JAK redux: a second look at the regulation and role of JAKs in the heart

Abstract

A number of type 1 receptor cytokine family members protect the heart from acute and chronic oxidative stress. This protection involves activation of two intracellular signaling cascades: the reperfusion injury salvage kinase (RISK) pathway, which entails activation of phosphatidylinositol 3-kinase (PI3-kinase) and ERK1/2, and JAK-STAT signaling, which involves activation of transcription factor signal transducer and activator of transcription 3 (STAT3). Obligatory for activation of both RISK and STAT3 by nearly all of these cytokines are the kinases JAK1 and JAK2. Yet surprisingly little is known about how JAK1 and JAK2 are regulated in the heart or how they couple to PI3-kinase activation. Although the JAKs are linked to antioxidative stress programs in the heart, we recently reported that these kinases are inhibited by oxidative stress in cardiac myocytes. In contrast, others have reported that cardiac JAK2 is activated by acute oxidative stress by an undefined process. Here we summarize recent insights into the regulation of JAK1 and JAK2. Besides oxidative stress, inhibitory regulation involves phosphorylation, nitration, and intramolecular restraints. Stimulatory regulation involves phosphorylation and adaptor proteins. The net effect of stress on JAK activity in the heart likely represents the sum of both inhibitory and stimulatory processes, along with their dynamic interaction. Thus the regulation of JAKs in the heart, once touted as the paragon of simplicity, is proving rather complicated indeed, requiring a second look. It is our contention that a better understanding of the regulation of this kinase family that is implicated in cardiac protection could translate into effective therapeutic strategies for preventing myocardial damage or repairing the injured heart.

a number of type 1 receptor cytokine family members, namely the interleukin (IL)-6-type cytokines [IL-6, IL-11, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM)] (40, 78, 58, 85), growth hormone (GH; Ref. 91) erythropoietin (Epo; Refs. 20, 134, 138), and granulocyte colony-stimulating factor (G-CSF; Refs. 26, 61, 62, 72, 159, 166), as well as insulin (3, 23, 54, 64, 99, 164, 186), have been shown to protect the heart from acute oxidative stress, viz., ischemia-reperfusion injury. LIF (121), GH (56), Epo (77), and insulin (141) have also been reported to protect cardiac myocytes from chronic oxidative stress. Notably, a number of clinical trials have been completed or are underway assessing the efficacy of Epo or G-CSF in treating myocardial infarction and heart failure (13, 87, 88, 93).

The protective action of the type 1 receptor cytokines (see Fig. 1 for IL-6 cytokine signaling) and insulin has been shown to involve activation of two intracellular signaling cascades: 1) the reperfusion injury salvage kinase (RISK) pathway (17, 27, 40, 41, 64, 66, 77, 99, 101, 102, 108, 121, 126, 128, 133, 134, 142, 156, 159, 160, 161, 166) and 2) JAK-STAT signaling (20, 40, 61, 85, 161, 174). The former, which involves the sequential activation of phosphatidylinositol 3-kinase (PI3-kinase) and AKT, as well as ERK1/2, has as a major target inhibition of the mitochondrial permeability transition pore (MPTP) and plays a dominant role in the mediator phase of both early (classical) preconditioning and postconditioning of the heart (29, 164). The JAK-STAT pathway for the most part involves activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and upregulation of cyclooxygenase-2 (COX-2) and nitric oxide synthase 2 (NOS2) (involved in MPTP inhibition), vascular endothelial growth factor (VEGF) (angiogenic and cardioprotective agent), the antioxidants manganese superoxide dismutase (MnSOD) and metallothioneins (MT1 and MT2), and matrix metalloproteases that are important in repair or scar formation (85). The JAK-STAT pathway has been shown to be essential to late (delayed) preconditioning of the heart, also referred to as the second window of protection (SWOP), which is initiated in vivo by the autocrine/paracrine production of IL-6 (15, 18, 24, 133, 175–178).

Fig. 1.

Overview of interleukin (IL)-6 cytokine signaling focusing on the basics of cardiac protective signaling. See text for details. Modified from ScienceSlides 2007 software version 2.0 (VisiScience, Chapel Hill, NC).

