- Email: [email protected]
Role of Raf Proteins in Cardiac Hypertrophy and Cardiomyocyte Survival
Role of Raf Proteins in Cardiac Hypertrophy and Cardiomyocyte Survival Anthony J. Muslin* Cardiomyocyte survival and growth are regulated by the action of extracellular ligands that activate intracellular signaling cascades. The Raf family of protein serine/threonine kinases plays a critical role in the regulation of cardiomyocyte survival and growth. The three Raf family members, Raf-1, B-Raf, and A-Raf, are highly homologous, and they are all expressed in the heart. Protein kinases of the Raf family phosphorylate and activate the mitogen-activated protein kinase kinases (MKKs, also known as MEKs). MKKs, in turn, are dualspecificity threonine and tyrosine kinases that phosphorylate and activate extracellular signal-regulated kinases (ERKs). ERKs phosphorylate a variety of substrates, including nuclear transcription factors, which regulate cell physiology. Raf proteins also have antiapoptotic activity that is independent of MKK and ERK. In this review, the role of Raf family members in the regulation of cardiomyocyte survival and growth will be discussed. (Trends Cardiovasc Med 2005;15:225 – 229) D 2005, Elsevier Inc. The growth and survival of adult cardiomyocytes are regulated by extracellular ligands, growth factors, and cytokines that bind to cell-surface receptors and activate intracellular signal transduction cascades. These signaling pathways control essential processes in cardiomyocytes, including gene transcription, protein translation, cytoskeletal remodeling, contractile function, cell metabolism, and cell survival. The analysis of the function of specific signaling proteins in cardiomyocyte biology is a major goal for cardiovascular scientists.
The Raf family of protein kinases is known to regulate cell survival, differentiation, and growth in many cell types and model organisms. There are three members of the Raf family, A-Raf, B-Raf, and Raf-1 (Hagemann and Rapp 1999, Wellbrock et al. 2004). The role of Raf proteins in human cancer is well known, and activating mutations in B-Raf are observed in two thirds of malignant melanomas and also in many ovarian, colorectal, and papillary thyroid cancers (Davies et al. 2002, GraySchopfer et al. 2005).
Anthony J. Muslin is at the Center for Cardiovascular Research, Washington University School of Medicine, St Louis, Missouri. * Address correspondence to: Anthony J. Muslin, Center for Cardiovascular Research, Washington University School of Medicine, 660 South Euclid Avenue, Box 8086, St. Louis, MO 63110, USA. Tel.: (+1) 314-7473525; fax: (+1) 314-747-3545; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
TCM Vol. 15, No. 6, 2005
Activation of Raf Family Members
Activation of Raf-1 is a complex multistep process (Wellbrock et al. 2004). In general, GTP loading of the ras GTPase is required to initiate Raf-1 activation (Figure 1). Ras activation can occur in response to growth factor receptor-mediated relocation of protein complexes that include guanine nucleotide exchange factors, such as a protein called Son of
sevenless (SOS), to the plasma membrane of cells where ras is constitutively located (Nimnual and Bar-Sagi 2002). SOS-mediated GTP loading alters the conformation of ras, and this promotes the interaction of ras with a variety of effectors, including Raf-1. Inactive Raf-1 is bound to 14-3-3 proteins and heat shock proteins in the cytosol of cells. In the inactive conformation, the amino-terminal portion of Raf-1 is thought to bind to and inactivate the carboxy-terminal kinase domain. 14-3-3 Protein dimers, by binding simultaneously to phosphorylated serine residues in both the aminoand carboxy-terminal portion of Raf-1 (serines 259 and 621), may stabilize this inactive conformation (Figure 1) (Muslin et al. 1996). GTP-bound ras triggers Raf-1 activation by binding to the amino-terminal portion of Raf-1 (Moodie et al. 1993, Vojtek et al. 1993). Ras–GTP binding to Raf-1 displaces 14-3-3 from its amino-terminal binding site at serine 259 (Rommel et al. 1997), and this may promote the dissociation of the amino-terminal portion of Raf-1 from the carboxy-terminal kinase domain (Figure 1). Dephosphorylation of serine 259 in Raf-1 by protein phosphatase 2A (PP2A) also promotes the dissociation of 14-3-3 from the amino-terminal portion of Raf-1 (Ory et al. 2003). After ras–GTP binding, Raf-1 is further modified by a series of phosphorylation events. In particular, Src tyrosine kinase and possibly p21-activated kinase (PAK) are thought to phosphorylate Raf1 in the hinge region that separates the amino-terminal portion from the carboxy-terminal region (Chaudhary et al. 2000, King et al. 1998, Mason et al. 1999). Src family members phosphorylate tyrosine 341 of Raf-1, and PAK or an unidentified kinase phosphorylates serine 338 of Raf-1 (Figure 1). Phosphorylation of the hinge region is predicted to disrupt the interaction of the amino- and carboxy-terminal portion of Raf-1. Activation of Raf-1 also depends on the phosphorylation of two additional sites within the carboxy-terminal kinase domain, threonine 491 and serine 494, and these residues may be autophosphorylation sites (Chong et al. 2001). Activation of A-Raf is similar to that of Raf-1 and also depends on recruitment to the plasma membrane by ras GTP, displacement of 14-3-3 binding
225
Figure 1. A schematic model of Raf-1 activation. In the inactive conformation, Raf-1 is bound to 14-3-3 dimers and is phosphorylated on serines 259 and 621, but is not phosphorylated at the hinge region (serine 338, tyrosine 341). When ras is loaded with GTP, it binds to the aminoterminal portion of Raf-1, displacing 14-3-3, and serine 259 is dephosphorylated by PP2A. Raf-1 is then phosphorylated by PAK or an identified kinase at serine 338 and by Src at tyrosine 341. Finally, phosphorylation of two additional residues in the kinase domain, threonine 491 and serine 494, perhaps by autophosphorylation, results in maximal kinase activation of Raf-1.
from the amino-terminal regulatory domain, phosphorylation of a threonine residue and a tyrosine residue in the hinge region, and phosphorylation of a threonine and a serine residue in the kinase domain (Wellbrock et al. 2004). In contrast, activation of B-Raf is somewhat simpler than that of Raf-1. B-Raf does not have a tyrosine residue that corresponds to tyrosine 341 in the hinge region of Raf-1 but instead contains an aspartic acid residue at this position (aspartic acid 448). In addition, the threonine residue in B-Raf (serine 445) that corresponds to threonine 338 in the hinge region of Raf-1 is constitutively phosphorylated in B-Raf (Mason et al. 1999). As a result of these differences, B-Raf has higher basal kinase activity than Raf-1. Moreover, B-Raf is more robustly activated by ras– GTP than Raf-1 (Mason et al. 1999). Indeed, Wellbrock proposed that B-Raf is the major family member that couples ras to extracellular signal-regulated kinase (ERK) activation in most cell types, and that Raf-1 and A-Raf play only supplementary roles in this process (Wellbrock et al. 2004).
