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Growth Factors and Cytokines – Introduction

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Video Summary

Growth factors and cytokines are signaling molecules that control cell activities in an autocrine, paracrine or endocrine manner. They exert their biological functions by binding to specific receptors and activating associated downstream signaling pathways which in turn, regulate gene transcription in the nucleus and ultimately stimulate a biological response (1) (Figure 1). A growth factor or cytokine can have various functions on different cell types while distinct growth factor or cytokines can exert similar or overlapping functions on certain cells. Growth Factors and cytokines affect a wide variety of physiological processes such as cell proliferation, differentiation, apoptosis, immunological or hematopoietic response, morphogenesis, angiogenesis, metabolism, wound healing, and maintaining tissue homeostasis in adult organisms. The abnormal production or regulation of growth factors and cytokines can cause various diseases such as cancer (2), liver fibrosis (3) and bronchopulmonary dysplasia (4). Historically, growth factors were thought to be biological moieties that have a positive effect on cell growth and proliferation while cytokines were typically considered to have an immunological or hematopoietic response. However, as different lines of research have converged, it has been found that ‘cytokines’ and ‘growth factors’ can have similar functions and therefore, these terms are now used interchangeably.

Signal Transduction Mechanism

Figure 1 – Communication between neighbor cells using signaling molecules.

Growth Factor Signaling Mechanisms

Paracrine Signaling

Paracrine signaling occurs between neighboring cells where the signals elicit quick responses and last only a short while due to the degradation of the paracrine ligands.

Autocrine Signaling

As the name suggests, in autocrine signaling, a cell signals itself through a moiety that it synthesizes, ultimately leading to a biological response within the same cell. Autocrine signaling can either occur within the cytoplasm of the cell or, by a secreted growth factor/cytokine interacting with receptors on the surface of the same cell.

Endocrine Signaling

In endocrine signaling, growth factor/cytokine moieties are secreted into the blood and carried by blood and tissue fluids on to the target cells whereby subsequent responses are triggered.

Growth Factor Classification

Based on structural and functional characteristics, growth factors can be divided into various families/superfamilies. Major growth factor families include:

The Transforming Growth Factor Beta (TGF-beta) Superfamily

The TGF-beta superfamily includes the TGF-beta proteins, Bone Morphogenetic Proteins (BMPs), Growth Differentiation Factors (GDFs), Glial-derived Neurotrophic Factors (GDNFs), Activins, Inhibins, Nodal, Lefty, and Mülllerian Inhibiting Substance (MIS) (Figure 2). Common to all family members are their dimeric structures and their heterodimeric receptor complexes consisting of type I and type II receptor subunits with serine/threonine kinase domains. Following ligand binding, the type II receptor phosphorylates and activates the type I receptor which then activates a Smad-dependent signaling pathway that regulates gene transcription. The TGF-beta superfamily members are multifunctional regulators of various biological processes such as morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing, inflammation and cancer. (5).

The TGF-beta superfamily

Figure 2 – The TGF-beta superfamily.

Epidermal Growth Factors (EGFs) Family

The EGF family members include EGF, TGF-α, Neuregulins, Amphiregulin, Betacellulin, and others. All family members contain one or more repeats of the conserved amino acid sequence containing 6 cysteine residues that form three intra-molecular disulfide bonds. The EGF family members work via EGFR/ErbB receptor tyrosine kinases. The members of the EGF family are best known for their ability to stimulate cell proliferation, differentiation and survival. Deregulation of the members of this family and their receptors is closely associated with tumorigenesis (6).

Platelet-Derived Growth Factors (PDGFs)

Platelet-derived growth factors (PDGFs) are potent mitogenic and chemotactic proteins. There are currently four known PDGF proteins encoded by four genes (PDGFA, PDGFB, PDGFC and PDGFD). PDGFs are produced by distinct populations of cells that include, activated macrophages, epithelial and endothelial cells, smooth muscle cells and activated platelets (7,8). PDGFs are secreted as disulfide-linked homodimers or heterodimers that includes PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB. There are two known PDGF receptors with intrinsic tyrosine kinase activity; PDGFRα and PDGFRβ; both of which can form heterodimers and homodimers. Ligand binding promotes receptor dimerization, autophosphorylation, and the consequent activation of multiple downstream intracellular signaling cascades. Signaling via PDGFRα is important for the development of the facial skeleton, hair follicles, spermatogenesis oligodendrocytes and astrocytes, as well as for the development of the lung and intestinal villi while signaling via PDGFRβ is crucial for the development of blood vessels, kidneys and white adipocytes (9). Thus, PDGFs are essential for early development, wound healing and angiogenesis. It is noteworthy that the abnormal regulation and production of PDGF isoforms may cause tumor, vascular disease, and fibrotic disease.

