP2 Shuttling:- An electromicroscopy using immunogold labelling shows that under hydrated conditions, AQP2 is present beneath the surface of the cell.
  - When AVP is added (due to dehydration), AQP2 is now on the surface of the cell so the membrane becomes permeable. If pre-treated with OPC for 15 or 30 minutes then there is a loss of AQP2 concentration so the vesicles can be seen throughout the cytoplasm.
  

Actions of Vasopressin

• Signalling via cGMP: - When AVP binds to the V2 receptor there is an increase in cAMP production which activates a protein kinase to phosphorylate AQP2. This increases membrane permeability.
  - AQP2 distribution is disorganised (away from the membrane) by microtubule disruption with colchicine.
  
  

  Tubular Protein Motors:
  - Dynein has been colocalized with AQP2 in the IMCD vesicles and it has been found that Dynein walks the vesicle down the microtubule to the membrane.
  - Therefore it promotes trafficking of vesicles along microtubules.

  SNARE Proteins:
  - Entrap the vesicle onto the membrane.
  - vSNAREs (vesicular) are located on the vesicle which interact with a tSNARE (terminal) located within the apical membrane, enabling the vesicle to fuse with the membrane presenting AQP's onto the membrane.
  

Actions of Vasopressin

• Summary of the Renal Actions of AVP:
  
  - Under normal conditions , there is a large osmotic difference between the lumen (300 mOsm) and the blood (700 mOsm). When the membrane is impermeable, urine is dilute.
  
  

  - Dehydration causes AVP to be produced which binds to the V2 receptor to activate adenylyl cyclase. ATP is then converted to cAMP which activates Protein Kinase A to phosphorylate the AQP2 protein channel.
  - Phosphorylation results in dynein-mediated trafficking along the microtubule to the apical membrane where the SNARE proteins attach.
  - AQP2 is then incorporated into the membrane to increase permeability. H2O is then moved from the lumen into the blood to dilute it.
  

Oxytocin

• Actions of Oxytocin:
  Parturition:
  - Uterine contraction activates cervical stretch receptors which send afferents to the brainstem. This relays axons to the hypothalamus to stimulate the production of oxytocin from the posterior pituitary lobe.
  - The secretion of oxytocin then enhances uterine contraction for child birth.
  
  

  Lactation:
  - Suckling activates mechanoreceptors in the ****** which projects afferents to the brainstem. This relays axons to the supraoptic and paraventricular nuclei of the hypothalamus to stimulate the production of oxytocin.
  - This causes the contraction of myoepithelial cells to eject milk.
  - Mechanoreceptor afferents also inhibit the release of dopamine from the arcuate nucleus to prevent the inhibition of prolactin secretion. They also inhibit GnRH from secreting LH and FSH from the anterior pituitary to prevent ovulation.
  

Oxytocin

• The Oxytocin System:
  - The expression of oxytocin mRNA increases in the paraventricular nucleus throughout pregnancy. This change in mRNA is regulated by steroids.
  - Progesterone and oestrogen increase to drive the production of oxytocin. At the end of a pregnancy the progesterone levels drop off. If this does not happen then the change in mRNA expression is inhibited to prolong pregnancy.
  - Naloxone acts as an antagonist for opioids.
  
  

  Oxytocin Accumulation:
  - Progesterone drives an increased production of endogenous opioids which hyperpolarize neurones.
  - Oestrogen activates the nucleus of the neuronal cell body to produce more oxytocin which is transported down the axon to accumulate at the axon terminal.

  - If you record from a series of oxytocin neurones at the same time there is a striking degree of action potential synchrony, i.e. the neurones fire at the same time.

Oxytocin

• The Oxytocin System: - Glial cells are identified with a GFAP marker as sending projections in between neurones containing oxytocin to insulate them and prevent the activation of neurones by others.
  - This action of one neurone becoming excited causing the depolarisation of a neighbouring neurone is known as syncitium.
  
  

  - Towards the end of pregnancy, as progesterone levels fall, hyperpolarisation of the cell does not occur so local oxytocin causes the glial cells to withdraw and thus synchrony occurs.

• Oxytocin Receptor:
  - The oxytocin receptor is a 389 amino acid GPCR (class 1) from the OXY/AVP subfamily.
  - The gene for this receptor was first cloned in 1994 by Kimura et al and is located on chromosome 3.
  

Oxytocin

• Oxytocin Receptor:
  
  Regulation:
  - The receptor is sensitive to dimerization. It is delivered to the membrane where it can form a dimer with another oxytocin receptor, or form a heterodimer with a different GPCR.
  
  

  Signalling:
  - When oxytocin binds to its receptor it activates phospholipase C through the dissociation of the Gaq subunit.
  - This hydrolyses PIP2 into IP3 and DAG. The IP3 molecule binds to its receptor in the sarcoplasmic reticulum to increase the intracellular calcium concentration.
  

Oxytocin

• Mammary Gland Sensitivity:Structure:
  - The milk duct opens into glandular tissue containing myoepithelial cells which are lined with epithelial milk-secreting cells.
  - The oxytocin receptors are expressed in the myoepithelial cells and increase during pregnancy.
  
  

  - Oxytocin receptor expression is differentially regulated by oestrogen. Oestrogen potentially increases the number of receptors, whereas progesterone interacts with the receptor by allosteric hindrance.
  - i.e. it acts as an antagonist to prevent oxytocin binding. At the end of pregnancy, progesterone will be removed from the receptor to prepare.
  

Growth Hormone

• GH Gene Structure and Regulation:- The GH gene has 5 extracellular transmembrane proteins.
  - GHRH stimulates a receptor to activate adenylyl cyclase via the Gas/Gsa protein. This causes an increase in cAMP to activate Protein Kinase A.
  - PKA activates CREB for somatotroph proliferation, plus GHF-1 to induce somatotroph proliferation, increase GH mRNA and increase GHRH-R mRNA expression. Therefore elevating GH secretion.
  - Somatostatin (SRIF) binds to a receptor on the somatotroph which activates a Gi protein to inhibit adenylyl cyclase.
  
  
• Growth Hormone:
  - Human GH is a 191 amino acid protein containing 4 alpha helices arranged in an 'up-up-down-down' topology with two disulphide bridges and two non-identical binding domains.
  - GH is secreted in pulses (non continuous). Episodes of GH secretion are generated by integrated SRIF and GRF secretion. SRIF is inhibitory; GRF and GHRH are excitatory.
  