226
The ERK Cascade
Three- and four-component intracellular protein kinase cascades are thought to exist as a mechanism for massive signal amplification in response to an initiating stimulus. The best-described three-component kinase cascade is the ERK pathway that includes the initiating kinase Raf-1, B-Raf, or A-Raf, the middle kinase mitogen-activated protein kinase kinase (MKK) 1 or MKK2 (hereafter referred to as MKK), and the terminal kinase ERK1 or ERK2 (hereafter referred to as ERK) (Guan 1994, Seger and Krebs 1995). Once activated, Raf proteins phosphorylate MKK on two serine residues, resulting in increased MKK kinase activity. Activated MKK, in turn, activates ERK by phosphorylating it on a threonine residue and a tyrosine residue. Scaffolding proteins such as KSR-1 may be required for the proper coordination, localization, and efficiency of this triple kinase cascade (Roy et al. 2002, Xing et al. 1997). Activated ERK phosphorylates a variety of cytosolic and nuclear target proteins, including transcription factors, cytoskeletal proteins, protein kinases,
translation factors, and proapoptotic proteins. In particular, ERK phosphorylates the transcription factors Elk-1 and GATA-4 that are involved in a variety of growth responses in the heart (Babu et al. 2000, Purcell et al. 2004). Furthermore, ERK phosphorylates eIF4E-binding protein 1 (4E-BP1) and indirectly activates the ribosomal S6 kinase to promote the translation of proteins (Wang et al. 2001, Wang and Proud 2002). Moreover, ERK phosphorylates the proapoptotic proteins caspase 9 and BIM, reducing the ability of these proteins to trigger programmed cell death (Allan et al. 2003, Harada et al. 2004). ERK identifies its substrates based on the presence of a target serine or threonine residue adjacent to a proline residue, typically in the motif P-x-S-P, where x is any amino acid. In addition, ERK identifies substrates based on the presence of docking sites that are near to the P-x-S-P motif (Jacobs et al. 1999).
MKK-Independent Functions of Raf Proteins
Although the role of Raf kinases in the ras–Raf–MKK–ERK cascade is well established, additional MKK- and ERKindependent functions of Raf kinases may exist. For example, mice deficient in Raf-1 exhibit normal ERK activation in many tissues but do not survive embryonic development and die by embryonic day 16.5 (Mikula et al. 2001). Raf-1 / embryos display growth retardation with vascular defects in the yolk sac and placenta (Huser et al. 2001, Mikula et al. 2001). In addition, Raf-1 / embryos display markedly increased apoptosis in a variety of embryonic tissues, including fetal liver. Raf-1 / embryonic fibroblasts are sensitized to apoptosis induced by serum withdrawal or Fas activation. Raf-1 / embryonic fibroblasts exhibit normal MKK and ERK activation in response to EGF stimulation, but B-Raf kinase activity is increased in these cells (Mikula et al. 2001), and this may compensate for the absence of Raf-1 to maintain normal ERK signaling. Murine knockout studies show that Raf-1 has an antiapoptotic function independent of the ERK cascade, but the antiapoptotic targets of Raf-1 are not well defined. Two potential antiapoptotic targets of Raf-1 are the apoptosis TCM Vol. 15, No. 6, 2005
signal-regulating kinase 1 (ASK1) and the mammalian sterile 20-like kinase 2 (MST2) (O’Neill et al. 2004, Chen et al. 2001). ASK1 is a critical mediator of cell death in response to several stimuli, including oxidative stress, DNA damage, TNF-a treatment, and Fas activation. Activated ASK1 promotes activation of the JNK and p38 MAPK cascades. Coimmunoprecipitation studies were performed revealing that Raf-1 binds to the amino-terminal portion of ASK1 and inactivates its kinase activity (Chen et al. 2001). Raf-1-mediated inhibition of ASK1 is not dependent on Raf-1 kinase activity. It is not apparent whether A-Raf or B-Raf also interacts with ASK1. ASK1 is expressed in cardiomyocytes and is activated in response to proapoptotic stimuli in heart (Yamaguchi et al. 2003). Neonatal murine cardiomyocytes lacking ASK1 are resistant to H2O2induced apoptosis. Raf-1 was also found to interact with MST2 in a protein interaction screen (O’Neill et al. 2004). Catalytically inactive forms of Raf-1 were found to also bind to MST2. MST2 is a kinase that is activated by proapoptotic agents, including staurosporine and anti-Fas antibodies (Taylor et al. 1996). Raf-1 and MST2 were found to form a complex in COS cells, and Raf1 binding prevents MST2 dimerization and activation. Interestingly, Raf-1 / embryonic fibroblasts treated with siRNAs directed against MST2 reduced the sensitivity of these cells to proapoptotic stimuli. Furthermore, acute depletion of Raf-1 from several different cell lines resulted in increased MST2 activity. MST2 is widely expressed but is only present at low levels in cardiomyocytes. It is not known whether A-Raf and B-Raf also interact with MST2. Another potential MKK-independent function of Raf family members is to activate the transcription factor nuclear factor nB (NF-nB) (Baumann et al. 2000, Ikenoue et al. 2003). Both Raf-1 and B-Raf activate NF-nB in noncardiac cells through an unknown mechanism, and dominant negative MKK does not block Raf-1-mediated NF-nB activation (Baumann et al. 2000). Finally, the phosphatase CDC25C and the retinoblastoma tumor suppressor protein (Rb) have been proposed to be direct Raf-1 substrates (Wellbrock et al. 2004), although the in vivo significance of these targets is uncertain. TCM Vol. 15, No. 6, 2005
Analysis of Raf Function in Cultured Cardiomyocytes and in Rodent Model Systems
A large number of investigators previously examined the role of Raf-1, MKK, and ERK in the growth of neonatal rat cardiomyocytes in culture. Various studies relied on the expression of dominant negative forms of these signaling proteins or on the use of chemical inhibitors of various components of this cascade. Many—but not all—of these studies support the model that Raf, MKK, and ERK activation are required for the ligandinduced growth of cardiomyocytes (Bueno and Molkentin 2002). In particular, Yue et al. (2000) treated rat neonatal cardiomyocytes with a selective MKK inhibitor, U0126, a Raf family member inhibitor, SB-386023, or an adenovirus encoding dominant negative Raf-1. All three reagents blocked endothelin1-induced and phenylephrine-induced cardiomyocyte growth, sarcomeric reorganization, and atrial natriuretic factor gene expression. Similarly, Ueyama et al. (2000) showed that adenovirus-mediated overexpression of dominant negative MKK1 in cultured neonatal cardiac myocytes blocked endothelin-1-, phenylephrine-, isoproterenol-, leukemia inhibitory factor-, and mechanical stretch-induced growth and sarcomeric reorganization. The role of the ras– Raf–MKK–ERK cascade in cardiac hypertrophy is supported by studies with transgenic mice, in which cardiacspecific overexpression of activated MKK1 resulted in profound cardiac hypertrophy with preserved contractile function (Bueno et al. 2000). To evaluate the role of Raf kinases in cardiac disease, we generated transgenic mice with cardiac-specific expression of a dominant negative form of Raf-1 (DNRaf) that is mutated at the ATP binding site in the kinase domain (Harris et al. 2004). Multiple transgenic lines were obtained, and the highest-expressing line, with integration of four copies of the transgene (DN-Raf-4x), exhibited twice as much DN-Raf protein as native Raf-1 in cardiac lysates. DN-Raf-4x mice appeared normal at baseline and had normal cardiac structure and contractile function. In response to pressure overload by transverse aortic constriction (TAC), ERK activation was reduced in DN-Raf-4x transgenic mice when com-
