Stat5a 5b Crystallization

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Excessive angiogenesis (high level of VEGF)

->- Infertility

Angiogenesis

Psoriasis

Tumor growth Ocular disease

Gynecological disorders

Macular ^degeneration "

Blindness

Diabetic -retinopathy-

Figure 5 Implications of vascular endothelial growth factor receptors (VEGFRs) level of expression in pathologic angiogenesis.

3.10.5.2 Vascular Endothelial Growth Factor Receptors (VEGFRs)

The VEGF family, composed of VEGF-A, VEGF-B,72 VEGF-C,73 VEGF-D,74 and placental growth factor (PlGF),75 is highly implicated in the growth and survival of the vascular endothelium. VEGF-A is a key regulator of blood vessel growth, PlGF is known to mediate arteriogenesis, whereas VEGF-C and VEGF-D are shown to regulate lymphatic angiogenesis. Here we will mainly focus on the biology, signaling pathways, and clinical implications of the VEGF-A member, also called VEGF.

VEGF is a heparin-binding glycoprotein secreted as a homodimer of 45 kDa. The human VEGF gene is organized as eight exons separated by seven introns. Alternative exon splicing results in the generation of different VEGF isoforms composed of 121-206 amino acids that differ in their expression patterns and in their biochemical and biological properties depending on the vascular endothelial growth factor receptor (VEGFR) they are binding to.78

The VEGFR family is composed of three signaling tyrosine kinase (TK) receptors. VEGFR-1/Flt-1 and VEGFR-2/ Flk-1 are expressed at the cell surface of most blood endothelial cells, while VEGFR-3/Flt-4 is largely restricted to lymphatic endothelial cells.79 These VEGFRs consist of seven immunoglobulin G (IgG)-like extracellular domains, a single transmembrane region, and a consensus TK sequence that is interrupted by a kinase insert domain (Figure 6). They are distinguished from each others by an inserted sequence.80 Neuropilins, heparan-sulfated proteoglycans, cadherins, and aVb3 integrin serve as coreceptors for some, but not all, VEGF proteins.81

VEGFR-1 and VEGFR-2, located on vascular endothelium, are the two high-affinity binding sites of VEGF. VEGFR-2 mediates most of the downstream effects of VEGF in angiogenesis, including endothelial cell proliferation, invasion, survival, and microvascular permeability; whereas VEGFR-1 does not mediate an effective mitogenic signal in endothelial cells. This receptor is critical for physiologic and developmental angiogenesis and it also acts predominantly as a ligand-binding molecule, sequestering VEGF from VEGFR-2 signaling.

As for a variety of receptor TKs, the activation of these growth factor receptors is preceded by the formation of receptor dimers and subsequent receptor phosphorylation.82 The recognition site for VEGF is located on the first three N-terminal Ig-like loops, whereas dimerization of the receptor is stabilized due to an additional domain located on

Ig-like loop 1

Ig-like loop 7

VEGF-binding domain

VEGF-binding domain

Juxtamembrane domain

TBBB5SBE

I uuiuuuuj

Juxtamembrane domain

1st kinase domain

Y-951

Y-951

Y-996

Y-996

Kinase insert domain

Y-1054

Y-1059

Y-1054

Y-1059

2nd kinase domain

C-terminal tail

Endothelial cell proliferation, migration, and survival

Figure 6 Schematic representation of the VEGFR-2 tyrosine kinase receptor. The VEGFR-2 tyrosine kinase receptor consists of seven immunoglobulin-like structures in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase domain interrupted by a kinase insert domain.

Ig-like loop 4.83 Therefore, the binding of VEGF to VEGFR-2 causes receptor dimerization, kinase activation, and autophosphorylation of specific tyrosine residues within the dimeric complex. VEGFR-2 consists of Tyr801, Tyr951, and Tyr996 present in the kinase-insert domain, Tyr1054 and Tyr1059 located in the kinase domain, and Tyr1175 and Tyr1214 in the C-terminal tail; all have been identified as autophosphorylation sites.84

3.10.5.3 Signaling Pathways Activated by Vascular Endothelial Factor Receptor-2

Several proteins have been found to associate, via their Src homology-2 (SH2) domain, with these specific autophosphorylated tyrosine residues, so that they are activated to take part in signal transduction cascades84'85 (Figure 7).