Growth Hormone

In the hypothalamus, the periventricular nucleus contains somatostatin neurones and the arcuate nucleus contains GHRH neurones.
- When GHRH is released in bursts, SRIF release is suppressed and GH release is stimulated.

Factors stimulating GH secretion:

•  Deficiency in energy substrate
  - Hypoglycaemia (OGIT)
  - Fasting
  - Vigorous exercise
• Increase in circulating amino acids (especially arginine).
• Leptin (energy storage)
• Oestrogens (females have higher than males)
• Sleep
• GH increase breaks down fat and is produced with fasting or exercise.

Growth Hormone

Factors inhibiting GH secretion:
- Glucose
- Adrenal function
- Sleep (REM)
- Growth Hormone/IGF-1
- Obesity
- Age

A decrease in cortisol decreases the number of receptors for GHRH so there is desensitization of the pituitary gland, thus there is a GH decrease.

Cytokine Receptors

• The Cytokines:- Erythropoletin is found in the kidney and is involved in the proliferation of red blood cells.
  
  

  - Interleukin 2 (IL-2) is involved in the proliferation of T cells, whereas Interleukin-4 (IL-4) is involved in the proliferation of B cells.

  - Granulocyte colony stimulating factors are involved in the proliferation of granulocyte progenitor cells

  - Growth hormone is produced by the anterior pituitary and is involved in the differentiation of cells in the germinal zone.

  - Prolactin is also produced by the anterior pituitary and is involved in the differentiation of acinar cells and milk production.
  

Cytokine Receptors

• Cytokine Receptor Subfamily:
  Class 1 (Hematopoietin Receptor) - Receptors for erythropoietin, growth hormone, prolactin, leptin and IL-13 form homodimers.
  - Receptors for IL-3, IL-5 and GM-CSF share a common chain CD132 or beta.
  - Receptors for IL-2, IL-4, IL-7, IL-9 and IL-15 share a common chain, CD132 or common gamma chain. The IL-2 receptor also has a third chain, a high affinity subunit IL-2Ra.
  
  

  Class 2 Cytokine Receptor - Interferon alpha, beta and gamma receptors; IL-10 receptor.

  TNF-Receptor Family - Tumour Necrosis Factor receptors 1 and 2; CD40; Apo 1; CD30; CD27; Nerve growth factor receptors all are monomers.

  Chemokine-Receptor Family - CCR1-5; CXCR1-4 have several transmembrane spanning domains.
  

Cytokine Receptors

• Growth Hormone Receptor:Gene and Protein Structure - The human GH receptor gene contains 10 exons.
  - The extracellular binding domain takes place across 3-7 transmembrane domains.
  - Rodents have an extra 8A exon which contains a stop code. This produces the truncated protein Growth Hormone Binding Protein.
  - The receptor has a hormone binding domain on the outside which has a higher affinity for site 1; a dimerisation domain where 2 receptors interact (the receptors are present in the membrane as loose, inactive dimers); a transmembrane domain and an activation domain.
  
  

  Receptor Dimerization
  - The binding of GH to site 1 induces sequential receptor dimerization. A conformational change in the dimerization domain bends GH over so that the receptor can bind to site 2. The close affinity between intracellular domains allows the activation of the receptor.

  - Site 2 GH mutants have antagonist activity. GH-induced receptor dimerization elicits activation.
  

Cytokine Receptors

• Growth Hormone Receptor:Signal Transduction Pathways
  - GH induced receptor dimerization results in the phosphorylation of JAK2 causing the receptor tyrosines to also phosphorylate.
  
  

  - Stat5 becomes associated with the receptor and phosphorylates to form a dimer. It is then translocated to the nucleus.

  - The phosphorylation of Shc activates the Ras-Raf-MEK-MAPK pathway where MAP Kinase is phosphorylated to increase the mRNA expression.

  - The phosphorylated JAK2 also activates phospholipase C to hydrolyse PIP2 into IP3 and DAG. DAG then activates PKC to open calcium channels and increase calcium concentration.

  - The resulting receptor gene has pleiotropic effects and also stimulates the release of Suppressors of Cytokine Signalling (SOCs) which bind to Stat5 and prevent it from being phosphorylated.
  

Actions of Growth Hormone

• GH-Binding Protein:- In humans the GHBP and GH receptor are produced as one splice product from the same gene. When this is enzymatically cleaved, the extracellular portion floats away in the cytoplasm to form the binding protein.
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  - In rodents however, there is a unique etitope that occurs at the tail end of the binding protein which allows it to be identified as a separate product.
  
  

  - Circulating GHBP is regulated by long term changes in GH. In normal males, there are large bursts of GH but a small, steady level of GHBP. In dwarf males there are smaller GH peaks.
  - In normal females there is an irregular number of GH peaks that arent large but in total the GH concentration is higher than in males. GHBP concentration is also higher than in males. In dwarf females it decreases.
  - GHBP increases the half life of GH; When mixing GH with the BP before injecting it into the circulation, the presence of GH is maintained.
  

Actions of Growth Hormone

• Growth Hormone Receptor Distribution:
  - Growth Hormone receptors are widely distributed, in e.g. Adipose tissue, Bone (chondrocytes, osteoblasts and osteoclasts), Brain, Cartilage, Corpus luteum, Heart, Intestine, Kidney, Liver, Lung, Lymphatic cells (lymphocytes and thymocytes), Pancreas, Skeletal muscle and Testis.
  
  

  - Therefore, GH has pleiotropic actions.

  Skeletal growth promotion
  Metabolic effects:
  - Increases protein synthesis;
  - Reduces plasma glucose/utilization (IGF-1).
  - Mobilization of fatty acids (lipolysis).
  Anabolic effects:
  - DNA synthesis.
  Other:
  - Activation of prolactin receptors.

Actions of Growth Hormone

• Regulation of Skeletal Growth:
  Dual Effector Theory:
  - GH is released from the anterior pituitary gland to stimulate the epiphyseal plate directly to induce growth.
  - It also stimulates receptors in the liver to produce IGF-1 which also acts on the bone to stimulate growth and releases more IGF-1.
  
  

  Epiphyseal Growth Plate
  - Contains a germinal (resting) zone which contains GH receptors. This is where resting cells differentiate to become the cells on top of the proliferative zone. GH induces the appearance of IGF-1 mRNA which also induces the expansion of the proliferative zone.
  - Then there is the hypertrophic zone where cells become engorged with calcium and become hypertrophic (i.e. T3) at the bottom of the plate.
  - The cells then rupture and the calcified substructure of the cell becomes the basis of tubercle formation in the calcifying zone.
  