Obligatory for activation of both the RISK pathway and STAT3 by these cytokines (with the possible exception of G-CSF) (40, 85, 180), and presumably to some extent the former by insulin (144, 146, 169), are the Janus kinases JAK1 and JAK2. Yet surprisingly little is known about how JAK1 and JAK2 are regulated in the heart or how they couple to PI3-kinase activation (85). Although the JAKs are linked to antioxidative stress programs in the heart, we recently reported (86) that these kinases, JAK1 especially, are inhibited by oxidative stress in cardiac myocytes, consistent with evidence by others for a redox-switch in their JH1 catalytic domain (106). In contrast, others have reported that JAK2 is activated by acute oxidative stress in cardiac myocytes or the heart (24, 57, 63, 111, 181), although the mechanism for this activation is unknown. Here we present an update on the regulation of the JAKs with particular focus on the heart.

Activation of the JAKs

The JAKs are nonreceptor tyrosine kinases and consist of four mammalian members, one of which (JAK3) is strictly expressed in hematopoietic cells (85). For cardiac myocytes, JAK1 and JAK2 are predominant. The JAKs are characterized by seven highly conserved JAK homology (JH) domains (Fig. 2), which, starting at the COOH terminus, are the JH1 kinase domain; the JH2 pseudokinase domain, which appears to serve a complex regulatory function specific for each JAK family member; the JH3–JH4 region, which has some similarity to SH2 domains but does not have phosphotyrosine binding ability; and the JH4–JH7 region, which constitutes a divergent FERM domain (four-point-one, ezrin, radixin, moesin) and is involved in the interactions of JAKs with receptors.

Fig. 2.

Domain structure of JAK1 and JAK2. FERM, four-point-one, ezrin, radixin, moesin domain; JH, JAK homology; P, phosphorylation site. Adapted with permission from J Cardiovasc Pharmacol 50: 126–141, 2007. Copyright 2007 Lippincott Williams & Wilkins.

Of the JAKs, the molecular events underlying JAK2 activation have been best studied. Activation occurs upon dimerization of the JAKs and the transautophosphorylation of conserved tandem tyrosines (Tyr1007/Tyr1008) found in the activation loop of JAK2's catalytic domain (38). JAK2 dimerization and activation is an immediate consequence of cytokine-induced receptor dimerization (172). Without phosphorylation of Tyr1007/Tyr1008, JAK2 exists in a basal activity state characterized by inefficient ATP use and restricted substrate recognition (21). Upon phosphorylation of these tyrosines, JAK2 exists in a high-activity state in which its enzymatic efficiency with respect to ATP increases by at least 4 orders of magnitude and its substrate recognition spectrum broadens. In the inactive state, the JH2 domain is thought to physically interact with the JH1 domain of JAK2 to block its catalytic activity. Recent evidence indicates that for receptor-unbound JAK2, the FERM domain may fold back on the JH1 and JH2 domains to provide an additional physical constraint to activation (45). The JAK1 FERM domain is involved in autoinhibition as well (59).

Canonical JAK activation.

In the classical system, JAKs are activated upon ligand-induced dimerization of receptor subunits, which allows for the transautophosphorylation of the activation loop of juxtapositioned JAKs that are preassociated via their FERM domain with cytoplasmic membrane-proximal Box1/Box2 regions of these receptor subunits. This is the case for the hematopoietin receptor superfamily, which includes receptors for over two dozen different cytokines (80). Upon receptor stimulation, the associated JAK becomes a fully active protein tyrosine kinase and phosphorylates its tyrosines and nearby tyrosines, such as those on the cytoplasmic regions of the receptor, that serve as docking sites for various scaffold or signaling molecules including the STAT (signal transducers and activators of transcription) transcription factors.

Noncanonical JAK activation.

The JAKs can be activated in an atypical fashion, i.e., independently of receptor dimerization or even receptor association. This has been well documented for JAK2, but to the best of our knowledge not for JAK1. Examples of such atypical activation involve 1) G protein-coupled receptors (GPCRs), 2) oxidative stress, and 3) hypertonicity. Given the plethora of autocrine/paracrine factors that are released, and the reactive oxygen species (ROS) produced, both canonical and noncanonical JAK activation likely occur with cardiac ischemia-reperfusion and preconditioning.

g protein-coupled receptors.

