Identification of Cell Cycle Regulatory and Inflammatory Genes As Predominant Targets of p38 Mitogen-Activated Protein Kinase in t
http://www.100md.com
Olli Tenhunen, Jaana Rys, Mika Ilves, Yl
参见附件。
the Departments of Pharmacology and Toxicology (O.T., J.R., H.R., H.L.) and Physiology (M.I.), Biocenter Oulu
the Department of Pathology (Y.S.), University of Oulu, Finland.
Abstract
Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38 in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion, and transcription. A large number of previously unknown p38 target genes were found. Using gel mobility-shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene-expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression-induced gene expression results in myocardial cell proliferation, inflammation, and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart.
Key Words: mitogen-activated protein kinases inflammation proliferation
Introduction
Cardiac myocytes, as virtually all other eukaryotic cells, possess a complex network of parallel intracellular signal transduction pathways that are activated in response to extracellular stimulation. Among these, MAPKs represent a central converge point, which are activated by a variety of stimuli and are able to activate diverse cellular processes.1,2 The MAPK family consists of a cascade of sequentially acting upstream kinases, MAPK kinase kinases and MAPK kinases (MKK), and 3 terminal kinases p38 MAPK, extracellular signal-regulated kinase (ERK1/2), and c-jun N-terminal kinase (JNK).1,2 Accumulating evidence has indicated that p38 MAPK is involved in the pathophysiology of inflammatory diseases, such as arthritis, septic shock, or pulmonary disease.3 Yet, persistent controversy underlies the functional role of p38 MAPK in cardiac pathology. In particular, loss-of-function and gain-of-function studies in transgenic animals have reported exceedingly controversial findings.4 For example, both animals overexpressing constitutively active p38 MAPK upstream kinases and animals with dominant-negative isoforms of these kinases exhibit enhanced fibrosis.4a,5 Further, transgenic mice expressing dominant-negative mutants of p38 or MKK6 were reported to be protected from ischemia-reperfusion injury via antiapoptotic mechanisms, but, equally, functional recovery and protection were observed in response to MKK6 overexpression.6,7 Similarly, pharmacological inhibition of p38 MAPK has been shown to attenuate hypertensive end-organ damage, but transgenic animals overexpressing p38 MAPK do not show significant cardiomyocyte hypertrophy.4a,8
Activation of p38 MAPK leads to diverse cellular responses via activation and phosphorylation of several kinases and transcription factors (TFs). Among the well-characterized in vitro downstream targets of p38 MAPK are MAPK-activated protein kinases 2 and 3, MAPK-interacting kinases, p38-regulated/activated kinase, and activating transcription factor-2 (ATF-2).1 In addition, TFs such as GATA-4, myocyte enhancer factor-2 (MEF-2), serum response factor (SRF), activator protein-1 (AP-1), nuclear factor B (NF-B), and ELK-1 have been shown to be activated directly by p38 MAPK within several tissue types,1,9–11 but whether they are sensitive to p38 MAPK in vivo is unknown. At the level of cardiac gene expression, p38 MAPK activation has been reported to be involved eg, in the regulation of A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and cyclooxygenase-2 (COX-2) gene expression in vitro.11–13 However, the global gene-expression profile and transcriptional mechanisms controlled by p38 MAPK remains largely unknown.
This study was designed to characterize the underlying molecular mechanism for the p38 MAPK activation in the heart in vivo. To reach this aim, p38 MAPK was selectively activated by adenovirus-mediated gene transfer of wild-type p38 (WTp38) and constitutively active upstream kinase MKK3b into the left ventricle (LV) of adult rats. The global gene-expression profile was analyzed by DNA microarrays, and the changes of a subset of genes were confirmed by quantitative real-time RT-PCR and Northern blots. The gene list generated by microarray analysis was analyzed using PathwayAssist software to identify potential interactions between p38 MAPK downstream targets. We also identified p38 MAPK sensitive TFs by gel mobility-shift assays. Finally, we determined the functional significance of local p38 MAPK overexpression in the normal adult rat heart by using histological analysis and echocardiography. Our results reveal p38 as a critical regulator of cardiac gene expression in vivo and demonstrate that increased p38 MAPK activity causes massive cell proliferation and inflammation with fibrosis in the heart.
