RELATIONSHIP BETWEEN THE JNK PATHWAY AND APOPTOSIS IN HEART FAILURE.

JNKs belong to mitogen activated protein kinases family and are involved in regulation of various cellular responses including cell survival, cell proliferation and differentiation. They can be activated by several factors such as radiation, stress, growth factors , cytokines and extracellular signals. JNKs activation results in phosphorylation of downstream molecules involve in normal cellular processes. Furthermore the recent have linked JNK signaling with different and hypertrophy. Gq signaling hypertrophic growth of neonatal rat cultured myocytes and is with the over expression of c-jun through Ap-1 and ATF-2 transcripting factor, it can cause decompensatory hypertrophy which can proceed to cardiac failure. This review analyzes our present understanding of the role of JNK in apoptotic signaling in heart failure and some other mechanism which induces apoptosis in cardiomyocytes.

387 delayed, the growth factor induced JNK-activation was rapid and transient. Subsequent studies expanded the role of JNK in TNF-α, Fas-L, X-ray, and UV-ray induced apoptosis in different cellular and physiological contexts. Unequivocal evidence that JNK1 and JNK2 are involved in apoptotic signaling came from studies using embryonic fibroblasts derived from JNK1-/-JNK2-/-mice that showed resistance to apoptosis in response to diverse genotoxic and cytotoxic stress [34,35]. In general the mechanism through which JNKs regulates the apoptotic pathway involves expression of pro-apoptotic genes by activating the specific transcrition factors such as the c-Jun, p73 and p53 [36].

Nuclear signaling of JNK in the regulation of apoptosis
Upon activation by the upstream MAP2Ks, the phosphorylated JNK translocates to the nucleus where it phosphorylates and transactivates c-Jun [36] resulting in the formation of AP-1. The AP-1 is involved in the transcription of a wide variety of proteins, including several pro-apoptotic proteins [37] such as TNF-α, Fas-L, and Bak [38]. JNK can also phosphorylate several other transcription factors including JunD, ATF2, ATF3, Elk 1, Elk-3, p53, RXRα, RARα, AR, NFAT4, HSF1, and c-Myc [39]. Thus, in the context of apoptosis, the nuclear activity of JNK can potentially lead to an increase in the expression of pro-apoptotic genes and/or a decrease in the expression of pro-survival genes. There is sufficient evidence that the nuclear activity of JNK and the transactivation of c-Jun are required for its apoptotic activity. It has been observed that JNK is required for the apoptosis of central nervous system neurons and the expression of dominant negative inhibitors of nuclear JNK confer resistance to their apoptosis following trophic support [40]. These findings suggest an important role of nuclear JNK in promoting apoptotic signaling. It is significant to note here that the non-phosphorylatable mutants of c-Jun confer resistance to apoptosis of MEFs in response to UV-irradiation. Together, these results may be indicative of a role for JNK-c-Jun/Ap1 mediated expression of pro-apoptotic genes in JNK-mediated apoptosis. An alternate pathway contributing to JNKmediated apoptosis involves the phosphorylation of the p53 family of proteins by JNK [41]. It has been suggested that the phosphorylation of p53 at Ser6 by JNK inhibits ubiquitin-mediated degradation of p53 and thereby stabilizing the levels of p53. Recent studies have shown that the phosphorylation of p53 at Ser6 by JNK2 is critically required for the apoptotic pathway [42].