Several extracellular stimuli that directly activate GPCR pathways have been reported to stimulate JAK2 and to require JAK2 activation in order to evoke a complete biological response. These GPCRs include angiotensin II type 1 (AT₁) receptor (137), platelet-activating factor receptor (105), CXCR4 (SDF-1α receptor; Refs. 170, 187), CCK2 (cholecystokinin receptor; Ref. 39), CCR2B (MCP-1 receptor; Ref. 118), Mas [angiotensin-(1–7) receptor; Ref. 49], and—notably—receptors for two agonists that have been linked to ischemic preconditioning, viz., bradykinin B₂ receptors (94, 183) and opioid receptors (55).

The exact mechanism by which GPCRs activate JAK2 remains poorly defined but has been best studied with the AT₁ receptor. Evidence has shown that AT₁-mediated JAK2 activation occurs before, and is not dependent on, association with AT₁ (4, 50); nor does it involve EGF receptor transactivation (42). At least in vascular smooth muscle cells (VSMCs), AT₁-mediated JAK2 activation involves PKCδ, preassociated PYK (a nonreceptor tyrosine kinase), and most likely a Src family member (42). Superoxide generation from NADPH oxidase is required (151), while conflicting results on the role of intracellular calcium have been reported (42, 150). In any event, the likely scenario for GPCR-mediated JAK2 activation involves the intermediacy of a nonreceptor tyrosine kinase, without a requirement for JAK2-receptor association (Fig. 3). This model likely applies to insulin-induced JAK2 activation also (146). Whether atypical JAK2 activation is part of the repertoire of IL-6-type cytokine signaling is unknown but is a possibility given that these cytokines also activate JAK2, although the JAK associated with their receptor subunits is JAK1 (85, 143). One feature that distinguishes GPCR-mediated, and likely noncanonical, JAK2 activation from cytokine-mediated activation is an absolute requirement for Tyr972 autophosphorylation, which is postulated to stabilize the active conformation of the protein (117).

Fig. 3.

Noncanonical JAK2 activation by G protein-coupled receptors (GPCRs) and oxidative stress. Evidence implicates PKCδ, the tyrosine kinase PYK2, and an NADPH oxidase (NOX). A likely scenario to explain their involvement in JAK2 activation is proposed in this figure. Redox-sensitive PKCδ can be activated via GPCR angiotensin II type 1 (AT₁) receptor-induced phospholipase C activation or through oxidative stress. In turn, PKCδ can activate NOX directly or through a Src family member. NOX stimulation may further enhance PKCδ activity. Both reactive oxygen species (ROS) and PKCδ can inhibit SHP-1, a protein tyrosine phosphatase that dephosphorylates and thereby inactivates PYK2 and JAK2. PYK2 and JAK2 are preassociated. SHP-1 inhibition, PYK2 activation, or Src activation may create docking sites on some scaffold or membrane receptor protein that recruits preassociated PYK2-JAK2, thereby bringing two JAK2 molecules into close position to allow for their transautophosphorylation and activation. Src may also associate with PYK2. Increased cellular Ca²⁺ may enhance PYK2 activity, but its importance in noncanonical JAK2 activation is not clear.

oxidative stress.

ROS (most commonly hydrogen peroxide) have been documented to stimulate JAK2 activity (114, 155). No direct molecular mechanism would seem to explain how this occurs; most likely the effect is indirect, possibly involving other biomolecules that interact with JAK2. Supporting this conclusion is the demonstration that JAK2-mediated signaling following hydrogen peroxide exposure is dependent on the cell line used (76). Indirect activation of JAK2 may result in part from oxidative inhibition of the active site cysteine in protein tyrosine phosphatases (53, 119, 165, 168), which dramatically increases phosphotyrosine content of JAK2 (60, 92). Alternatively, indirect activation of JAKs may require intervention of a transphosphorylating Src kinase family member (1), as H₂O₂-stimulated JAK2 activity was inhibited in Fyn^−/− cells. Finally, JAK2 activation has also been reported to cause ROS generation in atypical GPCR-mediated systems (149), but for the most part these studies relied on the so-called JAK2 inhibitor AG490. Not only is AG490 not a specific inhibitor of JAK2 (135), but AG490 may itself act as a ROS scavenger (52).