In this way, Tyr951 creates a binding site for the VEGFR-associated protein, VRAP, which in turn activates PI3K and phospholipase Cyl(PLC-yl), whereas Tyr1175 creates one for ScK (Shc-related adaptor protein)87 and PLC-g1.88 VRAP and Sck are adaptor proteins that facilitate and regulate the interaction of KDR (kinase insert domain-containing receptor) with cytoplasmic effector proteins important to endothelial cell survival and proliferation. Binding of PLC-g1 activates PKC that stimulates the Ras/Raf/MEK pathway,89 leading to the subsequent induction of the extracellular kinase (Erk) pathway (p42/44 mitogen-activated protein kinase). Erk can then translocate to the nucleus, where it phosphorylates and activates transcription factors, including c-Jun and the ternary complex factor, which in turn induce immediate transcription of the c-fos gene90-92 leading to cell proliferation. Tyr1059 is responsible for ligand-mediated intracellular Ca2 + mobilization, MAPK activation, and endothelial cell proliferation.93

Phosphorylated Tyr1214 presents a binding site for the focal adhesion kinase (FAK) that will then interact with PI3K and paxillin in order to ensure focal adhesion and cell migration.94 Such VEGF-induced cytoskeletal reorganization and cell migration are also established by the interaction of VEGFR-2 with p38MAPK.95

VEGFR-2 also activates PI3K, resulting in an increase of the lipid phosphatidylinositol, (3,4,5)P3, leading to the activation of protein kinase B (Akt/PKB)96 and the small guanosine triphosphate (GTP)-binding protein Rac. These proteins are known to be implicated in the regulation of vascular permeability and cellular migration.97 On the one hand Akt/PKB induces Bcl-2 antagonist of cell death (BAD) and inhibits Bcl-2-associated death promoter homolog and caspase-9, thereby promoting cell survival.98 On the other hand, the Akt pathway activates prostacyclins (PGI2) and the endothelial nitric oxide synthase (eNOS), with the subsequent production of nitric oxide. These two intracellular mediators are predicted to have a vascular protective effect but also to mediate angiogenic and vascular permeability, increasing the effects of VEGF.99 Several other important intracellular signaling elements, such as Src, are also activated by VEGFR-2.100 VEGF is also implicated in the permeabilization of the extracellular membrane of endothelial cells, corresponding to the initial step of angiogenesis. In fact, VEGF induces a variety of enzymes and proteins, such as matrix-degrading metalloproteinases, metalloproteinase interstitial collagenase, and urokinase-type plasminogen (uPA), leading to this process of membrane degradation.101 Moreover, uPA itself has been shown to increase the production of a variety of different angiogenic factors, including VEGF, suggesting that an autocrine regulatory loop may exist.

3.10.5.4 Regulation of Vascular Endothelial Growth Factor Gene Expression

Many cytokines and growth factors such as PDGF TNF-a, epidermal growth factor (EGF), transforming growth factor (TGF), interleukins, and also MAP kinases and certain oncogenes such as ras, V-src, and HER2 are known to upregulate VEGF mRNA expression or to induce VEGF release.92,102-105

Hypoxia has been shown to be one of the major inducers of VEGF expression through both increased transcription and stabilization of VEGF.106,107 In fact, in hypoxic conditions, the two subunits, HIF-1a and HIF-1a/ARNT, of the hypoxic inducible factor-1 (HIF-1) bind to a hypoxia-responsive element (HRE) located in the 5' flanking region of the VEGF promoter gene in order to enhance its transcription and stabilization.108,109 This induction could be inhibited by the inhibitory PAS (Per/ARNT/Sim)110 and has been shown to implicate PI3K, which is a downstream activator of Ras.90

Moreover, a posttranscriptional regulation of the VEGF expression is enhanced by the VHL (von Hippel-Lindau) suppressor gene product.111

3.10.5.5 Physiological versus Pathological Angiogenesis Linked to Vascular Endothelial Growth Factor Expression

VEGF is implicated not only in physiological but also in pathological angiogenesis.112,113 As previously described, it takes part in many intracellular functions, such as endothelial cell permeability, migration, proliferation, and survival. Therefore the development of pharmacological treatments for disorders characterized by inadequate tissue perfusion has been the central point of interest and investigation of many researchers and pharmaceutical firms over the last decades.