It has been observed that the expression of FDH (10-formyltetrahydrofolate dehydrogenase) is drastically reduced in tumors and its elevation induces p53-dependent apoptosis. Further analyses of this apoptotic event indicated that FDH induces direct phosphorylation of p53 at Ser6 involving both JNK1 and JNK2. Treatment of FDH-expressing cells with JNK inhibitor SP600125 or silencing of JNK1/2 or inhibited the phosphorylation of p53 at Ser6 as well as p53-dependent apoptosis in response to DNA-damage. Since p53 is involved in the expression of several proapoptotic genes such as Bax (Bcl2-associated X protein) and PUMA (p53 up-regulated modulator of apoptosis), it is quite possible that apoptotic pathway activated by JNK involves p53-mediated upregulation of pro-apoptotic genes. DNA damage that activates JNK also causes the stabilization and activation of p73, a member of the p53 family of transcription factors [43].Similar to p53, p73 induces apoptosis by increasing the expression of proapoptotic genes including Bax and PUMA. Analysis of this pathway has shown that JNK is required for p73-mediated apoptosis induced by the DNA damaging agent cisplatin and it involves the phosphorylation of p73 at several serine and threonine residues. Consistent with the notion that JNK and its activity are required for p73-mediated apoptosis, mutation of JNK-phosphorylation site in p53 abrogates cisplatin-induced stabilization of p73 along with a marked reduction in p73-transcriptional activity, and cisplatin-induced apoptosis. Thus, JNK can stimulate the expression of pro-apoptotic genes and decrease the expression pro-survival genes via multiple transcription factors in cell-type and stimuli specific manner.

JNK in Intrinsic Apoptotic Pathway
Cytoplasmic stress and the damage to the DNA activates pro-apoptotic protein Bak or Bax throgh the Bh3 protein, which in turn it induces the mitochondrial outer membrane permeabilization, resulting in release of the pro-apoptotic proteins such as smac/diablo and omi/htra2 and the Cytochrome C from mitochondrial intermembrane space. Release cytocrome c, together with Apaf-1 and caspase 9 from the apoptosomes, that triggers caspase-9 leading to the activation of caspase3 and -7 which results in to cellular apoptosis.Further more the following process potentiated by the OMI and SMAC that inhibits the anti apoptotic protein XIAP. The observation in which the MOMB and the consequesnt release of cytochrome c in reponse to UV irradiation were inhibited in JNK1-/-and JNK2/-MEFsSuggests a role for the kinases in the intrinsic pathway [44].One mechanism through which JNK mediates the release of mitochondrial cytochrome C is through its ability to increase the expression of BAX via transcriptional activation of c-Jun leading to MOMB [45]. JNK triggers the release of cytochrome c through stimulating traslocation of Bax to mitochondria [46]. JNK mediates translocation of Bax by phosphorylation of the 388 phosphorylation of 14-3-3α and/ or 14-3-3ζ protein. Phosphorylation of the 14-3-3 proteins leads to dissociation of BAX following which it translocates to mitochondria to induce MOMB and cytochrome c release. Moreover, to promote its translocation to mitochondria and subsequent release of cytochrome c JNK directly phosphorylates 14-3-3 associated Bad. To promote intrinsic apoptosis further JNK targeting BH3-only family of pro-apoptotic proteins [47]. JNK phosphorylates the pro-apoptotic Bim, BH3 and Bmf during the UV induced apoptosis to release release them from sequestering myosinV motor and dynein complexes -only proteins Bim and Bmf to release them from sequestering dynein and myosin V motor complexes respectively [48]. Further more release of Bim and Bmf can activate Bax or Bax to intitate the apoptosis. [49].To neurtrilize the anti apoptotic activites of BCL2 and BCL2 homologs, the Jnk phosphorylated Bim can bind and neutralize the anti apoptoic-activites of BCL2 homologs and BCL2 [50].
JNK can also promote apoptosis through the phosphorylation of pro-apoptotic protein Bad JNK has also been shown to promote apoptosis through the phosphorylation of the pro-apoptotic protein Bad [51].It was reported that JNK phosphorylates the Ser 123 of BAD and promotes its apoptotic effect of BAD in primary granule neurons of rat cerebellum [51]. During the absence of apoptotic stimuli, the pro survical kineases PAK-1 AK1 and PKA inhibit apoptotic activity of BAD by phosphorylating it at Ser112/136/155, following which the phosphorylaed BAD is sequestered by 14-3-3 family of proteints. Through the phosphorylation of the BAD at Ser 128,an event which inhibits Bad 14-3-3 interaction JNK promotes apoptosis moreover it also potentiates the mechanism through phosphorylating 14-3-3 protein at Ser184 following which 14-3-3 relaease the sequestered Bad [52]. Some evidance showes that JNK promotes apoptosis through the direct phosphorylation of the Bcl-2 to inhibit its anti apoptotic activity.During the paclitaxel induced apoptosis of the breast cancer cells, it has been shown that JNK is activated and the activated JNK phosphorylates Bcl2 at Ser70 to induce apoptosis [53].

