Properties of STATs.
Glomerulonephritis is a renal disease characterized by inflammation of the glomeruli, i.e., small blood vessels, in the kidneys. It may present with isolated hematuria and/or proteinuria or as nephritic syndrome, acute renal failure, or chronic renal failure. Glomerulonephritis has several different pathological patterns that can be broadly grouped into non-proliferative and proliferative types. Non-proliferative types include minimal change glomerulonephritis, focal segmental glomerulosclerosis, and membranous glomerulonephritis. Proliferative types include IgA nephropathy, post-infectious glomerulonephritis, membranoproliferative/mesangiocapillary glomerulonephritis, and rapidly progressive glomerulonephritis (Miller et al., 2010). Of the many factors involved in the inflammatory response, cytokines are the most important factors that bind to their receptors and activate signal transduction pathways. Progress in our understanding of inflammatory signaling pathways has led to the identification of the involvement of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs) such as p38, and Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) pathways (O’Neill, 2006). The NF-κB and p38 pathways are activated by the stimulation of interleukin-1 (IL-1) and tumor necrosis factor (TNF). The principal signaling pathways activated by IL-1 and TNF are the NF-κB and stress-activated MAPK pathways, whereas the JAK-STAT pathway is activated by many other cytokines.
2. STAT family
JAK-STAT is an important tyrosine kinase pathway activated by almost all cytokines (Ihle, 2001) that are well known for their role in the progression of renal diseases (Johnson, 1997). The STAT family consists of seven members (Table 1). The STAT proteins are unique transcription factors containing Src homology (SH) 2 and phosphotyrosine-binding domains. The SH2 domain interacts with sites of tyrosine phosphorylation to recruit STATs to receptor complexes. Following tyrosine phosphorylation of STATs, dimerization occurs between the SH2 domains. Tyrosine phosphorylation around amino acid position 700 (STAT1, tyrosine 701; STAT3 tyrosine 705) is essential for dimerization of STATs and the concomitant nuclear translocation of the dimer (Kaptein et al., 1996; Shuai et al., 1993). The basic model of cytokine pathways depends on tyrosine phosphorylation by JAK proteins non-covalently bound to specific receptors. The JAK family consists of four members: JAK1, JAK2, JAK3, and Tyk2. In addition, the activity of the C-terminal transactivation domain of STATs is at least partially regulated by serine phosphorylation (serine 727 in STAT1 and STAT3) that does not involve the JAK protein (Wen et al., 1995). The kinase responsible for this serine phosphorylation depends on the signaling pathway and cellular context.
|Family member||Chromosomal location||Activating cytokines||Phenotype of knockout mice|
|STAT1||1||2q12–33||IFNs, IL-6||Viable, normal development, IFN functions eliminated, tumors increased, growth control impaired.|
|STAT2||10||12q13–14.1||IFNs||Viable, normal development, IFN functions affected.|
|STAT3||11||17q11.2–22||IL-6 family||Embryonic lethal, extraembryonic endoderm defects, cell survival impaired.|
|STAT4||1||2q12–33||IL-12||Viable, normal development, IL-12 functions eliminated, Th1 differentiation impaired.|
|STAT5A||11||17q11.2–22||Numerous||Viable, mammary gland deficiency, prolactin responsiveness eliminated.|
|STAT5B||11||17q11.2–22||Numerous||Viable, partial loss of growth hormone functions.|
|STAT6||10||12q13–14.1||IL-4, IL-13||Viable, normal development, IL-4 functions eliminated. Th2 differentiation impaired.|
STAT3 is a member of the JAK-STAT signaling pathway (Zhong et al., 1995). Cytoplasmic STAT3 in unstimulated cells is activated by recruitment through the SH2 domain to phosphorylated motifs within complexes of cytokine receptors, growth factor receptors, and non-receptor tyrosine kinases. Determination of STAT3 functions based on knockout mice studies have been difficult because mouse embryos die early in embryogenesis, prior to gastrulation (Takeda et al., 1997). STAT3 is essential for the early development of mouse embryos. Recently, some studies have succeeded in isolating STAT3 from individual tissues by the Cre-loxP method (Akira, 2000), which circumvents the problem of embryonic lethality, and revealed the roles of STAT3 in a wide variety of tissues (Table 2). A surprising result from such studies has been the identification of a multitude of and sometimes contradictory roles of STAT3 in biological processes including cell growth, cell growth suppression and apoptosis, and cell motility. The phenotypes resulting from the loss of STAT3 in adult tissues include failure of cell survival, impaired apoptosis, loss of negative feedback regulation, and impaired cell migration and wound healing (Levy & Lee, 