High levels of glucose, as occur in diabetes, have been shown to enhance basal activity of JAK2 in aortic VSMCs and perhaps cardiac myocytes and to potentiate JAK2 activation by angiotensin II and endothelin-1 (5, 9, 10, 122, 153). Like GPCR-mediated JAK2 activation, the effect of high glucose involves a PKC family member (in this case activated via the polyol pathway) and subsequent activation of NADPH oxidase (153). Evidence is reported suggesting that subsequent ROS generation enhances JAK2 activity by inhibiting the tyrosine phosphatase SHP-1, which mediates JAK2 dephosphorylation (153). Whether a similar scenario explains AT₁-mediated JAK2 activation is unclear. Although angiotensin II was reported to induce a rapid decrease in SHP-1 activity (within 5 min) in VSMCs, the decrease was very transient and followed by increased SHP-1 activity (109). Furthermore, the transient decrease in SHP-1 activity was accompanied by decreased association of JAK2 with SHP-1, while the angiotensin II-induced increase in SHP-1 activity was accompanied by increased association. Overall, evidence would indicate that SHP-1 is responsible for JAK2 deactivation in response to AT₁ stimulation.

In contrast, oxidative stress has been shown to attenuate or inhibit JAK activity in several “classical” cytokine systems, including IL-6 and LIF (85), leptin (74), IL-3 (32), IL-2 (14, 116), ciliary neurotrophic factor (CNTF) (76), and interferon-α (28). Important evidence provided by the Halvorsen lab (76, 123, 124) showed that neurons treated with oxidative stress reagents do not effectively respond to JAK2-stimulating cytokines such as CNTF. Besides the possible cell type-specific differences in the contribution of phosphatases to JAK2 activation, we think it highly likely that structural/molecular differences between JAK1 and JAK2 that impact on their regulation, together with relative differences in their contributions to a particular signaling system, contribute to the confusion in the literature as to the impact of oxidative stress on JAK activity.

osmotic stress.

There are two reports that osmotic stress induced by hyperosmolarity can activate the JAKs in various cell lines; the basis for this activation is unexplained, but it does not appear to involve a Src kinase family member (47, 48). Demonstration of the in vivo occurrence of this mechanism has not been reported.

Additional Control Mechanisms Regulating JAK Activity

Superimposed on the basic process of transautophosphorylation that activates JAK1 and JAK2 are several distinct mechanisms, as discussed here.

Redox sensitivity.

Oxidants such as nitric oxide (NO) can inhibit Janus kinases under cellular conditions, and the oxidative inhibition of Janus kinases is reversible under in vitro conditions (32). Recently, individual and combinatorial cysteine-to-serine mutagenesis led to the identification of four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competence, an observation that might explain why the enzyme is catalytically inactive when oxidized and maximally active when reduced (106). On the basis of their positions in the JAK2 catalytic domain, one might speculate that Cys866 and Cys917 coordinate the metal center of the magnesium/manganese ATP and therefore have a direct role in catalysis. The two sulfur centers of these cysteine residues are ∼9 Å apart and might be able to form a disulfide bond in an oxidizing environment. Because of the high homology between JAK1 and JAK2 in their JH1 domain, it is likely that Cys891 and Cys943 perform a similar function in JAK1.

Phosphorylation and nitration.

An early study reported evidence for direct PKCδ-mediated phosphorylation of JAK2 on serine and threonine residues, which was inhibitory; however, the residues involved were not identified (84). Subsequent studies have shown that cytokine-induced Ser523 phosphorylation, within the linker region between the JH3 and JH2 domains, most likely by ERK1/2, inhibits JAK2 activity (73, 115).

Cytokine-induced JAK2 phosphorylation of Tyr570 within the JH2 domain is also inhibitory and would seem to function as a classical negative feedback mechanism (37). Autophosphorylation of Tyr913, which was observed as a result of Epo stimulation, also seems to serve as a negative feedback mechanism (46). Whether Tyr913 phosphorylation is a general feature of JAK2 activation or specific for certain activators is not known. In contrast, JAK2-mediated phosphorylation of Tyr813 within the JH1 domain enhances its catalytic activity by creating a docking site for the cytoplasmic adaptor protein SH2B1 (89). Finally, two recent reports documented that agonist-induced nitration of Tyr1007/Tyr1008 within the regulatory epitope of JAK2 inhibits its catalytic activity (35, 36). Nitration was studied in the context of GH signaling of liver cells and was interpreted as the basis for endotoxin-induced resistance and as a natural counterpoise to JAK2 activation that was linked to a parallel signaling pathway. Whether nitration has functional relevance for JAK activation in the heart awaits exploration.

