stem cells, cytokines and their receptors

1AsPac J. Mol. Biol. Biotechnol., Vol. 11 (1), 2003 Stem Cells, Cytokines And Their ReceptorsAsia Pacific Journal of Molecular Biology and Biotechnology, 2005Vol. 13(1) : 1-13

*Author for Correspondence.Mailing address:Center for Gene Analysis and Technology,School ofBiosciences and Biotechnology,Faculty of Science and Technology43600Universiti Kebangsaan Malaysia, Selangor.Tel: 03-89213245; Email:[email protected]

Stem Cells, Cytokines And Their Receptors

Shahrul Hisham Zainal Ariffin1*, Rohaya Megat Abdul Wahab2, Ismanizan Ismail4,Nor Muhammad Mahadi1 and Zaidah Zainal Ariffin3

1Center for Gene Analysis and Technology, School of Biosciences and Biotechnology,Faculty of Science and Technology, UKM, 43600 Bangi, Selangor.

2Department of Orthodontic, Faculty of Dentistry, UKM, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur. 3Department of Microbiology, Faculty of Applied Sciences, UiTM, 40450 Shah Alam, Selangor.

4Plant Biotechnology Laboratory, School of Biosciences and Biotechnology,Faculty of Science and Technology, UKM, 43600 Bangi, Selangor.

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 stageduring embryonic development whereas adult stem cells are derived from bone marrow. Stem cells have the ability todifferentiate into mature cells or transdifferentiate into other tissues partly due to cellular signals triggered by the growthfactors 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 onlyto haemopoietic stem cells but also to liver stem cells might be a potential factor for transdifferentiation. IL-3, GM-CSF andIL-5 receptors are members of a common â subfamily because they share the same â subunit known as â common (âc). Thisreview focuses on the â subfamily and in particular on their potential signalling pathways, i.e. proliferation, differentiationand survival that triggers at the cytoplasmic domain of both subunits (α subunits and âc) on the stem cells.

Keywords. stem cells, âc subunit, ααααα subunit, cytoplasmic domain, cellular signals

INTRODUCTION

Stem cells, previously known as mother cells, can be categorisedinto two types: adult stem cells and embryonic stem cells.Both these types can be defined as undifferentiated cells, capableof proliferating and differentiating into more than onespecialised cell type (Weissman, 2000). However, the potentialdegree of cellular differentiation varies among stem cellpopulation, a feature which has led to their categorisation.These cell populations can be categorised into three categoriesas totipotent, pluripotent or multipotent. A totipotent cell isthe most primitive cell such as embryonic cells. On the otherhand, although the pluripotent cells are more differentiatedthen the totipotent cells, the total number of these cells in atissue is extremely small. The haemopoietic stem cells in adultbone marrow for example, comprise of only 0.01-0.05% ofthe total bone marrow population. These cells are able tosustain adult needs such as an increase production ofeosinophils during parasitic infections or production ofosteoblasts and osteoclasts in maintaining the dynamics of

bone formation and resorption. The multipotent cells are moredifferentiated and more committed and give rise to lineage-restricted, tissue specific cell types. The respective cells are moredifferentiated and committed compared to pluripotent cells,are also known as progenitor or precursor cells and can producefew cell types (Lackie and Dow, 1999) Stem cells demonstrate two methods of proliferativeactivity, i.e. unlimited symmetrical division and asymmetricaldivision. Symmetrical self-renewal produces two identicalprogeny. On the other hand, asymmetrical division gives riseto one daughter cell resembling its mother and anotherdaughter cell able to give rise to multiple types of differentiatedcells representing all embryonic germ layer or progenitor offully differentiated mature cells (Stojkovic et al., 2004).