Fibroblast Growth Factors (FGFs) Family

In humans, twenty-two members of the FGF family have been identified all of which are heparin-binding proteins. High affinity interactions with cell-surface-associated heparan sulfate proteoglycans are essential for FGF signal transduction as mediated by receptor tyrosine kinases (10). FGFs are pluripotent proteins that are primarily mitogenic but also have regulatory, morphological, and endocrine effects. FGFs are involved in embryonic developmental processes (9), mature tissues/systems angiogenesis (11), keratinocyte organization (12) and wound healing processes (13).

Insulin-like Growth Factors (IGFs)

The Insulin-like Growth Factors (IGFs) are proteins with high sequence similarity to Insulin. The IGF receptor is a disulfide-linked heterotetrameric transmembrane protein with a cytoplasmic tyrosine kinase domain. There are two types of IGF receptor: IGFI-R and IGFII-R. The availability of IGFs can be regulated by IGF Binding Proteins 1-6 (14). The major action of IGFs is on cell growth. Indeed, most of the actions of pituitary growth hormone are mediated by IGFs, primarily IGF-1. Growth hormone stimulates many tissues, particularly the liver, to synthesize and secrete IGF-1, which in turn stimulates both hypertrophy (increase in cell size) and hyperplasia (increase in cell number) in most tissues, including bone. IGFs can also induce neuron survival, protect cartilage cells and activate osteocytes (15).

Vascular Endothelial Growth Factors (VEGFs)

VEGFs are homodimeric, glycoprotein growth factors that are specific to endothelial cells (16). They regulate angiogenesis and vascular permeability, especially during embryogenesis, skeleton growth and reproductive functions. They also play important roles in hematopoiesis. VEGFs signal mainly through tyrosine kinases VEGFR1 and VEGFR2 and stimulate cell survival, proliferation, migration, and/or adhesion. (17). Deregulation of VEGFs has been associated with tumors, intraocular neovascular disorders and other diseases (16). Members of the VEGF gene family include VEGF/VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and Placental Growth Factor (PlGF) (18).

Hepatocyte Growth Factors (HGFs)

HGF is secreted by mesenchymal cells and acts as a multi-functional cytokine on cells that are mainly of epithelial and endothelial origin. It regulates cell growth, cell motility and morphogenesis by activating a tyrosine kinase signaling cascade via HGFR (19). HGF has been shown to have a major role in embryonic organ development, adult organ regeneration, and wound healing. Furthermore, its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis and tumorigenesis (20).

Tumor necrosis factors (TNFs)

Cytokines that were known to be involved in tumor cell apoptosis were initially classified as Tumor Necrosis Factors (or under the TNF family). All TNF family members share a trimeric, conserved C-terminal domain called the ‘TNF homology domain’ or THD. Responsible for receptor binding, THD shares ~20–30% sequence identity amongst family members. Although most ligands are synthesized as membrane-bound proteins, soluble forms can be generated by limited proteolysis (21). The first two members of the family to be identified were TNFα and TNFβ. To date, 19 TNF superfamily ligands have been identified along with 32 TNF superfamily receptors. While many TNF superfamily members promote or inhibit apoptosis, they also regulate critical functions of both the innate and adaptive immune system including natural killer cell activation, T-cell co-stimulation, and B-cell homeostasis and activation (22). In addition, several TNF superfamily members regulate cell type-specific responses such as follicle apoptosis (23) and osteoclast development (24).