Actions of Growth Hormone

• Regulation of Skeletal Growth:- The effect of GH on bone in skeletal growth can be examined using a genetically modified mouse where the bone produces GH.
  - The effect of GH on skeletal growth is pattern-dependent. If you give it in slow profusion there is a continuous effect so there are more pulses, thus males grow bigger and faster.
  - There is a small but significant increase in the length of the tibia and femur but not body weight. Thus the removal of hepatic IGF-1 does not significantly impair skeletal growth.
  
  
• Lipolysis:- GH inhibits preadipocyte differentiation, and thus reverses bone marrow adipogenesis in GH-deficient rats. Without GH the number of fat cells increases, and there is a larger reservoir for fat to be stored in.
  - The lipolytic effect of GH in humans is pattern dependent. In GH deficient patients on an intermittent GH replacement the serum non-esterified fatty acid concentration increases with an increase in serum GH so it promotes lipolysis.
  

Actions of Growth Hormone

• Disruption of the GH Axis:Acromegaly
  - Is caused by adult onset GH excess usually as a result of a GH-secreting tumour.
  - The symptoms of which include coarsening of facial features; enlarged hands and feet; thickening of palms and soles; Carpel Tunnel Syndrome (tingling sensation or pain in the hands); Sweating and oily skin; Headaches; Visual disturbance; Lethargy and reduced fertility.
  
  

  Gigantism
  - Is caused by childhood GH excess. The symptoms include some features of acromegaly and excess skeletal growth as the epiphyseal growth plate is still intact. After puberty there is apoptosis of the chondrocytes in the plate which leads to less ossification so growth slows and later stops when the entire cartilage is replaced by bone. 

  - Excesses in GH can be treated by somatostatin which is a GH antagonist.
  

Actions of Growth Hormone

• Disruption of the GH Axis:
  Dwarfism
  - GH deficiency in childhood can cause dwarfism where symptoms include increased adiposity; reduced muscle mass; increased fracture rate; lethargy; anxiety/depression and social isolation. This can also be caused by craniopharyngiomas, chromophobe tumours or thrombosis of pituitary vessels.
  With hypothyroid dwarfism the dwarf is not proportional in features.
  - This can be treated with recombinant hGH.
  - Mutations of the GHRH receptor can also produce profound dwarfism as seen in the dwarfs of Sindh in Pakistan. This can be treated with GH.
  - Laron Syndrome is when the dwarf has a square or flat face due to a mutation in the GH receptor. This can be treated with IGF-1.
  - Pygmy dwarfism is caused by a disrupted IGF-1 gene which can also be treated with IGF-1.

Actions of Growth Hormone

• Modelling Disruption of the GH Axis:
  - A mouse was injected with rat GH and the overexpression of this hormone produced gigantism so the mouse was twice the size of a normal mouse.
  
  

  - A mouse that has no copies of the growth hormone receptor or GHBP gene produces profound dwarfism.

  - Guinea pigs are naturally occurring Laron dwarves which have non functional GH receptors.
  

Ghrelin

• Growth Hormone Secretagogues (GHSs):- Bowers, C.Y (1984) described a group of synthetic compounds that stimulate GH release in a specific way. These compounds include Met-Enkephalin and GHRP-6.
  - GHS's act mainly via the arcuate nucleus GRF neurones. GHRP-6 stimulates Fos expression in the arcuate nucleus.
  - They act through a specifc G protein coupled receptor known as GHS-R1a, first cloned in 1966, which is present in the anterior pituitary but expressed mostly in the hypothalamus.
  
  

  - There are two forms of the receptor: a truncated version (GHSR1b) which doesnt have an intracellular C3 loop so doesnt interact with G. It is then dimerized with 1a to regulate cell sensitivity.
  - The Gaq subunit dissociates from the receptor and activates phospholipase C to hydrolyse PIP2 into DAG and IP3. DAG activates Protein Kinase C to open calcium channels in the cell membrane to cause an influx of calcium ions and IP3 opens calcium channels in the sarcoplasmic reticulum to increase the intracellular calcium concentration.

  - MKO677 is an orally active GHS which induces GH release in humans.

Ghrelin

• Discovery:
  - A cellular screen was generated: A cell is taken which does not express GHSR's but has the internal gizmos for responding to activation of Gaq (i.e. it cant activate Gaq but can respond to it). The cells are injected with GHSR cDNA so that the receptor is expressed on the surface of the cell. When it is activated the intracellular calcium concentration increases, and if Fluo4 is injected it will fluoresce to show the presence of calcium ions.
  
  

  - The endogenous GHS-R1a ligand is found in the stomach shown as there is an increased fluorescence from a stomach fraction and the purified ligand increases fluorescence. Thus purifying the stomach fraction found Ghrelin.

Ghrelin

• Structure:
  - Ghrelin has 28 amino acids, the third of which is serine with a unique C8 chain from octanoic acid. This side chain is necessary for Ghrelin to bind to and activate GHSR. It has a unique structural modification octanoylation.
  - Octanoylation is essential for binding to GHS-R1a. GOAT (Ghrelin O-acyltransferase) is found in the stomach and cells that line the brain, and adds a side chain to Ghrelin to convert unacylated Ghrelin (UAG) into Ghrelin. The majority of Ghrelin is present in circulation as UAG.
  
  
• Distribution:
  - The stomach is the main site of Ghrelin production, but a network of ghrelinergic neurones is present in the hypothalamus (arcuate nucleus and periventricular nucleus) too.

Ghrelin

• Regulation:
  - Circulating Ghrelin is increased by fasting and suppressed by refeeding. Filling the stomach with nutrients suppresses Ghrelin.
  - Ghrelin concentration increases before breakfast and decreases after, and for all other meals it does this.
  
  
• Actions of Ghrelin:
  - Ghrelin is secreted in response to undernutrition.
  - Acute ghrelin stimulates GH secretion via the activation of GRF neurones. (i.e. the same neurones that GHRP-6 activates).
  - Chronic exposure to Ghrelin suppresses GH secretion. Growth promotion is pattern dependent. Continuous Ghrelin exposure suppresses GH secretion.
  - Ghrelin suppresses LH and FSH secretion.
  - Ghrelin stimulates feeding behaviour. This is not dependent on the pattern of infusion.
  - Ghrelin activates orexigenic neurones (Neuropeptide y) in the arcuate nucleus inducing fos expression. These neurones run up to the paraventricular nucleus.
  