pared to wild-type littermates (Harris et al. 2004). Furthermore, DN-Raf-4x transgenic mice exhibited markedly reduced survival, with 65% of mice alive 7 days after TAC compared to a survival rate of 100% for nontransgenic littermates. Reduced survival in DN-Raf-4x transgenic mice was associated with increased cardiomyocyte apoptosis. DNRaf-4x transgenic mice that survived TAC had reduced cardiac hypertrophy when compared to nontransgenic littermates, despite similar pressure gradients achieved by aortic constriction. Reduced cardiac hypertrophy in DN-Raf-4x transg e n i c m i c e w a s a s s o c i a t e d w i th decreased expression of the atrial natriuretic factor and h-myosin heavy-chain genes. These results suggest that Raf-1 regulates cardiomyocyte survival and also hypertrophic growth. However, this mouse model relies on the overexpression of a mutant form of Raf-1. DN-Raf binds to MKK1/2 and likely blocks activation of MKK by Raf-1, B-Raf, and ARaf (Hindley and Kolch 2002). Yamaguchi et al. (2004) recently generated a murine model with cardiacspecific disruption of the Raf-1 gene (Raf CKO). In this model system, Raf-1 floxed-allele mice were bred with transgenic mice with cardiac-specific overexpression of Cre recombinase. Raf CKO mice were born at mendelian ratios and were fertile, with a normal life span. The cardiac function of Raf CKO mice, however, was not normal, and by 10 weeks of age, they displayed markedly reduced systolic function with left ventricular dilatation by echocardiography. Furthermore, posterior wall thickness was reduced in Raf CKO mice at 10 weeks of age, suggestive of reduced cardiomyocyte growth or increased apoptosis. Cardiac catheterization of 10-week-old Raf CKO mice showed reduced systolic and diastolic function. Increased cardiomyocyte apoptosis was observed in Raf CKO heart at 3 to 5 weeks of age. Activation of cardiac MKK and ERK was not reduced in Raf CKO mice after infusion of endothelin 1 when compared to control mice. Interestingly, cardiac ASK1, JNK, and p38 MAPK activities were increased in Raf CKO mice at baseline. To determine whether increased ASK1 activity was responsible for the cardiac phenotype, Raf CKO mice were bred with ASK1 / mice. Raf CKO ASK1 / mice exhibited improved con-
227
Figure 2. Model of Raf kinase action in cardiomyocyte growth and survival. Simplified model of intracellular signaling cascades regulated by Raf family members.
tractile function and reduced chamber dilatation when compared to Raf CKO ASK1+/+ mice. Furthermore, the incidence of cardiomyocyte apoptosis was reduced in Raf CKO ASK1 / mice when compared to Raf CKO ASK1+/+ mice. These results suggest that Raf-1 has a critical antiapoptotic effect in cardiomyocytes that is independent of MKK and ERK, and that depends, at least in part, on the inhibition of ASK1.
regulate cardiomyocyte growth in response to pressure overload via the ERK signaling cascade (Figure 2). Analysis of mice with cardiac-specific disruption of B-Raf, MKK, or ERK will provide important additional information to elucidate the specific role of these proteins in the cardiac hypertrophic growth program and also in cardiomyocyte survival. This proposed signaling model of Raf family member activity in cardiac biology has implications for human disease. The potent antiapoptotic activity of Raf family members suggests that agents that reduce Raf protein levels or kinase activity in heart may sensitize patients to the development of cardiac dysfunction and congestive heart failure while antagonizing the development of cardiac hypertrophy. Pharmacological agents that inhibit the kinase activity of Raf proteins exist, and some have been used in clinical studies for the treatment of cancer (Strumberg and Seeber 2005). On the other hand, agents that increase Raf protein levels or kinase activity may promote the development of cardiac hypertrophy and may also block the development of cardiac dysfunction. A variety of growth factors, cytokines, and ligands, including some that are commercially available, activate Raf proteins in tissues.
Conclusion
Recent studies with genetically modified mice have investigated the role of Raf proteins in cardiomyocyte growth and survival (Harris et al. 2004, Yamaguchi et al. 2004). One conclusion that emerges from these studies is that Raf-1 has potent antiapoptotic activity in cardiomyocytes that is independent of MKK and ERK activation and that may be dependent on its ability to block ASK1 (Figure 2). It is important to note that MKK and ERK also have antiapoptotic activity in cardiomyocytes that is distinct from the ability of Raf-1 to inhibit ASK1 (Bueno and Molkentin 2002), and this antiapoptotic activity may be due to the ability of ERK to phosphorylate and inactivate caspase 9 and BIM (Allan et al. 2003; Harada et al. 2004). Therefore, Raf family members have antiapoptotic activity that is both dependent and independent of ERK activation (Figure 2). Another conclusion that emerges from studies with genetically modified mice is that Raf family members, including Raf-1, B-Raf, and possibly A-Raf,
228
References Allan LA, Morrice N, Brady S, et al.: 2003. Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat Cell Biol 7:647–654. Babu GJ, Lalli MJ, Sussman MA, et al.: 2000. Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy. J Mol Cell Cardiol 32:1447–1457. Baumann B, Weber CK, Troppmair J, et al.: 2000. Raf induces NF-nB by membrane shuttle kinase MEKK1, a signaling pathway critical for transformation. Proc Natl Acad Sci USA 97:4615– 4620. Bueno OF, Molkentin JD: 2002. Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res 91:776–781. Bueno OF, De Windt LJ, Tymitz KM, et al.: 2000. The MEK1–ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 19: 6341– 6350. Chaudhary A, King WG, Mattaliano MD, et al.: 2000. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 10:551–554.