Materials and Methods
Adenoviruses containing the coding regions of the constitutively active MKK3b (RAdMKK3bE) and WTp38 (RAdp38) and recombinant replication-deficient adenovirus RAdlacZ were transferred into the LV as a local intramyocardial injection. Expression profiling was done with GeneChip Rat Expression Set 230_2.0 Array (Affymetrix). Data analysis was done using GeneSpring 7.2 (Silicon Genetics) and PathwayAssist software 3.0 (Stratagene). All methods used in this work are described in detail in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org
Results
Adenovirus-Mediated Gene Transfer of MKK3bE and WTp38 Increases p38 MAPK Activity in the Heart
To study the downstream targets of p38 MAPK in the heart, we established a protocol to locally increase myocardial p38 MAPK activity in vivo. The direct local injection of adenovirus constructs into the LV used in this study has previously been documented as an efficient cardiac-specific approach of gene delivery.14 This protocol targets high expression of the transgene in the LV without affecting other organs and thereby provides a delicate approach to study the direct effects of the selected transgene on the gene-expression profile. The activation of p38 isoforms can be specifically controlled through different upstream regulators, the p38 isoform being preferentially phosphorylated by MKK3.4,4a To achieve maximal upregulation of p38 kinase activity, we examined adenoviruses encoding constitutively active MKK3 (MKK3bE) and MKK6 (MKK6bE) as well as adenoviruses encoding WTp38 and WTp38 in several different combinations (data not shown). Of these combinations, the strongest p38 activation was observed when WTp38 at 2x108 infectious units was coinjected with 6x108 units of MKK3bE. The control animals were injected with adenovirus expressing the Escherichia coli -galactosidase (LacZ) gene at 8x108 infectious units. The combination of MKK3bE and WTp38 produced a 2.9-fold increase in LV phospho-p38 levels (Figure 1A; P<0.01, n=8 to 9) 3 days after injection. ATF-2–based kinase assays showed a corresponding activation in p38 kinase activity (data not shown). No change was observed in the ERK1/2 or JNK activity (Figure 1B and 1C). As assessed by echocardiography, cardiac function of the MKK3bE+WTp38-treated animals was similar to that of LacZ-injected group 3 days after gene transfer (Table).
Analysis of p38 MAPK Target Genes and Transcription Factors
To identify genes that are regulated by p38 MAPK in the heart in vivo, the LV gene-expression profiles 3 days after MKK3bE+WTp38 gene transfer were compared with those of LacZ-treated animals. We confirmed selected microarray results by comparison with mRNA levels obtained by Northern blot analysis or real-time quantitative RT-PCR. As shown in Table II of the online data supplement, similar fold changes in mRNA levels, as measured by both microarray and Northern/RT-PCR, were observed. The microarray analysis identified 264 transcripts that were upregulated more than 2-fold in the LV overexpressing p38 MAPK compared with LacZ-injected hearts according to our selection criteria (supplemental Table III). Upregulated genes were further organized into groups representing their known biological functions: apoptosis, cell adhesion, cell division, cell signaling/communication (subgrouped into hormones and growth factors, intracellular transducers, protein modification, and receptors), cell structure/motility, cell/organism defense, channel/transport proteins, inflammatory and immune response, metabolism (subgrouped into amino acid, carbohydrate, lipid, nucleotide, and nucleic acid metabolism), protein synthesis/turnover/posttranslational modification, TFs, translation factors, unclassified genes and expressed-sequence tags (ESTs), as well as other unknown transcripts (Figure 2 and supplemental Table III). Of note, only 2 genes (pyruvate dehydrogenase kinase isoenzyme-4 and gap junction membrane channel protein-1) were downregulated more than 2-fold. When DNA microarray analysis was extended to include genes that were upregulated 1.5- to 2.0-fold in hearts with p38 MAPK overexpression, 651 additional genes (521 upregulated, 130 downregulated) were identified, the majority of which belonged to the groups of unknown function and ESTs (data not shown).
The major functional groups of known genes affected by p38 MAPK overexpression were genes related to cell division (31 genes) and cell signaling/communication (28 genes) as well as genes related to inflammatory and immune response (23 genes). Yet, the most transcripts to be upregulated in the p38 MAPK overexpressing hearts were genes with unknown function or ESTs and related sequences (Figure 2 and supplemental Table III). Strikingly, the individual genes upregulated by p38 MAPK included critical regulators of cell cycle and many fundamental inflammation-associated genes like interleukin-6 (IL-6) and Toll-like receptor-2. Furthermore, the group of cell adhesion genes included well-known fibrosis related factors, such as selectin E, connective tissue growth factor (CTGF), tenascin C, and osteopontin (supplemental Table III). Most of the genes we uncovered have not previously been known to be regulated by p38 MAPK in any tissue types. These novel p38 MAPK target genes include genes involved in the regulation of cell cycle, such as cyclins A2, B1, and B2, kinesin family member-23, and polo-like kinases. Interestingly, a number of them belong to transcription and cell signaling/communication-related functional groups, including TFs ATF-3, Runx1, and Krox20. However, our microarray analysis also identified several genes that have previously been suggested as cardiac p38 MAPK target genes, such as COX-2, ANP,11,13 and cell cycle target histone H3.15
Finally, the complete list of 266 differentially expressed genes was loaded into PathwayAssist. Of those genes, 73 were recognized by the software, and 39 of them (printed in bold in supplemental Table III) were found to have pathway relationships in published research literature. The interaction map showing potential connections among genes regulated by p38 MAPK overexpression in the heart is shown in supplemental Figure I. A central signaling molecule in this pathway is IL-6, which has been implicated in the regulation of 20 genes found to be affected by p38 MAPK. In addition, chemokine (C-C motif) ligand-2 (CCL2), myelocytomatosis oncogene (myc), and cyclin-dependent kinase inhibitor 1A (CDKN1A) also figure prominently in this network.