JNK in Extrinsic Apoptotic Pathway
Extrinsic apoptotic pathway can be induced through the apoptotic signaling initiated through the activation of death receptors, such as Trail-R1 (DR4), CD95 (Fas,APO-1), TRAIL-R2 (DR5), TNFR1, DR3 (TRAMP) and DR6 through their respective ligands [54].Those death receptors which are activated recruit the adaptor molecules such as FAS assoicated death domain protein that in turn activates caspase 8, which directly activates the caspase 3 and caspase 7 that initiates apoptosis. Activated death receptors recruit the adaptor molecules such as FAS associated death domain protein, which activates caspase 8. Activated caspase 8 directly activates caspase 3 and caspase 7 that initiates apoptosis.However, some of the death receptors can also induce activation of JNKs. Throgh the AP1 mediated increased expression of death receptors, pro-apoptotic proteins such as Bim, Bak [55], Bax [56], Trail-R2 [57] and ligands the JNKs contribute to extrinsic pathway. In HeLa cells TNF-α induced apoptosis, JNK mediated phosphorylation causes the cleavage of Bid.The 21kDa cleaved fragment of the Bid translocates to mitochondria and selectively induces the release of OMI, SMAC/DIABLO, the two of the mitochondria intermembrane proteins that can inhibit the apoptotic protein XIAP, an inhibitor of caspase 8. In addition, OMI inhibits cellular-inhibitor of apoptosis protein 1 or cIAP1, another inhibitor of caspase 8 [58]. Inhibition of XIAP and cIAP1 leads to the activation of the executioner caspases 3 and 7, leading to apoptosis [59]. Even in the absence of such a cleavage, JNK-phosphorylated full length Bid translocates to mitochondria and potentiates extrinsic signaling during TNFinduced apoptosis in PC12 cells [60].Cumulatively, the multiple signaling inputs generated by JNK promotes extrinsic apoptotic pathway JNK signaling in heart failure Heterotrimeric guanine nucleotide-binding proteins (G proteins) of the Gq family transduce signals from a different of widely expressed membrane receptors to generate diverse, tissue-specific effects [61]. In variety of target tissues, receptor mediated activation of Gq regulates physiological responses such as contraction and secretion. A role for Gq-coupled receptors in regulation of cell growth has become apparent only more recently [62]. Cardiac muscle expresses Gq-coupled receptors that do not appear to play a primary role in modulating cardiac contractile function. Rather, the relevant physiological role for Gq-coupled receptor agonists may be stimulation of cardiac hypertrophy. Indeed, multiple Gqcoupled receptor agonists stimulate hypertrophy of cultured neonatal rat cardiac myocytes [63]. In the intact heart, cardiac hypertrophy is typically a compensatory response to increased hemodynamic load. The resulting increase in cardiac mass improves cardiac performance in the short term by reducing wall stress [64]. When the hemodynamic load is not relieved however, the hypertrophied heart ultimately dilates and fails in a phenomenon termed ""decompensation."" A role for Gq-signaling in the development and decompensation of hypertrophy is supported by theeffects of transgenic overexpression of α 1 adrenergic and angiotensin II receptors in cardiomyocytes [65]. In fact, heterozygous transgenic overexpression of Gaq in cardiomyocytes induces cardiac 389 enlargement with many of the molecular ,structural and functional characteristics of pressure overload hypertrophy [66].higher level of Gaq overexpression in dual heterozygous mice causes a lethal dilated congestive cardiomyopathy with myocyte loss in the absence of inflammation, suggesting a role for apoptosis. Although cardiomyocyte apoptosis has been implicated in human cardiomyopathy [67] and animal models of myocardial injury [68], a role for Gq signaling in apoptotic cardiomyocyte death has not been explored, and the relationship between apoptosis and cardiac failure has not been defined adequately. It was explore the role of Gq signaling in cardiomyocyte hypertrophy and apoptosis by using cultured adenovirus-infected neonatal cardiac myocytes or transgenic mice with enhanced G a q signaling. The results demonstrate that sustained high level activation of Gaq can produce apoptotic cardiomyocyte death and can lead to heart failure.In the heart, JNK is activated in response to a variety of pathological insults such as hypertension, ischemia/reperfusion injury, and myocardial infarction [69]. Several studies have implicated JNK signaling as a key mediator of pathological cardiac remodeling and heartfailure.An important role for JNK signaling has been demonstrated in cardiac hypertrophy [70] as well as cardiomyocyte apoptosis [71]. JNK activation to a specific aspect of pathological cardiac remodeling, stress-induced down-regulation of the gap junction protein connexin 43 (Cx43) [72]. Loss of gap junctions is a common feature of the failing heart [73].