Chen J, Fujii K, Zhang L, et al.: 2001. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism. Proc Natl Acad Sci USA 98:7783–7788. Chong H, Lee J, Guan KL: 2001. Positive and negative regulation of Raf kinase activity by phosphorylation. EMBO J 20:3716–3727. Davies H, Bignell GR, Cox C, et al.: 2002. Mutations of the BRAF gene in human cancer. Nature 417:949–954. Gray-Schopfer VC, Da Rocha Dias S, Marais R: 2005. The role of B-RAF in melanoma. Cancer Metastasis Rev 24:165–183. Guan KL: 1994. The mitogen activated protein kinase signal transduction pathway: From the cell surface to the nucleus. Cell Signal 6:581–589. Hagemann C, Rapp UR: 1999. Isotype-specific functions of Raf kinases. Exp Cell Res 253:34 – 46. Harada H, Quearry B, Ruiz-Vela A, Korsmeyer SJ: 2004. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. Proc Natl Acad Sci USA 43:15313–15317. Harris IS, Zhang S, Treskov I, et al.: 2004. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 110:718–723. Hindley A, Kolch W: 2002. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J Cell Sci 115 (Pt. 8): 1575–1581. Huser M, Luckett J, Chiloeches A, et al.: 2001. MEK kinase activity is not necessary for Raf-1 function. EMBO J 20:1940–1951. Ikenoue T, Hikiba Y, Kanai F, et al.: 2003. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res 63:8132–8137. Jacobs D, Glossip D, Xing H, et al.: 1999. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev 13:163–175. King AJ, Sun H, Diaz B, et al.: 1998. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature 396:180–183. Mason CS, Springer CJ, Cooper RG, et al.: 1999. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18:2137–2148. Mikula M, Schreiber M, Husak Z, et al.: 2001. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 20:1952–1962. Moodie SA, Willumsen BM, Weber MJ, et al.: 1993. Complexes of Ras. GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658–1661.
TCM Vol. 15, No. 6, 2005
Muslin AJ, Tanner JW, Allen PM, Shaw AS: 1996. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84:889–897.
Yamaguchi O, Watanabe T, Nishida K, et al.: 2004. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114:937–943.
Nimnual A, Bar-Sagi D: 2002. The two hats of SOS. Sci STKE 145:PE36.
Yue TL, Gu JL, Wang C, et al.: 2000. Extracellular signal-regulated kinase plays an
essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy. J Biol Chem 275:37895–37901. PII S1050-1738(05)00089-7
TCM
O’Neill E, Rushworth L, Baccarini M, Kolch W: 2004. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306:2267–2270. Ory S, Zhou M, Conrads TP, et al.: 2003. Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol 13:1356–1364. Purcell NH, Darwis D, Bueno OF, et al.: 2004. Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes. Mol Cell Biol 24:1081–1095. Rommel C, Radziwill G, Moelling K, Hafen E: 1997. Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo. Mech Dev 64:95–104. Roy F, Laberge G, Douziech M, et al.: 2002. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16:427– 438. Seger R, Krebs EG: 1995. The MAPK signaling cascade. FASEB J 9:726–735. Strumberg D, Seeber S: 2005. Raf kinase inhibitors in oncology. Onkologie 28:101–107. Taylor LK, Wang HC, Erikson RL: 1996. Newly identified stress-responsive protein kinases, Krs-1 and Krs-2. Proc Natl Acad Sci USA 93:10099–10104. Ueyama T, Kawashima S, Sakoda T, et al.: 2000. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy. J Mol Cell Cardiol 32:947–960. Vojtek AB, Hollenberg SM, Cooper JA: 1993. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74: 205–214. Wang L, Proud CG: 2002. Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res 91:821–829. Wang L, Gout I, Proud CG: 2001. Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes. J Biol Chem 276:32670–32677. Wellbrock C, Karasarides M, Marais R: 2004. The RAF proteins take centre stage. Nat Rev Mol Cell Biol 5:875–885. Xing H, Kornfeld K, Muslin AJ: 1997. The protein kinase KSR interacts with 14-3-3 protein and Raf. Curr Biol 7:294 –300. Yamaguchi O, Higuchi Y, Hirotani S, et al.: 2003. Targeted deletion of apoptosis signalregulating kinase 1 attenuates left ventricular remodeling. Proc Natl Acad Sci USA 100:15883 –15888.
TCM Vol. 15, No. 6, 2005
Cardiac Stem and Progenitor Cell Biology for Regenerative Medicine Daniele Torella*, Georgina M. Ellison, Bernardo Nadal-Ginard, and Ciro Indolfi* Stem cell therapy is a new and promising treatment of heart disease. However, the race is still on to find the bbestQ cell to reconstitute the myocardium and improve function after myocardial damage. The recent discovery in the adult mammalian myocardium of a small cell population with the phenotype, behavior, and regenerative potential of cardiac stem and progenitor cells has proposed these cells as the most appropriate for cell therapy. The existence of these cells has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. These findings have placed the heart squarely among other organs with regenerative potential despite the fact that the working myocardium is mainly constituted of terminally differentiated cells. Although CSCs (cardiac cells proven to have stem and/or progenitor characteristics) can be isolated and amplified in vitro or stimulated to differentiate in situ, it has become reasonable to exploit this endogenous regenerative potential to replace the lost muscle with autologous functional myocardium. Therefore, it is imperative to obtain a better understanding of the biology and regenerative potential of the endogenous CSCs. This will enable us to design better protocols for the regeneration of functional contractile mass after myocardial injury. (Trends Cardiovasc Med 2005;15:229 – 236) D 2005, Elsevier Inc.