Even though the microarray analysis identified increased gene expression of several TFs, such as Egr-1 and Egr-2, the expression levels of many well-characterized cardiac TF genes remained unaffected. Because these factors are known to be subject to posttranscriptional and posttranslational modification by p38 MAPK,1 and the cardiac-restricted TF GATA-4 is regulated by p38 MAPK in vitro,9 we first measured LV GATA-4 DNA binding from the MKK3bE+WTp38-injected hearts by gel mobility-shift assay using a 30-bp oligonucleotide probe containing the –90 GATA sites of the rat BNP promoter. Consistently with previous in vitro results, the gene transfer resulted in a 1.6-fold increase (Figure 3A) in BNP GATA-4 DNA binding. However, the transcriptional effects of p38 MAPK were not restricted to GATA-4, because significant increases were also observed in AP-1 (Figure 3B; 4.5-fold versus LacZ), NF-B (Figure 3C; 1.7-fold versus LacZ), and SRF (Figure 3D; 1.4-fold versus LacZ) DNA binding in MKK3bE+WTp38-treated hearts 3 day after injection. In contrast, MKK3bE+WTp38 gene transfer did not affect MEF-2 binding (data not shown).
To confirm the specificity of the DNA-binding reactions, we performed supershift and competitor analysis using specific antibodies and competitor DNAs. An antibody-induced supershift was seen for GATA-4 but not for GATA-5 or GATA-6 complexes, and GATA-4 DNA binding was effectively inhibited by unlabeled self DNA but unaffected by nonrelated competitor DNA Oct-1 or competitor DNA-containing mutated GATA site (Figure 4A). Similarly AP-1, NF-B, and SRF binding was attenuated by unlabeled self DNAs but not by mutated or nonrelated competitor DNAs. Antibody induced supershifts were observed for AP-1, NF-B, and SRF, demonstrating that the complexes bound by oligonucleotide probe contained AP-1, NF-B, and SRF proteins (Figure 4B through 4D).
Functional Effects of p38 MAPK Overexpression in the Adult Heart
To determine the functional significance of changes in cell cycle regulatory and inflammatory genes identified by microarray analysis, we studied the morphological changes in histological analysis and hemodynamic consequences by echocardiography 1 week after MKK3bE+WTp38 gene transfer. Because microarray analysis revealed several genes that are associated with cell division and proliferation, we first performed immunohistochemical staining against Ki-67 antigen to identify cells undergoing proliferation. Ki-67 is considered as a specific marker of cellular proliferation, as all cells that express Ki-67 go on to divide.16 Ki-67 immunostaining 1 week after gene transfer showed large numbers of Ki-67–positive nuclei in the anterior wall of the MKK3bE+WTp38-treated hearts. In contrast, only few Ki-67–positive cells were observed in the LacZ-treated hearts (Figure 5A). Next, histological sections from both experimental groups were stained with hematoxylin/eosin, Sirius Red, and Masson’s trichrome. Remarkably, the histological sections showed a massive infiltration of inflammatory cells in the MKK3bE+WTp38-treated hearts. As shown in Figure 5A, large numbers of mononuclear inflammatory cells were present in the interstitium of the p38 MAPK overexpressing myocardium. Furthermore, Masson’s trichrome and Sirius Red stainings showed large stained areas in the p38 MAPK overexpressing hearts, indicating massive cardiac fibrosis. The area covered by collagen as assessed from Masson’s trichrome-stained sections was significantly higher in p38 MAPK overexpressing than control hearts (11.1% versus 4.3%; P<0.05; n=5 to 6). Histological sections also revealed a notable thickening of the anterior wall (Figure 5B). At day 3 after gene transfer, only minor fibrotic lesions and inflammatory cell infiltration were observed, suggesting that upregulation of gene expression of inflammatory and fibrosis promoting factors preceded the functional changes (data not shown).
To determine the effects of p38 MAPK overexpression on cardiac function, we performed echocardiographic analysis 1 week after gene transfer. These measurements showed preserved cardiac function in the MKK3bE+WTp38-treated animals, as no statistically significant changes in LV diameter, fractional shortening, or ejection fraction were observed (Figure 6). Consistently with the anterior wall thickening observed in histological analysis, the diameter of the interventricular septum was significantly increased. Moreover, the E/A-ratio in MKK3bE+WTp38-treated animals was raised suggesting development of diastolic dysfunction. The LV weight versus body weight (LVW/BW) ratio was also increased in the MKK3bE+WTp38-treated animals in comparison to that of LacZ-injected group 1 week after gene transfer (Figure 6).
