Stem Cells - Semantic Scholar

Asia Pacific AsPac J. Mol.Journal Biol. Biotechnol., of Molecular Vol.Biology 11 (1),and 2003 Biotechnology, 2005 Vol. 13(1) : 1-13

Stem Cells, Cytokines And Their Receptors

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Stem Cells, Cytokines And Their Receptors Shahrul Hisham Zainal Ariffin1*, Rohaya Megat Abdul Wahab2, Ismanizan Ismail4, Nor Muhammad Mahadi1 and Zaidah Zainal Ariffin3 Center for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, UKM, 43600 Bangi, Selangor. 2 Department of Orthodontic, Faculty of Dentistry, UKM, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur. 3 Department of Microbiology, Faculty of Applied Sciences, UiTM, 40450 Shah Alam, Selangor. 4 Plant Biotechnology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology, UKM, 43600 Bangi, Selangor. 1

Received 10 March 2005 / Accepted 20 May 2005 Abstract. Stem cells that have totipotent, pluripotent and multipotent abilities can be divided into two main categories: embryonic stem cells and adult stem cells. Embryonic stem cells originate from the inner cell mass of the blastocyst stage during embryonic development whereas adult stem cells are derived from bone marrow. Stem cells have the ability to differentiate into mature cells or transdifferentiate into other tissues partly due to cellular signals triggered by the growth factors such as cytokines. Cytokines produce cellular signals through the cytoplasmic domain of their cognate receptor. Cytokine receptors have been categorised into several superfamilies followed by subfamilies partly due to structural similarities (extracellular and cytoplasmic domains) and combination of subunits. The ability of IL-3 to trigger differentiation not only to haemopoietic stem cells but also to liver stem cells might be a potential factor for transdifferentiation. IL-3, GM-CSF and IL-5 receptors are members of a common â subfamily because they share the same â subunit known as â common (âc). This review focuses on the â subfamily and in particular on their potential signalling pathways, i.e. proliferation, differentiation and survival that triggers at the cytoplasmic domain of both subunits (α subunits and âc) on the stem cells. Keywords.

stem cells, âc subunit, α subunit, cytoplasmic domain, cellular signals

INTRODUCTION Stem cells, previously known as mother cells, can be categorised into two types: adult stem cells and embryonic stem cells. Both these types can be defined as undifferentiated cells, capable of proliferating and differentiating into more than one specialised cell type (Weissman, 2000). However, the potential degree of cellular differentiation varies among stem cell population, a feature which has led to their categorisation. These cell populations can be categorised into three categories as totipotent, pluripotent or multipotent. A totipotent cell is the most primitive cell such as embryonic cells. On the other hand, although the pluripotent cells are more differentiated then the totipotent cells, the total number of these cells in a tissue is extremely small. The haemopoietic stem cells in adult bone marrow for example, comprise of only 0.01-0.05% of the total bone marrow population. These cells are able to sustain adult needs such as an increase production of eosinophils during parasitic infections or production of osteoblasts and osteoclasts in maintaining the dynamics of

bone formation and resorption. The multipotent cells are more differentiated and more committed and give rise to lineagerestricted, tissue specific cell types. The respective cells are more differentiated and committed compared to pluripotent cells, are also known as progenitor or precursor cells and can produce few cell types (Lackie and Dow, 1999) Stem cells demonstrate two methods of proliferative activity, i.e. unlimited symmetrical division and asymmetrical division. Symmetrical self-renewal produces two identical progeny. On the other hand, asymmetrical division gives rise to one daughter cell resembling its mother and another daughter cell able to give rise to multiple types of differentiated cells representing all embryonic germ layer or progenitor of fully differentiated mature cells (Stojkovic et al., 2004). *Author for Correspondence. Mailing address:Center for Gene Analysis and Technology,School of Biosciences and Biotechnology,Faculty of Science and Technology 43600Universiti Kebangsaan Malaysia, Selangor. Tel: 03-89213245; Email:[email protected]

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AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005


Table 1. Differentiated cells and culture condition that are conducive to differentiation of mouse embryonic stem cells.

Cell type

Culture Conditions

Reference

Blood cells

Haemopoietic growth factors: IL-3, IL-6, G-CSF, erythropoietin and thrombopoietin.

Spangrude et al., 1991; Morrison et al., 1995

Osteoblast (bone cell)

Co-cultured with foetal mouse osteoblast, dexamethsone, retinoic acid, ascorbic acid and β-glycerophosphate.