2 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (1), 2005 Stem Cells, Cytokines And Their Receptors

Embryonic Stem Cells. Embryonic stem cells were firstidentified in 1981 as cell populations capable of differentiatinginto cell types of all embryonic germ layers (Martin, 1981). Thecells were derived from the inner cell mass (ICM) of theblastocyst. Experimentally, the derivation process involvesplating the blastocysts on mouse embryonic fibroblasts andexpansion of the outgrowth into established cell lines (Smith,2001). In order for the cells to remain primitive, the new cellline was cultured in the presence of leukaemia inhibitory factor(LIF) or fibroblast feeder layer (Smith et al., 1988; Williams etal., 1988). In the absence of LIF or feeder cell layer, theembryonic stem cells would differentiate into embryoid bodiesand finally differentiate into three cell types, each representingthe germ layers. On the other hand, with specific media andgrowth factors, the embryonic stem cells would differentiateinto specific cells such as skeletal muscle, endothelium,chondrocytes, cardiac muscle, blood (haematopoietic) cells andadipocytes (Table 1).

Adult Stem Cells. The ability of most tissues to regenerateis already well known. The ability is necessary to replace senescent

Cell type Culture Conditions Reference

Blood cells Haemopoietic growth factors: IL-3, Spangrude et al., 1991;IL-6, G-CSF, erythropoietin and Morrison et al., 1995thrombopoietin.

Osteoblast (bone cell) Co-cultured with foetal mouse osteoblast, Buttery et al., 2001dexamethsone, retinoic acid, ascorbic acidand β-glycerophosphate.

Keratinocyte (skin) β-mercapthoethanol, implanted Bagutti et al., 1996of embryonic stem cells in mice.

Skeletal Muscle 5-azacytidine, amphotericin B. Wakitani et al., 1995Endothelial Cultured over collagen-IV matrix, Yamashita et al., 2000

absence of LIF, vascular endothelialgrowth factor.

Chondrocytes Bone Morphological Protein-2 and Kramer et al., 2000Bone Morphological Protein –4.

Cardiac muscle Injected labelled Haemopoietic Orlic et al., 2001stem cells into mice injured heart muscle.

Astrocyte Neuron Epidermal growth factor, Sanchez-Ramos et al., 2000brain-derived neutrophic factor,β-mercaptoethanol, retinoic acids.

Pancreatic islet-like Serum free media, absence of feeder Lumelsky et al., 2001cell layer, basic fibroblastgrowth factor, Nicotinamide.

Adipocytes Retinoic acid; Insulin, Dani et al., 1997Thyroid hormone and LIF.

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

cells or restore the lost function of cells due to disease ortrauma (Sadiq and Gerber, 2004). Examples of regenerationability are shown by haemopoietic stem cells that give rise tomultiple haemopoietic phenotypes (Baum et al., 1992), CNS-derived stem cells in producing neuronal cells (Kondo et al.,2000; Gritti et al., 1999; Morshead et al., 1994; Thompson etal., 1990) and intestinal stem cells that develope into multiplecell types in the gut (Bjerknes and Cheng, 1999; Thompson etal., 1990). Current reports show that adult stem cells may not belineage restricted. These cells are able to cross lineage boundariesand differentiate into different types of cells or tissue. Thisphenomenon is known as metaplasia and is defined as theconversion of one cell or tissue type into another. This includestransdifferentiation and also conversion betweenundifferentiated stem cells of different tissues (Slack and Tosh,2001). Recent studies show that haemopoietic stem cellsderived form bone marrow cells are capable oftransdifferentiating into liver cells (Theise et al., 2000a; Theiseet al., 2000b; Krause et al., 2001; Fujii et al., 2002) whereasneural progenitor cells can differentiate into muscle cells (Galli

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

3AsPac J. Mol. Biol. Biotechnol., Vol. 11 (1), 2003 Stem Cells, Cytokines And Their Receptors