Identification of Cell Types Affected by p38 MAPK Overexpression
To illuminate the cell types affected by p38 MAPK overexpression, immunohistochemistry with specific antibodies was performed. p38 MAPK, GATA-4, and IL-6 immunostaining was localized on cardiac myocytes, whereas inflammatory cells and endothelial cells were stained with Ki-67 (Figure 7).
Discussion
The family members of the MAPKs play a vital role in numerous biological processes in response to extracellular stimuli. p38 MAPK was originally identified as a protein kinase that underwent tyrosine phosphorylation in response to osmotic shock and endotoxin treatment.1 Subsequent studies demonstrated that p38 MAPK responds strongly to proinflammatory cytokines, tumor necrosis factor (TNF)-, heat stress, and growth factors.1 Within the cardiomyocytes, p38 MAPK has been documented to be activated in response to G protein-coupled receptor (GPCR) agonists, such as phenylephrine, endothelin-1, and angiotensin II as well as ischemia-reperfusion injury, mechanical stretch and pressure overload.12,17–19 Considering its central position in signal transduction, it is surprising to note that the p38 MAPK-dependent gene-expression pattern has remained fundamentally unknown. This study provides a comprehensive analysis regarding the target genes and TFs of p38 MAPK in the myocardium as well as the functional effects of p38 MAPK activation in the adult heart.
Our findings demonstrate that p38 MAPK controls a wide array of genes at transcriptional level in the heart, the majority of which are related to cell division, inflammation, cell signaling, cell adhesion, and transcription. All of these categories include individual genes that have been previously suggested to be regulated by p38 MAPK in other cell types but also a number of new, previously nondescribed p38 MAPK targets. Although this study was aimed to characterize the gene-expression profile controlled by p38 MAPK in the heart, it is likely that the significance of our findings can be extended beyond the cardiovascular system. So far, p38 MAPK-related genome profiling has been performed by using pharmacological p38 MAPK inhibitors in transformed follicular lymphoma cells and in cultured cardiomyocytes.20,21 Given the diverse effects of pharmacological inhibitors, these studies do not provide direct evidence concerning genes that are controlled by p38 MAPK. In addition, gene-expression profiles in transgenic adult mouse hearts have been determined at 4- to 7-day and 2- to 4-week time points following MKK3b transgene induction by tamoxifen.22 In the current experimental design, the gene-expression profile was analyzed at a time point when no or only minor functional or structural changes were observed, suggesting that the present results represent targets that are directly regulated by p38 MAPK. Furthermore, the profile of the genes that were upregulated by p38 MAPK in our data were not cardiac restricted, as it included a number of conserved genes that are widely expressed in different tissue types. It is also noteworthy that our results, like those following MKK3b transgene induction by tamoxifen,22 represent p38 MAPK targets in several types of cells, such as cardiac myocytes, fibroblasts, and endothelial cells, because adenovirus-mediated gene transfer into the LV was performed as a local intramyocardial injection. On the other hand, the effects of chronic and potent activation of the p38 MAPK pathway achieved with adenovirus-mediated gene transfer may differ from those of more transient activation observed in many experiment models.1–4
A key finding of the present study is the role of p38 MAPK in the cell cycle control in the heart. Previous studies suggest that p38 MAPK may play either a positive or a negative role in this process, depending on cell types and stimuli.1,21 Our microarray data identified important regulators of diverse cell cycle events ranging from DNA replication (DNA topoisomerase ), the control of the cell cycle at the G2/mitosis transition (cyclins A2, B1, and B2 and CDC2) to centrosome function and chromosome segregation in mitosis (kinesins, pituitary tumor-transforming 1, Aurora kinase B). Specifically, p38 MAPK seems to play a major role in the regulation of cell division through transcriptional upregulation of central molecules needed in G2/mitosis cell cycle checkpoint activation. Functionally, the upregulation of the cell cycle regulatory genes was associated with cellular proliferation, as the Ki-67 immunostaining revealed a large number of replicating cells in the myocardium overexpressing p38 MAPK. Based on histological stainings, the majority of the proliferating cells belong to other cell types than cardiomyocytes, such as inflammatory and endothelial cells. However, a recent study showed that constitutively expressed cyclin A2 is able to induce cardiomyocyte mitosis in postmitotic myocardium,23 and downregulation of polo-like kinase has been reported to correlate with loss of proliferative ability of cardiac myocytes.24 In view of these findings, it is tempting to speculate that p38 MAPK-induced gene expression may also promote cardiomyocyte proliferation. Even though p38 was originally reported as a proapoptotic protein kinase in cultured cardiomyocytes, our microarray analysis identified virtually no apoptosis-related genes.25 The only gene upregulated in this category was Bcl-related protein A2, suggesting that the in vitro observations regarding proapoptotic effects of p38 MAPK may not be extended to the adult heart. Taken together, cardiac p38 MAPK is a growth promoting molecule, likely playing a key role in G2/mitosis checkpoint activation. Interestingly, microarray analysis further revealed activation of several genes related to amino acid and nucleotide metabolism, probably also reflecting the vigorous cell growth in the heart.