Buttery et al., 2001

Keratinocyte (skin)

β-mercapthoethanol, implanted of embryonic stem cells in mice.

Bagutti et al., 1996

Skeletal Muscle

5-azacytidine, amphotericin B.

Wakitani et al., 1995

Endothelial

Cultured over collagen-IV matrix, absence of LIF, vascular endothelial growth factor.

Yamashita et al., 2000

Chondrocytes

Bone Morphological Protein-2 and Bone Morphological Protein –4.

Kramer et al., 2000

Cardiac muscle

Injected labelled Haemopoietic stem cells into mice injured heart muscle.

Orlic et al., 2001

Astrocyte Neuron

Epidermal growth factor, brain-derived neutrophic factor, β-mercaptoethanol, retinoic acids.

Sanchez-Ramos et al., 2000

Pancreatic islet-like

Serum free media, absence of feeder cell layer, basic fibroblast growth factor, Nicotinamide.

Lumelsky et al., 2001

Adipocytes

Retinoic acid; Insulin, Thyroid hormone and LIF.

Dani et al., 1997

Abbr: IL-3, Interleukin 3; IL-6, Interleukin 6; G-CSF, Granulocyte Stimulating Factors; LIF, Leukaemia Inhibitory Factor

Embryonic Stem Cells. Embryonic stem cells were first identified in 1981 as cell populations capable of differentiating into cell types of all embryonic germ layers (Martin, 1981). The cells were derived from the inner cell mass (ICM) of the blastocyst. Experimentally, the derivation process involves plating the blastocysts on mouse embryonic fibroblasts and expansion of the outgrowth into established cell lines (Smith, 2001). In order for the cells to remain primitive, the new cell line was cultured in the presence of leukaemia inhibitory factor (LIF) or fibroblast feeder layer (Smith et al., 1988; Williams et al., 1988). In the absence of LIF or feeder cell layer, the embryonic stem cells would differentiate into embryoid bodies and finally differentiate into three cell types, each representing the germ layers. On the other hand, with specific media and growth factors, the embryonic stem cells would differentiate into specific cells such as skeletal muscle, endothelium, chondrocytes, cardiac muscle, blood (haematopoietic) cells and adipocytes (Table 1). Adult Stem Cells. The ability of most tissues to regenerate is already well known. The ability is necessary to replace senescent

cells or restore the lost function of cells due to disease or trauma (Sadiq and Gerber, 2004). Examples of regeneration ability are shown by haemopoietic stem cells that give rise to multiple haemopoietic phenotypes (Baum et al., 1992), CNSderived stem cells in producing neuronal cells (Kondo et al., 2000; Gritti et al., 1999; Morshead et al., 1994; Thompson et al., 1990) and intestinal stem cells that develope into multiple cell types in the gut (Bjerknes and Cheng, 1999; Thompson et al., 1990). Current reports show that adult stem cells may not be lineage restricted. These cells are able to cross lineage boundaries and differentiate into different types of cells or tissue. This phenomenon is known as metaplasia and is defined as the conversion of one cell or tissue type into another. This includes transdifferentiation and also conversion between undifferentiated stem cells of different tissues (Slack and Tosh, 2001). Recent studies show that haemopoietic stem cells derived form bone marrow cells are capable of transdifferentiating into liver cells (Theise et al., 2000a; Theise et al., 2000b; Krause et al., 2001; Fujii et al., 2002) whereas neural progenitor cells can differentiate into muscle cells (Galli