Interleukins (ILs)

Interleukins are a large group of immunomodulatory proteins that regulate growth, differentiation and activation of cells in the immune or haematopoietic systems during immune response. ILs are different from (a) chemokines - the predominant function of which is to direct immune cells to the site of inflammation via chemotaxis and from (b) interferons (IFNs) - which mainly mediate cellular response to viral infection. Since ILs can exert pro- and anti-inflammatory effects, they are essential for host defense against pathogens. Based on distinguishing structural features, the known ILs can be divided into four major groups that include; the IL1-like cytokines, the class I helical cytokines (IL4-like, γ-chain and IL6/12-like), the class II helical cytokines (IL10-like and IL28-like) and the IL17-like cytokines (Table 1). In addition, there are a number of ILs that have their unique/uncharacterized structural features. Different interleukins have different functions. For example, IL1 and IL2 are primarily responsible for activating T and B lymphocytes, while IL2 serves to stimulate T- and B-cell growth and maturation. Along with IL6, IL1 is also a mediator of inflammation. IL4 often leads to an increase in antibody secretion by B lymphocytes, while IL12 stimulates the production of the leukocytes cytotoxic T cells and natural killer cells (25). While ILs are secreted primarily by leukocytes, they can also be secreted by other related, non-immune cells such as keratinocytes, chondrocytes, fibroblasts and endothelial, epithelial and smooth muscle cells.

Table 1 – Classification of Interleukins

Group Genes Common features and structural motifs
IL1-like IL1A, IL1B, IL1RN, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1F10, IL18, IL33 Fold rich in Β-strand known as a Β-trefoil
Γ-chain utilizing IL2, IL4, IL7, IL9, IL15, IL21, TSPL Four tightly packed α-helices known as “four- helix bundle” motif; receptor complex contains γc chain subunit
IL4-like IL3, IL4, IL5, IL13, CSF2 Four tightly packed α-helices known as “four- helix bundle” motif; shorter core helices
IL6/12-like IL6, IL11, IL12A, IL23A, IL27A, IL31, CLCF1, CNTF, CTF1, LIF, OSM, CSF3 Four tightly packed α-helices known as “four- helix bundle” motif; longer core helices
IL10-like IL10, IL19, IL20, IL22, IL24, IL26 “Bundle helix” structural motif containing six or seven stacked helices
IL28-like IL28A, IL28B, IL29 “Bundle helix” structural motif containing six or seven stacked helices
IL17-like IL17A, IL17B, IL17C, IL17D, IL25, IL17F Neurotrophin-like cysteine-knot fold
Non-classified IL8, TXLNA, IL16, IL32, IL34, CSF1 Varies

(Adapted from: Human IL classification. Human Genomics website. Accessed September 1, 2015)

Interferons (IFNs)

IFNs are a group of signaling proteins that are made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. IFNs don’t work directly on pathogens but rather stimulate the infected cells and those nearby, to produce proteins that prevent the replication and growth of pathogens. Interferons also have immunoregulatory functions; they inhibit B-cell activation, enhance T-cell activity, and increase the cellular-destruction capability of natural killer cells. More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided into two classes: Type I IFN and Type II IFN. Type I IFNs are also known as viral IFNs and include IFN-α, IFN-β and IFN-ω. Type II IFN is also known as immune IFN (IFN-γ). The viral IFNs are induced by virus infection, whereas type II IFN is induced by mitogenic or antigenic stimuli. Most types of virally infected cells are capable of synthesizing Type I IFN in cell culture. By contrast, IFN-γ is synthesized only by certain cells of the immune system including natural killer cells, CD4 Th1 cells, and CD8 cytotoxic suppressor cells (26).

Colony-Stimulating Factors (CSFs)

Colony-Stimulating Factors (CSFs) are secreted glycoproteins that bind to receptor proteins on the surfaces of hemopoietic stem cells and activate intracellular signaling pathways that can cause the cells to proliferate and differentiate into a specific kind of blood cell (usually white blood cells). In this way, they lessen some of the side effects of cancer treatments and reduce the risk of infection. They are 3 types of CSFs: CSF1, CSF2 and CSF3. CSF1 promotes the growth and maturation of monocytes and macrophage precursors. It also enhances the phagocytic and tumoricidal activity of human macrophage/monocytes and induces them to secrete a variety of different cytokines. CSF2 is known to stimulate the growth and differentiation of hematopoietic precursor cells while CSF3 is involved in hematopoiesis by controlling the production, differentiation, and function of white cell populations of the blood, the granulocytes and the monocytes-macrophages.

abm has a repertoire of 300+ growth factor and cytokines from Human, Mouse and Rat that span all major and minor families.
Applications of Growth Factors and Cytokines