et al., 2000). Other examples involve transdifferentiation ofliver stem cells into pancreatic (Yang et al., 2002) and cardiacphenotypes (Malouf et al., 2001). According to Slack and Tosh(2001), there are three reasons that make metaplasia anintresting phenomenon. First, understanding the molecularbasis of tissue-type switching can increase knowledge ofnormal development mechanisms. Second, some metaplasiasare precursors of cancer are thus important in humanpathology. Third, by understanding the tissue type switchingwe can improve our knowledge and capability to reprogrammestem cells for therapeutic transplantation purposes (Tosh andSlack, 2002). The ability of cells to switch from one cell’s phenotype intoanother might be controlled by growth factors. For example,IL-3 is known to act on haemopoietic progenitor cells topromote proliferation and terminal differentiation intomegakaryocytic, neutrophil/macrophage, late erythroidprogenitor cells and eosinophils (Sparrow et al., 1987; Harrantand Lindley, 1998). IL-3 was also found to triggerdifferentiation signals on other progenitor cells such as liverprogenitor cell. Studies by Inderbitzin et al. (2005), showedthat adult liver stem cells derived from bone marrow can beinduced to differentiate qualitatively and quantitatively, in thepresence of IL-3. Therefore the cytosolic events, i.e. pathwaystriggered by growth factors are important in stem celldevelopment. At the begining of this review we discussedtwo types of stem cells, i.e. embryonic stem cells and adultstem cells. We also discussed the specific media involved inculturing stem cells for proliferation and maintaining the stemcell’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 receptorsuperfamily (CRS) that are involved in stem cellsdevelopment.

2) More defined important domains at the cytoplasmic domainof multisubunit receptors with focus on GM-CSF, IL-3and IL-5 cytokine receptors subfamily that are important intransducing two different signals of progenitor stem cells,i.e. proliferation and differentiation signals.

3) Signalling proteins and pathways involved during GM-CSF, IL-3 and IL-5 inductions to progenitor stem cells.

Cytokine Receptor Superfamily. Cytokines are polypeptideswhich transmit signals between cells. They stimulate cell cycleprogression, cell proliferation and more importantlydifferentiation as well as inhibiting apoptosis of haemopoieticstem cells (Arai et al., 1990; Wang et al., 1997a; 1997b). Cytokinesin their native form is glycosylated, although this feature inmost cases does not have an essential role in biological activity.In order for the cytokines to give all their respective biologicalfunction, they bind to cognate receptors, which are expressedon the surface of the target cells and transduce their signalsupon activation. Upon binding, these receptors initiate a

complex series of intercellular reactions, ultimately resultingin multiple cellular response. Basically there are two majorclasses of cytokine receptors, i.e. the receptor tyrosine kinase(RTK), which has an intrinsic tyrosine kinase activity; and thecytokine receptor superfamily (CRS), which associatesnoncovalently with cytoplasmic tyrosine kinase (Gonda andD’Andrea, 1997). Cytokine receptors lacking intrinsic tyrosine kinase activityare members of the cytokine receptor superfamily (CRS). TheCRS contains an extracellular binding domain that is specificfor various cytokines. The binding of specific cytokines totheir receptors initiates a transduction signal in the cell throughcytoplasmic subunits which are often common among thevarious receptors. For example, GM-CSF, IL-3 and IL-5 bindto their specific receptors on the α subunit, and initiates thebiological response through both the α and common âsubunit on the cytoplasmic side of the membrane. Amongthe CRS similiar association of the various specific receptorswith common subunit exist, and thus have allowed thesubdivision onto several subfamilies shown in (Table 2). Theextracellular domain, i.e. the cytokine-binding domain of mostmembers, is characterised by a 200 amino acid region (at least)composed of two modified fibronectin III modules. Thismotif consists of four positionally-conserved Cys residues atthe amino terminus and the signature of Trp-Ser-X-Trp-Ser(WSXWS; where X represented any amino acid residue) motiflocated proximal to the cell membrane (O’Neal and Yu-Lee,1993). On the other hand, the cytoplasmic domains of thereceptors are more diverse, the only conserved regions beingare two short stretches of amino acid residues located at themembrane-proximal region. These regions are referred to asbox 1 and box 2 (Hibi and Hirano, 1998). Additional regionsof class I CRS are found in gp130, LIFα and G-CSF, locatedat the middle of the cytoplasmic region called box 3 (Baumannet 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 intransducing proliferation-suppressing and differentiation-suppressing signals in embryonic stem cells (Ernst et al., 1999).This conserved region also been defined as the minimaldomain required to support proliferation as shown for thereceptor of G-CSF (Avalos, 1996), EPO and IL-2 (Jiang et al.,1996). The CRS forms oligomeric complexes typically consistingof two or four receptor subunits in order to induce signalstransduction. Although CRS members lack intrinsic tyrosinekinase activity, the receptors produce signals via associationand activation of various members of the cytoplasmic tyrosinekinase (Wells and de Vos, 1996) such as JAK, Src, Fps/Fes andTec/Btk families. In cytokine multisubunit receptors, alldifferent subunits are able to transduce cellular signals. Themulti subunit receptors such as GM-CSF, IL-3 and IL-5 consistof two different subunits; in this case, α and âc. Both havebeen shown to be necessary for signal transduction. Featuresof the CRS, such as having a common subunit associated