et al., 2000). Other examples involve transdifferentiation of liver stem cells into pancreatic (Yang et al., 2002) and cardiac phenotypes (Malouf et al., 2001). According to Slack and Tosh (2001), there are three reasons that make metaplasia an intresting phenomenon. First, understanding the molecular basis of tissue-type switching can increase knowledge of normal development mechanisms. Second, some metaplasias are precursors of cancer are thus important in human pathology. Third, by understanding the tissue type switching we can improve our knowledge and capability to reprogramme stem cells for therapeutic transplantation purposes (Tosh and Slack, 2002). The ability of cells to switch from one cell’s phenotype into another might be controlled by growth factors. For example, IL-3 is known to act on haemopoietic progenitor cells to promote proliferation and terminal differentiation into megakaryocytic, neutrophil/macrophage, late erythroid progenitor cells and eosinophils (Sparrow et al., 1987; Harrant and Lindley, 1998). IL-3 was also found to trigger differentiation signals on other progenitor cells such as liver progenitor cell. Studies by Inderbitzin et al. (2005), showed that adult liver stem cells derived from bone marrow can be induced to differentiate qualitatively and quantitatively, in the presence of IL-3. Therefore the cytosolic events, i.e. pathways triggered by growth factors are important in stem cell development. At the begining of this review we discussed two types of stem cells, i.e. embryonic stem cells and adult stem cells. We also discussed the specific media involved in culturing stem cells for proliferation and maintaining the stem cell’s potential to differentiate. The rest of this review discusses the following: 1) Features of cytokine receptors’ extracelullar domain and subfamilies of the multisubunit cytokine receptor superfamily (CRS) that are involved in stem cells development. 2) More defined important domains at the cytoplasmic domain of multisubunit receptors with focus on GM-CSF, IL-3 and IL-5 cytokine receptors subfamily that are important in transducing two different signals of progenitor stem cells, i.e. proliferation and differentiation signals. 3) Signalling proteins and pathways involved during GMCSF, IL-3 and IL-5 inductions to progenitor stem cells.

Cytokine Receptor Superfamily. Cytokines are polypeptides which transmit signals between cells. They stimulate cell cycle progression, cell proliferation and more importantly differentiation as well as inhibiting apoptosis of haemopoietic stem cells (Arai et al., 1990; Wang et al., 1997a; 1997b). Cytokines in their native form is glycosylated, although this feature in most cases does not have an essential role in biological activity. In order for the cytokines to give all their respective biological function, they bind to cognate receptors, which are expressed on the surface of the target cells and transduce their signals upon activation. Upon binding, these receptors initiate a


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complex series of intercellular reactions, ultimately resulting in multiple cellular response. Basically there are two major classes of cytokine receptors, i.e. the receptor tyrosine kinase (RTK), which has an intrinsic tyrosine kinase activity; and the cytokine receptor superfamily (CRS), which associates noncovalently with cytoplasmic tyrosine kinase (Gonda and D’Andrea, 1997). Cytokine receptors lacking intrinsic tyrosine kinase activity are members of the cytokine receptor superfamily (CRS). The CRS contains an extracellular binding domain that is specific for various cytokines. The binding of specific cytokines to their receptors initiates a transduction signal in the cell through cytoplasmic subunits which are often common among the various receptors. For example, GM-CSF, IL-3 and IL-5 bind to their specific receptors on the α subunit, and initiates the biological response through both the α and common â subunit on the cytoplasmic side of the membrane. Among the CRS similiar association of the various specific receptors with common subunit exist, and thus have allowed the subdivision onto several subfamilies shown in (Table 2). The extracellular domain, i.e. the cytokine-binding domain of most members, is characterised by a 200 amino acid region (at least) composed of two modified fibronectin III modules. This motif consists of four positionally-conserved Cys residues at the amino terminus and the signature of Trp-Ser-X-Trp-Ser (WSXWS; where X represented any amino acid residue) motif located proximal to the cell membrane (O’Neal and Yu-Lee, 1993). On the other hand, the cytoplasmic domains of the receptors are more diverse, the only conserved regions being are two short stretches of amino acid residues located at the membrane-proximal region. These regions are referred to as box 1 and box 2 (Hibi and Hirano, 1998). Additional regions of class I CRS are found in gp130, LIFα and G-CSF, located at the middle of the cytoplasmic region called box 3 (Baumann et al., 1994). Studies on the truncated carboxyl-terminal domain, which lack an intact box homology domain of receptor gp130, LIF and G-CSF, demonstrate that this region is essential in transducing proliferation-suppressing and differentiationsuppressing signals in embryonic stem cells (Ernst et al., 1999). This conserved region also been defined as the minimal domain required to support proliferation as shown for the receptor of G-CSF (Avalos, 1996), EPO and IL-2 (Jiang et al., 1996). The CRS forms oligomeric complexes typically consisting of two or four receptor subunits in order to induce signals transduction. Although CRS members lack intrinsic tyrosine kinase activity, the receptors produce signals via association and activation of various members of the cytoplasmic tyrosine kinase (Wells and de Vos, 1996) such as JAK, Src, Fps/Fes and Tec/Btk families. In cytokine multisubunit receptors, all different subunits are able to transduce cellular signals. The multi subunit receptors such as GM-CSF, IL-3 and IL-5 consist of two different subunits; in this case, α and âc. Both have been shown to be necessary for signal transduction. Features of the CRS, such as having a common subunit associated

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Table 2. Combination of subunits of three major CRS.