JH2 domain and SH2B1.

The JH2 domain of JAK2, which is highly conserved among human, mouse, and rat (95.7% identity), is thought to function like a pseudokinase domain to maintain basal JAK2 in a low-activity state (147, 188). Exactly how this is accomplished is not known. On the basis of dimer formation in crystals of the tyrosine kinase domain of the FGF receptor, Lindauer et al. (98) postulated two regions of interaction between JH1 and JH2. Interface 1 would involve the region Tyr590 to Gln603 of JH2, which is predicted to form an α-helix, and interface 2 would comprise JH2 residues Val617 to Glu621 (Fig. 4). Interface 1 was predicted to form a helix-helix interaction with the analogous region of JH1, while interface 2 was predicted to interact with the activation loop of JH1. Using a deletion mutational strategy, Saharinen et al. (148) identified three inhibitory regions (IR) of JH2 against JH1 catalytic activity. IR3 (Asp758–Ser807) directly inhibited JH1 activity (indicating a likely physical interaction with JH1) by lowering the V_max for enzymatic activity with no effect on substrate affinity (Fig. 4). IR3 is predicted to comprise three α-helices and lies proximal to Tyr813, the phosphorylation of which enhances JAK2 activity. IR1 (Gly619–Ile670) and IR2 (Glu725–Gly757) were found to enhance the inhibitory actions of IR3.

Fig. 4.

Mouse JAK2 JH2 domain. The amino acids comprising the JH2 domain are shown in black. Regions postulated to be important in the inhibition of JAK2 catalytic activity are highlighted. Highlighted in blue (from NH₂ to COOH terminus) are interface 1 and interface 2. Highlighted in yellow (from NH₂ to COOH terminus) are inhibitory regions (IR)1, 2, and 3. Tyr813, which is important in recruiting SH2B1, is highlighted in red. h, α-Helix; e, extended strand; t, β-turn; c, random coil. Analysis was by the secondary structure prediction method (SOPM) at http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html (21a).

SH2B1 (previously named SH2-B) is a ubiquitously expressed cytoplasmic adaptor protein that selectively enhances catalytic activity of JAK2 (113, 132). SH2B1 contains an NH₂-terminal dimerization domain, a central pleckstrin homology (PH) domain, and a COOH-terminal SH2 domain. The latter binds phosphorylated Tyr813 in the JH2 domain and somehow enhances JAK2 activity. Contrary to original thinking, SH2B1 dimerization, with subsequent JAK2-JAK2 apposition, is not how SH2B1 enhances JAK2 activity. Rather, the SH2 domain of SH2B1 alone appears to be sufficient in this regard (89). One explanation for this observation would be that SH2B1 physically prevents JH2 from reassociating with and inhibiting the JH1 catalytic domain.

Recent evidence indicates that in the case of leptin and insulin signaling, insulin receptor substrate (IRS)1 and IRS2 are recruited to the SH2B1·JAK2 complex to be phosphorylated by JAK2, thereby promoting their association with the p85 regulatory subunit of PI3-kinase and subsequent AKT activation (30, 95, 97, 140). A similar scenario may occur with opioid receptors in the heart (55).

Role of JAKs in the Heart

To date, no cardiac myocyte-specific JAK1 or JAK2 knockout mice have been generated (85). Therefore, no definitive conclusions can be made as to what role(s) the JAKs play in the stressed heart. However, JAK-STAT (in particular STAT3) signaling has been associated with the responses of the heart and isolated cardiac myocytes to various stresses (Fig. 5), and most evidence indicates that JAK signaling linked to STAT3 activation elicits a protective response. Transgenic and knockout mouse models have clearly shown that STAT3 activation in the heart is protective (85). Since class I cytokine receptors that activate STAT3 initially activate JAK1 and/or JAK2, it would seem that the JAKs are protective as well. In fact, the JAKs that are associated with class I cytokine receptors activate other protective signaling pathways besides STAT3, such as PI3-kinase/Akt (156).

Fig. 5.

Role of the JAKs or JAKs-signal transducer and activator of transcription 3 (STAT3) in the heart.

Preconditioning or pretreatment.