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

The Common βββββ Receptor The IL-3, GM-CSF and IL-5receptors are members of the common β receptor subfamilybecause the respective receptors are multisubunit receptorscomprising a unique ligand-specific α subunit and a commonβ chain called βc. Both the α and βc subunits possess thecharacteristics of members of CRS, i.e. conserved structuralfeatures such as four conserved residues at the N-terminal andthe WSXWS motif at the region proximal to the plasmamembrane (Miyajima et al., 1993). The βc is considered to playa major role in signal transduction. It has a long cytoplasmicdomain of about 420 amino acids. The cytoplasmic domainof α subunit of human IL-3, GM-CSF and IL-5 are 56, 57and 61 amino acids in length respectively. There are distinctregions that contribute to multiple functions transduced bythe receptor such as cell proliferation, survival anddifferentiation (Nicola et al., 1997). Although the α subunitof the cytoplasmic domain is relatively short, several studieshave shown that mutation or deletion of this short cytoplasmicdomain abolishes signalling but has no effect on ligand-binding abilities (Polotskaya et al., 1993; Kouro et al., 1996;Barry et al., 1997). The binding of the ligand to the α subunitis a prequisite to its functional interaction involving thecommonβ subunit (Bagley et al., 2001). In the mouse but not

in humans or rats, an additional βc chain, known as βIL-3, hasbeen identified. The βIL-3 chain or subunit does not participatein interaction with α subunit of GM-CSF and IL-5 but interactsspecifically with IL-3Rα only. This shows that in mouse, thereare two types of β subunits, i.e. one type of β subunit that caninteract with all three α subunits and another is specific to IL-3 α subunit. In terms of tranducing cell signals, both types ofβ subunit are capable to transduce similar signals. This is showsby in vivo study involved in mice with null mutation of thecommon βc (βc-/-). The respective bone marrow cells areunresponsive to IL-5 and GM-CSF but not to IL-3(Nishinakamura et al., 1995; Robb et al., 1995). This is anindication that in mice βc and βIL-3 are redundant in terms ofIL-3 signalling. The βc-/- mice referred to above showed areduction in eosinophils in both peripheral blood and bonemarrow similar to that in IL-5-/- (Matthaei et al., 1997) and IL-5 α subunit-/- mice (Kopf et al., 1996; Yoshida et al., 1996).This is an indication that signals mediating eosinophildevelopment and supposedly triggered by IL-5, is interruptedbut not fully abolished since eosinophils are not completelyabsent. Mutated mice of βIL-3

-/- showed normal developmentof bone marrow that responded to IL-5, GM-CSF as well asIL-3. Furthermore, double knockout mice of βc, βIL-3

-/-

showed similar defects to the βc mutant mice (Nishinakamuraet al., 1996b). This indicates that βc and βIL-3 are functionallyredundant.

IL-6 Receptor (Geijsen et al., 2001) Combinations of SubunitsIL-6 IL-6Rα and gp130IL-11 IL-11Rα and gp130LIF LIFR and gp130 OSM (Type 1 or 2) (LIFR or OSMRβ) and gp130CNTF CNTFRα, LIFR and gp130CT-1 CT-1Rα, LIFR and gp130

IL-2 Receptor (Leonard and Lin, 2000) Combinations of SubunitsIL-2 IL-2Rα (Low affinity); IL-2Rα and γ (Intermediate

affinity); IL-2Rα, IL-2Rβ and γ (High affinity)IL-15 IL-15Rα, IL-2Rβ and γIL-4 (type 1) IL-4Rα and γIL-7 IL-7Rα and γIL-9 IL-9Rα and γIL-4 (type 2) IL-4Rα and IL-13Rα1IL-13 IL-4Rα and IL-13Rα1

The common βc receptor (Geijsen et al., 2001) Combinations of SubunitsGM-CSF GM-CSFRα and βcIL-3 IL-3Rα and βcIL-5 IL-5Rα and βc

Table 2. Combination of subunits of three major CRS.