Daniele Torella and Ciro Indolfi are at the Division of Cardiology, Magna Graecia University, Catanzaro, Italy. Georgina M. Ellison and Bernardo Nadal-Ginard are at the Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York. * Address correspondence to: Ciro Indolfi, MD, FACC, FESC, Professor of Cardiology and Daniele Torella, MD, are to be contacted at Laboratory of Molecular and Cellular Cardiology, Division of Cardiology, Department of Clinical and Experimental Medicine, Magna Graecia University, Via Tommaso Campanella, 115, Catanzaro 88100, Italy. Tel.: (+39) 0961-712-310; fax: (+39) 0961-712-445; e-mails: [email protected], [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
Nowadays, the prevention and treatment of atherosclerotic heart disease have substantially reduced cardiovascular morbidity. Nonetheless, once damage to the heart is established and heart failure is overt, no therapies can improve cardiac function in the long term (Jessup and Brozena 2003). Owing to the present era of regenerative medicine and the recent excitement regarding stem cell therapies, attention has focused on the possibility of regenerating myocardial tissue to treat cardiac diseases (Nadal-Ginard et al. 2003, Korbling and Estrov 2003). The majority of cardiac myocytes exit the cell cycle after a terminal round of
229
The Raf family of protein kinases is known to regulate cell survival, differentiation, and growth in many cell types and model organisms. There are three members of the Raf family, A-Raf, B-Raf, and Raf-1 (Hagemann and Rapp 1999, Wellbrock et al. 2004). The role of Raf proteins in human cancer is well known, and activating mutations in B-Raf are observed in two thirds of malignant melanomas and also in many ovarian, colorectal, and papillary thyroid cancers (Davies et al. 2002, GraySchopfer et al. 2005).
Anthony J. Muslin is at the Center for Cardiovascular Research, Washington University School of Medicine, St Louis, Missouri. * Address correspondence to: Anthony J. Muslin, Center for Cardiovascular Research, Washington University School of Medicine, 660 South Euclid Avenue, Box 8086, St. Louis, MO 63110, USA. Tel.: (+1) 314-7473525; fax: (+1) 314-747-3545; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
TCM Vol. 15, No. 6, 2005
Activation of Raf Family Members
Activation of Raf-1 is a complex multistep process (Wellbrock et al. 2004). In general, GTP loading of the ras GTPase is required to initiate Raf-1 activation (Figure 1). Ras activation can occur in response to growth factor receptor-mediated relocation of protein complexes that include guanine nucleotide exchange factors, such as a protein called Son of
sevenless (SOS), to the plasma membrane of cells where ras is constitutively located (Nimnual and Bar-Sagi 2002). SOS-mediated GTP loading alters the conformation of ras, and this promotes the interaction of ras with a variety of effectors, including Raf-1. Inactive Raf-1 is bound to 14-3-3 proteins and heat shock proteins in the cytosol of cells. In the inactive conformation, the amino-terminal portion of Raf-1 is thought to bind to and inactivate the carboxy-terminal kinase domain. 14-3-3 Protein dimers, by binding simultaneously to phosphorylated serine residues in both the aminoand carboxy-terminal portion of Raf-1 (serines 259 and 621), may stabilize this inactive conformation (Figure 1) (Muslin et al. 1996). GTP-bound ras triggers Raf-1 activation by binding to the amino-terminal portion of Raf-1 (Moodie et al. 1993, Vojtek et al. 1993). Ras–GTP binding to Raf-1 displaces 14-3-3 from its amino-terminal binding site at serine 259 (Rommel et al. 1997), and this may promote the dissociation of the amino-terminal portion of Raf-1 from the carboxy-terminal kinase domain (Figure 1). Dephosphorylation of serine 259 in Raf-1 by protein phosphatase 2A (PP2A) also promotes the dissociation of 14-3-3 from the amino-terminal portion of Raf-1 (Ory et al. 2003). After ras–GTP binding, Raf-1 is further modified by a series of phosphorylation events. In particular, Src tyrosine kinase and possibly p21-activated kinase (PAK) are thought to phosphorylate Raf1 in the hinge region that separates the amino-terminal portion from the carboxy-terminal region (Chaudhary et al. 2000, King et al. 1998, Mason et al. 1999). Src family members phosphorylate tyrosine 341 of Raf-1, and PAK or an unidentified kinase phosphorylates serine 338 of Raf-1 (Figure 1). Phosphorylation of the hinge region is predicted to disrupt the interaction of the amino- and carboxy-terminal portion of Raf-1. Activation of Raf-1 also depends on the phosphorylation of two additional sites within the carboxy-terminal kinase domain, threonine 491 and serine 494, and these residues may be autophosphorylation sites (Chong et al. 2001). Activation of A-Raf is similar to that of Raf-1 and also depends on recruitment to the plasma membrane by ras GTP, displacement of 14-3-3 binding
225
Figure 1. A schematic model of Raf-1 activation. In the inactive conformation, Raf-1 is bound to 14-3-3 dimers and is phosphorylated on serines 259 and 621, but is not phosphorylated at the hinge region (serine 338, tyrosine 341). When ras is loaded with GTP, it binds to the aminoterminal portion of Raf-1, displacing 14-3-3, and serine 259 is dephosphorylated by PP2A. Raf-1 is then phosphorylated by PAK or an identified kinase at serine 338 and by Src at tyrosine 341. Finally, phosphorylation of two additional residues in the kinase domain, threonine 491 and serine 494, perhaps by autophosphorylation, results in maximal kinase activation of Raf-1.
from the amino-terminal regulatory domain, phosphorylation of a threonine residue and a tyrosine residue in the hinge region, and phosphorylation of a threonine and a serine residue in the kinase domain (Wellbrock et al. 2004). In contrast, activation of B-Raf is somewhat simpler than that of Raf-1. B-Raf does not have a tyrosine residue that corresponds to tyrosine 341 in the hinge region of Raf-1 but instead contains an aspartic acid residue at this position (aspartic acid 448). In addition, the threonine residue in B-Raf (serine 445) that corresponds to threonine 338 in the hinge region of Raf-1 is constitutively phosphorylated in B-Raf (Mason et al. 1999). As a result of these differences, B-Raf has higher basal kinase activity than Raf-1. Moreover, B-Raf is more robustly activated by ras– GTP than Raf-1 (Mason et al. 1999). Indeed, Wellbrock proposed that B-Raf is the major family member that couples ras to extracellular signal-regulated kinase (ERK) activation in most cell types, and that Raf-1 and A-Raf play only supplementary roles in this process (Wellbrock et al. 2004).