Pretreatment of rats in vivo with G-CSF (65) or IL-6 (112) improved hypothermic preservation ex vivo and reduced infarct size and apoptosis immediately after ischemia-reperfusion, respectively. The former response was blocked by AG490 and was associated with increased cardiac capillary density. JAK activation was also implicated by means of AG490 in early preconditioning in several studies using the isolated rat heart (63, 70, 181). Blocking JAK2 attenuated preconditioning-induced improved contractile function and reduced infarct size, and this was associated with reduced upregulation and downregulation of antiapoptotic (bcl-2) and apoptotic (Bax) genes, respectively. Using knockout mice, Bolli and colleagues (24, 175, 176) found that IL-6 plays an essential role in the late phase of preconditioning, although the lack of IL-6 did not modulate infarct size in naïve myocardium. IL-6 was found to be obligatory for the upregulation of NOS2 and COX-2, comediators of late preconditioning. With AG490, JAK2 was implicated in the upregulation of COX-2.

Recovery.

Evidence indicates that the JAKs contribute to the recovery of the heart from myocardial infarction (MI) or ischemia-reperfusion. An early study done on an acute rat MI model reported that blocking JAK activation with AG490 resulted in deterioration of myocardial viability (130). A number of cytokines that signal via gp130 and/or a receptor closely related to gp130 have been shown to improve cardiac function and reduce myocardial damage when introduced at the time of MI in either mouse or rat. These include leptin (2), LIF (12), CT-1 (145), and G-CSF (61). Of note, leptin treatment preserved systolic function but caused eccentric dilatation of the heart. A number of studies either in vivo or on isolated hearts have demonstrated that Epo delivered acutely during ischemia or reperfusion or chronically after MI reduces infarct size, oxidative stress, inflammation, and apoptosis and improves cardiac function (20, 96, 136, 138). Thrombopoietin (Tpo), another class I hematopoietic cytokine that shares sequence homology with Epo, was shown to preserve cardiac structure and function when given before ischemia-reperfusion assault of the rat heart and to reduce infarct size when given at the onset of ischemia or at reperfusion (8).

There are several reports that JAK2 activation is bad in rat hearts (isolated and in vivo) subjected to ischemia (and reperfusion) or in isolated adult rat cardiac myocytes treated with angiotensin II or subjected to hypoxia-reoxygenation (Fig. 5; Refs. 34, 71, 110, 111). Besides increased infarct size and worsened functional hemodynamics, adverse effects were associated with increased angiotensinogen gene and Bax protein expression, increased caspase and protein phosphatase 1 activity, and decreased levels of p16-phospholamban. The discrepancy between these studies and those showing a beneficial role for JAK signaling after MI may perhaps be explained by differences in experimental approach. However, those studies reporting that JAK activity is detrimental relied solely on the use of the inhibitor AG490 to draw their conclusions, and because of the demonstrable lack of specificity of this inhibitor these conclusions cannot be viewed as definitive.

Various stresses.

Multiple in vitro studies have shown that JAK-STAT protects cardiac myocytes against various stresses. Most of these studies have utilized neonatal rat ventricular myocytes (19, 22, 44, 69, 104, 128, 154, 156, 179), although a few have used adult [e.g., mouse (179) and cat (171)] cardiac myocytes. The gp130-related cytokines IL-6 (156), LIF (129, 171), and CT-1 (22) were shown to protect against hypoxia-reoxygenation-induced cell damage via the activation of a number of signaling molecules downstream of the JAKs, including p38 MAP kinase, the ERKs, AKT, NF-κB, and NO. Specifically how these cytokines mediated protection was not defined, although one study reported an upregulation of MnSOD (129). LIF also protects cardiac myocytes against oxidative stress initiated by H₂O₂ (19), which may represent a manifestation of an innate JAK2 apoptosis resistance mechanism in cardiac muscle (104). An innate protective mechanism against viral infection linked to gp130 has been postulated as well (179). Finally, LIF and CT-1 protect cultured cardiac myocytes against serum deprivation through survival signaling involving, for example, the ERKs and AKT and by upregulating bcl-xL expression (44, 69, 154).

Hypertrophy.