VEGF plays an important role in embryonic and early postnatal development,114,115 skeletal growth, endochondral bone formation,98 and in ovarian angiogenesis,116 as well as a partial inhibition of VEGF-achieved impaired organ

VEGF

Vegfr Intracellular Signalling

Figure 7 VEGFR-2 intracellular signaling. VEGF binding to the extracellular domain of VEGFR-2 induces the dimerization and autophosphorylation of specific tyrosine residues present in <i the catalytic domain of the receptor. Several proteins associate by their SH2 domain to these tyrosine residues and are thus activated. Downstream signal transduction molecules then lead O

to several endothelial cell functions such as vasculopermeability, proliferation, migration, and survival. Arrows indicate positive stimulation and bold lines show inhibition. i n

Figure 7 VEGFR-2 intracellular signaling. VEGF binding to the extracellular domain of VEGFR-2 induces the dimerization and autophosphorylation of specific tyrosine residues present in <i the catalytic domain of the receptor. Several proteins associate by their SH2 domain to these tyrosine residues and are thus activated. Downstream signal transduction molecules then lead O

to several endothelial cell functions such as vasculopermeability, proliferation, migration, and survival. Arrows indicate positive stimulation and bold lines show inhibition. i n ho o development and an increased mortality. Disorders of the vascular system are known to be linked to the overexpression but also to the downregulation ofVEGF (Figure 4). We will thereafter give details of the implications ofVEGFin some diseases: however, the following list is not exhaustive.

3.10.5.5.1 Vascular endothelial growth factor in inflammatory disorders

Psoriasis is a chronic inflammatory skin disorder characterized by dermal angiogenesis and overexpression of VEGFand VEGFR,117'118 supposedly stimulated by TGF- a and EGF, since receptors of these factors are overexpressed in psoriatic skin.119 In the case of severe skin lesions, neovastat (AE-941, Aeterna) has shown a promising therapeutic outcome for the treatment of psoriasis.120

3.10.5.5.2 Pathology of the female reproductive tract

High levels of VEGFare also involved in pathologic angiogenesis and are responsible for several gynecological disorders, such as endometriosis, dysfunctional uterine bleeding, endometrial hyperplasia, and polycystic syndrome, which is known to be a leading cause of infertility.121-123 Antiangiogenic compounds are under investigation in order to provide novel therapeutic approaches for such diseases.124'125

3.10.5.5.3 Intraocular neovascular syndromes

Retinal hypoxia and excessive secretion of VEGF can lead to an inappropriate retinal vascularization126 and hemorrhages contributing to visual loss observed in retinopathy of prematurity, diabetic retinopathy, as well as age-related macular degeneration.127 Antiangiogenic therapies are now under investigation in order to prevent such retinal neovascularization. In the case of wet age-related macular degeneration, such treatment consists of lucentis, an antibody fragment designed to bind all forms of VEGF (ranibizumab, rhuFabV2, Genentech) and in the angiostatic steroid, Retaane (anecortave acetate, Alcon Laboratories),128 which are both in phase III of clinical trials,129 whereas Macugen (pegaptanib sodium injection, Eyetech Pharmaceuticals), an aptamer specific for the VEGF165 isoform, has recently received US FDA approval.130 Various others drugs for the treatment of age-related macular degeneration, such as AdPEDF (Genvec),131 squalamine (Genaera),132 and combrestatin A4 prodrug (Oxigene),133 are in early stages of investigation.

3.10.5.5.4 Vascular endothelial growth factor and cancer

Angiogenesis is essential for the growth of most primary tumors and their subsequent metastatic spread. In fact, tumors can absorb sufficient nutriments and oxygen by simple diffusion up to a size of 1-2 mm, at which point their further growth requires connections with the existing blood vessels in order to permit vascular supply. Consequently, VEGF expression is increased and takes part in the angiogenesis process,112 leading to cell survival by preventing endothelial cell apoptosis.134 This action is mediated by the PI3K/Akt pathway,96,98,135 but also by the induction of the expression of antiapoptotic proteins such as Bcl-2 and Bcl-A1, by an increased phosphorylation of FAK, and by the stimulation of prostaglandin I2 and nitric oxide production by endothelial cells.

Moreover, consistent with its role in tumor angiogenesis, the expression of VEGF is upregulated by the common genetic events leading to malignant transformation. These include the activation of oncogenes such as ras, V-src,fos, and HER2,92 but also the loss of tumor suppressor genes, such as p53, known to cause cell cycle arrest, degradation of HIF-1a and to inhibit the production of VEGF.59,136,137

VEGF expression is increased in the majority of cancers, including hematological malignancies,138 colon and rectal cancers,139 lung,140 breast,141 kidney and bladder cancers,142 ovary and uterine cervix carcinomas,143 intracranial tumors,144 and others.