Ghrelin

• Actions of Ghrelin:- Ghrelin stimulates adipogenesis for fat deposition, the sensitivity to Ghrelin is depot-dependent. It increases visceral fat and the fat in bone marrow.
  - Only if its given continuously, there is no increase in fat mass with pulses. UAG Ghrelin does the same thing.
  - Ghrelin induced adipogenesis in bone marrow is not mediated by the GHS-R1a, but in intra-abdominal fat it is. This is found by genetically modified mice that without the receptor are not affected by Ghrelin.
  
  
• Physiological Significance:- Mice with a deletion of the gene for Ghrelin show normal skeletal growth, whereas you would expect them to be skinny and small. Thus under normal conditions it may not regulate this.
  - Mice with this deletion may also show normal food intake, thus under normal conditions it may not regulate food intake.
  - These also show normal fat deposition under normal conditions. Thus, Ghrelin is only released in response to undernourishment. (an energy deficit signal).
  

Insulin and IGF-1 are similar in structure and sequence, and so bind to the same receptor. Insulin is involved in metabolism, and IGF is involved in cell proliferation and growth.

• Receptor:
  - Members of the type II receptor tyrosine kinase (RTK) family function as heterotetramers, i.e. two ligand binding alpha subunits and two beta subunits containing the TK activity.
  - The IGF-1 receptor has a high affinity to IGF-1, a medium affinity for IGF-2, and a low affinity for Insulin.
  - The Insulin A receptor has a high affinity for insulin, medium affinity for IGF-2 and a low affinity for IGF-1; whereas the Insulin B receptor has a high affinity for insulin and a low affinity for IGF-1.
  
  
• Signal Transduction and Role:- Plasma glucose levels are narrowly contained throughout the day and are dependent on the balance of hypoglycaemic actions of insulin and hyperglycaemic actions of anti-insulin hormones.
  - Glucose does not have a high spike, but insulin secretion is shown as a 2 phase release (with two spikes). Glucose promotes insulin production and decreases glucagon release.
  

• Insulin:
  - It is produced in the pancreas endocrine portion. The exocrine portion is responsible for producing pancreatic juice.
  - Within the endocrine portion are cells known as Islets of Langerhans which make up 2% of pancreatic tissue and are made up of 70% B-cells which make insulin, and 30% a-cells that make glucagon. 
  
  

  The structure of an insulin molecule:
  - 2 polypeptide chains linked by 2 disulphide bridges. The alpha chain is 21 amino acids long and the beta chain is 30.
  - Initially there is a signal peptide attached to the end of the insulin molecule which directs it into the endoplasmic reticulum, and a connecting peptide (C-peptide) which holds it all together.
  - Preproinsulin is split into proinsulin and the signal peptide in the endoplasmic reticulum, and then proinsulin is packaged into secretory granules in the Golgi. Before secretion, this is split further into the C-peptide and insulin.
  

• Insulin Signalling:
  - Insulin is made and stored in granules until required.
  - Via other cell signalling pathways, it is released into the blood and travels to its target cells.
  - Insulin binds to its receptor and via signal transduction, results in glucose transporters moving to the plasma membrane.This increases glucose uptake.
  - GLUT1 is found in erythrocytes, GLUT2 is found in liver and pancreatic cells, GLUT3 is found predominantly in the brain, GLUT4 is found in intracellular vesicles of muscle and adipose tissue, GLUT5 is found on the apical brush border of enterocytes in the small intestine.
  - Insulin is released in two phases: 1) In response to high glucose and 2) Independently of glucose, new insulin is produced from amino acid stimulation.
  
  
• Phase 1 Insulin Secretion:- This is determined by glucose metabolism in the B cell.
  1) Glucose enters the cell via GLUT2 and is converted to pyruvate producing ATP.
  2) ATP then binds to K+ channels to close them causing a depolarization of the membrane.
  3) This opens voltage-gated Ca2+ channels allowing calcium ions to enter the cell.
  4) Calcium ions activate phospholipase C to hydrolyse PIP2 into IP3 + DAG. IP3 binds to receptors on the ER to release more Calcium. Elevated cytosolic Ca2+ stimulates the release of secretory vesicles containing insulin.
  

• Phase 2 Insulin Secretion: 
  - Is determined by glucose independent mechanisms.
  - Insulin is newly synthesized in response to amino acids (e.g. leucine, arginine and lysine); Gastrointestinal hormones (e.g. glucose-dependent insulinotropic peptide (GIP), cholecystokinin, glucagon-like peptide-1 (GLP-1) and vasoactive intestinal peptide (VIP);
  and Acetylcholine stimulation of the vagus nerve.
  
  
• Signalling via the Insulin Receptor:- In the absence of a ligand 'Cam loops' keep the a-subunits apart which forces the B-subunits to be apart, and thus the receptor is inactive.
  - In the presence of a ligand, the cam loops rotate and allow the a-subunits to close around one ligand molecule.The B-subunits now move together.
  - Tyrosine kinase binds ATP and phosphorylates itself for activation.
  - TK can now transphosphorylate target tyrosines on the B-subunit of opposite receptors and also some adaptors that bind to the receptor.
  - In most kinases, an area known as the activation loop is often blocking the target binding site of the kinase and it needs to be phosphorylated to be moved.
  

• Signalling via the InsR:
  The activated receptor kinase initiates a kinase cascade:
  - When the tyrosine kinase is activated, the two units of the receptor are held in close proximity to one another so that additional sites are also phosphorylated.
  - These sites act as docking sites for other substrates, including a class of molecules referred to as insulin-receptor substrates (IRS).
  - IRS1 and IRS2 are homologous proteins with a common structure.
  
  

  - The phosphotyrosine residues in the IRS proteins are phosphorylated by the insulin receptor kinase, and also recognised by other proteins containing a class of domain called Src homology 2. These domains bind to stretches of polypeptide containing phosphotyrosine residues.
  - Phosphoinositide 3-Kinase contains the SH2 domain allowing it to bind to the IRS protein, drawing it towards the membrane where it can phosphorylate PIP2 to form PIP3.

  - PIP3 activates PDK1 by a PIP-specific pleckstrin homology domain present in the kinase.
  - PDK1 can then go on to activate Akt which moves through the cell to phosphorylate target cells including components that control the trafficking of GLUT4 to the cell surface, as well as enzymes that stimulate glycogen synthesis.
  