226
The ERK Cascade
Three- and four-component intracellular protein kinase cascades are thought to exist as a mechanism for massive signal amplification in response to an initiating stimulus. The best-described three-component kinase cascade is the ERK pathway that includes the initiating kinase Raf-1, B-Raf, or A-Raf, the middle kinase mitogen-activated protein kinase kinase (MKK) 1 or MKK2 (hereafter referred to as MKK), and the terminal kinase ERK1 or ERK2 (hereafter referred to as ERK) (Guan 1994, Seger and Krebs 1995). Once activated, Raf proteins phosphorylate MKK on two serine residues, resulting in increased MKK kinase activity. Activated MKK, in turn, activates ERK by phosphorylating it on a threonine residue and a tyrosine residue. Scaffolding proteins such as KSR-1 may be required for the proper coordination, localization, and efficiency of this triple kinase cascade (Roy et al. 2002, Xing et al. 1997). Activated ERK phosphorylates a variety of cytosolic and nuclear target proteins, including transcription factors, cytoskeletal proteins, protein kinases,
translation factors, and proapoptotic proteins. In particular, ERK phosphorylates the transcription factors Elk-1 and GATA-4 that are involved in a variety of growth responses in the heart (Babu et al. 2000, Purcell et al. 2004). Furthermore, ERK phosphorylates eIF4E-binding protein 1 (4E-BP1) and indirectly activates the ribosomal S6 kinase to promote the translation of proteins (Wang et al. 2001, Wang and Proud 2002). Moreover, ERK phosphorylates the proapoptotic proteins caspase 9 and BIM, reducing the ability of these proteins to trigger programmed cell death (Allan et al. 2003, Harada et al. 2004). ERK identifies its substrates based on the presence of a target serine or threonine residue adjacent to a proline residue, typically in the motif P-x-S-P, where x is any amino acid. In addition, ERK identifies substrates based on the presence of docking sites that are near to the P-x-S-P motif (Jacobs et al. 1999).
MKK-Independent Functions of Raf Proteins
Although the role of Raf kinases in the ras–Raf–MKK–ERK cascade is well established, additional MKK- and ERKindependent functions of Raf kinases may exist. For example, mice deficient in Raf-1 exhibit normal ERK activation in many tissues but do not survive embryonic development and die by embryonic day 16.5 (Mikula et al. 2001). Raf-1 / embryos display growth retardation with vascular defects in the yolk sac and placenta (Huser et al. 2001, Mikula et al. 2001). In addition, Raf-1 / embryos display markedly increased apoptosis in a variety of embryonic tissues, including fetal liver. Raf-1 / embryonic fibroblasts are sensitized to apoptosis induced by serum withdrawal or Fas activation. Raf-1 / embryonic fibroblasts exhibit normal MKK and ERK activation in response to EGF stimulation, but B-Raf kinase activity is increased in these cells (Mikula et al. 2001), and this may compensate for the absence of Raf-1 to maintain normal ERK signaling. Murine knockout studies show that Raf-1 has an antiapoptotic function independent of the ERK cascade, but the antiapoptotic targets of Raf-1 are not well defined. Two potential antiapoptotic targets of Raf-1 are the apoptosis TCM Vol. 15, No. 6, 2005
signal-regulating kinase 1 (ASK1) and the mammalian sterile 20-like kinase 2 (MST2) (O’Neill et al. 2004, Chen et al. 2001). ASK1 is a critical mediator of cell death in response to several stimuli, including oxidative stress, DNA damage, TNF-a treatment, and Fas activation. Activated ASK1 promotes activation of the JNK and p38 MAPK cascades. Coimmunoprecipitation studies were performed revealing that Raf-1 binds to the amino-terminal portion of ASK1 and inactivates its kinase activity (Chen et al. 2001). Raf-1-mediated inhibition of ASK1 is not dependent on Raf-1 kinase activity. It is not apparent whether A-Raf or B-Raf also interacts with ASK1. ASK1 is expressed in cardiomyocytes and is activated in response to proapoptotic stimuli in heart (Yamaguchi et al. 2003). Neonatal murine cardiomyocytes lacking ASK1 are resistant to H2O2induced apoptosis. Raf-1 was also found to interact with MST2 in a protein interaction screen (O’Neill et al. 2004). Catalytically inactive forms of Raf-1 were found to also bind to MST2. MST2 is a kinase that is activated by proapoptotic agents, including staurosporine and anti-Fas antibodies (Taylor et al. 1996). Raf-1 and MST2 were found to form a complex in COS cells, and Raf1 binding prevents MST2 dimerization and activation. Interestingly, Raf-1 / embryonic fibroblasts treated with siRNAs directed against MST2 reduced the sensitivity of these cells to proapoptotic stimuli. Furthermore, acute depletion of Raf-1 from several different cell lines resulted in increased MST2 activity. MST2 is widely expressed but is only present at low levels in cardiomyocytes. It is not known whether A-Raf and B-Raf also interact with MST2. Another potential MKK-independent function of Raf family members is to activate the transcription factor nuclear factor nB (NF-nB) (Baumann et al. 2000, Ikenoue et al. 2003). Both Raf-1 and B-Raf activate NF-nB in noncardiac cells through an unknown mechanism, and dominant negative MKK does not block Raf-1-mediated NF-nB activation (Baumann et al. 2000). Finally, the phosphatase CDC25C and the retinoblastoma tumor suppressor protein (Rb) have been proposed to be direct Raf-1 substrates (Wellbrock et al. 2004), although the in vivo significance of these targets is uncertain. TCM Vol. 15, No. 6, 2005
Analysis of Raf Function in Cultured Cardiomyocytes and in Rodent Model Systems
A large number of investigators previously examined the role of Raf-1, MKK, and ERK in the growth of neonatal rat cardiomyocytes in culture. Various studies relied on the expression of dominant negative forms of these signaling proteins or on the use of chemical inhibitors of various components of this cascade. Many—but not all—of these studies support the model that Raf, MKK, and ERK activation are required for the ligandinduced growth of cardiomyocytes (Bueno and Molkentin 2002). In particular, Yue et al. (2000) treated rat neonatal cardiomyocytes with a selective MKK inhibitor, U0126, a Raf family member inhibitor, SB-386023, or an adenovirus encoding dominant negative Raf-1. All three reagents blocked endothelin1-induced and phenylephrine-induced cardiomyocyte growth, sarcomeric reorganization, and atrial natriuretic factor gene expression. Similarly, Ueyama et al. (2000) showed that adenovirus-mediated overexpression of dominant negative MKK1 in cultured neonatal cardiac myocytes blocked endothelin-1-, phenylephrine-, isoproterenol-, leukemia inhibitory factor-, and mechanical stretch-induced growth and sarcomeric reorganization. The role of the ras– Raf–MKK–ERK cascade in cardiac hypertrophy is supported by studies with transgenic mice, in which cardiacspecific overexpression of activated MKK1 resulted in profound cardiac hypertrophy with preserved contractile function (Bueno et al. 2000). To evaluate the role of Raf kinases in cardiac disease, we generated transgenic mice with cardiac-specific expression of a dominant negative form of Raf-1 (DNRaf) that is mutated at the ATP binding site in the kinase domain (Harris et al. 2004). Multiple transgenic lines were obtained, and the highest-expressing line, with integration of four copies of the transgene (DN-Raf-4x), exhibited twice as much DN-Raf protein as native Raf-1 in cardiac lysates. DN-Raf-4x mice appeared normal at baseline and had normal cardiac structure and contractile function. In response to pressure overload by transverse aortic constriction (TAC), ERK activation was reduced in DN-Raf-4x transgenic mice when com-