Growth factors are used extensively in a wide variety of cell models (cell culture) to study their functions and the intricacies of the associated signaling pathways. TGFs, EGFs, TNFs and PDGFs have been used on various cancer cell lines to study their effects on cancer cell growth, migration and invasion (9,27,28,29). VEGFs have been reported to promote growth of vascular endothelial cells derived from arteries, veins and lymphatics, and to induce microvascular endothelial cells to invade collagen gels and form capillary-like structures in tri-dimensional cell culture models (17). IL2 has been found to stimulate the growth and differentiation of B-cells, natural killer cells, lymphocyte activated killer cells, monocytes/macrophages and oligodendrocytes in vitro (30). Growth factors are also used to maintain cells in a certain status. For examples, Basic FGF is a critical component for maintaining embryonic stem cells in culture in an undifferentiated state (31).

Based on their crucial roles, many growth factors and cytokines have been utilized for preclinical and clinical applications. Recombinant Human Platelet-Derived Growth Factor (PDGF), for example, has been used to stimulate angiogenesis, migration and mitosis of mesenchymal cells in preclinical studies. Recombinant human PDGF-BB has been demonstrated to be effective and safe for use in regenerating periodontal tissue and has been approved by the FDA to treat the loss of periodontal structures due to chronic inflammation (32). Granulocyte Colony-Stimulating Factor (G-CSF; filgrastim) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF; sargramostim) are used to stimulate the production of white blood cells in patients with cancer. These agents also can be used to mobilize hematopoietic progenitor (stem) cells into the peripheral blood circulation in order to generate cells that can be harvested and used for autologous bone marrow transplant (33).

Additionally, some cytokines also play important roles in immunotherapy. Immunotherapy works by boosting the body’s own immune defenses and is considered one of the standard treatment options for kidney cancer patients with advanced metastatic disease. In 1992, Interleukin 2 (IL2) was approved by the FDA for the treatment of metastatic renal cell carcinoma. In some cases, IL2 therapy produces responses lasting greater than 10 years in a small percentage of treated patients and therefore, represents a significant milestone in the treatment of kidney cancer. Interferon alpha (IFN-α) is also widely used to treat kidney cancer. IFN-α works by interrupting cancer cell biological processes, preventing its growth and making the cell vulnerable to the immune system. IFN-α has also been approved for treating viral infections; hepatitis B, hepatitis C (non-A, non-B hepatitis), and genital warts. The beta form of interferon is mildly effective in treating the relapsing-remitting form of multiple sclerosis while the gamma form is used to treat chronic granulomatous disease, a hereditary condition in which white blood cells fail to kill bacteria.

However, there are also side effects accompanied with growth factor/cytokine application. For example, the administration of G-CSF has been reported to cause osteoporosis, bone marrow necrosis and acute coronary syndrome (33). The intake of IL2 and interferons has also been associated with symptoms of depression (34,35). Therefore, the use of growth factors/cytokines as a treatment option should always be carefully monitored clinically for both short - and long term scenarios.

Quality Control Measures

To ensure high quality for growth factors and cytokines, rigorous quality control standards need to be implemented.

1. Purity

Purity helps define the amount of extraneous matter in the finished product, whether or not harmful to the recipient or deleterious to the product. In case of growth factor and cytokine products, purity is measured from two perspectives:

a. With respect to background/non-specific proteins and;

b. With respect to endotoxins

a. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE): is a technique used to visualize (and quantity) different proteins in a sample and thus estimate protein purity. SDS-PAGE helps separate proteins according to their size. SDS is an anionic detergent that denatures secondary and non-disulfide-linked tertiary structures while applying a negative charge to each protein in proportion to its mass. When an electric field is applied across the gel matrix, depending on their size, each biomolecule moves differently through the gel matrix: small molecules are able to travel faster through the pores in the gel while larger ones move relatively slower. Following electrophoresis, the gel is stained (Coomassie Brilliant Blue R-250, silver stain or fluorescent dye), allowing visualization of the entire subset of protein(s) in the mix.