IL-6 Receptor (Geijsen et al., 2001) IL-6 IL-11 LIF OSM (Type 1 or 2) CNTF CT-1 IL-2 Receptor (Leonard and Lin, 2000) IL-2 IL-15 IL-4 (type 1) IL-7 IL-9 IL-4 (type 2) IL-13 The common βc receptor (Geijsen et al., 2001) GM-CSF IL-3 IL-5

Combinations of Subunits IL-6Rα and gp130 IL-11Rα and gp130 LIFR and gp130 (LIFR or OSMRβ) and gp130 CNTFRα, LIFR and gp130 CT-1Rα, LIFR and gp130 Combinations of Subunits IL-2Rα (Low affinity); IL-2Rα and γ (Intermediate affinity); IL-2Rα, IL-2Rβ and γ (High affinity) IL-15Rα, IL-2Rβ and γ IL-4Rα and γ IL-7Rα and γ IL-9Rα and γ IL-4Rα and IL-13Rα1 IL-4Rα and IL-13Rα1 Combinations of Subunits GM-CSFRα and βc IL-3Rα and βc IL-5Rα and βc

IL: Interleukin; R: Receptor; LIF: Leukaemia Inhibitory Factor; OSM: Oncostatin M; CNTF: Ciliary Neurotrophic Factor; CT-1: Cardiotrophin1; GM-CSF: Granulocytes Macrophage Stimulating Factor.

with a unique subunit to confer binding specificity with a particular ligand, have allowed division into several subfamilies, each of which is represented in Table 2.

The Common β Receptor The IL-3, GM-CSF and IL-5 receptors are members of the common β receptor subfamily because the respective receptors are multisubunit receptors comprising a unique ligand-specific α subunit and a common β chain called βc. Both the α and βc subunits possess the characteristics of members of CRS, i.e. conserved structural features such as four conserved residues at the N-terminal and the WSXWS motif at the region proximal to the plasma membrane (Miyajima et al., 1993). The βc is considered to play a major role in signal transduction. It has a long cytoplasmic domain of about 420 amino acids. The cytoplasmic domain of α subunit of human IL-3, GM-CSF and IL-5 are 56, 57 and 61 amino acids in length respectively. There are distinct regions that contribute to multiple functions transduced by the receptor such as cell proliferation, survival and differentiation (Nicola et al., 1997). Although the α subunit of the cytoplasmic domain is relatively short, several studies have shown that mutation or deletion of this short cytoplasmic domain abolishes signalling but has no effect on ligandbinding abilities (Polotskaya et al., 1993; Kouro et al., 1996; Barry et al., 1997). The binding of the ligand to the α subunit is a prequisite to its functional interaction involving the commonβ subunit (Bagley et al., 2001). In the mouse but not

in humans or rats, an additional βc chain, known as βIL-3, has been identified. The βIL-3 chain or subunit does not participate in interaction with α subunit of GM-CSF and IL-5 but interacts specifically with IL-3Rα only. This shows that in mouse, there are two types of β subunits, i.e. one type of β subunit that can interact with all three α subunits and another is specific to IL3 α subunit. In terms of tranducing cell signals, both types of β subunit are capable to transduce similar signals. This is shows by in vivo study involved in mice with null mutation of the common βc (βc-/-). The respective bone marrow cells are unresponsive to IL-5 and GM-CSF but not to IL-3 (Nishinakamura et al., 1995; Robb et al., 1995). This is an indication that in mice βc and βIL-3 are redundant in terms of IL-3 signalling. The βc-/- mice referred to above showed a reduction in eosinophils in both peripheral blood and bone marrow similar to that in IL-5-/- (Matthaei et al., 1997) and IL5 α subunit-/- mice (Kopf et al., 1996; Yoshida et al., 1996). This is an indication that signals mediating eosinophil development and supposedly triggered by IL-5, is interrupted but not fully abolished since eosinophils are not completely absent. Mutated mice of βIL-3-/- showed normal development of bone marrow that responded to IL-5, GM-CSF as well as IL-3. Furthermore, double knockout mice of βc, βIL-3-/showed similar defects to the βc mutant mice (Nishinakamura et al., 1996b). This indicates that βc and βIL-3 are functionally redundant.