As tumors require new vasculature to grow and survive, the inhibition of angiogenesis145 could be an effective strategy to eradicate cancer. First, monoclonal antibodies can be used to neutralize the ligands VEGF or receptors VEGFR-2 that are key molecular targets. These neutralizing antibodies block the binding ofVEGF to its receptor and cause apoptosis of the endothelial cells, taking part of newly formed immature vessels that are dependent on VEGF to maintain cell adhesion. Bevacizumab (Avastin, rhu-Mab-VEGF Genentech), a specific VEGF monoclonal antibody, is the first antiangiogenic agent to be approved by the FDA for the treatment of colorectal cancer in conjunction with chemotherapy and is still under trial for other cancer types.113,146 In addition, another VEGF antibody strategy using VEGF trap (Regeneron) has shown promising results in the case of solid tumors in advanced stage, as this compound exhibits a higher affinity for VEGF than other monoclonal antibodies tested.147,148

Indirect strategies, such as the use of IMC-1C11 and neovastat (AE941, Aeterna) are also tested to target VEGF and its receptors. Neovastat is in phase III clinical trials for the treatment of renal cell carcinoma and non-small-cell lung cancer. It prevents the binding of VEGF to its receptors but also inhibits metalloproteinases and induces endothelial cell apoptosis.150

Small molecules that block or prevent the activation of VEGFR-2 TK are also used to inhibit VEGF function and the subsequent signaling pathways.151'152 The 'target' of these TK inhibitors consists of the ATP-binding site located within the kinase domain of these receptors. Such inhibitor compounds include PTK787 (Vatalanib, Novartis),153 ZD6474 (AstraZeneca)'154'155 SU11248 (sunitinib malate, Pfizer),156 SU5416 (semaxinib, Sugen),157 VGA1102,158 SU6668 (Sugen),159 AZD2171(AstraZeneca),154 and many others.

Finally, VEGF and VEGFR can also be targeted by antisense therapy. This strategy consists of short sequences of oligonucleotides that are designed to be complementary to a region of mRNA that encodes VEGF160 or VEGFR,161 as well as blocking the subsequent translation of mRNA into protein and thus the proliferation of endothelial cells in tumor angiogenesis. It can also consist of catalytic antisense RNAs, called ribozymes such as RPI4610 (Ribozyme Pharmaceuticals) that cleave RNA substrates in a sequence-specific manner.162,163

The number and variety of novel targeted agents as well as the ongoing clinical evaluation offer a realistic hope for significant advances in cancer treatment. To date, the most successful results of antiangiogenic therapy have been obtained when they are used in combination with certain conventional chemotherapies.164 Moreover, the combined inhibition of VEGF/EGFR has shown encouraging antitumoral activity.140

3.10.5.6 Vascular Endothelial Growth Factor as a Therapeutic Tool

In the previous cited diseases, VEGF was responsible for vascular system disorders leading to acute angiogenesis. But in some others, VEGF may be useful for attempts to increase the collateral vessel formation in order to overcome inadequate tissue perfusion or ischemia, observed in cardiovascular diseases but also in neonatal respiratory distress syndrome165 and in amyotrophic lateral syndrome.166,167 Several trials, such as the administration of VEGF recombinant protein168 or gene transfer using nonviral delivery vector,139,169 are under investigation and aim to avoid the potential risk of subsequent pathological angiogenesis.

VEGF and its receptors play an important role in the development and regulation of angiogenesis by initiating several signal transduction cascades. In this way, they are highly implicated in several pathological disorders, and various therapeutic approaches aiming to inhibit the function of VEGF/VEGFR are currently under investigation. These antiangiogenic strategies consist of neutralizing antibodies, aptamer and antisense therapy directed against VEGF and VEGFR, and also small-molecular-weight compounds preventing VEGFR TK activity. Currently, clinical trials have a promising outcome and generating hope for the treatment of cancer and ocular disease.