pared to wild-type littermates (Harris et al. 2004). Furthermore, DN-Raf-4x transgenic mice exhibited markedly reduced survival, with 65% of mice alive 7 days after TAC compared to a survival rate of 100% for nontransgenic littermates. Reduced survival in DN-Raf-4x transgenic mice was associated with increased cardiomyocyte apoptosis. DNRaf-4x transgenic mice that survived TAC had reduced cardiac hypertrophy when compared to nontransgenic littermates, despite similar pressure gradients achieved by aortic constriction. Reduced cardiac hypertrophy in DN-Raf-4x transg e n i c m i c e w a s a s s o c i a t e d w i th decreased expression of the atrial natriuretic factor and h-myosin heavy-chain genes. These results suggest that Raf-1 regulates cardiomyocyte survival and also hypertrophic growth. However, this mouse model relies on the overexpression of a mutant form of Raf-1. DN-Raf binds to MKK1/2 and likely blocks activation of MKK by Raf-1, B-Raf, and ARaf (Hindley and Kolch 2002). Yamaguchi et al. (2004) recently generated a murine model with cardiacspecific disruption of the Raf-1 gene (Raf CKO). In this model system, Raf-1 floxed-allele mice were bred with transgenic mice with cardiac-specific overexpression of Cre recombinase. Raf CKO mice were born at mendelian ratios and were fertile, with a normal life span. The cardiac function of Raf CKO mice, however, was not normal, and by 10 weeks of age, they displayed markedly reduced systolic function with left ventricular dilatation by echocardiography. Furthermore, posterior wall thickness was reduced in Raf CKO mice at 10 weeks of age, suggestive of reduced cardiomyocyte growth or increased apoptosis. Cardiac catheterization of 10-week-old Raf CKO mice showed reduced systolic and diastolic function. Increased cardiomyocyte apoptosis was observed in Raf CKO heart at 3 to 5 weeks of age. Activation of cardiac MKK and ERK was not reduced in Raf CKO mice after infusion of endothelin 1 when compared to control mice. Interestingly, cardiac ASK1, JNK, and p38 MAPK activities were increased in Raf CKO mice at baseline. To determine whether increased ASK1 activity was responsible for the cardiac phenotype, Raf CKO mice were bred with ASK1 / mice. Raf CKO ASK1 / mice exhibited improved con-
227
Figure 2. Model of Raf kinase action in cardiomyocyte growth and survival. Simplified model of intracellular signaling cascades regulated by Raf family members.
tractile function and reduced chamber dilatation when compared to Raf CKO ASK1+/+ mice. Furthermore, the incidence of cardiomyocyte apoptosis was reduced in Raf CKO ASK1 / mice when compared to Raf CKO ASK1+/+ mice. These results suggest that Raf-1 has a critical antiapoptotic effect in cardiomyocytes that is independent of MKK and ERK, and that depends, at least in part, on the inhibition of ASK1.
regulate cardiomyocyte growth in response to pressure overload via the ERK signaling cascade (Figure 2). Analysis of mice with cardiac-specific disruption of B-Raf, MKK, or ERK will provide important additional information to elucidate the specific role of these proteins in the cardiac hypertrophic growth program and also in cardiomyocyte survival. This proposed signaling model of Raf family member activity in cardiac biology has implications for human disease. The potent antiapoptotic activity of Raf family members suggests that agents that reduce Raf protein levels or kinase activity in heart may sensitize patients to the development of cardiac dysfunction and congestive heart failure while antagonizing the development of cardiac hypertrophy. Pharmacological agents that inhibit the kinase activity of Raf proteins exist, and some have been used in clinical studies for the treatment of cancer (Strumberg and Seeber 2005). On the other hand, agents that increase Raf protein levels or kinase activity may promote the development of cardiac hypertrophy and may also block the development of cardiac dysfunction. A variety of growth factors, cytokines, and ligands, including some that are commercially available, activate Raf proteins in tissues.
Conclusion
Recent studies with genetically modified mice have investigated the role of Raf proteins in cardiomyocyte growth and survival (Harris et al. 2004, Yamaguchi et al. 2004). One conclusion that emerges from these studies is that Raf-1 has potent antiapoptotic activity in cardiomyocytes that is independent of MKK and ERK activation and that may be dependent on its ability to block ASK1 (Figure 2). It is important to note that MKK and ERK also have antiapoptotic activity in cardiomyocytes that is distinct from the ability of Raf-1 to inhibit ASK1 (Bueno and Molkentin 2002), and this antiapoptotic activity may be due to the ability of ERK to phosphorylate and inactivate caspase 9 and BIM (Allan et al. 2003; Harada et al. 2004). Therefore, Raf family members have antiapoptotic activity that is both dependent and independent of ERK activation (Figure 2). Another conclusion that emerges from studies with genetically modified mice is that Raf family members, including Raf-1, B-Raf, and possibly A-Raf,
228
References Allan LA, Morrice N, Brady S, et al.: 2003. Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat Cell Biol 7:647–654. Babu GJ, Lalli MJ, Sussman MA, et al.: 2000. Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy. J Mol Cell Cardiol 32:1447–1457. Baumann B, Weber CK, Troppmair J, et al.: 2000. Raf induces NF-nB by membrane shuttle kinase MEKK1, a signaling pathway critical for transformation. Proc Natl Acad Sci USA 97:4615– 4620. Bueno OF, Molkentin JD: 2002. Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res 91:776–781. Bueno OF, De Windt LJ, Tymitz KM, et al.: 2000. The MEK1–ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 19: 6341– 6350. Chaudhary A, King WG, Mattaliano MD, et al.: 2000. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 10:551–554.