2002). Of great current interest is the persistently active STAT3, which occurs in a wide variety of human tumors (Bowman & Jove, 1999). Overexpression and/or elevated protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR), Src, and other protein kinases is associated with the progression of numerous human cancers. As a consequence, growing evidence indicates that abnormal STAT3 signaling in response to hyperactive protein tyrosine kinase activity is frequent in human tumors and is associated with the progression of oncogenesis (Garcia & Jove, 1998). Furthermore, STAT3 can be converted into an oncogene by experimental mutation (Bromberg et al., 1999). A persistently active protein is required because introduction of a dominant-negative form of STAT3 into head and neck cancer cells or multiple myeloma cells causes apoptosis of recipient cancer cells (Bowman et al., 2000). Persistent STAT3 activation in head and neck cancer is associated with mutations in EGFR or mutations that result in the production of excess ligands or normal receptors (Song & Grandis, 2000). In some multiple myelomas, excess production of IL-6 might be the underlying defect (Catlett-Falcone et al., 1999). IL-6-dependent accumulation of long-lived plasma cells occurs due to elevated levels of a key regulatory protein, Bcl-xL, a member of the Bcl-2 family of proteins that prevent apoptosis. Constitutive activation of STAT3 signaling, an important component of the IL-6 pathway, directly contributes to the induction of Bcl-xL gene expression. Thus, constitutive activation of STAT3 signaling in response to IL-6 promotes tumor cell survival and malignant progression of multiple myelomas by directly inducing expression of a key apoptosis regulatory protein. All these results suggest that STAT3 may be essential for many cell functions.
|Skin||Impaired second hair cycle, wound repair and keratinocyte migration|
|Thymic epithelium||Age-dependent thymic hypoplasia, hypersensitivity to stress|
|T lymphocytes||Impaired IL-6-dependent survival and IL-2Rα expression|
|Monocytes/neutrophils||Enhanced inflammatory responses and Th1 differentiation, chronic colitis|
|Granulocytes||Enhanced proliferation owing to impaired negative feedback|
|Mammary epithelium||Defective apoptosis, delayed mammary involution|
|Liver||Impaired acute phase response|
|Neurons||Impaired cell survival|
4. STAT3 in gp130 cytokine signaling
Several cytokines including IL-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin share a gp130 subunit that is required to activate JAKs and STATs (Stahl et al., 1994). These cytokines are competent to activate STAT3 and induce various pleiotrophic responses that include hematopoiesis regulation, immune response, and neuronal differentiation (Snick, 1990). gp130-associated kinases JAK1, JAK2, and Tyk2 become activated on stimulation, and the cytoplasmic tail of gp130 is phosphorylated (Heinrich et al., 1998). STAT3 is recruited to phosphorylated tyrosine residues in the YXXQ motif including the tyrosine residue Y705 of gp130, where it is activated and dimerized. It subsequently enters the nucleus and regulates gene expression (Hirano et al., 2000). STAT3 is essential for gp130-mediated cell survival and G1 to S cell cycle transition signals. Both
The SHP-2 signal-deficient mice (gp130F759/F759) were born normally but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXXQ/FXXQ) died perinatally, similar to the gp130-deficient mice. The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation. Importantly, these mice in a mixed background with 129 and C57BL/6 spontaneously develop a rheumatoid arthritis-like autoimmune disease in old age (Atsumi et al., 2002). The mice show severe immunological abnormalities, including autoantibody production, increased memory/activated T cells, impaired thymic negative selection, and peripheral clonal deletion. Development of a rheumatoid arthritis-like disease is entirely dependent on mature lymphocytes, but abnormally enhanced homeostatic proliferation of CD4 T cells is caused by augmented production of IL-7 by non-hematopoietic stromal cells through a STAT3-dependent process (Sawa et al., 2006). In mice showing hyperactivation of STAT1/STAT3, T cell recruitment and CCL5/RANTES expression is enhanced (McLoughlin et al., 2005). A recent study described an IL-17A-triggered positive-feedback loop of IL-6 signaling that involved activation of the transcription factors NF-κB and STAT3 in fibroblasts (Ogura et al., 2008). Although the underlying mechanisms by which STAT3 regulates tissue fibrosis are not fully understood, its activation is required for upregulation of transforming growth factor (TGF)-β signaling, activation/proliferation of myofibroblasts, and deposition of extracellular matrix proteins (Ma & Zhuang, 2011). All these results suggest that STAT3 plays an important role in the function of multiple cells.
