Dr. Tímea Berki, Dr. Ferenc Boldizsár, Dr. Mariann Szabó, Dr. Gergő Talabér, Dr. Zoltán Varecza (2011)
University of Pécs
Growth factors (GFs) are small molecular weight soluble mediators controlling proliferation, survival, metabolism and tissue differentiation. They have also important implications in tumor.
Their description and isolation were closely linked to the development of in vitro tissue/cell culturing techniques. Propagation of cells under in vitro conditions began at the turn of the 19th-20th century. Rous made experiments with chicken tumor (sarcoma) cells (RSV). Carrel’s experiments showed that in simple buffered salt solution the cells did not proliferate, he made initial trials with diluted plasma/serum. Temin and Dulbecco worked out the precise requirements for tissue culturing and found reduced serum need of tumor cells which they interpreted as an enhanced capacity of tumor cells to respond to proliferation signals (“growth factors”). They also observed that serum supported cell growth better than plasma, which, as later turned out, was due to PDGF coming from activated platelets. R. Levi-Montalcini and S. Cohen described NGF and EGF, the first growth factors.
PDGF: platelet-derived growth factor; EGF: epithelial growth factor; NGF: neuronal growth factor; FGF: fibroblast growth factor; TGF: transforming growth factor, IGF: insulin-like growth factor (Figure II.2-1 and Table II.2-1).
Figure II.2-1: Growth factor (GF) receptors
Table II.2-1: Receptor classes
Growth factor receptors belong to the receptor tyrosine kinase family (for the details of growth factor receptor signaling see chapter I.2.3.1 Receptor tyrosine kinases).
Ligand binding leads to receptor dimerization, which induces phosphorylation of the kinase domain and its activation (Figure II.2-2 – Figure II.2-5). Different receptors utilize different dimerization/activation strategies: for example PDGF is a dimer, which cross-links two cell surface PDGF receptor monomers; the binding of EGF to its receptor induces a conformational change, which promotes dimerization; FGF is complexed by heparin and cross links two FGF monomers; in case of insulin the receptor is already dimerized on the cell surface, ligand binding causes a conformational change and autophosphorylation (for more details on insulin signaling see next chapter).
Figure II.2-2: Autophosphorylation of RTKs
Figure II.2-3: Overview of EGF signaling
Figure II.2-4: General characteristics of GF signaling
Figure II.2-5: GF receptors as therapeutic targets
Growth factors and receptor tyrosine kinases and their signaling pathways are not only involved in the physiological regulation of cell proliferation and differentiation but also in the development of malignant tumors. They serve as pathogenic or prognostic markers and are also promising targets of tumor therapies. EGFR is expressed in several malignant tumor types e.g. non-small cell lung cancer (NSCLC), head & neck squamous cell carcinoma (SCCHN), colorectal carcinoma, glioblastoma, prostate-, ovarian- and breast cancer. For example, HER2 (human epidermal growth factor receptor-2) positive breast cancer can be successfully treated with a monoclonal antibody, produced against the receptor (Herceptin). The antibody inhibits EGF signal transduction and consequently the proliferation of the tumor. Signals mediated by EGFR are also important in the angiogenesis of the tumors, leading to tumor growth and higher metastasis ratio. PDGFR and VEGFR are also involved in tumor development; their inhibitors prevent tumor proliferation and inhibit angiogenesis.
Insulin is a hormone produced by pancreatic beta cells in response to elevated blood glucose level, which regulates carbohydrate and fat metabolism of the body. Insulin induces glucose uptake of liver, muscle and fat tissue cells from the blood and glycogen storage. In addition to promoting glucose storage, insulin inhibits the production and release of glucose by the liver controlling the activities of a set of metabolic enzymes by phosphorylation and dephosphorylation events and also regulating the expression of genes encoding hepatic enzymes involved in gluconeogenesis. In the absence of insulin or when insulin-response is impaired („insulin resistance”) a serious metabolic disorder, diabetes mellitus develops.
Insulin like growth factor is a 7.6 kDa peptide secreted mainly by the liver stimulated by growth hormon.
Insulin receptor is a trans-membrane protein dimer consisting of 2 alpha and 2 beta chains covalently bound by disulfide bridges.
