Role of Fibroblast growth factor (FGF) in neural stem cell growth

Fibroblast growth factor (FGF) constitutes a large family of polypeptide growth factors found in a variety of multicellular organisms, including invertebrates.

The function of FGFs is not restricted to cell growth. Instead, FGFs are involved in diverse cellular processes including chemotaxis, cell migration, differentiation, cell survival, and apoptosis. The common feature of FGFs is that they are structurally related and generally signal through receptor tyrosine kinases. FGFs play an important role in embryonic development in invertebrates and vertebrates.

The human FGF protein family consists of 22 members that share a high affinity for heparin as well as a high-sequence homology within a central core domain of 120 amino acids, which interacts with the FGFR.

Structure of a generic FGF protein contains a signal sequence and the conserved core region that contains receptor- and HSPG-binding sites. The main structural features of FGFRs including Ig domains, acidic box, heparin-binding domain, CAM-homology domain (CHD), transmembrane domain, and a split tyrosine kinase domain are illustrated with respective functions: CAM, Cell adhesion molecule; ECM, extracellular matrix; PKC, protein kinase C.

FGFR signal transduction

The signal transduction starts soon after the FGF binds to the Ig domain III. Binding of FGFs causes receptor dimerization and triggers tyrosine kinase activation leading to autophosphorylation of the intracellular domain. Tyrosine autophosphorylation controls the protein tyrosine kinase activity of the receptor but also serves as a mechanism for assembly and recruitment of signaling complexes. These phosphorylated tyrosines function as binding sites for Src homology 2 and phosphotyrosine binding domains of signaling proteins, resulting in their phosphorylation and activation. A subset of Src homology 2-containing proteins such as Src-kinase and phospholipase Cγ (PLCγ) possesses intrinsic catalytic activities, whereas others are adapter proteins. FGF signal transduction, as analyzed during early embryonic development, can proceed via three main pathways and i.e.

Ras/MAPK pathway

The most common pathway employed by FGFs is the MAPK pathway. This involves the lipid-anchored docking protein FRS2 (also called SNT1) that constitutively binds FGFR1 even without receptor activation. Several groups have demonstrated the importance of FRS2 in FGFR1-mediated signal transduction during embryonic development. After activation of the FGFR, tyrosine phosphorylated FRS2 functions as a site for coordinated assembly of a multiprotein complex activating and controlling the Ras-MAPK signaling cascade and the Phosphatidylinositol 3 (PI3)-kinase/Akt pathway. The FRS2 tyrosine phosphorylation sites are recognized and bound by the adapter protein Grb2 and the protein tyrosine phosphatase (PTP) Shp2. Grb2 forms a complex with the guanine nucleotide exchange factor Son of sevenless (SOS) via its SH3 domain. Translocation of this complex to the plasma membrane by binding to phosphorylated FRS2 allows SOS to activate Ras by GTP exchange due to its close proximity to membrane-bound Ras. Once in the active GTP-bound state, Ras interacts with several effector proteins, including Raf leading to the activation of the MAPK signaling cascade. This cascade leads to phosphorylation of target transcription factors, such as c-myc, AP1, and members of the Ets family of transcription factors.

PLCγ/Ca²⁺pathway

The PLCγ/Ca²⁺ pathway involves binding of PLCγ to phosphorylated tyrosine 766 of FGFR1.Upon activation, PLCγ hydrolyzes phosphatidylinositol-4,5-diphosphate to form two second messengers, inositol-1,4,5-triphosphate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas inositol-1, 4, 5-triphosphate stimulates the release of intracellular Ca²⁺. This cascade has been implicated in the FGF2-stimulated neurite outgrowth (48, 49) and in the caudalization of neural tissue by FGFR4 in Xenopus.

PI3 kinase/Akt pathway

The PI3 kinase/Akt pathway can be activated by three mechanisms after FGFR activation, and the phospholipids thereby generated regulate directly or indirectly the activity of target proteins such as Akt/PKB.

Among other processes, the PI3 kinase signaling branch is involved during Xenopus mesoderm induction acting in parallel to the Ras/MAPK pathway. Overexpression of a dominant negative form of the PI3 kinase-regulatory subunit p85 interferes with Xenopus mesoderm formation. Conversely, co expression of activated forms of MAPK and PI3 kinase leads to synergistic mesoderm induction. 

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Hedgehog protein – signaling pathway in vertebrate neural development

Hedgehog family of proteins is signaling proteins which control the cell growth, survival, fate and pattern (almost every aspect) of the vertebrate body plan.  The name was derived from the short and spiked phenotype of the cuticle of the Hh mutant Drosophila larvae.

Except in C.elegans, It is found in both vertebrates and invertebrates. In C.elegans, it has several proteins homologous to the Hh receptor Ptc.  