Activation of IL-3, GM-CSF and IL-5 Receptors. The activation of IL-3, IL-5 and GM-CSF receptor complexes occurs through heterodimerisation of receptor α and βc subunits through non-covalent and covalent processes (Bagley et al., 1997). The exact subunit stoichiometry of the receptor complexes remains unclear but studies suggest that the active receptor complex consist of α2β2 heterodimers (Lia et al., 1996; D’Andrea and Gonda, 2000). In IL-3, IL-5 and GM-CSF receptor complexes, tyrosine phosphorylation can only occur in the presence of a sulphide-linkage between the α and βc subunits (Stomski et al., 1998). Mutation of either Cys86 or Cys91 residues of the βc subunit (located at the extracellular domain) to alanine abolished tyrosine phosphorylation of the βc cytoplasmic domain thus activation of cellular signals was to be abolished. Preventing of the formation of the disulphide cross-linkage between α and βc subunits would prevent any cellular signalling. Further studies on over expression of this βc mutant (βc subunit mutated at the Cys86 and Cys 91 residues) in CTLL-2 cells, i.e. in cells that are free from any endogenous mβc and mβIL-3 triggers proliferative signals similar to transfected CTLL cells that expressed the wild type βc subunit (Le et al., 2000). This indicates that tyrosine phosphorylation events are not absolutely required for IL-3, IL-5 and GM-CSF proliferation signals. However, D’Andrea and Gonda (2000) suggested that mutation of Cys86 and Cys91 causes an incomplete disulphide linkage within the α2β2 complex but does not affect binding and proliferation signals. This suggests that there may be two forms of α2β2 complexes during receptor activation, i.e. immature α2β2, which does not have a disulphide linkage, and mature α2β2 complex which consists of disulphide linkage. D’Andrea and Gonda (2000) proposed that ligand interaction alters the conformation of the human βc subunit and induces complex formation of βc with the α subunit. Those authors proposed that the IL-3 receptor complex in unstimulated conditions (intermediate complexes) can exist in two states. There are two alternative models proposed for the structure of the formation of mature α2β2 complex due to ligand interaction, i.e. preformedβc 2 homodimers and αβc heterodimers. The formation of α2β2 complex form by disulphide bonds produced a more efficient receptor complex. Cytoplasmic Domain of βc subunit. Compared to the α subunit, the βc subunit has a relatively large cytoplasmic domain of around 420 amino acids and plays an essential role in signal transduction (Sakamaki et al., 1992). Like all cytoplasmic domains of Class I CRS, theβc subunit does not possess any known intrinsic enzymatic activities and contains the conserved motif of box 1 and box 2 (Figure 1). Therefore, association with cytoplasmic tyrosine kinase in the cytoplasmic region is essential for triggering cell signals. The Janus kinases family (Jak) plays a major role in cytokine signalling. One of the members of Jak that has been activated in response to IL-3, GM-CSF and IL-5 is Jak2 (de Groot et al., 1998). Box 1 of the βc subunit has been shown to be its


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binding site (Quelle et al., 1994). Jak2 has been shown to be constitutively associated with βc through its own N-terminal domain and kinase activity is only activated after ligand binding (Zhao et al., 1995). It is therefore appears that Jak2 is activated upon ligand binding. Guthridge et al. (1998) suggested that Jak2 is activated by transphosphorylation of two bound Jak molecules for transmision of cell signals from the cell surface to the nucleus. Deletion analysis of the box 1 region showed that it is essential in c-myc mRNA expression associated with cell proliferation (Sato et al., 1993) when activated by Jak2 (Guthridge et al., 1998). Activation of Jak2 triggers phosphorylation of tyrosine residues of the βc subunit. The cytoplasmic domain of human βc contains 8 tyrosine residues, 6 of which are conserved between human and mouse subunits (Figure 1). These six conserved tyrosine residues are located at positions 577, 612, 695, 750, 806 and 866. Mutagenesis studies have shown that phosphorylation of tyrosine at position 577, 612 and 695 are involved in the SHP-2 (tyrosine phosphatase), Raf/ERK cascade, and c-fos transcription with tyrosine at position 577 being essential for Shc phosphorylation. Investigations of human GM-CSF receptor showed that phosphorylation of each tyrosine residue within the cytoplasmic domain of βc subunit can act as docking sites for STATs and facilitate their activation (Sakurai et al., 2000). The STATs family are the cytoplasmic tyrosine kinase proteins that will be activated upon phosphorylation of JAKs. There are a total of seven STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6 (Takeda and Akira, 2000). STATs bind to phosphotyrosine residues of the receptors through the src homology region 2 (SH2) domain and will be phosphorylated by the activated JAK. The STAT protein that is predominantly activated by human βc subunit is STAT5 (STAT5a or STAT5b) that binds to phosphorylated tyrosine through its SH domain (Pawson and Scott, 1997). The activated STAT5 will dimerise and translocate to the nucleus, where it is directly involved in regulating gene transcription (Itoh et al., 1998). Mutagenesis studies show that box 2 located downstream of box 1 will enhance the ligand response triggered by box 1 but is not absolutely required for either Jak2 activation or proliferation. Jak2-/- mice were embryonically lethal due to lack of formation of blood cells and failure to respond to IL-3, IL-5 and GMCSF (Parganas et al., 1998; Aringer et al., 1999). STAT5a-/- mice exhibited a defective response to GM-CSF and thus reduced macrophage proliferation (Feldman et al., 1997). Furthermore, STAT5a and STAT5b deficient (STAT5a/b-/-) mice produced smaller colonies of various types of blood cells in response to IL-3, IL-5 and GM-CSF or G-CSF. This represent the reduction in the number of colonies. This indicates that IL-3, IL-5 and GM-CSF are mediating STAT5 activation and regulating proliferation of haemopoietic stem cells (Coffer et al., 2000). The cytoplasmic domain of human βc subunit also regulates cell survival and differentiation. There are several critical domains that are essential for cell survival and differentiation