Ligand-induced tyrosine-phosphorylation of the insulin/IGF receptors leads to the cytoplasmic recruitment of Insulin receptor substrate 1 (IRS-1) through its SH2 domains. IRS-1 transmits signals from the insulin/IGF-1 receptors towards the PI3K/Akt and the ERK/MAPK pathways. IRS-1 is an important mediator of both metabolic and growth promoting pathways: IRS-1-/- mice have only mild diabetes but pronounced growth retardation (50% of the weight of normal mice). IRS-1 overexpressing transgenic mice develop breast cancer.
PI3-kinases control an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Many of these functions relate to the ability of class I PI 3-kinases to activate protein kinase B.
Akt/PKB, a serine/threonine protein kinase, is involved in multiple cellular processes for example glucose metabolism, proliferation, apoptosis, transcription and cell migration. Activated Akt phosphorylates glycogen synthase kinase 3 (GSK3). A major substrate of GSK3 is glycogen synthase, an enzyme catalyzing the final step in glycogen synthesis. Phosphorylation of glycogen synthase by GSK3 inhibits glycogen synthesis; therefore the inactivation of GSK3 by Akt promotes glucose storage as glycogen.
Epinephrine (also known as adrenaline) is a catecholamine hormone produced by the adrenal medulla from phenylalanine or tyrosine. It increases heart rate, constricts blood vessels, dilates air passages and participates in the complex adaptation to danger situations (“fight-or-flight”). Epinephrine comes from epi- and nephros (Greek), whereas the term adrenaline comes from ad- and renes (Latin), both meaning “on the kidney” referring to the anatomic location of the adrenal glands.
Adrenal extracts containing adrenaline as well as other catecholamines were first isolated by the Polish physiologist N. Cybulski in 1895. J. Takamine K. Uenaka isolated adrenaline in 1901. Adrenaline was first synthesized by F. Stolz and H. D. Dakin, independently, in 1904.
Adrenaline receptors (Figure II.2-6) belong to the G-protein coupled receptors (7-TM); subtypes include α1/2, β1/2/3. α1 receptors are Gq coupled and activate PLC, α2 are Gi coupled, while β receptors are Gs coupled inhinibiting or activating adenylyl-cyclase, respectively. For a more detailed description of the G-protein coupled receptor signaling see Chapters I.2.2 and I.4.1.
Figure II.2-6: Adrenergic receptors
Glucagon is a hormone produced by pancreatic α-cells in the Langerhans islets, which elevates blood glucose level, thus, has an opposing effect to insulin.
The glucagon receptor is a 62 kDa protein belonging to the G protein coupled receptor family (class B). The glucagon receptor associates with Gs, activating adenylyl –cyclase causing cAMP elevation and PKA activation. For a more detailed description of the G-protein coupled receptor signaling see Chapters I.2.2 and I.4.1.
Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter (not hormone) which is synthesized from tryptophan. 5-HT can be found in a wide variety of tissues: gastrointestinal tract (source: enterochromaffin cells), platelets and the central nervous system. One of its important effects is the induction of positive feelings; hence its other name "happiness hormone". An important consequence is that the modulation of serotonin at synapses could be used to treat depression and other mood disorders. Moreover, 5-HT also participates in the regulation of appetite, sleep, muscle contraction, and some cognitive functions, including memory and learning.
Serotonin receptors, also known as 5-HT receptors, belong either to the G protein-coupled receptors (GPCRs) or the ligand-gated ion channels in the central or peripheral nervous system where they might exert either excitatory or inhibitory neurotransmission.
5-HT 1/5 receptors are Gi coupled, with inhibitory functions; 5-HT 2 is Gq coupled with excitatory function, 5-HT 4/6/7 receptors are Gs coupled with excitatory functions; 5-HT 3 receptor is a ligand-gated Na + /K + channel. For a more detailed description of the G-protein coupled receptor signaling see Chapters I.2 and I.4.1.
Intracellular/nuclear receptors are also considered as ligand-dependent transcription factors. Their structural organization is highly conserved, but their function is very diverse.
The first observation was made by the Scottish surgeon G.T. Beatson who found that inoperable breast tumors showed regression after ovaryectomy. Other observations included that castration of animals improves meat; ancient Chinese medicine used placental extracts in different diseases. Kendall and Reichstein described cortisone and thyroxine in 1926. Butenandt and Doisy discovered estrogen in the urine of pregnant women. The discovery of androsteron and progesteron (first isolated from the corpus luteum of pigs) followed. In 1961 Jensen described the estrogen receptor, in the 1980s: cloning of estrogen (ER), glucocorticoid (GR) and thyroxine (TR) receptors were done by Chambon, Evans and Vennström.