JNK signaling and apoptosis in the heart JNK activation begin at the cell membrane by the activation of the Cdc42/Ras/Rac.these these GTP binding proteins directly induce MAPK kinase kinase 1,2,3 and apoptosis signal-regulating kinase-1(ASK1) activation which in turn promote phosphorylation and activation of MAPK kinases, MKK4 and MKK7 that directly phosphorylate and activate JNK [77].There are three different types JNK genes that encode over 10 JNK isoforms through alternative splicing [78]. JNK 1 and JNK2 can be produced in mouse tissues including the heart, while JNK3 can not be produced in heart [79].JNKs can be activated by the vareity of stree and mitogenic stimuli, such as growth factors, cytokines, UV irradiation, toxic compounds, osmotic shock, hypoxia, ischemia and oncogenes [80].Activation of JNK results in activation of several trascription factors such as the as c-jun activating trascription factor-2 (ATF-2),P53,Elk1, nuclear factor of activated T-cells(NFAT) and Sap-1 [77].JNK is the first MAPK activated in the rat heart by production of chronic angiotensin II infusion and in culture by mechanial stretch [81].It was shown that JNK can be activated by Ras in transgenic hearts, by ischemia reperfusion injury and by pressure overload [82]. Moreover JNK can aslo activated by the Gaq couple receptor agonist, such as phenylephrine(PE) which is required for atrial natriuretic factor(ANF) promoter activity in cardiac myocytes [82].Activation of JNK signaling pathway by expression of an activated MKK7 mutant protein in cardiac myocytes induced the characteristic feature of hypertrophy in vitro [83]. Furthermore, expression of MKK4 which is dominant negative attenuated the hypertrophic response of neonatal rat in culture of endothelin 1 in cardiomyocytes [84].Similarly the adenoviral gene transformation of MKK4 protein to the adult rat heart blocked activation of JNK and attenuate the cardiac hypertrophy by pressure overload [85].However , some recent studies in mekk1 gene targeted mice or jnk1 or jnk2 gene targeted mice showed no positive correlation with the cardiac hypertrophic respose in vivo [86].