881

783

Y

Negative regulation

866

Y

806

Y

750

Y

695

612

Y

Survival 2

Survival 1

577

585

S

763

626

544

541 Box 2

Y

Differentiation 2

Differentiation 1

Proliferation

450 452

YY

Box 1

517

CYTOPLASM

PLASMA MEMBRANE


CYTOSOL

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Figure 1. Schematic diagram of the cytoplasmic domain of the âc. Conserved tyrosine (bold Y) and nonconserved tyrosine (nonbold Y) together with serine (S) are numbered below the protein. The domains that are important for cell proliferation and differentiation as well as for survival are indicated and numbered above each protein. The area represented by showed 75% homology between mouse and human âc subunits and coincided with their receptor survival roles. The numbering system is that of Sakamaki and colleagues (1992).

signals (Figure 1). Studies by Inhorn et al. (1995) showed that the region between amino acids at position 544 to 763 was identified to be important for cell survival, such as in BaF3 cells. The deletion of the region between amino acids 626 to 763 can effectively be overcome by the presence of serum. However, region 544 to 626 is essential for cell survival. Interestingly, amino acids at positions 570 to 626 exhibit unusually high sequence identity (75%) of βc between mouse and human compared to 55% identity for the βc subunit overall (Figure 1). Subsitution of two tyrosine residues at positions 577 and 612 in the 570 to 626 region or even replacement of all the eight tyrosine at the cytoplasmic domain by phenylalanine residues results in receptors that are capable of suppressing apoptosis and maintaining cell survival (Itoh et al., 1998). This indicates that cell survival signals are not strictly dependent on receptor tyrosine phosphorylation and other means of cellular activation usually needed for cell survival. Mutation analysis of serine residues provides an answer to cell survival signals. Mutation analysis of serine residue at position 585 of the cytoplasmic domain βc subunit in response

to GM-CSF was shown to recruit the adaptor protein 14-3-3æ and phosphatidyl inositol 3-OH kinase (Guthridge et al., 2000). This signal pathway is initially independent of the tyrosine phosphorylation pathway, i.e. phosphorylation of tyrosine residues will lead to the JAK-STAT pathway whereas serine phosphorylation will trigger activation PI-3 kinase and Akt (Guthridge et al., 2004). Therefore, the PI-3 kinase pathway (activated through phosphorylation of Ser 585) is essential for cell survival. Cytokines are important in stimulating differentiation of stem cells. The cells must be able to differentiate into various types of cells such as liver, bone or blood cells. The ability of the βc subunit to stimulate differentiation is dependent on its cytoplasmic domain. The most prominent studies involving the differentiation signal of the cytoplasmic domain of βc are those of Smith and colleagues (1997). Smith et al. (1997) observed cellular differentiation signals of M1 and myeloid leukaemic cell lines using morphological analysis, induction of macrophage cellular surface markers as well as macrophage migratory activity and proliferation activity. Their data indicate that induction of cellular differentiation may not be a single


process but rather the results of cumulative signals emanating from different regions on the βc subunit, i.e. 541 and 541 to 656 (Figure 1). Smith et al. (1997) also demonstrated that the C-terminal region of the βc subunit (from 783-897) is important in mediating regulatory signals. This is because truncation of βc at 783 to 626 from C-terminus (Figure 1) produced an approximately 10 fold increase in cell proliferation and survival compared cells with the wild type βc subunit.