Table II.2-2: Intracellular receptor families
There are 48 known receptors in human, but 270 (!) in C. elegans; note: several orphan receptors.
Figure II.2-7: Nuclear receptor superfamily
The receptors are made-up from 6 domains (Figure II.2-8). The N-terminal region (A/B domains) of the molecule is variable (50-500 AA); the central (C domain) DNA binding domain (DBD) is highly conserved (70 AA) double zinc finger. The moderately conserved (200-250 AA) ligand-binding domain (LBD; domain E) is situated between the hinge domain (D) and the C-terminal (F) domain of variable length. Activation function (AF)-1/2 sequences are found in the N-/C-terminal domains, with ligand-dependent or –independent regulatory functions, respectively. Many members of the nuclear receptor family form homo- or heterodimers, the DNA and the ligand binding domains are important in these processes.
Figure II.2-8: Functional domains of transcription factors
The inactive (unliganded) Class I receptors (e.g. GR) form a cytoplasmic receptor complex with heat shock proteins (Hsp90, 70, 40), co-chaperone p23 and immunophilins (e.g. FKBP52 which links the complex to dynein). In the absence of ligand there is dynamic assembly-disassembly of this complex. Upon ligand binding the receptor dissociates from the complex and transported to the nuclear pores along microtubules (Figure II.2-9).
Class II receptors (e.g. RXR, TR), on the other hand, localize in the nucleus, already in unliganded state.
Figure II.2-9: Mechanism of steroid receptor action
DNA binding sites of intracellular receptors are called response elements (RE) usually comprise 2x6 base pair sequences. Members of the steroid receptor family form homodimers and bind to palindromic, inverted repeats separated by 3bp spacer (IR3)
(e.g. GR, MR, PR, AR: 5’-AGAACA-3’; ER: 5’-AGGTCA-3’). Non-steroid receptors bind to direct repeats of the sequence 5’-AGGTCA-3’ (DRn, n=number of spacers), and can both form homodimers (e.g. TR, VDR) or heterodimers (e.g. TR, VDR, RAR, LXR, FXR, PXR, CAR, PPAR).
Activated intracellular receptors can act as trans-activators (Figure II.2-10):
(1) The ligand-bound receptor recruits co-activators up-regulating transcription of the target genes through the interaction with the general transcription factors. Importantly, chromatin has to be “opened up” (ATP-dependent chromatin remodeling / histone acetylation) for the transcription initiation.
(2) Ligand binding can also lead to co-repressor dissociation, enabling co-activators to bind to the transcription initiation complex.
In case of trans-repression without ligand transcription proceeds constitutively, and ligand binding inhibits transcription. For more details on transcription factors see Chapter I.4.4.
Figure II.2-10: Genomic steroid actions
Transcriptional activity of intracellular receptors can be up-regulated by phosphorylation of Ser residues in the N-terminal A/B domains by cyclin-dependent kinases, PKC, PKA, ERK, PKB/Akt, JNK/SAPK, p38-MAPK. AF-1 can be phosphorylated by CDK, ERK, JNK, p38-MAPK, PKB, while AF-2 by Src in case of ER. Down-regulation of transcriptional activity can be caused by phosphorylation of the DBD by PKC or PKA.
Several hormone analogues are used for the treatment of a wide variety of diseases. Synthetic glucocorticoid analogues are used as anti-inflammatory, immunosuppressive drugs (e.g. autoimmune diseases, transplantation, some leukemias). Sex steroids are used as substitution therapy (endocrine diseases), birth control and breast cancer. Thyroxin can be used as substitution therapy after thyroidectomy, while Vitamin A/D to treat vitamin deficiency.
The above described intracellular receptor signaling pathway is considered as “classical” or genomic (see Chapter II.2.3), since it acts via the regulation of gene-transcription (Figure II.2-11). Relatively long time (hours) is needed from the translocation of the active hormone receptor into the nucleus and then the transcription and translation, so the net effect appears only slowly.