• Signalling via the InsR:
  Is terminated by:
  - Adaptor proteins, e.g. IRS-1 is dephosphorylated.
  - Receptor internalization in endosomes
  - Dissociation of ligand from receptor.
  - Dephosphorylation of receptor by tyrosine phosphatases.
  - Receptor is recycled back to the cell surface.
  
  
• Metabolic Effects of Insulin Signalling and Glucose Uptake:
  - Glucose transporters are stored in the walls of cytoplasmic vesicles.
  - Insulin induced IRS-1/PI-3 kinase/PKB signalling triggers vesicle translocation to the plasma membrane.
  - The vesicles fuse with the membrane where they take up glucose and pass in to the cell.
  - The Cbl-CAP complex interacts with flotillin in a lipid raft. Then there is further signalling, i.e. the Crk/C3G/TC10 cascade is promoted.
  - Together with PI3-K signalling, this facilitates GLUT4 translocation.
  

• Regulation of Metabolic Responses:
  - Insulin binding on the insulin receptors causes the uptake of glucose through GLUT4 into muscle cells, where it either undergoes glycogenesis to be stored as glycogen, or undergoes glycolysis to produce ATP.
  
  

  - Glucose is also taken up into adipose cells through GLUT4 where it undergoes glycolysis to produce Acetyl coenzyme A which forms dihydroxyacetone phosphate and also undergoes lipogenesis to form fatty acids.
  - Dihydroxyacetone phosphate is then reduced to glycerol-3-phosphate which, along with the fatty acid, can be converted into triglyceride where glucose is stored until required.

  - Glucose is taken up from the blood into the liver via GLUT2 transporter where it either undergoes glycogenesis and is stored as glycogen, or undergoes glycolysis where it is converted to acetyl coenzyme A. This then also undergoes lipogenesis to produce fatty acids, and dihydroxyacetone-phosphate to be reduced into glycerol-3-phosphate which is converted to triglyceride, then to a very-low-density lipoprotein that can exit the liver cell and be converted to low-density lipoprotein which is either taken into the adipose cell to form fatty acids, or is converted to glycogen outside of the cell.
  

• Bone Structure:
  - Bones consist of the epiphysis located at the end of the bone, which runs into the metaphysis (the neck) and then the diaphysis which is the shaft.
  - On the outer surface is the compact/cortical bone whilst on the inside the bone marrow cavity is the spongy/trabecular bone.
  
  
• Bone Development: 
  Bone formation process varies at different skeletal sites:
  Intramembranous Ossification - When the bone forms within the membrane; Occurs in flat bones such as the skull, scapula, mandible and ileum. This has no intermediate phase and so is direct.
  Endochondral Ossification - Occurs in long bones such as the tibia, femur and humerus which involved an intermediate cartilage phase.
  
  
• Intramembranous Ossification:1) The development of the centre of ossification - mesenchymal cells divide and differentiate into osteoblasts (bone forming cells) which form a woven bone with irregular collagen orientation, large and numerous osteocytes and patchy calcification.

• Intramembranous Ossification:
  2) Calcification
  3) Formation of trabeculae - The blood vessels are trapped between mineralised bone and the trabeculae to form a bone marrow cavity.
  4) Development of the periosteum - Woven bone is remodelled and replaced by lamella bone.
  
  
• Endochondral Ossification:1) Cartilage formation - Mesenchymal cells divide and differentiate into chondroblasts which secrete cartilage and become embedded in the lacunae within the matrix.
  2) Vascular Invasion and Longitudinal Growth - A ring of woven bone is formed in the midshaft, and osteoclasts allow vascular invasion of woven bone and cartilage, allowing differentiation of osteoblasts. The growth plate is also formed which allows the bone to extend.
  
  
• Cells of the Bone:
  Osteoblasts - Derived from mesenchymal cells; Are non-dividing and mononuclear; are metabolically active (RER and Golgi): genes for matrix proteins (Type I collagen = 20%, and osteocalcin = 1%) and regulatory factors (BMPs, TGFbs, IGFs)
  - Have a key role in directing bone resorption and make the bone matrix.

• Cells of Bone:
  Osteocytes: - Osteoblast (15%) embedded within lacunae in bone matrix.
  - Have a reduced metabolic activity.
  - Connected by canaliculi (canals within mineralised matrix) which allow nutrient diffusion and cell to cell communication.
  
  

  Osteoclasts: - Derived from mononuclear cells in the bone marrow.
  - Are large (100um) and multinucleate (20).
  - Involved in bone resorption (lysosomes, mitochondria, ruffled border)

• Functions of Bone:1) Mechanical - The bone provides a rigid skeleton which protects vital organs and facilitates locomotion.
  2) Calcium Homeostasis - Calcium is stored in bone matrix and released from bone when there is low blood calcium concentration.
  3) Bone marrow - Red bone marrow is found within the bone trabeculae which is where blood cells and their precursors reside.
  4) Mechanical uses and calcium homeostasis act with osteoclasts for bone turnover.
  

• Bone Modelling and Remodelling:
  - Bone modelling is when there are changes in the shape and diameter of the bone during growth.
  - Bone remodelling is when there is a constant bone turnover, i.e. old bone is replaced by new via the activation-resorption-formation sequence.
  
  
• Bone Remodelling Cycle:

• Development vs Remodelling vs Differentiation:
  Development - Bone cells need to differentiate in order to lay down mineralised skeleton.
  Growth - Skeleton is modelled to produce adult shape and size.
  Adult skeleton - Continually remodelled according to metabolic and mechanical requirements.
  Pathology - Too much or too little bone.
  These are all affected by factors that control differentiation and activity of bone cells.
  
  
• Osteoblast Lineage:- A mesenchymal stem cell can become either fat, muscle, cartilage or a preosteoblast. If it differentiates into the latter, then this can then differentiate into an osteoblast which will eventually become an osteocyte.
  - 3 genes are known to be essential for osteoblast differentiation, i.e. the development of bony skeleton.
  These genes are as follows:
  - Indian hedgehog (Ihh). Without this gene there would be a disorganised growth plate, no osteoblasts at the sites of endochondral ossification so that children die at birth, and osteoblasts are present in intramembranous bones therefore it is not essential for intramembranous ossification.
  - Cbfa, where knockouts have bee shown to have cartilagenous skeletons and osteoblasts completely absent; - And Osx.
  