Cytoplasmic Domain of α subunit IL-3, GM-CSF and IL-5. The α subunit receptor binds specifically to its ligand with low affinity in the absence of the common βc. The α subunit binds directly to the cognate ligand at the extracellular domain and the key elements of the binding site of each receptor α subunit have been identified through mutagenesis studies. Mutagenesis studies indicate that a truncated cytoplasmic domain of the IL-3 receptor α subunit is capable of binding IL-3 cytokine with high-affinity similar to that of the wild type receptor (Barry et al., 1997). This result suggests that any interaction between the cytoplasmic region of both the common βc and α subunit is not necessary for stabilisation of the high-affinity receptor complex. The lengths of the cytoplasmic domains of human IL-3, GM-CSF and IL-5 are variable, i.e. 56, 57 and 61 amino acids respectively. Further studies by Barry et al. (1997) at the truncated cytoplasmic domain of the IL-3 receptor α subunit shows that although IL-3 cytokine binds with high-affinity similar to wild type receptor, the receptor is unable to stimulate proliferation, neither inducing phosphorylation activity at the cytoplasmic domain of βc subunit nor activation of STAT5 (Barry et al., 1997). This indicates the importance of the α subunit cytoplasmic domain in inducing cellular signals. Studies by Takaki et al. (1993) in mice lacking the entire cytoplasmic domain of IL-5 receptor α subunit were not able to elicite any signalling response (Takaki et al., 1993). Further analysis of a truncated C-terminal mutant of the IL-5 receptor α subunit has revealed that Jak2 is constitutively associated with the cytoplasmic domain of the α subunit receptor of IL5 (Ogata et al., 1998) similar to the cytoplasmic domain of βc (Takaki et al., 1994). Jak2 is a cytoplasmic kinase that upon activation of the βc subunit cytoplasmic domain will transphosphorylate the tyrosine residue of Jak2. Thus, the transphosphorylation of Jak2 will activate its pathways. The activated Jak2 phosphorylates the tyrosine residue of the cytoplasmic domain which will be formed as the docking sites for the recruitment of STAT such as STAT5. The recruitment of STAT5 will trigger the JAK-STAT pathway to produce cellular signals. Several studies have already shown that the cytoplasmic domain of the GM-CSF receptor α subunit is essential for receptor functions (Polotskaya et al., 1993; Ronco et al., 1995; Matsuguchi et al., 1997). They showed that deletion of the cytoplasmic domain of GM-CSF prevents receptor-mediated cell growth and differentiation. Moreover, this domain proves


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to be essential for the phosphorylation of various signalling molecules (Matsuguchi et al., 1997). Matsuguchi et al. (1997) also demonstrated that it is the membrane-proximal prolinerich region of CRS α subunits and β subunit receptors that is most conserved (Figure 2). In addition, the 16 amino acids adjacent to the α subunit were shown to be important for both proliferation and differentiation signals (Matsuguchi et al., 1997). These 16 amino acid residues are also conserved in the IL-5 receptor and deletion of 6 amino acids downstream of the proline-rich region abolished the capability of IL-5 to induce Jak activation. Latest findings show that approximately five amino acids after the proline-rich region is important in controlling differentiation and proliferation signals of haemopoietic stem cells. Studies by Shahrul Hisham et al. (2005) showed that this region which consists of three amino acids, exhibits the cell’s differentiating signal of the GM-CSF receptor and can be changed into a proliferative signal. This region of three amino acids is located at the cytoplasmic domain next to the proline-rich region. Exchange of tripeptides between human GM-CSF and the IL-3 α subunit receptor has shown that these regions contribute to the critical differences between functions of GM-CSF and IL-3 receptor, which are respectively the differentiation and proliferation signals (Evans et al., 2002; Shahrul Hisham et al., 2005). However, the C-terminal region of the GM-CSF α subunit apparently gives slightly different effects. This region showed only partial inhibition of cell proliferation (Matsuguchi et al., 1997). Studies by Matsuguichi et al. (1997) showed clones that expresssed GM-CSF α chain which lacked of 18 amino acid residues at the C-terminal did not lead to an increase in the cell numbers. Instead, the respective cells died more slowly than clones that have been deleted the entire α chain cytoplasmic domain. However, MTS cell proliferation assay showed that the clones which lacked the C terminal domain still produced proliferation signal but weaker than the wild type. On the other hand, the clone that lacked the entire cytoplasmic domain did not produced any proliferation signal in MTS cell proliferation assay. This is an indication that the C-terminal region of the α subunit is not essential for cellular signals and that this region is not conserved between α-receptors of the βc common subfamily. Futhermore, studies by Lilly et al. (2001) showed that two types of human GM-CSF, isoforms α1 and α2, which differ at the receptor’s C-terminal end appeared to be a region that was able to modulate Jak2 activation (Lilly et al., 2001). The results mentioned in this section confirm that the cytoplasmic domain of the α subunit is important for cell signalling.