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


Activation of IL-3, GM-CSF and IL-5 Receptors. Theactivation of IL-3, IL-5 and GM-CSF receptor complexes occursthrough heterodimerisation of receptor α and βc subunitsthrough non-covalent and covalent processes (Bagley et al.,1997). The exact subunit stoichiometry of the receptorcomplexes remains unclear but studies suggest that the activereceptor complex consist of α2β2 heterodimers (Lia et al., 1996;D’Andrea and Gonda, 2000). In IL-3, IL-5 and GM-CSFreceptor complexes, tyrosine phosphorylation can only occurin the presence of a sulphide-linkage between the α and βcsubunits (Stomski et al., 1998). Mutation of either Cys86 orCys91 residues of the βc subunit (located at the extracellulardomain) to alanine abolished tyrosine phosphorylation ofthe βc cytoplasmic domain thus activation of cellular signalswas to be abolished. Preventing of the formation of thedisulphide cross-linkage between α and βc subunits wouldprevent any cellular signalling. Further studies on overexpression of this βc mutant (βc subunit mutated at the Cys86and Cys 91 residues) in CTLL-2 cells, i.e. in cells that are freefrom any endogenous mβc and mβIL-3 triggers proliferativesignals similar to transfected CTLL cells that expressed thewild type βc subunit (Le et al., 2000). This indicates that tyrosinephosphorylation events are not absolutely required for IL-3,IL-5 and GM-CSF proliferation signals. However, D’Andrea and Gonda (2000) suggested thatmutation of Cys86 and Cys91 causes an incomplete disulphidelinkage within the α2β2 complex but does not affect bindingand proliferation signals. This suggests that there may be twoforms 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 interactionalters the conformation of the human βc subunit and inducescomplex formation of βc with the α subunit. Those authorsproposed that the IL-3 receptor complex in unstimulatedconditions (intermediate complexes) can exist in two states.There are two alternative models proposed for the structureof the formation of mature α2β2 complex due to ligandinteraction, i.e. preformedβc2 homodimers and αβcheterodimers. The formation of α2β2 complex form bydisulphide bonds produced a more efficient receptor complex.

Cytoplasmic Domain of βββββc subunit. Compared to the αsubunit, the βc subunit has a relatively large cytoplasmic domainof around 420 amino acids and plays an essential role in signaltransduction (Sakamaki et al., 1992). Like all cytoplasmicdomains of Class I CRS, theβc subunit does not possess anyknown intrinsic enzymatic activities and contains the conservedmotif of box 1 and box 2 (Figure 1). Therefore, associationwith cytoplasmic tyrosine kinase in the cytoplasmic region isessential for triggering cell signals. The Janus kinases family (Jak) plays a major role in cytokinesignalling. One of the members of Jak that has been activatedin 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