• Osteoclast Differentiation: 
  - A pluripotent mononuclear precursor found in bone marrow and circulating the blood is converted into a mononuclear cell by M-CSF.
  - This cell contains appropriate signals and fuses at the bones surface to differentiate into a preosteoclast cell using PTH, IL-1 and 1,25vit D.
  - The preosteoclast is a multi nucleate cell which differentiates into a mature osteoclast that polarises on the bones surface to resorb bone.
  

There are various transcription factors controlling osteoclast differentiation:
PU.1 - Regulates the c-fms receptor (i.e. the receptor for M-CSF).
- Knockouts of this factor are osteopetrotic (too much bone) with no macrophage so no osteoclasts.
c-fos - Is an early response gene and a component of AP-1 TF. Knockouts of this factor are also osteopetrotic but they do have macrophages, therefore they have no mature osteoclasts.
NFkB - Is a dimer composed of proteins containing a 'Rel' domain (p50 & p52). Knockouts of these two domains are again osteopetrotic and have no mature osteoclasts.
mi - Mi mice with a mutation in this gene are osteopetrotic. There are mature osteoclasts present, but they do not resorb the bone.

• Secreted Factors Controlling Osteoclast Differentiation:
  M-CSF - an op/op mouse has a mutation in the M-CSF gene so they become osteopetrotic and have no macrophages or osteoclasts.
  OPG - Osteoprotegerin (osteoclast inhibitory factor) was discovered in 1997 in a genomic screen for novel secreted. An overexpression of this causes osteopetrosis with no mature osteoclasts. OPG -/- mice have osteoporosis where there are holes in the bones due to an increase in the number of osteoclasts. It has a soluble ligand known as RANKL.
  
  
• RANKL:- Are expressed on the cell surface of osteoblast progenitor and secreted in bone microenvironment. Systemic administration in vivo increases the bone resorption. In vitro there is an increased number of osteoclasts and activity.
  - Knockouts have osteopetrosis.
  RANK signals TRAF6 which stimulates Src to activate PI3K; or ERK to activate myc in the nucleus; or p38 to activate mi in the nucleus; or JNK to activate Jun/Fos in the nucleus; and also NFkB to activate p50/RelA in the nucleus.
  - OPG prevents RANKL binding to the osteoclast.
  

Nuclear receptors are members of a large superfamily of evolutionary related DNA-binding transcription factors that regulate programs involved in a broad spectrum of physiological phenomena.
- The transcription factor activate 4 genes which will go on to produce 4 proteins, and they also turn off one gene to prevent the production of a protein.

• Types of Receptor-Effector Linkage:
  1) Ligand-gated ion channels (ionotropic receptors) - When activated/opened, ions enter the cell to cause either hyperpolarisation or depolarisation of the cell which causes cellular effects. This is extremely quick taking milliseconds. E.g. Nicotinic or ACh receptors.
  2) G-Protein Coupled Receptor (metabotropic) - The ligand binds to the receptor which is associated with a G protein which can either activate or inactivate an ion channel to cause a change in excitability and thus cellular effects; or it can activate or inactivate an enzyme to either inhibit or excite the production of second messengers to either cause/inhibit calcium release, protein phosphorylation or other effects to stimulate cellular effects.
  3) Kinase-linked Receptors - The ligand binds to the receptor or enzyme causing protein phosphorylation and then gene transcription, protein synthesis then cellular effects.
  4) Nuclear Receptors - The ligand binds to the receptor within the nucleus which causes gene transcription, then moving out of the nucleus causing protein synthesis. This takes hours.

The predominant sex hormone in females is Oestradiol, an oestrogen, which is also present in males but at lower levels. It represents the major oestrogen in humans. Oestradiol not only has a critical impact on reproductive and sexual functioning but also affects other organs including the bones. It has a general steroid shape.

The predominant sex hormone in males is Testosterone, an androgen, which plays a key role in the development of male reproductive tissues such as the testis and prostate as well as promoting secondary sexual characteristics such as increased muscle mass and hair growth. It is also important to prevent osteoporosis.

A stress hormone which is also a steroid that is produced in response to stress and anxiety is Cortisol, a glucocorticoid. This is essential for life and regulates or supports a variety of important cardiovascular, metabolic, immunologic, and homeostatic functions.

• Steroid Biosynthesis:
  - Within the mitochondria, cholesterol is converted to pregnenolone by an enzyme in the inner membrane called CYP11A1.
  - Pregnenolone itself is not a hormone, but is the immediate precursor for the synthesis of all the steroid hormones.

Thyroxine is a hormone produced by the thyroid gland which is a secosteroid. The thyroid hormones, thyroxine (T4) and triiodothyronine (T3) are tyrosine-based hormones produced in the thyroid gland primarily responsible for the regulation of metabolism.

Vitamin D3 is produced in the skin of vertebrates after exposure to ultraviolet B light, and occurs naturally in a small range of foods. Retinoic acid is the oxidised form of Vitamin A.

These substances all function via the nuclear receptor superfamily.

• Nuclear Receptors:
  - Each receptor has a crucial and non-redundant role in processes such as growth, development and homeostasis.
  - They modulate transcription through several distinct mechanisms.
  - These mechanisms make nuclear receptor signalling remarkably complex, in terms of gene repression, release of gene repression and gene activation etc.
  - Nuclear receptors consist of six domains (A-F) based on regions of conserved sequence and function. The evolutionarily conserved regions are C and E, and the divergent regions A/B, D and F regions.

• N-terminal Regulatory Domain:
  - A-B: Contains the activation function 1 (AF1) whose action is independent of the presence of ligand. The transcriptional activation of AF1 is normally very weak, but it does synergize with AF2 in the E-domain to produce a more robust upregulatin of gene expression. The A-B domain is highly variable in sequence between various nuclear receptors.
• DNA-Binding Domain (DBD):
  - C: Is highly conserved, contains two zinc fingers which bind to specific sequences of DNA called hormone response elements (HRE).
• Hinge Region:
  - Thought to be a flexible domain which connects the DBD with the LBD. Influences intracellular trafficking and subcellular distribution.
• Ligand Binding Domain (LBD):
  - Moderately conserved in sequence and highly conserved in structure between the various nuclear receptors. The structure of the LBD is referred to as an alpha helical sandwich fold in which 3 anti paralell alpha helices are flanked by 2 alpha helices on 1 side and 3 on the other.
  - Along with the DBD, the LBD contributes to the dimerization interface of the receptor and bind coactivator and copressor proteins. It also contains the activation function 2 (AF2) whose action is dependent on the presence of bound ligands.