Although loss of MEKK1 attenuated hypertrophy, cardiomyopathy associaged with Gaq overexpression [87],although loss of mekk1 reduced hypertrophic cardiomyopathy associated with Gaq overexpression [87].While some disagreement are exists to the hypertrophic regulatory role for JNK signaling in the heart. In some studies it has shown a critical role for JNK factors and their upstream activators as affectors of apoptosis in mamalian cells. For example , the nul fibroblasts JNK were demonstrated to be deficient in mitochondria driven apoptosis, suggest that jnk has critical role in this process [88].Similarly, jnk3 null mice showed significant reductions in excitoxicity-induced hippocampal neuron apoptosis [89]. Jnk 2 null mice showed the defect in apoptosis in immature thymocytes [90].While jnk1 or jnk 2 double null mice indicated alterations in region specific apoptosis in developing mouse brain [91]. Up stream of jnk1/2/3, ask null cells indicated significant impairment in H2O2 and TNF a induced cell death [92].Null embryonic mekk1 stem cells can be differentiated in to the cardiac myocytes indicated enhanced oxidative stress induced apoptosis [93].Moreover, jnk2 null mice also showed apoptotic defects in immature thymocytes [90], Consistent with the observation that mekk1 null mice have more cardiac apoptosis following pressure overload stimulation [71]. However,the results observed in mekk1 deficient cells suggest that the JNK signaling branch can serve a protective role, in contrast to data obtained in ask1, mekk1, andjnkgene-targeted mice.However,MEKK1can also influence ERK activation, as well as effect other regulatory events that are independent of JNK signaling,suggesting that greater emphasis should be placed on the jnk gene targeting studies for cause-and-effect assessment [94]. Further investigation into the causality between JNK signaling and cardiac myocyte apoptosis has revealed additional discordance. For example, expression of dominant negative JNK1 or dominant-negative MKK4 in cultured cardiacmyocytes increased nitricoxide-390 inducedcardiomyocyte apoptosis, suggesting a protective role for JNK signaling [95]. Consistent with this study, ischemia-reperfusion induced apoptosis in cultured neonatal cardiomyocytes was increased by expression of JNK inhibitory mutants [96]. In contrast, JNK1 inhibition was recently reported to actually protect cardiac myocytes from ischemia-induced apoptosis, while JNK2 inhibition had no effect [96]. Similarly, inhibition of JNK in H9c2 myocytes blocked apoptosis induced by stressst imulation [97] , while infection of adult cardiacmyocytes with a dominant-negative JNK-expressing adenovirus antagonized H2O2-induced apoptosis [98].Upstream of JNK signaling,targeted deletion of ask1 in mice attinuated the myocyte apoptosis following pressure overload and myocardial infarction, however the expression of an activated ASK1 mutant protein stimulated myocyte apoptosis in culture [99]. The b-adrenergic stimulated apoptosis of cultured adult cardiomyocyte was inhibited through expression of dominant negative JNK by a mitochondrial dependent mechanism [100].In a study by, Aoki et al. It showed that activated JNK and MKK4 localized to the mitochondria,induced cytochrome c release, and promoted apoptosis of cardiac myocytes [101]. Baines et al. Reported that fraction of JNK protein was localized to mitochondria [102], and is consistent with an earlier observation made in fibroblasts, whereas JNK was required for cytochrome c release and mitochondrial-driven cell death [43]. Apoptosis can be induced by JNK signaling through a transcription dependent mechanism which involves the activation of the c-jun and induction of pro apoptotic genes [103].It was also reported that JNK directly phoshphorylate the pro-survival factor Bcl-2, restuling in its activation [104]. With respect to the Bcl-1 family JNK can directly phosphorylate the serine 128 on the pro apoptotic factor Bad, enhancing cell the death through the intrinsic pathway [105]. JNK activation was also recently shown to directly activate Bid cleavage, independent of caspase-8, thus further facilitating the mitochondrial death pathway [106].It was also reported that JNK directly phsophorylate two additional pro -apoptotic memebers of the BH3-only subgroup of bim bcl-2 and bmf, facilitating their translocation to mitchondria [107].

Conclusion:-
We found out that the JNK signaling pathway activated by the biologic stimuli such as the TNF,Gowth factors ,Stress ,Cytokines, ischemia and hypoxia .the overexpression of the following molecule is belived that it causes to the progression of HF by virtue of the deleterious effects they exert on the heart and the circulation. JNK is a master protein kinase that plays an important role in osteoblast proliferation, differentiation and apoptosis. An overview of apoptotic pathway indicates that JNK signaling is involved in the extrinsic apoptotic pathway initiated by death receptors as well as the intrinsic pathway initiated at the mitochondria. In response to both the extrinsic and intrinsic apoptotic stimuli, JNK plays an essential role through its ability to interact and modulate the activities of diverse pro-and anti-apoptotic proteins.