binding site (Quelle et al., 1994). Jak2 has been shown to beconstitutively associated with βc through its own N-terminaldomain and kinase activity is only activated after ligand binding(Zhao et al., 1995). It is therefore appears that Jak2 is activatedupon ligand binding. Guthridge et al. (1998) suggested thatJak2 is activated by transphosphorylation of two bound Jakmolecules for transmision of cell signals from the cell surfaceto the nucleus. Deletion analysis of the box 1 region showedthat it is essential in c-myc mRNA expression associated withcell proliferation (Sato et al., 1993) when activated by Jak2(Guthridge et al., 1998). Activation of Jak2 triggers phosphorylation of tyrosineresidues of the βc subunit. The cytoplasmic domain of humanβc contains 8 tyrosine residues, 6 of which are conservedbetween human and mouse subunits (Figure 1). These sixconserved tyrosine residues are located at positions 577, 612,695, 750, 806 and 866. Mutagenesis studies have shown thatphosphorylation of tyrosine at position 577, 612 and 695 areinvolved in the SHP-2 (tyrosine phosphatase), Raf/ERKcascade, and c-fos transcription with tyrosine at position 577being essential for Shc phosphorylation. Investigations ofhuman GM-CSF receptor showed that phosphorylation ofeach tyrosine residue within the cytoplasmic domain of βcsubunit can act as docking sites for STATs and facilitate theiractivation (Sakurai et al., 2000). The STATs family are thecytoplasmic tyrosine kinase proteins that will be activated uponphosphorylation of JAKs. There are a total of seven STATproteins: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5band STAT6 (Takeda and Akira, 2000). STATs bind tophosphotyrosine residues of the receptors through the srchomology region 2 (SH2) domain and will be phosphorylatedby the activated JAK. The STAT protein that is predominantly activated byhuman βc subunit is STAT5 (STAT5a or STAT5b) that bindsto phosphorylated tyrosine through its SH domain (Pawsonand Scott, 1997). The activated STAT5 will dimerise andtranslocate to the nucleus, where it is directly involved inregulating gene transcription (Itoh et al., 1998). Mutagenesisstudies show that box 2 located downstream of box 1 willenhance the ligand response triggered by box 1 but is notabsolutely required for either Jak2 activation or proliferation.Jak2-/- mice were embryonically lethal due to lack of formationof blood cells and failure to respond to IL-3, IL-5 and GM-CSF (Parganas et al., 1998; Aringer et al., 1999). STAT5a-/- miceexhibited a defective response to GM-CSF and thus reducedmacrophage proliferation (Feldman et al., 1997). Furthermore,STAT5a and STAT5b deficient (STAT5a/b-/-) mice producedsmaller colonies of various types of blood cells in response toIL-3, IL-5 and GM-CSF or G-CSF. This represent the reductionin the number of colonies. This indicates that IL-3, IL-5 andGM-CSF are mediating STAT5 activation and regulatingproliferation of haemopoietic stem cells (Coffer et al., 2000). The cytoplasmic domain of human βc subunit alsoregulates cell survival and differentiation. There are several criticaldomains that are essential for cell survival and differentiation


signals (Figure 1). Studies by Inhorn et al. (1995) showed thatthe region between amino acids at position 544 to 763 wasidentified to be important for cell survival, such as in BaF3cells. The deletion of the region between amino acids 626 to763 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 exhibitunusually high sequence identity (75%) of βc between mouseand human compared to 55% identity for the βc subunitoverall (Figure 1). Subsitution of two tyrosine residues atpositions 577 and 612 in the 570 to 626 region or evenreplacement of all the eight tyrosine at the cytoplasmic domainby phenylalanine residues results in receptors that are capableof suppressing apoptosis and maintaining cell survival (Itohet al., 1998). This indicates that cell survival signals are notstrictly dependent on receptor tyrosine phosphorylation andother means of cellular activation usually needed for cellsurvival. Mutation analysis of serine residues provides an answer tocell survival signals. Mutation analysis of serine residue atposition 585 of the cytoplasmic domain βc subunit in response

to GM-CSF was shown to recruit the adaptor protein 14-3-3æand phosphatidyl inositol 3-OH kinase (Guthridge et al., 2000).This signal pathway is initially independent of the tyrosinephosphorylation pathway, i.e. phosphorylation of tyrosineresidues will lead to the JAK-STAT pathway whereas serinephosphorylation 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 essentialfor cell survival. Cytokines are important in stimulating differentiation ofstem cells. The cells must be able to differentiate into varioustypes of cells such as liver, bone or blood cells. The ability ofthe βc subunit to stimulate differentiation is dependent on itscytoplasmic domain. The most prominent studies involvingthe differentiation signal of the cytoplasmic domain of βc arethose of Smith and colleagues (1997). Smith et al. (1997)observed cellular differentiation signals of M1 and myeloidleukaemic cell lines using morphological analysis, inductionof macrophage cellular surface markers as well as macrophagemigratory activity and proliferation activity. Their data indicatethat induction of cellular differentiation may not be a single