However, some steroid effects can already be detected within minutes e.g. ion-currents change, membrane changes, phosphorylation changes (Figure II.2-11). Importantly, glucocorticoid analogues are widely used for the treatment of acute conditions: asthma, allergies or shock where high dose steroids exert rapid effects. Accumulating evidence proves that the apoptosis-inducing capacity of glucocorticoid hormone within the thymus might be, at least partially, also independent from genomic effects. The rapid nature of these effects excludes the possibility that the classical, genomic pathway could mediate them. Hence, these steroid responses, appearing within minutes after hormone exposure, are mediated by “non-genomic” or “alternative” signaling pathways (Figure II.2-11). Most of our knowledge about non-genomic steroid effects was drown from research on glucocorticoids and estrogen.
Figure II.2-11: Genomic and non-genomic GC effects
Figure II.2-12: Summary of genomic and non-genomic glucocorticoid effects
(1) Direct membrane effects
Glucocorticoids (GCs), especially at high doses, could change the physico-chemical properties of the plasma membrane due to their lipid soluble nature. Such effects were observed on human red blood cells. In a mammary cancer cell line, high-dose steroid treatment influenced the membrane lipid mobility, and also increased membrane lipid mobility in LPS treated B lymphocytes. Inhibition of Na+ and Ca2+ transport through the plasma membrane and increased H+ uptake into the mitochondria was also described. In canine kidney epithelial cell system dexamethasone (a synthetic glucocorticoid analogue) had a direct effect on tight junction formation. 20 minutes of cortisol treatment caused changes in the excitability of principal basolateral amygdala neurons.
(2) Membrane GR
Membrane bound GR (mGR) was identified in rodent and human lymphoid cell lines and amphibian brain. Moreover, there was a correlation between the mGR expression and the cell cycle-dependent GC-induced apoptosis sensitivity of a human leukaemia cell line, so, the presence of the mGR correlates with GC-resistance of a cell type. mGR was also found on human blood monocytes and B cells; importantly, mGR+ monocyte frequency increased in rheumatoid arthritis, SLE and ankylosing spondylitis patients indicating that the mGR expression might have had pathogenetic consequences. However, intracellular signalling pathways activated by the mGR are still unknown.
(3) Interaction between the GR and other cytoplasmic signaling proteins
As discussed in Chapter II.2.3, the unliganded GR forms a multimolecular complex in the cytoplasm. Recent studies in human T cells showed that, besides the chaperon molecules (heat shock proteins and immunophilins), the GR associates with cytoplasmic signaling proteins, too. For example, the ligand bound glucocorticoid receptor associates or increases its association with many signaling molecules of the T cell receptor-signaling pathway (e.g. Lck, Fyn or ZAP-70). Moreover, this association can induce phosphorylation changes in Lck, Fyn or ZAP-70, for example. This cross-talk between the GR and the TcR signaling pathway could account for the immunosuppressive action of some glucocorticoid analogues.
(4) Mitochondrial GR
Upon ligand binding the glucocorticoid receptor can directly translocate to the mitochondria in both lymphoid and non-lymphoid cells where it can initiate the apoptotic cascade. The ligand-induced mitochondrial GR translocation showed a close correlation with the GC-induced apoptosis sensitivity of several cell types. In case of CD4+CD8+ (DP) thymocytes the GR translocates to the mitochondria rather than to the nucleus upon short-term in vitro GC treatment correlating with their high GC-induced apoptosis sensitivity. In the mitochondria, the GR might act through diverse mechanisms:
a) Acts as mitochondrial transcription factor.
b) Interaction with other mitochondrial transcription factors.
c) Interaction with pro- and anti-apoptotic proteins (e.g. Bcl-2 family proteins).
d) Decreases the mitochondrial membrane potential.
Estrogens have been shown to induce multiple changes in intracellular signaling cascades. Membrane estrogen receptor (mER) was also identified and structural data support that it is a G-protein coupled receptor. Mitochondrial translocation of the ER has also been described.
Progesterone might influence cell membrane permeability and stimulate progesterone membrane component 1 or its complexes. Progesterone receptor localized near the plasma membrane induces phosphorylation and intracellular calcium level changes. Membrane bound progesterone receptor has also been identified.
Androgens can activate the MAPK cascade through non-receptor tyrosine kinase c-Src, and might act through PKA as well. The membrane bound form of testosterone receptor is thought to take part in non-genomic androgen actions.
Non-genomic aldosterone induces phosphorylation and calcium level changes, and influence the Na+-K+-2Cl- -transporter. The membrane aldosterone receptor is also thought to be a G-protein coupled receptor.
Thyroid hormones and Vitamin-D can both induce phosphorylation of signaling molecules and elicit intracellular calcium signal.