CONCLUSIONS Essentially, stem cells can be defined as undifferentiated cells, capable of proliferating and differentiating into more than one specialised cell type. Mouse stem cells can be isolated in a

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Granulocytes Macrophage Colony Factor αsubunit.

P

P

V

P

Q

I

Interleukin 5 α subunit.

P

P

I

P

A

P

Growth Hormone.

P

P

V

P

G

P

Interleukin 6 α subunit.

P

P

Y

P

L

H

Granulocytes Colony Stimulating Factor.

P

S

V

P

D

P

Leukaemia Inhibitory Factor α subunit.

P

D

I

P

N

P

GP130.

P

N

V

P

D

P

Interleukin-3 α subunit.

P

R

I

P

H

M

Interleukin 2 â subunit.

C

N

T

P

D

P

Interleukin 4 â subunit.

D

Q

I

P

T

P

â common subunit.

E

K

I

P

N

P

Figure 2. Conserved region of membrane-proximal proline-rich region of various cytokine receptors. Bold letter P shows proline residue that are conserved in the region.

proliferative undifferentiated state in vitro by growing them in a feeder cell layers of mouse fetal cells. An alternative to culturing on feeder layers is the addition of leukemia inhibitory factor (LIF) to the growth medium. LIF is produced by feeder cells and allows mouse stem cells to continue proliferating in vitro without differentiation. In the absence of LIF, the cultured stem cells will differentiate into several types of mature cells. Despite the use of specialized media as shown in Table 1, growth factors such as IL-3 can also be involved in stem cell differentiation. This indicates that growth factors such as cytokines are important in inducing either differentiation or proliferation signals. Different combinations of growth factors will trigger different stem cells’ cellular signals, thus producing different mature cells such as liver, haemopoietic or nerves cells. IL-3 as well as GM-CSF and IL-5 receptors are members of the βc subfamily of CRS because they share the same β subunit. The α subunit is specific to ligand binding at the extracellular domain and receptor activation requires a complex heterodimerisation consisting of two molecules of the α subunits and two molecules of the βc subunits (α2β2). D’Andrea and Gonda (2000) proposed a mechanism of receptor activation. There are two intermediate complexes formed: the homodimer βc2 and the heterodimer αβc. Interaction of ligand produces heterodimerisation of the α2β2 complex and subsequently formation of covalent bonds by disulphide linkages which results in a functionally more efficient

signalling complex. This shows that βc plays a central role in cellular differentiation. However, several studies have also shown that the α subunit is capable of producing cell signals. The cytoplasmic domains of the cytokine receptors are essential in producing cellular signals. Motifs that control cellular signals such as proliferation, differentiation and survival have already been determined for the α subunit and βc. Jak2, the cytoplasmic tyrosine kinase protein is the main signal protein activated by both βc and α subunits. Activation of Jak2 would activate phosphorylation of STAT5a or STAT5b and eventually cellular signals. However, phosphorylation of cytoplasmic tyrosine residues are not enough to produce cell signals. Phosphorylation of other amino acid residues such as serine is shown to be capable of producing different cellular signals, in this case, the cell’s survival signal. This shows that due to stem cell’s complex cellular signals, the respective cells signals might not be restricted by tyrosine phosphorylation of amino acids. This is shown by the addition of serine phosphorylation as one of the event during cells’ differentiation and proliferation. Phosphorylation of amino acids also might not be complex enough for the cells to produce signals. There should be other events beside phosphorylation to induce all types of the cellular signals. However, futher studies on cellular signals are needed to prove this statement.


ACKNOWLEDGEMENTS The authors would like to thank Professor D.A. Luke for reading the manuscript and Intan Zarina Zainol Abidin in organising references of this review.

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