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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 anddifferentiation 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 ofSakamaki and colleagues (1992).


process but rather the results of cumulative signals emanatingfrom different regions on the βc subunit, i.e. 541 and 541 to656 (Figure 1). Smith et al. (1997) also demonstrated that the C-terminalregion of the βc subunit (from 783-897) is important inmediating regulatory signals. This is because truncation of βcat 783 to 626 from C-terminus (Figure 1) produced anapproximately 10 fold increase in cell proliferation and survivalcompared cells with the wild type βc subunit.

Cytoplasmic Domain of ααααα subunit IL-3, GM-CSF andIL-5. The α subunit receptor binds specifically to its ligandwith low affinity in the absence of the common βc. The αsubunit binds directly to the cognate ligand at the extracellulardomain and the key elements of the binding site of eachreceptor α subunit have been identified through mutagenesisstudies. Mutagenesis studies indicate that a truncatedcytoplasmic domain of the IL-3 receptor α subunit is capableof binding IL-3 cytokine with high-affinity similar to that ofthe wild type receptor (Barry et al., 1997). This result suggeststhat any interaction between the cytoplasmic region of boththe common βc and α subunit is not necessary for stabilisationof 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 acidsrespectively. Further studies by Barry et al. (1997) at the truncatedcytoplasmic domain of the IL-3 receptor α subunit showsthat although IL-3 cytokine binds with high-affinity similar towild type receptor, the receptor is unable to stimulateproliferation, neither inducing phosphorylation activity at thecytoplasmic domain of βc subunit nor activation of STAT5(Barry et al., 1997). This indicates the importance of the αsubunit cytoplasmic domain in inducing cellular signals.Studies by Takaki et al. (1993) in mice lacking the entirecytoplasmic domain of IL-5 receptor α subunit were not ableto elicite any signalling response (Takaki et al., 1993). Furtheranalysis of a truncated C-terminal mutant of the IL-5 receptorα subunit has revealed that Jak2 is constitutively associatedwith the cytoplasmic domain of the α subunit receptor of IL-5 (Ogata et al., 1998) similar to the cytoplasmic domain of βc(Takaki et al., 1994). Jak2 is a cytoplasmic kinase that uponactivation of the βc subunit cytoplasmic domain willtransphosphorylate the tyrosine residue of Jak2. Thus, thetransphosphorylation of Jak2 will activate its pathways. Theactivated Jak2 phosphorylates the tyrosine residue of thecytoplasmic domain which will be formed as the docking sitesfor the recruitment of STAT such as STAT5. The recruitmentof STAT5 will trigger the JAK-STAT pathway to producecellular signals. Several studies have already shown that the cytoplasmicdomain of the GM-CSF receptor α subunit is essential forreceptor functions (Polotskaya et al., 1993; Ronco et al., 1995;Matsuguchi et al., 1997). They showed that deletion of thecytoplasmic domain of GM-CSF prevents receptor-mediatedcell growth and differentiation. Moreover, this domain proves