5. STAT3 activation in renal cells
Various factors show STAT3 activation in glomerular mesangial and proximal tubule cells. High glucose activates the growth-promoting enzyme JAK2 and its latent STAT transcription factors (STAT1, STAT3, and STAT5) and increases TGF-β and fibronectin synthesis in mesangial cells (Wang et al., 2002). High glucose-induced tyrosine phosphorylation of JAK2, STAT1, and STAT3 as well as TGF-β and fibronectin synthesis are abolished by a specific JAK2 inhibitor, AG-490. However, antisense oligonucleotide studies have shown that STAT1 activation is more important than STAT3 activation for TGF-β and fibronectin synthesis. Angiotensin II induces phosphorylation of JAK2, STAT1, STAT3, STAT5A/STAT5B and SHP-2, and angiotensin II-induced phosphorylation is enhanced by high glucose levels (Amiri et al., 2002). Angiotensin II-induced growth and collagen IV synthesis are also increased under high glucose conditions. Transfection of glomerular mesangial cells with JAK2 antisense oligonucleotides blocks angiotensin II-induced growth and collagen IV synthesis under both normal and high glucose conditions.
In renal proximal tubule cells, IL-6 increases phosphorylation of STAT3, phosphoinositide-3 kinase (PI3K)/Akt, MAPKs, and NF-κB (Lee et al., 2007). IL-6 also stimulates α-methyl-D-[14C]glucopyranoside uptake, which is indicative of active transport in renal proximal tubule cells. The increased uptake can be blocked by pretreatment with a STAT3 inhibitor, a PI3K inhibitor, an Akt inhibitor, MAPK inhibitors, and NF-κB inhibitors. High glucose also induces activation of Raf-1, p42/p44 MAPK, JAK2, STAT1, and STAT3 (but not STAT5) in renal tubular epithelial cells (Huang et al., 2007). Moreover, severe oxidative stress leads to tyrosine phosphorylation of STAT3 at Y705 (Arany et al., 2006). This event depends on the activation of EGFR and JAK2 and is directly linked to cell death because inhibition of STAT3 function enables cells to survive severe oxidative stress.
6. STAT3 activation in an animal model of glomerulonephritis
In an animal model of mesangial proliferative glomerulonephritis induced by the injection of anti-Thy1.1 antibody, STAT3 is phosphorylated in mesangial cells (Yanagita et al., 2001a). STAT3 phosphorylation peaks 8 days after the anti-Thy1.1 injection. Inhibition of growth arrest-specific gene 6 by the extracellular domain of its receptor Axl or warfarin abolishes STAT3 phosphorylation (Yanagita et al., 2001a) and inhibits mesangial cell proliferation (Yanagita et al., 2001b)
We have generated a series of knock-in mouse lines with several genetic backgrounds in which gp130-mediated STAT3 or SHP-2 signals are selectively disrupted. gp130 is a shared receptor of the IL-6 cytokine family (Ohtani et al., 2000). We found that characteristic abnormalities in C57BL/6 background gp130F759/F759 mice included not only spontaneous polyarthritis but also glomerulonephritis (Fig. 3, only in females) (Tsuji et al., 2009). However, spontaneous glomerulonephritis did not develop in DBA/1J background mice, indicating that genetic background may affect the development of disease.
7. STAT3 activation in human glomerulonephritis
The JAK-STAT pathway is a pleiotropic cascade essential to cytokine and growth hormone receptor signaling. Signaling through the JAK-STAT pathway is important for the kidney’s response to injury in disease. STAT3 activation is observed in an animal model of glomerulonephritis and human glomerulonephritis, and the STAT3 pathway inhibition ameliorates the renal conditions in some animal models. Further studies are needed to clarify the role of STAT3 activation in human glomerulonephritis, but the STAT3 pathway inhibition may be one of the potential therapeutic approaches for renal diseases.