STAT3 and the JAKs have been implicated as well in cardiac hypertrophy (Fig. 5). Whether this is good or bad is arguable, and consequently we note it as a gray area Fig. 5. Pathological hypertrophy as observed during hypertension eventually proves detrimental to heart function and may lead to heart failure (16). gp130 stimulation was shown to induce hypertrophy of isolated human atrial myocytes (6) and cultured neonatal rat ventricular myocytes (see, e.g., Ref. 83) and to lead to left ventricular hypertrophy in a transgenic mouse model of chronic gp130 stimulation (68). Furthermore, the JAK2 inhibitor AG490 was reported to block development of concentric hypertrophy in mice subjected to transverse aortic constriction (11). Which signaling pathway (ERK, STAT3, or PI3-kinase) links gp130 to cardiac hypertrophy is less than clear; however, that issue is irrelevant, as JAK1 and JAK2 (to a lesser extent) are critically important in gp130 signaling overall (85, 143). Nonetheless, Kodama et al. (83) reported that JAK2 inhibition with AG490 markedly attenuated LIF-induced myofilament reorganization and atrial natriuretic peptide (ANP) expression but had minimal effects on protein synthesis itself.

Some have suggested that gp130 and JAK signaling in the heart activates a response that is protective during cardiac hypertrophy. With the use of human transvenous endomyocardial biopsies, an association was found between depression of gp130 protein expression and myocardial apoptosis in hypertensive patients who develop heart failure, but not in those who did not (51). Unlike wild-type mice that develop compensatory cardiac hypertrophy in response to aortic pressure overload, mice with cardiac myocyte-restricted gp130 deletion developed rapid-onset dilated cardiomyopathy and heart failure marked by marked myocardial apoptosis (67). But is this primarily due to the absence of hypertrophy or the loss of cardiac protective signaling? Finally, the hypertrophic agonist endothelin-1 was recently reported to upregulate the cytoprotective stress protein αβ-crystallin in neonatal rat cardiac myocytes, and JAK2 was implicated in this effect through the use of AG490 (107). As suggested by others, gp130 signaling, and by extension JAK activation, may be viewed as a precarious balance between compensatory (but ultimately) maladaptive hypertrophy and cytoprotection (67).

Evidence that acute oxidative stress activates JAK2 in cardiac myocytes.

Ischemia-reperfusion and preconditioning have been shown to activate JAK2 in vivo and in the isolated working heart (24, 63, 111, 160, 190). Moreover, JAK2/JAK1 signaling has been implicated in the protective effects of classical (63, 157, 181) and delayed (24) preconditioning. The basis for this activation is not established but could result from the generation of ROS and/or from the local release of angiotensin II, growth factors, or cytokines. In cultured human cardiac myocytes, angiotensin II was reported to couple to JAK2 activation via ROS generation (122). Overall, however, evidence that JAK2 is in fact activated by ROS per se in cardiac myocytes is scant; at this time a more accurate statement would be that JAK2 may be active during exposure of the heart or cardiac myocytes to ROS.

Evidence that chronic oxidative stress inhibits JAK1 and JAK2.

We recently reported (85) that parthenolide-induced oxidative stress inhibits JAK1, and to a lesser degree JAK2, activation by the IL-6-type cytokines in isolated cardiac myocytes. Perhaps JAK2 is less affected by ROS because of the opposing actions of ROS on JH2 and JH1. Consistent with the possibility that chronic oxidative stress inhibits JAK activation in the human heart is a report that JAK2 phosphorylation in the left ventricles is markedly reduced (by 72%) in patients with dilated cardiomyopathy (139).

Recent evidence for a role of JAK2 activation in cardiac protection: Epo and G-CSF.

Involvement of the JAKs in the cardioprotective action of the IL-6 family cytokines in the heart was reviewed recently elsewhere (85) and is not further discussed here. Rather, we focus on two agents that also activate receptors belonging to the type 1 cytokine receptor family and that have lately generated much clinical interest, namely, Epo and G-CSF.

Besides its ability to increase the number of red blood cells and consequently oxygen delivery to underperfused organs, Epo has other actions that are beneficial under conditions of perfusion dysfunction, including stimulation of new blood vessel formation and a direct protective action on cells, most notably cardiac myocytes (13, 93). Epo is able to protect the heart against ischemia-reperfusion injury and reduce infarct size via activation of its receptor on cardiac myocytes. Epo-initiated intracellular signaling involves formation of a receptor homodimer with subsequent activation via transphosphorylation of juxtaposed JAK2 molecules. This preconditioning-like effect of Epo involves in part Akt-mediated targeting of mitochondrial glycogen synthase kinase (GSK)-β (82, 134). Although postinfarct ventricular remodeling impairs this protective mechanism, the protective actions of Epo are retained because of a compensatory ERK-mediated mechanism (120). In addition, STAT3 activation likely contributes to the protective effects of Epo on cardiac myocytes (81, 138). Another way Epo protects the heart is through increased expression and activation by phosphorylation of NOS3 (33). The subsequent increase in NO acts to improve blood flow and oxygen delivery, as well as to quench superoxide formation.