to be essential for the phosphorylation of various signallingmolecules (Matsuguchi et al., 1997). Matsuguchi et al. (1997)also demonstrated that it is the membrane-proximal proline-rich region of CRS α subunits and β subunit receptors that ismost conserved (Figure 2). In addition, the 16 amino acidsadjacent to the α subunit were shown to be important forboth proliferation and differentiation signals (Matsuguchi etal., 1997). These 16 amino acid residues are also conserved inthe IL-5 receptor and deletion of 6 amino acids downstreamof the proline-rich region abolished the capability of IL-5 toinduce Jak activation. Latest findings show that approximatelyfive amino acids after the proline-rich region is important incontrolling differentiation and proliferation signals ofhaemopoietic 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 receptorand can be changed into a proliferative signal. This region ofthree amino acids is located at the cytoplasmic domain next tothe proline-rich region. Exchange of tripeptides betweenhuman GM-CSF and the IL-3 α subunit receptor has shownthat these regions contribute to the critical differences betweenfunctions of GM-CSF and IL-3 receptor, which are respectivelythe differentiation and proliferation signals (Evans et al., 2002;Shahrul Hisham et al., 2005). However, the C-terminal region of the GM-CSF α subunitapparently gives slightly different effects. This region showedonly partial inhibition of cell proliferation (Matsuguchi et al.,1997). Studies by Matsuguichi et al. (1997) showed clones thatexpresssed GM-CSF α chain which lacked of 18 amino acidresidues at the C-terminal did not lead to an increase in the cellnumbers. Instead, the respective cells died more slowly thanclones that have been deleted the entire α chain cytoplasmicdomain. However, MTS cell proliferation assay showed thatthe clones which lacked the C terminal domain still producedproliferation signal but weaker than the wild type. On theother hand, the clone that lacked the entire cytoplasmic domaindid not produced any proliferation signal in MTS cellproliferation assay. This is an indication that the C-terminalregion of the α subunit is not essential for cellular signals andthat this region is not conserved between α-receptors of theβc common subfamily. Futhermore, studies by Lilly et al. (2001)showed that two types of human GM-CSF, isoforms α1 andα2, which differ at the receptor’s C-terminal end appeared tobe a region that was able to modulate Jak2 activation (Lilly etal., 2001). The results mentioned in this section confirm thatthe cytoplasmic domain of the α subunit is important for cellsignalling.

CONCLUSIONS

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


proliferative undifferentiated state in vitro by growing them ina feeder cell layers of mouse fetal cells. An alternative to culturingon feeder layers is the addition of leukemia inhibitory factor(LIF) to the growth medium. LIF is produced by feeder cellsand allows mouse stem cells to continue proliferating in vitrowithout differentiation. In the absence of LIF, the culturedstem 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 celldifferentiation. This indicates that growth factors such ascytokines are important in inducing either differentiation orproliferation signals. Different combinations of growth factorswill trigger different stem cells’ cellular signals, thus producingdifferent mature cells such as liver, haemopoietic or nervescells. IL-3 as well as GM-CSF and IL-5 receptors are members ofthe βc subfamily of CRS because they share the same β subunit.The α subunit is specific to ligand binding at the extracellulardomain and receptor activation requires a complexheterodimerisation consisting of two molecules of the αsubunits and two molecules of the βc subunits (α2β2).D’Andrea and Gonda (2000) proposed a mechanism ofreceptor activation. There are two intermediate complexesformed: the homodimer βc2 and the heterodimer αβc.Interaction of ligand produces heterodimerisation of the α2β2complex and subsequently formation of covalent bonds bydisulphide linkages which results in a functionally more efficient

signalling complex. This shows that βc plays a central role incellular differentiation. However, several studies have alsoshown that the α subunit is capable of producing cell signals.The cytoplasmic domains of the cytokine receptors are essentialin producing cellular signals. Motifs that control cellular signalssuch as proliferation, differentiation and survival have alreadybeen determined for the α subunit and βc. Jak2, the cytoplasmictyrosine kinase protein is the main signal protein activated byboth βc and α subunits. Activation of Jak2 would activatephosphorylation of STAT5a or STAT5b and eventually cellularsignals. However, phosphorylation of cytoplasmic tyrosineresidues are not enough to produce cell signals.Phosphorylation of other amino acid residues such as serine isshown to be capable of producing different cellular signals, inthis case, the cell’s survival signal. This shows that due to stemcell’s complex cellular signals, the respective cells signals mightnot be restricted by tyrosine phosphorylation of amino acids.This is shown by the addition of serine phosphorylation asone of the event during cells’ differentiation and proliferation.Phosphorylation of amino acids also might not be complexenough for the cells to produce signals. There should be otherevents beside phosphorylation to induce all types of the cellularsignals. However, futher studies on cellular signals are neededto prove this statement.

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 prolineresidue that are conserved in the region.


ACKNOWLEDGEMENTS

The authors would like to thank Professor D.A. Luke forreading the manuscript and Intan Zarina Zainol Abidin inorganising references of this review.

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stem cells, cytokines and their receptors

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