There are three subgroups in hedgehog protein family:

                    A.  Desert hedgehog (Dhh)

                    B.  Sonic hedgehog (Shh)

                    C.  Indian hedgehog (Ihh)

Both the vertebrates and invertebrates, hedgehog binds to the receptor patched (Ptc) and activates a signaling cascade which ultimately drives the activation of the zinc finger transcription factor (Ci in Drosophila and GLI-3 in mammals) leading to activation of specific target genes.

 A.      Desert hedgehog protein:

The expression of this protein is restricted to gonads only which includes sertoli cells of testis and granulose cells of ovaries. Its deficiency in mice has not shown any significant phenotypic changes, but in males it leads to infertile since there is absence of mature sperm. Dhh is required for organogenesis, Leydig cell formation and sex cord formation. In the absence of Dhh signaling, the size of the precursor Leydig cell population is unaffected but these cells have reduced expression of the transcription factor steroidogenic factor 1 (SF-1). This is the likely cause of impaired expression of steroidogenic enzymes and ultimately testosterone, which is required for virilisation and spermatogenesis.

B.      Sonic hedgehog protein:

Basically it’s a mammalian hedgehog signaling molecule which is expressed during the early vertebrate embryogenesis in midline tissues such as node, notochord and floor plate to control the patterning of the left –right and dorso ventral axes of the embryo. In the zone of polarizing activity (ZPA) of the limb bud, Shh is expressed and critically involved in patterning of the distal elements of the limbs. During organogenesis also, it is expressed in and affects the development of most epithelial tissues. In the absence of the Shh protein will lead to cyclopia, and defects in ventral neural tube, somite and foregut patterning. The protein is very important for hypothalamic and pituitary development.

C.      Indian hedgehog protein:

The expression of this protein is limited to number of tissues which includes primitive endoderm and prehypertrophic chondrocytes in the growth plates of bones. During embryogenesis its absence in the embryo has lead to its death due to poor development of yolk sac vasculature. 

Hedgehog signaling pathway:

A general outline of the hedgehog signaling pathway in the cell is shown. After the translation of hedgehog, it undergoes multiple processing steps that are required for generation and release of the active ligand from the producing cell.

Maturation of the protein (Hedgehog)

1.       The signaling molecule undergoes a self cleavage soon after the signal sequence is removed. The cleavage is catalyzed by its own C terminal domain that occurs between the conserved glycine and Cysteine residues.

2.       The peptide between the two residues is rearranged to form a thioester. Now, oxygen of the –OH group of cholesterol attacks the -CHO of the thioester. This lead to displacing of the sulfur and cleaving of the Hh protein into two parts:

 Ø   A C -terminal processing domain with no known signaling activity.

 Ø  An N- terminal Hh signaling domain (HhN) of approx 19 kDa that contains an ester linked cholesterol at its C terminus. This cholesterol modification will result in association of the HhN with the plasma membrane of the cell.

3.       A palmitic acid moiety which is required for the HhN activity is added to the N terminus of Hh by the acyl transferase skinny hedgehog, this process is called palmitylation.

Without the cholesterol modification, the protein can escape through the dispatched protein easily and can be palmitoylated later during the transport or at the receiving cell. Hence Cholesterol modification role is found to negligible or more study need to be done on it. But palmitylation role is found to be important for generating soluble multimeric Hh protein complexes and also for long range signaling in vertebrate.  

Signaling process: 

Finally the matured Hh protein binds to the membrane protein called Dispatched, which will let the protein to be secreted out of the cell. Loss of the dispatched protein will cause the accumulation of the Hh protein in the producing cells and failure of long range signaling occurs.

The concentration and transport of the Hh protein is controlled by the extracellular proteins such as you/Scube 2 (in the case of zebra fish) as well as proteins on the surface of cells situated between those producing and receiving hedgehog signals (for example, patched (PTC1) hedgehog interacting protein (HIP), exostosin (EXT1), CDO (interference hedgehog (IHOG) in Drosophila) and its relative BOC).

In the absence of the Hh protein, the patched receptor (Ptc) inhibits the Smo (GPCR smoothened) receptor present either on the cell surface or on a vesicle inside the cell. This inhibition will lead to the activation of GLI3R protein which indirectly inhibits the class I Transcription factors such as PAX6, PAX7.  Therefore Hh target genes are not transcribed.

But when the Hh protein is released by the secreting cells, it binds to the Ptc1 receptors leading to activation of the Smo receptor. This inturn will inhibit the GLI3R formation and supports the formation of GLI2/3A from GLI2/3 with the help of protein like wimple, flexo, KIF3A and Rab23.

Finally GLI2/3A will activate the transcription factor (class I and II) and also GLI1 leading to transcription of Hh targeted genes. 

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