Although it is also a member of the hematopoietin cytokine receptor superfamily, signaling via the G-CSF receptor involves prominently not only JAK2 but also Lyn kinase (189). Both of these nonreceptor kinases are recruited to the G-CSF homodimer and couple independently to distinct signaling pathways. Cardiac myocytes have been shown to express the G-CSF receptor, which directly couples to protective signaling. In addition, G-CSF, which is produced by many cells including endothelial and immune cells, indirectly promotes cardiac repair by mobilizing bone marrow-derived stem cells after myocardial infarction (87, 88).

JAK-AKT interface.

Numerous studies have reported that the IL-6-type cytokines couple to PI3-kinase and AKT activation, although the exact mechanism is unknown (101, 133, 156). LIF in particular has been shown to activate AKT in adult (41) and neonatal (83, 128) rat ventricular myocytes. Available evidence suggests a possible role for the adapter-protein GRB2 associated binder 1 (Gab1; Refs. 127, 160), which contains several highly conserved JAK2 substrate motifs as well as binding motifs for the PI3-kinase regulatory domain (although the 2 sets of motifs are distinct). In addition, activated JAK2 has been shown to form a complex with protein phosphatase 2A (PP2A), resulting in tyrosine phosphorylation and inactivation of this serine/threonine phosphatase (7, 25, 43, 79, 100, 152, 184, 185). PP2A appears to be the primary phosphatase downregulating AKT activity (142, 190). Both the receptors for Epo and G-CSF are also linked to Akt activation (134, 189).

An alternative inhibitory mediator of JAK2 action on PI3-kinase/AKT signaling is the protein tyrosine phosphatase SHP-1, which binds and is activated by JAK2 (75). It was reported that mice expressing functionally deficient SHP-1 are more glucose tolerant and insulin sensitive because of enhanced insulin receptor signaling in liver and muscle via the IRS-PI3-kinase-AKT pathway (31). SHP-1 has been shown to attenuate insulin signaling by binding and dephosphorylating the insulin receptor (31). Also, SHP-1 might attenuate PI3-kinase signaling to AKT by binding and dephosphorylating the phosphodiesterase PTEN, thus enhancing its binding to membrane phosphatidylinositols and effectiveness in inhibiting the AKT pathway (103). A recent report linked heightened JAK2 activity and insulin resistance in L6 myotubes to reduced AKT activity/phosphorylation at some level downstream of PI3-kinase (163). AKT activation (separate or together with downstream ERK1/2 activation) may in turn lead to attenuation of JAK2 activity, as well as STAT3 activity, although the details are not well defined. Finally, JAK2 and AKT signaling are likely to interact as well in cardiac myocytes downstream at the transcriptional level (104). AKT activation may lead to increased STAT3 expression via the intermediacy of NF-κB activation, while STAT3 activation increases AKT expression.

Summary and Perspectives

The regulation of the activities of JAK1 and JAK2 is now known to be more complicated than once thought only a few years ago. Our recent studies have revealed that the activity of these kinases in cardiac myocytes is dependent on cellular redox status and is inhibited by oxidative stress. A potential redox switch has been localized within the catalytic JH1 domain of JAK2. Based on primary sequence similarities, this redox switch is likely present in JAK1 as well. At the same time, the work of others has revealed a role for nitration and phosphorylation as well as scaffolding proteins in controlling JAK2 kinase activity. Altogether, recent studies support the conclusion that JAK1 and JAK2 function as part of integrated signaling networks. This raises several questions that challenge the limitations of current approaches. For instance, how do the various input signals that control JAK activity interact, and how does JAK signaling regulation change with cellular context and with changes in cellular environment that occur with cardiac remodeling? The answers will ultimately address the overarching question of whether the JAK kinases are a viable drug target relevant to cardiac remodeling and heart failure.

GRANTS

This work was supported by grants from the National Heart, Lung, and Blood Institute (1-R01-HL-088101, G. W. Booz) and Lebanese University (M. Kurdi).