• C-Terminal Domain:
  - Variable in sequence between various nuclear receptors.
  
  
• Mechanism of Nuclear Receptor Action:- In a class I nuclear receptor the hormone binds to a NR/HSP (heat shock proteins) complex which dissociates the HSP and dimerizes the NR with the hormone forming another complex. This then moves through the nuclear pore and binds to a specific sequence of nuclear DNA known as a hormone response element (HRE) to promote transcription. mRNA is then formed and moved out of the nucleus to be transcripted into a protein.
  - In a class II nuclear receptor the hormone binds to the receptor which is also bound to a corepressor protein. Ligand binding causes a dissociation of the corepressor and recruitment of a coactivator protein which in turn recruits additional proteins such as RNA polymerase that is responsible for the transcription of downstream DNA into RNA and eventually a protein.
  
  
• Nuclear Receptor Actions:- Regulate programs involved in a broad spectrum of physiological phenomena. They are responsible for the effect of approx 10% of all prescription drugs.
  - Many illnesses associated with malfunctioning of the nuclear receptor system, including inflammation, cancer and reproductive disorders.
  

• Mutations of Nuclear Receptors in Humans:
  - The androgen receptor is essential for androgen action. Mutations of the AR lead to the androgen insensitivity syndrome. Affected individuals are XY but can be externally female or intersex.
  - Androgens are essential for normal primary male sexual development before birth and for normal sexual development around puberty.
  

• Nuclear Receptor Superfamily:
  - Nuclear receptors are ligand activated transcription factors. Examples of the wide range of ligands that bind to these receptors: Steroids, retinoids, thyroid hormone, fatty acids and prostaglandins, unknown.
  
  

  Functional Domains - N terminal or A/B domain with an activation function (AF1) region important in modulating the transcription ability of a receptor; a DNA binding domain; Nuclear localisation domain; and Ligand binding domain with an AF region (AF2).

  Domain Structure - Highly conserved DNA binding region; Moderately conserved ligand binding domain; Highly variable N terminal region containing one or more activation domains, which is important for enhancing, regulating and suppressing transcription.

  - Nuclear receptors can be divided into two broad categories depending on their mechanism of action, and the distribution of receptor within the cell in the absence of a ligand.

• Category 1:
  - The hormone binds to a NR/HSP complex to dissociate the HSP (activating the receptor) and form a NR/hormone complex or a NR homodimer. This then enters the nucleus and binds to a hormone response element of the nuclear DNA of a target gene, then with the help of a coactivator, which opens up the DNA, and RNA polymerase the DNA can be transcribed into mRNa which leaves the nucleus and is translated into a protein.
  - E.g. the treatment of prostate cancer cells with the synthetic androgen R1881 promote the translocation of androgen receptors to the nucleus, and their ability to bind to DNA.
  
  
• Category 2:- The hormone enters the nucleus and binds as a heterodimer, usually with RXR, to its receptor on the nuclear DNA. In the absence of the hormone the nuclear receptor is associated with a corepressor, but when the hormone binds this dissociates from the receptor to allow a coactivator protein to bind to it. Other proteins including RNA polymerase will then bind and transcribe the DNA into mRNA which leaves the nucleus of the cell to be translated into a protein.

• Binding of the Nuclear Receptor to its Ligand:
  - The ligand binding domain is found towards the carboxyl terminus and consists almost entirely of alpha helices (3 layers). The ligand-binding pocket is found at the centre of the molecule and is hydrophobic.
  - When the ligand binds to the receptor, the conformation changes to surround the ligand within the ligand-binding pocket of the LBD.
  
  
• Binding of Nuclear Receptors to DNA:- The DNA binding domain contains 2 Cys4 zinc fingers and contains 9 conserved cysteines. The residues vary between nuclear receptors to confer specificity, and the DNA binding domain also contains a region that aids receptor dimerisation.
  - The zinc fingers bind and recognise specific DNA sequences called hormone response elements (HRE's), which are 2 short sequences of DNA separates by a variable number of nucleotides.
  - These exist as either inverted repeats (a sequence of nucleotides that is the reversed complement of another sequence downstream, e.g. GACGGCnnnGCCGTC);
  or direct repeats (two repeats of a specific nucleotide sequence, e.g. GGTCAAnnnnnGGTCAA).
  - Examples: The response elements for the glucocorticoid receptor and oestrogen receptor consist of inverted; for vitamin D, thyroid hormone receptor and retinoic acid consist of direct.
  

• Involvement of Coactivators in Initiating Transcription:
  - Coactivators, such as acetylate histones or methylate nucleosomes, bind to the AF2 portion of the ligand binding domain in ligand-activated nuclear receptors, which cause chromatin reorganization and the facilitation of transcription.
  - Acetylate's have two different families, p300 or p160, which catalyse the transfer of acetyl groups; Methylate's have two families of CARM1 (co-activator associated arginine methyltransferase 1), and PRMT1 (protein arginine methyltransferase 1).
  
  

   The domain structure of the p160 family of coactivators:
  - Has a basic helix-loop-helix structure with a PAS domain acting as a signal sensor. It also has a nuclear hormone receptor-interaction domain and a p300 and CBP interaction domain.
  - The p160 coactivator family include SRC1 (steroid receptor coactivator 1), GRIP1 (glucocorticoid receptor interacting protein 1), NcoA1 (nuclear hormone receptor coactivator).

  1) Transcription factor binds to DNA; 2) Coactivator binds to the transcription factor; 3) Acetylated lysine residues; 4) Remodeling engine binds to DNA; 5) Exposed site where RNA polymerase II can bind.
  

• Suppression of Transcription by Corepressors:
  - Corepressors bind to nuclear receptors and recruit histone deacetylases (HDAC) and/or interact with the basal transcription machinery.
  - Histone deacetylation results in a more compact chromatin structure with lower accessibility to transcriptional activators.
  - They block the site where coactivators bind to (AF2) and when the ligand binds it changes the conformational shape to release the corepressor.
  
  
• Classes of Nuclear Receptors:Class I - Form homodimers; HREs consist of inverted repeats; MOA and distribution as described for category 1.
  Class II - Form heterodimers (with retinoid X receptor); HREs consist of direct repeats; MOA and distribution as described for category 2.
  Class III (Orphan) - Form homodimers; HREs consist of direct repeats; MOA and distribution as described for category 2.
  Class IV - Can bind as monomers, homodimers or heterodimers; Bind to single half-site HREs; MOA and distribution can be category 1 or 2.
  