Our finding that cardiac p38 MAPK overexpression significantly increased IL-6, IL-18, COX-2, CCL2, and Toll-like receptor-2 mRNA levels together with massive inflammatory cell infiltration indicates p38 MAPK as a critical regulator of inflammatory responses of the heart. These observations are consistent with the strong link established between the p38 MAPK pathway and inflammatory responses and diseases in other organ systems. Pharmacological inhibition of p38 MAPK has been previously reported to attenuate the production of proinflammatory cytokines, including IL-1 and IL-6, as well as expression of COX-2.26 Consequently, transgenic mice overexpressing MKK6bE have been previously shown to exhibit increased plasma levels of IL-6.27 The importance of p38 MAPK in myocardial inflammation is further highlighted by the interaction analysis of our microarray results. When genes found to be regulated in the microarray study were used to create a network in which several of these genes are involved in or at least associated with in the literature, IL-6 appears to be a central signaling molecule in this network. Importantly, IL-6 is among the main downstream effectors of pharmacological p38 MAPK inhibition involved in cell proliferation and survival associated pathways.20
In addition to cell proliferation and inflammation, p38 MAPK overexpression caused cardiac fibrosis, which also was tightly coupled to the p38 MAPK-dependent gene-expression profile. p38 MAPK overexpression increased the expression levels of several fibrosis-associated genes, including basic fibroblast growth factor (bFGF), osteopontin, transforming growth factor-2 (TGF)-2, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), bone morphogenic protein-2 (BMP)-2, and CTGF. These findings are supported by previous gain-of-function studies, because transgenic mice with targeted overexpression of constitutively active MKK3 or MKK6 have been shown to exhibit increased interstitial fibrosis.4a,27 Moreover, microarray analysis in MKK3bE transgenic animals revealed changes in expression of genes particularly involved in extracellular matrix remodeling.22 Also, conflicting results have been reported, as cardiac-specific dominant-negative p38, MKK3, and MKK6 transgenic mice develop massive fibrosis in response to pressure overload.28
The upregulation of gene expression by p38 MAPK seems to be a result of transcriptional control, because gel mobility-shift assays demonstrated the activation of several cardiac TFs including GATA-4, AP-1, SRF, and NF-B. Regarding GATA-4, the present results extend our previous in vitro findings indicating that pharmacological p38 inhibition attenuates the DNA binding of GATA-4.9 This is of particular interest considering the central role of GATA-4 in the control of cardiac development and growth as well as in cardiac hypertrophy and heart failure.29 Notably, a very strong 4.5-fold increase was observed in AP-1 DNA binding, and NF-B was upregulated as well. Both of these factors are known to be involved in the inflammatory responses and control of cell cycle. Mechanistically, the activation of TFs in most cases is likely a consequence of direct phosphorylation, because p38 MAPK is known to phosphorylate, eg, GATA-4 and AP-1 components.29 Yet, other mechanisms may be involved as well, because the microarray analysis revealed that p38 MAPK overexpression increases the gene expression of several other intracellular signaling molecules (eg, Rho family GTPase-1 and dual-specificity phosphatase-2).
Our data reveal several novel aspects regarding the role of p38 MAPK and cardiac gene expression in cardiac pathophysiology. The present results demonstrate that in the adult heart, the physiological consequence of p38 MAPK overexpression, via distinct upregulation of gene expression, is cardiac cell proliferation and myocardial inflammation associated with fibrosis. Importantly, persistent inflammation is a characteristic of chronic heart failure. It includes increased levels of proinflammatory cytokines, which contribute to cardiac contractility, cardiomyocyte hypertrophy, and death. It is noteworthy that IL-6, which was strongly upregulated by p38 MAPK, is among the best characterized of these factors and particularly active soon after acute myocardial infarction.30 Fibrosis, in turn, is a critical determinant of postinfarction remodeling, diastolic heart failure, and hypertensive end-organ damage. However, p38 MAPK-induced inflammatory responses may not simply be detrimental to the heart, because under certain conditions, proinflammatory cytokines may also act in a cardioprotective manner by affecting extracellular matrix, integrins, and contribute to vascular and cardiac regeneration and myocardial angiogenesis.30 Accordingly, cardiac fibrosis and cell proliferation may be reparative or harmful depending on pathophysiological context. The cardioprotective role of p38 MAPK is indirectly supported by our observation that p38 MAPK-overexpressing hearts showed preserved systolic function despite the development of fibrosis, diastolic dysfunction, and massive anterior wall thickening. Finally, the findings regarding p38 MAPK and cardiac hypertrophy may be of functional significance. The expression of ANP was significantly increased by p38 MAPK overexpression, but genes encoding structural proteins, such as -myosin heavy chain (-MHC) and -skeletal actin (-SkA), were not upregulated. Furthermore, no morphological signs of cardiomyocyte hypertrophy were observed. These findings support the view that p38 MAPK activation is not prerequisite for the development cardiomyocyte hypertrophy but, in turn, may directly regulate the expression of selected hypertrophy-associated genes.