At abm, we ensure that our growth factor and cytokine products meet the minimum requirement of ≥95% purity using SDS-PAGE analysis.

b. Endotoxins: also known as lipoglycans and Lipopolysaccharides (LPS) are large molecules found in the outer membrane of Gram-negative bacteria. Presence of endotoxins in growth factors/cytokines can have undesirable effects on experimental processes like stem cell differentiation or angiogenesis. Thus, growth factor and cytokine products must be tested for contamination with endotoxins. The Limulus amebocyte lysate test (LAL test) is the gold standard for testing endotoxin level and comes in 3 different assay formats: Gel-Clot method, Turbidimetric Method and the Chromogenic method. The Gel-Clot method is based on the presence or absence of a clot of gel in the sample tube since endotoxins can trigger gelation. Turbidity testing determines the cloudiness of a solution when mixed with the LAL reagent using a spectrophotometer. The detection of bacterial endotoxins with the chromogenic method in the LAL test can be carried out in two ways: the chromogenic endpoint method and the kinetic chromogenic method. The lowest limit of detection (LLD) for a turbidimetric testing is 0.005 EU/mL, as compared to 0.03 EU/mL for the gel clot and 0.005 EU/mL for the chromogenic method. At abm, the endotoxin levels are measured using the LAL chromogenic end-point assay to make sure that the levels are <1.0 EU/μg of a growth factor/cytokine product.

2. Quantification

Accurate and consistent quantification is key to achieving reproducible product size. Some commonly used methods for protein quantification are:

  • Bradford colorimetric dye method
  • UV-Visible Spectroscopy
  • Densitometric comparison to standards on an SDS gel

The Bradford assay, as a colorimetric protein assay, based on an absorbance shift of the Coomassie Brilliant Blue G-250 dye in which the acidic red form of the dye is converted into its blue form (absorbs at 595nm) by binding to protein residues. This staining is not significantly affected by most mild chemicals and detergents. In UV-Visible spectroscopy the amount of light absorbed at any specific wavelength of electromagnetic spectrum is measured. The absorbance of a molecule depends linearly on its concentration while the wavelength of absorption and the strength of absorbance of a molecule depend on the chemical nature. UV protein spectroscopy does not require a standard curve of protein concentrations, and uses a UV absorbance coefficient determined from protein’s unique sequence of amino acid residues. The purity of a protein must be verified though on an SDS gel, and an appropriate blank must be used prior to UV measurement of an unknown protein sample. SDS-PAGE also allows for accurate measurements via densitometry software (Quantity One). Latter compares the unknown amount of a protein to the standard protein concentration curve. Usually known amounts of BSA (purified bovine serum albumin) are used to generate the standard curve.

3. Activity

Growth factors and cytokines are tested for their bio-activity through specific bioassays. Bioassays allow for the determination of the relative strength of a substance by comparing its effect on a test system with that of a standard preparation. A bioassay involves the use of live animal/plant (in vivo) or tissue/cell (in vitro) to determine the biological activity of a substance. The most commonly used systems for growth factors/cytokines are based on the measurement of responses of immortalized cell lines, which although not as functionally relevant as in vivo assays, are easier to use. Since the systems used for bioassay are themselves inherently variable, measurement of the growth factor's activity must be made relative to a reference/standard preparation to permit valid inter-assay and inter-laboratory comparisons (36). Bioassays can be qualitative and/or quantitative. Quantitative bioassays involve estimation of the dose-response curve; how the response changes with the increasing dose. The dose-response relation allows estimation of the dose or concentration of a substance associated with a specific biological response, such as the ED50 (Effective Dose 50). ED50 is the concentration at which the protein exhibits 50% of its maximum activity. By comparing the actual ED50 and the standardized ED50, one can know the activity of growth factors/cytokines.

Useful Terms

Mitogenic stimuli: a stimuli that can promote mitosis.

Antigenic stimuli: a stimuli that induces immune activity.

Tumoricidal: destroying tumor cells.

Heparin versus Heparan Sulfate (HS): Heparin and HS are glycosaminoglycans (GAGs) that are linear polysaccharides comprising repeats of an amino sugar and a uronic acid moiety. Heparin is different from HS as heparin is only produced by mast cells and functions solely as an anticoagulant. HS on the other hand is made by almost all cell types and also has anticoagulant activity. HS further varies from heparin in the degree of modification of the sugar residues. HS occurs as a proteoglycan (HSPG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligands such as FGF and cytokines and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation, abolishing detachment activity by GrB (Granzyme B) and tumour metastasis.

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