3.10.6 Signal Transducer and Activator of Transcription (STAT)

STAT proteins form a family of transcription factors that transduce signals from the extracellular milieu of cells to the nucleus. These proteins were first described in 1993 and are involved in a large number of diverse biologic processes, such as fetal development,173,174 cell growth,175 transformation,176 differentiation,176-178 immune response,179 inflammation,179,180 and apoptosis.181,182 The STAT transcription factor family is composed of seven different members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6.183

3.10.6.1 Chromosomal Localization and Structure of the Signal Transducers and Activators of Transcription

The chromosomal localizations of the human STATs were identified on three different chromosomal clusters, as STAT1 and STAT4 are situated on chromosome 2, STAT2 and STAT6 on chromosome 12 and STAT3, STAT5a and STAT5b on chromosome 17 (Table 1). Independently of the varying chromosomal localizations, the seven STAT proteins share the same overall structure that is organized into distinct functional domains (Figure 8). The highly conserved N-terminal domain, or oligomerization domain, is important for protein-protein as well as dimer-dimer interactions in order to form tetramer and oligomer STAT molecules.184,185 The DNA-binding domain determines the DNA-binding specificity of the different STAT proteins,186 while the SH2 domain is essential for the recruitment of the STAT proteins to the phosphorylated receptors. Furthermore, an interaction between the SH2 domain of one STAT monomer and the phosphorylated tyrosine of a second monomer is required for the formation of the STAT dimers.187 The position of the essential tyrosine residue for the STAT activation and dimerization is located in the transactivation domain and is specific for each family member (Table 1). Finally, the C-terminal transcriptional activation domain (also called transactivation domain) carries a conserved serine residue, except for STAT2 and STAT6. This phophorylation site has been described as regulating the transcriptional activity of the concerned STATs.188,189

Table 1 Signal transducers and activators of transcription

Chromosomal localization Molecular weight (kDa)

STAT1

2q32.2

91

STAT2

12q13.3

113

STAT3

17q21.2

89

STAT4

2q32.2

89

STAT5a

17q21.2

96

STAT5b

17q21.2

94

STAT6

12q13.3

94

Amino acids Phosphorylation sites Dimerization partners

750

Y701

S727

1, 2, 3

851

Y690

/

1

770

Y705

S727

1, 3

748

Y693

S721

4

794

Y694

S726

5a, 5b

787

Y699

S731

5a, 5b

847

Y641

/

6

Figure 8 Structure and functional domains of STAT proteins.

domain

Figure 8 Structure and functional domains of STAT proteins.

Following activation, all STAT proteins can associate as homodimers except for STAT2, which can only form a STAT1-2 heterodimer. Furthermore, STAT1-3 and STAT5a-5b heterodimers may be observed after STAT activation. The central role for this activation and dimerization is played by the tyrosine phosphorylation, as replacement of this residue results in an inactive STAT incapable of nuclear translocation or transactivation.

In addition to these full-length STAT isoforms, shortened STAT proteins lacking regions of the C-terminal transcriptional activation domain were identified in the cases of STAT1, STAT3 and STAT5. These truncated isoforms, termed STATp, present different transcriptional activities compared to the full-length STATa isoforms, and can act as dominant negative regulators.190,191 Two different mechanisms were described for the production of C-terminally truncated STAT isoforms. Alternative mRNA splicing could be observed in the case of STAT1, STAT3, and STAT5,191 while proteolytic cleavage was demonstrated for STAT3194 and STAT5.195

3.10.6.2 Signal Transducer and Activator of Transcription Proteins in Signal Transduction/Activation Mechanisms of Signal Transducer and Activator of Transcription Proteins

The activation of STAT proteins can be induced via the binding of cytokines, growth factors, or hormones to their cell surface receptors (Figure 9). The connection of these specific extracellular signaling proteins induces a receptor dimerization, which activates the Janus family of TKs (Jaks) by autophosphorylation (the Jaks family is composed of four members: Jak1, Jak2, Jak3, and Tyk2). In turn, the activated Jaks phosphorylate a tyrosine residue on the cytoplasmic part of the receptor, tyrosine residue, which then becomes the docking site for the STAT proteins.196 Tyrosine phosphorylation, by the Jaks, in the carboxy-terminal region of the STATs, induces the formation of STAT homo- or heterodimers via the interaction between the phosphotyrosine residue of one STAT and the SH2 domain of the other. Subsequently, the so-formed dimers translocate to the nucleus of the cell, where they bind to specific response elements, induce and regulate target gene transcription.