In summary, our transcriptional profiling results revealed a striking impact of p38 MAPK activation on cell cycle regulatory genes. In addition to cell proliferation, activation of p38 MAPK pathway involves inflammatory and profibrotic gene induction as prominent processes for pathological changes in the heart. We also show that p38 MAPK directly activates multiple cardiac TFs, further emphasizing the role of p38 MAPK as a key regulator of cardiac gene transcription. The results provide a novel link among intracellular signal transduction, gene expression, and cardiac pathophysiology in vivo, as well as an important source of information for development of p38-targeted therapies. The downstream target genes regulated by p38 MAPK suggest a wide variety of additional potential indications besides autoimmune disorders.
Acknowledgments
We thank Dr Jussi Vuoristo (Biocenter Oulu) and Sirpa Rutanen, Pirjo Korpi, Kaisa Penttil, Tuulikki Krn, Marja Arbelius, and Kati Viitala for expert technical assistance.
Sources of Funding
This work was financially supported by the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the National Technology Foundation TEKES, the Aarne Koskelo Foundation, the Ida Montin Foundation, and the Research and Science Foundation of Farmos.
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received April 2, 2006; revision received July 11, 2006; accepted July 17, 2006.
References
Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001; 81: 807–869.
Sugden PH, Clerk A. Regulation of mitogen-activated protein kinase cascades in the heart. Adv Enzyme Regul. 1998; 38: 87–98. [Order article via Infotrieve]
Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol. 2004; 4: 372–377. [Order article via Infotrieve]
Ravingerova T, Barancik M, Strniskova M. Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology. Mol Cell Biochem. 2003; 247: 127–138. [Order article via Infotrieve]
Liao P, Georgakopoulos D, Kovacs A, Zheng M, Lerner D, Pu H, Saffitz J, Chien K, Xiao RP, Kass DA, Wang Y. The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy. Proc Natl Acad Sci U S A. 2001; 98: 12283–12288.
Braz JC, Bueno OF, Liang Q, Wilkins BJ, Dai YS, Parsons S, Braunwart J, Glascock BJ, Klevitsky R, Kimball TF, Hewett TE, Molkentin JD. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J Clin Invest. 2003; 111: 1475–1486.
Kaiser RA, Bueno OF, Lips DJ, Doevendans PA, Jones F, Kimball TF, Molkentin JD. Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia-reperfusion in vivo. J Biol Chem. 2004; 279: 15524–15530.
Martindale JJ, Wall JA, Martinez-Longoria DM, Aryal P, Rockman HA, Guo Y, Bolli R, Glembotski CC. Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo. J Biol Chem. 2005; 280: 669–676.
Behr TM, Nerurkar SS, Nelson AH, Coatney RW, Woods TN, Sulpizio A, Chandra S, Brooks DP, Kumar S, Lee JC, Ohlstein H, Angermann CE, Adams JL, Sisko J, Sackner-Bernstein JD, Willette RN. Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction. Circulation. 2001; 104: 1292–1298.
Tenhunen O, Sarman B, Kerkela R, Szokodi I, Papp L, Toth M, Ruskoaho H. Mitogen-activated protein kinases p38 and ERK1/2 mediate the wall stress-induced activation of GATA-4 binding in adult heart. J Biol Chem. 2004; 279: 24852–24860.
Han J, Molkentin JD. Regulation of MEF2 by p38 MAPK and its implication in cardiomyocyte biology. Trends Cardiovasc Med. 2000; 10: 19–22. [Order article via Infotrieve]
Thuerauf DJ, Arnold ND, Zechner D, Hanford DS, DeMartin KM, McDonough PM, Prywes R, Glembotski CC. p38 mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6. J Biol Chem. 1998; 273: 20636–20643.
Liang F, Gardner DG. Mechanical strain activates BNP gene transcription through a p38/NF-kappaB-dependent mechanism. J Clin Invest. 1999; 104: 1603–1612.
Degousee N, Martindale J, Stefanski E, Cieslak M, Lindsay TF, Fish JE, Marsden PA, Thuerauf DJ, Glembotski CC, Rubin BB. MAP kinase kinase 6-p38 MAP kinase signaling cascade regulates cyclooxygenase-2 expression in cardiac myocytes in vitro and in vivo. Circ Res. 2003; 92: 757–764.
Szatkowski ML, Westfall MV, Gomez CA, Wahr PA. Michele DE, DelloRusso C, Turner II, Hong KE, Albayya FE, Metzger JM. In vivo acceleration of heart relaxation performance by parvalbumin gene delivery. J Clin Invest. 2001; 107: 191–198.