• Steroid Receptors:
  - Steroid receptors bind as symmetrical dimers. The AF1 and AF2 regions can act independently, where the AF2 region in the ligand binding domain forms an interacting surface for coactivators; whereas the AF1 region in the N-terminus interacts with (or can be modulated by) different cofactors.
   
  Oestrogen: - The term oestrogen encompasses 3 hormones, oestrone (post menopausal) which is made in the ovaries and peripheral tissues; Oestradiol (pre menopausal); and Oestriol (pregnancy).
  - Functions of oestrogens include growth and development of female sexual characteristics; Regulation of the menstrual cycle; Prevention of bone resorption and promotion of bone formation; Aids in the regulation of cholesterol levels.
  - AF2 interacts with coactivators p160, CBP and p300. AF1 interacts with other transcription factors and its activity can be modifiedby other molecules (e.g. phosphorylation).
  - About 70% of breast cancers depend on oestrogen for growth. It has agonistic effects in normal and cancerous breast tissues. Treatment for breast cancer is to suppress oestrogen action as it prevents oestrogen from binding to its receptor by means of an antagonist. Therapeutic antagonists in the treatment of breast cancer are the selective oestrogen receptor modulators (SERMs), Tamoxifen and Raloxifene.

Modular Structure of Oestrogen Receptor: - Also known as the tamoxifen complex.
- Tamoxifen binds to the same site in the receptor as oestradiol. Part of the tamoxifen molecule extends out from the ligand binding domain.
- Helix 12 is prevented from binding in its normal position and is forced to bind to the coactivator site.
- Thus, the binding of coactivators to the AF2 region is blocked and gene transcription is inhibited.
- Actions of Tamoxifen are gene and tissue specific. They have antagonistic effects as AF2 activity is blocked and both AF1 & AF2 are needed to drive transcription, e.g. in breast tissue they can stop breast cancer growth. They also have agonistic effects as only AF1 activity is required to drive transcription, e.g. in bone they lower the risk of osteoporosis, and the endometrium which increases the risk of endometrial cancer.

• Retinoid-X-Receptor:
  - All members of Class II form heterodimers with the RXR.
  - It can also form homodimers with Class III.
  - It is only activated by 9-cis-RA and all-trans RA (ATRA).
  - It exists in 3 isoforms: RXRalpha, found in liver, kidney, spleen, placenta and epidermis; RXRbeta found in most tissues; RXRgamma restricted to brain and muscle.
  

• RXR:
  - Consists of a 3 layered alpha helical structure; a highly conserved DNA binding domain; a variable N terminus with an AF1 region; and a ligand-binding domain (binds 9-cis-RA) containing an AF2 region, helix 12 seals the ligand pocket in presence of a ligand; a dimerisation portion; can form homodimers but heterodimerisation is the favoured state; cofactor binding sites, i.e. corepressor and coactivator sites.
  
  

  Model for RAR-RXR Heterodimer Function:
  - If the corepressor is bound to the RAR part of the heterodimer then the coactivator can bind to the RXR portion to activate it in the presence of an RXR agonist.
  - It could also bind to the RAR portion and replace the corepressor in the presence of an RAR agonist, then when an RXR agonist is added afterwards to also bind to the RXR portion.
  - With certain receptors, i.e. PPARs, FXR and LXR, the heterodimer can be activated from the RXR side i.e. permissive.

• Thyroid Hormones and Their Receptors:- Regulation of thyroid hormone production is controlled through the hypothalamus-pituitary-thyroid axis. Thyroxine (T4) is the main form of hormone produced by the thyroid gland, whereas Triiodothyronine (T3) is produced by deiodiation in peripheral tissues.

• Thyroid Hormones:
  - Growth and developmental actions include regulation of the rate of post natal growth, regulation of long bone growth and cell differentiation.
  - Metabolic actions include regulation of the basal metabolic rate, regulation of cholesterol and lipid metabolism, and nitrogen metabolism.
  - There are numerous forms of the thyroid hormone receptor (TR) that exist, i.e. TRalpha (of which there are two main isoforms with two truncaed versions); and TRbeta (of which there are 3 isoforms and one truncated version). The expression of TR isoforms is tissue dependent and developmentally regulated.
  
  

  Mechanism of Thyroid Hormone Receptor Action:
  - T3 and T4 are brought into the cell nucleus via a transporter and they bind to the receptor complex. There are also extracellular signals which activate kinases to phosphorylate the coactivator, corepressor and the receptor complex.
  - The activity of nuclear receptors can be altered by post-transcriptional modifications which result in enhancement of ligand-induced activity and ligand-independent activation of the receptor. Modifications include phosphorylation, acetylation and ubiquitinylation.
  

• Phosphorylation:
  - Can occur by tyrosine or serine/threonine kinases, e.g. MAPK and AKT.
  - This stabilises or enhances the activation of the receptor in the presence of a ligand. The AF1 region can be activated independently.
  - The mechanism by which receptors can modulate eachothers activity is known as cross talk.
  
  
• Acetylation:- Desensitises the receptor to the ligand and depends upon which part of the receptor is acetylated.
  - Aids binding to other proteins and enhances/reduces activity. It diminishes transcriptional responses to coactivators and aids nuclear localisation.
  
  
• Ubiquitinylation:- Poly-ubiquitinylation targets proteins for degradation. The rapid degradation of transcription factors allows sustained transcription.
  - Mono or bi-ubiquitinylation affects protein-protein interactions to stabilise the receptor molecule.
  

• Modulation of Orphan Receptor Activity:
  - Orphan receptors have no known ligand and may be activated by an as yet unknown ligand, in a ligand-independent manner by means of post-translational modifications. Such modifications include phosphorylation, and the mechanism by which cell surface receptors can influence nuclear receptors via signalling molecules such as MAPK.
  
  
• Summary:- The AF regions in nuclear receptors can co-operate or act independently to drive transcription. Independent AF activity and promoter context of target genes underpins the ability of certain compounds to have both antagonistic and conversely, agonistic effects which are tissue specific.
  - Heterodimers involving RXR are activated by ligand binding to the partner receptor and not from the RXR direction.
  - In addition to ligand binding, the activity of receptors can be modulated by post-translational modifications.