Alternatively, growth factor receptors possessing intrinsic TK activity, as EGF receptor or platelet-derived growth factor (PDGF) receptor, can also autophosphorylate their receptor cytoplasmic tail,197,198 leading to STAT activation without the implication of the Jaks. Besides, STAT proteins can be activated by nonreceptor TKs of the Src family.199

Growth factor receptor

Nonreceptor tyrosine kinases

Growth factor receptor

Nonreceptor tyrosine kinases

Gcsf Bcl2 Caspase

Gene expression

Figure 9 The STAT activation pathway. The binding of a cytokine or growth factor leads to intrinsic receptor tyrosine kinase activity or to the activation of the Janus family kinases associated with the receptor. Subsequent phosphorylation of the cytoplasmic tail of the receptor leads to the formation of docking sites for two STAT monomers. Tyrosine phosphorylation on the STAT proteins induces the dimerization of the latter, which induces the nuclear translocation of the activated STAT dimer. In addition, STAT proteins can also be activated by nonreceptor tyrosine kinases as Bcr or Abl. Once in the nucleus, the STAT dimers can bind to specific DNA response elements and activate transcription of target genes.

Gene expression

Figure 9 The STAT activation pathway. The binding of a cytokine or growth factor leads to intrinsic receptor tyrosine kinase activity or to the activation of the Janus family kinases associated with the receptor. Subsequent phosphorylation of the cytoplasmic tail of the receptor leads to the formation of docking sites for two STAT monomers. Tyrosine phosphorylation on the STAT proteins induces the dimerization of the latter, which induces the nuclear translocation of the activated STAT dimer. In addition, STAT proteins can also be activated by nonreceptor tyrosine kinases as Bcr or Abl. Once in the nucleus, the STAT dimers can bind to specific DNA response elements and activate transcription of target genes.

These different activation mechanisms control the activity of the STAT proteins and subsequently affect the numerous biologic processes influenced by these pathways. The numerous cytokines activating the Jak/STAT pathways highlight the central role of these proteins (Table 2).

3.10.6.2.1 STAT1 signaling

STAT1 is not only implicated in interferon (IFN-a, IFN-ß, and IFN-g) signaling, but it is also activated by IL6, IL10, IL11, IL21, or EGF (Table 2). STAT1 knockout mice proved to be viable and displayed no developmental defects.

Table 2

STAT-activating cytokines247 249 and STAT knockout mice

STAT

Activating cytokines

Phenotype of knockout mice

STAT1

IFN-a, IFN-ß, IFN-g, IL6, IL10, IL11, IL21, EGF

Defective IFN-dependent immune responses High sensitivity to viral and bacterial infections

STAT2

IFN-a, IFN-ß

Defective type I-dependent immune responses

STAT3

IL2, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL21, EGF, CSF1, G-CSF, PDGF, GH, OSM

Embryonic lethality

STAT4

IL12

Impaired Th1-cell development

STAT5a and/or

IL2, IL3, IL4, IL5, IL7, IL9, IL15, IL21, EPO

Deficient mammary gland development and lactogenesis

STAT5b

GH, PRL, EGF, PDGF, GM-CSF

Loss of sexually dimorphic growth and liver gene expression in males Female infertility

Slowed cell-growth after GM-CSF stimulation

Impaired IL2-induced splenocyte proliferation

Reduced number of NK cells and impaired IL2-induced

T-cell proliferation Fetal anemia

STAT6

IL4, IL13

Loss of tolerance and autoimmunity affecting multiple organs

Impaired Th2 differentiation and defective IgE class switch

However, these mice presented damaged responses to IFNs and their physiological functions related to the IFNs were absent.200,201 These defective IFN-dependent immune responses led to a high sensitivity toward viral infections and microbial pathogens. Interestingly, STAT1-deficient mice showed normal responses to EGFand other cytokines such as IL10, showing that even if STAT1 is activated by these cytokines, it is only essential for IFN-mediated signaling. More recently, STAT1-deficient mice were proven to be highly susceptible to pulmonary mycobacterial infection.202

3.10.6.2.2 STAT2 signaling

Until now, STAT2 activation has almost exclusively been described with type I IFNs (IFN-a and IFN-b). As observed with STAT1, STAT2 knockout mice are viable and develop normally. However, these mice present no response to type

I IFNs and are thus defective in type I IFN-dependent immune responses, leading to a high susceptibility to viral infections.203,204