Zhong SP, Ma WY, Dong Z. ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10. J Biol Chem. 2000; 275: 20980–20984.
Yuasa S, Fukuda K, Tomita Y, Fujita J, Ieda M, Tahara S, Itabashi Y, Yagi T, Kawaguchi H, Hisaka Y, Ogawa S. Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats. Mol Cell Biochem. 2004; 259: 177–181. [Order article via Infotrieve]
Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998; 83: 345–352.
Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC, Feuerstein GZ, Yue TL. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation. 1999; 99: 1685–1691.
Fischer TA, Ludwig S, Flory E, Gambaryan S, Singh K, Finn P, Pfeffer MA, Kelly RA, Pfeffer JM. Activation of cardiac c-jun NH(2)-terminal kinases and p38-mitogen-activated protein kinases with abrupt changes in hemodynamic load. Hypertension. 2001; 37: 1222–1228.
Lin Z, Crockett DK, Jenson SD, Lim MS, Elenitoba-Johnson KS. Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells. Mol Cell Proteomics. 2004; 3: 820–833.
Engel FB, Schebesta M, Duong MT, Lu G, Ren S, Madwed JB, Jiang H, Wang Y, Keating MT. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev. 2005; 19: 1175–1187.
Mitchell S, Ota A, Foster W, Zhang B, Fang Z, Patel S, Nelson SF, Horvath S, Wang Y. Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation. Physiol Genomics. 2006; 25: 50–59.
Chaudhry HW, Dashoush NH, Tang H, Zhang L, Wang X, Wu EX, Wolgemuth DJ. Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium. J Biol Chem. 2004; 279: 35858–35866.
Georgescu SP, Komuro I, Hiroi Y, Mizuno T, Kudoh S, Yamazaki T, Yazaki Y. Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes. J Mol Cell Cardiol. 1997; 29: 929–937. [Order article via Infotrieve]
Wang Y, Huang S, Sah VP, Ross J, Brown JH, Han J, Chien KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem. 1998; 273: 2161–2168.
Kumar S, Boehm J, Lee JC. p38 MAP kinases: Key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov. 2003; 2: 717–726. [Order article via Infotrieve]
Li M, Georgakopoulos D, Lu G, Hester L, Kass DA, Hasday J, Wang Y. p38 MAP kinase mediates inflammatory cytokine induction in cardiomyocytes and extracellular matrix remodeling in heart. Circulation. 2005; 111: 2494–2502.
Nishida K, Yamaguchi O, Hirotani S, Hikoso S, Higuchi Y, Watanabe T, Takeda T, Osuka S, Morita T, Kondoh G, Uno Y, Kashiwase K, Taniike M, Nakai A, Matsumura Y, Miyazaki J, Sudo T, Hongo K, Kusakari Y, Kurihara S, Chien KR, Takeda J, Hori M, Otsu K. p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload. Mol Cell Biol. 2004; 24: 10611–10620.
Pikkarainen S, Tokola H, Kerkela R, Ruskoaho H. GATA transcription factors in the developing and adult heart. Cardiovasc Res. 2004; 63: 196–207. [Order article via Infotrieve]
Nian M, Lee P, Khaper N, Liu P. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res. 2004; 94: 1543–1553.
the Departments of Pharmacology and Toxicology (O.T., J.R., H.R., H.L.) and Physiology (M.I.), Biocenter Oulu
the Department of Pathology (Y.S.), University of Oulu, Finland.
Abstract
Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38 in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion, and transcription. A large number of previously unknown p38 target genes were found. Using gel mobility-shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene-expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression-induced gene expression results in myocardial cell proliferation, inflammation, and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart.