Chen J, Fujii K, Zhang L, et al.: 2001. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism. Proc Natl Acad Sci USA 98:7783–7788. Chong H, Lee J, Guan KL: 2001. Positive and negative regulation of Raf kinase activity by phosphorylation. EMBO J 20:3716–3727. Davies H, Bignell GR, Cox C, et al.: 2002. Mutations of the BRAF gene in human cancer. Nature 417:949–954. Gray-Schopfer VC, Da Rocha Dias S, Marais R: 2005. The role of B-RAF in melanoma. Cancer Metastasis Rev 24:165–183. Guan KL: 1994. The mitogen activated protein kinase signal transduction pathway: From the cell surface to the nucleus. Cell Signal 6:581–589. Hagemann C, Rapp UR: 1999. Isotype-specific functions of Raf kinases. Exp Cell Res 253:34 – 46. Harada H, Quearry B, Ruiz-Vela A, Korsmeyer SJ: 2004. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. Proc Natl Acad Sci USA 43:15313–15317. Harris IS, Zhang S, Treskov I, et al.: 2004. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 110:718–723. Hindley A, Kolch W: 2002. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J Cell Sci 115 (Pt. 8): 1575–1581. Huser M, Luckett J, Chiloeches A, et al.: 2001. MEK kinase activity is not necessary for Raf-1 function. EMBO J 20:1940–1951. Ikenoue T, Hikiba Y, Kanai F, et al.: 2003. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res 63:8132–8137. Jacobs D, Glossip D, Xing H, et al.: 1999. Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev 13:163–175. King AJ, Sun H, Diaz B, et al.: 1998. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature 396:180–183. Mason CS, Springer CJ, Cooper RG, et al.: 1999. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18:2137–2148. Mikula M, Schreiber M, Husak Z, et al.: 2001. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 20:1952–1962. Moodie SA, Willumsen BM, Weber MJ, et al.: 1993. Complexes of Ras. GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658–1661.
TCM Vol. 15, No. 6, 2005
Muslin AJ, Tanner JW, Allen PM, Shaw AS: 1996. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84:889–897.
Yamaguchi O, Watanabe T, Nishida K, et al.: 2004. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 114:937–943.
Nimnual A, Bar-Sagi D: 2002. The two hats of SOS. Sci STKE 145:PE36.
Yue TL, Gu JL, Wang C, et al.: 2000. Extracellular signal-regulated kinase plays an
essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy. J Biol Chem 275:37895–37901. PII S1050-1738(05)00089-7
TCM
O’Neill E, Rushworth L, Baccarini M, Kolch W: 2004. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306:2267–2270. Ory S, Zhou M, Conrads TP, et al.: 2003. Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol 13:1356–1364. Purcell NH, Darwis D, Bueno OF, et al.: 2004. Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes. Mol Cell Biol 24:1081–1095. Rommel C, Radziwill G, Moelling K, Hafen E: 1997. Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo. Mech Dev 64:95–104. Roy F, Laberge G, Douziech M, et al.: 2002. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev 16:427– 438. Seger R, Krebs EG: 1995. The MAPK signaling cascade. FASEB J 9:726–735. Strumberg D, Seeber S: 2005. Raf kinase inhibitors in oncology. Onkologie 28:101–107. Taylor LK, Wang HC, Erikson RL: 1996. Newly identified stress-responsive protein kinases, Krs-1 and Krs-2. Proc Natl Acad Sci USA 93:10099–10104. Ueyama T, Kawashima S, Sakoda T, et al.: 2000. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy. J Mol Cell Cardiol 32:947–960. Vojtek AB, Hollenberg SM, Cooper JA: 1993. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74: 205–214. Wang L, Proud CG: 2002. Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res 91:821–829. Wang L, Gout I, Proud CG: 2001. Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes. J Biol Chem 276:32670–32677. Wellbrock C, Karasarides M, Marais R: 2004. The RAF proteins take centre stage. Nat Rev Mol Cell Biol 5:875–885. Xing H, Kornfeld K, Muslin AJ: 1997. The protein kinase KSR interacts with 14-3-3 protein and Raf. Curr Biol 7:294 –300. Yamaguchi O, Higuchi Y, Hirotani S, et al.: 2003. Targeted deletion of apoptosis signalregulating kinase 1 attenuates left ventricular remodeling. Proc Natl Acad Sci USA 100:15883 –15888.
TCM Vol. 15, No. 6, 2005
Cardiac Stem and Progenitor Cell Biology for Regenerative Medicine Daniele Torella*, Georgina M. Ellison, Bernardo Nadal-Ginard, and Ciro Indolfi* Stem cell therapy is a new and promising treatment of heart disease. However, the race is still on to find the bbestQ cell to reconstitute the myocardium and improve function after myocardial damage. The recent discovery in the adult mammalian myocardium of a small cell population with the phenotype, behavior, and regenerative potential of cardiac stem and progenitor cells has proposed these cells as the most appropriate for cell therapy. The existence of these cells has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. These findings have placed the heart squarely among other organs with regenerative potential despite the fact that the working myocardium is mainly constituted of terminally differentiated cells. Although CSCs (cardiac cells proven to have stem and/or progenitor characteristics) can be isolated and amplified in vitro or stimulated to differentiate in situ, it has become reasonable to exploit this endogenous regenerative potential to replace the lost muscle with autologous functional myocardium. Therefore, it is imperative to obtain a better understanding of the biology and regenerative potential of the endogenous CSCs. This will enable us to design better protocols for the regeneration of functional contractile mass after myocardial injury. (Trends Cardiovasc Med 2005;15:229 – 236) D 2005, Elsevier Inc.
Daniele Torella and Ciro Indolfi are at the Division of Cardiology, Magna Graecia University, Catanzaro, Italy. Georgina M. Ellison and Bernardo Nadal-Ginard are at the Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York. * Address correspondence to: Ciro Indolfi, MD, FACC, FESC, Professor of Cardiology and Daniele Torella, MD, are to be contacted at Laboratory of Molecular and Cellular Cardiology, Division of Cardiology, Department of Clinical and Experimental Medicine, Magna Graecia University, Via Tommaso Campanella, 115, Catanzaro 88100, Italy. Tel.: (+39) 0961-712-310; fax: (+39) 0961-712-445; e-mails: [email protected], [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
Nowadays, the prevention and treatment of atherosclerotic heart disease have substantially reduced cardiovascular morbidity. Nonetheless, once damage to the heart is established and heart failure is overt, no therapies can improve cardiac function in the long term (Jessup and Brozena 2003). Owing to the present era of regenerative medicine and the recent excitement regarding stem cell therapies, attention has focused on the possibility of regenerating myocardial tissue to treat cardiac diseases (Nadal-Ginard et al. 2003, Korbling and Estrov 2003). The majority of cardiac myocytes exit the cell cycle after a terminal round of
229