3.10.6.2.3 STAT3 signaling

Targeted disruption of STAT3 exposed that this protein is crucial for early embryonic development, as STAT3 knockout mice revealed to be embryonic lethal.205 Therefore, STAT3 knockout mice cannot assess the functions of STAT3 in adult tissues. In order to gain information about the different roles of STAT3, this protein was knocked out in a tissue- or cell-specific manner. STAT3-deficient T cells showed a reduced response to IL2 and IL6 treatment.206 Studies in STAT3-deficient macrophages and neutrophils indicated that IL10-induced STAT3 activation is important for antiinflammatory responses in these cells.207 Mammary glands lacking STAT3 expression presented a delay in involution, which was associated with decreased epithelial apoptosis,208 while keratinocyte-specific ablation of STAT3 led to compromised hair cycle and wound-healing processes, without affecting skin morphogenesis.209 Studies on mice with a cardiomyocyte-restricted knockout of STAT3 showed that this protein has a critical role in the protection of inflammation-induced heart damage. Furthermore, a dramatic increase in cardiac fibrosis could be observed in aged mice, although no signs of heart failure could be detected in young STAT3-deficient mice.210 STAT3 gene disruption in insulin-producing pancreatic beta cells in mice led to glucose intolerance and later to obesity.211 All these different studies tend to show that STAT3 deletion leads to very diverse effects, which seem to be strongly cell lineage-specific.

Furthermore, STAT3 was also shown to be strongly implicated in interleukin signaling, as an activation by IL2, IL6, IL7, IL9, IL10, IL11, IL12, IL15, and IL21 could be observed. Finally, epidermal growth factor (EGF), colony stimulating factor-1 (CSF-1), granulocyte-CSF (G-CSF), platelet derived growth factor (PDGF), growth hormone (GH), and oncostatin M (OSM) were described as STAT3 activators.

3.10.6.2.4 STAT4 signaling

STAT4 is primarily activated in response to IL12. The phenotype of STAT4 knockout mice is similar to that of IL12-deficient mice and the disruption of STAT4 overcomes the IL12 controlled T-helper cell differentiation along the Th1 pathway.177'212 The importance of STAT4 in the differentiation of CD4 + Tcells into Th1 effector cells was recently confirmed in STAT4-deficient CD4 + Tcells.213 Furthermore, experiments on STAT4-deficient mice revealed that the disruption of STAT4 prevents the development of spontaneous diabetes in nonobese diabetic mice, and reveals the important role of STAT4 in autoimmune diabetes pathogenesis.214

3.10.6.2.5 STAT5 signaling

Both STAT5a and STAT5b are known to become activated by a large number of cytokines. Among these, numerous interleukins (IL2, IL3, IL4, IL5, IL7, IL9, IL15, and IL21) implicate STAT5 signaling. Additionally, erythropoietin, GH, prolactin, and growth factor signaling (EGF, PDGF, granulocyte-macrophage colony-stimulating factor (GM-CSF)) display an action on STAT5 activation. First studies with STAT5 knockout mice showed that the deletion of STAT5a leads to deficient prolactin-dependent mammary gland development and lactogenesis in female mice,215 while STAT5b-deficient males lose the sexual dimorphism of body growth and liver gene expression as a result of impaired GH response.216 Furthermore, deletion of both STAT5a and STAT5b in female mice resulted in infertility.217

Experiments on STAT5a null mice also showed the implication of this protein in the signaling due to GM-CSF, as these mice displayed slowed cell growth after GM-CSF stimulation.218 Knockout mice models were also used to demonstrate the immunological role of STAT5. During these studies, an impaired IL2-induced splenocyte proliferation could be observed in STAT5a,219 STAT5b,220 and STAT5a/5b knockout mice.221 Furthermore, STAT5b null mice present a defective natural killer (NK) cell development,220 and thus a reduced number of NK cells, as well as an impaired IL2-induced T-cell proliferation.221 The loss of tolerance and autoimmunity affecting multiple organs in STAT5a/5b-deficient mice was also related to the signaling through IL2R.222 In embryos, STAT5a/5b deficiency was shown to lead to fetal anemia and apoptosis of red cell progenitors.223

3.10.6.2.6 STAT6 signaling

As STAT6 is essentially activated by IL4 and the related cytokine IL13,224,225 STAT6 knockout mice lack the physiological functions associated with these interleukins.224,226,227 Deficient IL4 signaling leads to impaired Th2 differentiation228 and defective IgE class switching.226

These results highlight the importance of STATs in the cellular signaling pathways and show the central role of these proteins in the normal functioning of the cells. However, STAT deregulation as well as aberrant STATactivation can have dramatic consequences, as increasing evidence is underlining the important role of STATs in oncogenesis,229 tumorigenesis,230 allergic inflammation, and autoimmune diseases.180

3.10.6.3 Pathological Variations of Signal Transducer and Activator of Transcription Expression

3.10.6.3.1 Signal transducers and activators of transcription in tumorigenesis

One decade ago, first studies indicated that STAT signaling is often activated by oncogenes and in tumor cells. First, STAT3 was identified to be activated by the oncogenic Src TK in rodent fibroblast cell lines.101 Further studies showed that a great number of oncogenes, including v-Abl.BCR-Abl, v-Eyk, and v-Fps can activate STAT molecules.231 It is also notable that constitutive activation of essentially STAT1, STAT3, and STAT5 has been demonstrated to be associated with malignant transformation induced by oncoproteins.232 Constitutive activation of one or more of these STATs was observed in many tumor cell lines and in primary tumors leading to the idea that the STATs play an important role in the malignant progression of human tumors.197,230

Different mechanisms were described to assign the transforming activity of constitutively activated STAT proteins. This activity could stem from the activation of antiapoptotic pathways and subsequent upregulation of apoptosis inhibitors such as Bcl-2, Bcl-xL, and Mcl-1, as described for example in breast cancer,233 multiple myeloma,197,234 head and neck cancer cell lines,235 as well as non-Hodgkin's lymphoma.236 STAT-induced upregulation of genes encoding for cell cycle regulators, such as cyclin D1, cyclin D2, or c-myc,232 was also described as playing a role in cell transformation. Furthermore, it was shown that activated STATs can increase the transcription of the VEGF gene, an inducer of angiogenesis.101,239 Thus it seems that activated STAT proteins can contribute to oncogenesis via its control of cell cycle progression and/or apoptosis by inducing genes coding for inducers of angiogenesis, cell cycle regulators, and apoptosis inhibitors. Far more detailed information about the role of STAT proteins in tumorigenesis can be found in several excellent reviews.197,229,230,233

3.10.6.3.2 Signal transducers and activators of transcription in allergic inflammation and autoimmune diseases

Asthma is a chronic allergic inflammation whose inflammatory process stems from an unsuitable immune response coordinated by Th2 cells. With regard to this mechanism, it is not surprising that STAT6 signaling, which plays an important role in Th2 differentiation, was investigated in asthma pathogenesis. The implication of STAT6 was shown by the deletion of the genes coding for its expression, as this left the mice resistant to the induction of experimental allergic asthma.240,241 Increased STAT6 expression was also monitored in subjects with allergic rhinitis after allergen challenge.242 Furthermore, STAT1, implicated in immune response, was related to asthma, as epithelial STAT1 was observed to be activated in asthmatic compared to normal control subjects.243

Supporting evidence for a STAT3 causal role in rheumatoid arthritis was affirmed, as activated STAT3 overexpression was reported in synovial tissues from patients with rheumatoid arthritis.244 Confirming information came from the observation that a mutation causing hyperactivation of STAT3 led to spontaneous development of autoimmune arthritis.245 In contrast, the function of STAT1 in rheumatoid arthritis is still controversial as inflammatory and protective roles of this protein have been described in the literature.246 It may thus be that the STAT1 function depends on the cell type and/or the stage of the inflammatory disease.

3.10.6.4 Future Directions

The various studies on STATs have demonstrated that these proteins are implicated in a large and very diverse number of cellular processes, which also means that a STAT deregulation can potentially affect or corrupt numerous cellular functions. A clear understanding of the molecular mechanisms implicated in the STAT-signaling pathway is thus of capital importance. The harmful effects of STAT deregulation can, for example, be observed in many tumors, where the STATs contribute to cellular dysfunctions in cell cycle regulation, apoptosis inhibitors, and angiogenesis, leading to uncontrolled cell cycle progression and apoptosis. The fact that STAT proteins present a point of convergence for TK signaling positions them as promising targets for future clinical treatment. Indeed, they represent a very limited number of targets, as compared to the multitude of kinases, and are additionally implicated in such diverse functions as cell growth, apoptosis, inflammation, and many others.

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