Key Words: mitogen-activated protein kinases inflammation proliferation
Introduction
Cardiac myocytes, as virtually all other eukaryotic cells, possess a complex network of parallel intracellular signal transduction pathways that are activated in response to extracellular stimulation. Among these, MAPKs represent a central converge point, which are activated by a variety of stimuli and are able to activate diverse cellular processes.1,2 The MAPK family consists of a cascade of sequentially acting upstream kinases, MAPK kinase kinases and MAPK kinases (MKK), and 3 terminal kinases p38 MAPK, extracellular signal-regulated kinase (ERK1/2), and c-jun N-terminal kinase (JNK).1,2 Accumulating evidence has indicated that p38 MAPK is involved in the pathophysiology of inflammatory diseases, such as arthritis, septic shock, or pulmonary disease.3 Yet, persistent controversy underlies the functional role of p38 MAPK in cardiac pathology. In particular, loss-of-function and gain-of-function studies in transgenic animals have reported exceedingly controversial findings.4 For example, both animals overexpressing constitutively active p38 MAPK upstream kinases and animals with dominant-negative isoforms of these kinases exhibit enhanced fibrosis.4a,5 Further, transgenic mice expressing dominant-negative mutants of p38 or MKK6 were reported to be protected from ischemia-reperfusion injury via antiapoptotic mechanisms, but, equally, functional recovery and protection were observed in response to MKK6 overexpression.6,7 Similarly, pharmacological inhibition of p38 MAPK has been shown to attenuate hypertensive end-organ damage, but transgenic animals overexpressing p38 MAPK do not show significant cardiomyocyte hypertrophy.4a,8
Activation of p38 MAPK leads to diverse cellular responses via activation and phosphorylation of several kinases and transcription factors (TFs). Among the well-characterized in vitro downstream targets of p38 MAPK are MAPK-activated protein kinases 2 and 3, MAPK-interacting kinases, p38-regulated/activated kinase, and activating transcription factor-2 (ATF-2).1 In addition, TFs such as GATA-4, myocyte enhancer factor-2 (MEF-2), serum response factor (SRF), activator protein-1 (AP-1), nuclear factor B (NF-B), and ELK-1 have been shown to be activated directly by p38 MAPK within several tissue types,1,9–11 but whether they are sensitive to p38 MAPK in vivo is unknown. At the level of cardiac gene expression, p38 MAPK activation has been reported to be involved eg, in the regulation of A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and cyclooxygenase-2 (COX-2) gene expression in vitro.11–13 However, the global gene-expression profile and transcriptional mechanisms controlled by p38 MAPK remains largely unknown.
This study was designed to characterize the underlying molecular mechanism for the p38 MAPK activation in the heart in vivo. To reach this aim, p38 MAPK was selectively activated by adenovirus-mediated gene transfer of wild-type p38 (WTp38) and constitutively active upstream kinase MKK3b into the left ventricle (LV) of adult rats. The global gene-expression profile was analyzed by DNA microarrays, and the changes of a subset of genes were confirmed by quantitative real-time RT-PCR and Northern blots. The gene list generated by microarray analysis was analyzed using PathwayAssist software to identify potential interactions between p38 MAPK downstream targets. We also identified p38 MAPK sensitive TFs by gel mobility-shift assays. Finally, we determined the functional significance of local p38 MAPK overexpression in the normal adult rat heart by using histological analysis and echocardiography. Our results reveal p38 as a critical regulator of cardiac gene expression in vivo and demonstrate that increased p38 MAPK activity causes massive cell proliferation and inflammation with fibrosis in the heart.
Materials and Methods
Adenoviruses containing the coding regions of the constitutively active MKK3b (RAdMKK3bE) and WTp38 (RAdp38) and recombinant replication-deficient adenovirus RAdlacZ were transferred into the LV as a local intramyocardial injection. Expression profiling was done with GeneChip Rat Expression Set 230_2.0 Array (Affymetrix). Data analysis was done using GeneSpring 7.2 (Silicon Genetics) and PathwayAssist software 3.0 (Stratagene). All methods used in this work are described in detail in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org
Results
Adenovirus-Mediated Gene Transfer of MKK3bE and WTp38 Increases p38 MAPK Activity in the Heart
To study the downstream targets of p38 MAPK in the heart, we established a protocol to locally increase myocardial p38 MAPK activity in vivo. The direct local injection of adenovirus constructs into the LV used in this study has previously been documented as an efficient cardiac-specific approach of gene delivery.14 This protocol targets high expression of the transgene in the LV without affecting other organs and thereby provides a delicate approach to study the direct effects of the selected transgene on the gene-expression profile. The activation of p38 isoforms can be specifically controlled through different upstream regulators, the p38 isoform being preferentially phosphorylated by MKK3.4,4a To achieve maximal upregulation of p38 kinase activity, we examined adenoviruses encoding constitutively active MKK3 (MKK3bE) and MKK6 (MKK6bE) as well as adenoviruses encoding WTp38 and WTp38 in several different combinations (data not shown). Of these combinations, the strongest p38 activation was observed when WTp38 at 2x108 infectious units was coinjected with 6x108 units of MKK3bE. The control animals were injected with adenovirus expressing the Escherichia coli -galactosidase (LacZ) gene at 8x108 infectious units. The combination of MKK3bE and WTp38 produced a 2.9-fold increase in LV phospho-p38 levels (Figure 1A; P<0.01, n=8 to 9) 3 days after injection. ATF-2–based kinase assays showed a corresponding activation in p38 kinase activity (data not shown). No change was observed in the ERK1/2 or JNK activity (Figure 1B and 1C). As assessed by echocardiography, cardiac function of the MKK3bE+WTp38-treated animals was similar to that of LacZ-injected group 3 days after gene transfer (Table).
Analysis of p38 MAPK Target Genes and Transcription Factors
To identify genes that are regulated by p38 MAPK in the heart in vivo, the LV gene-expression profiles 3 days after MKK3bE+WTp38 gene transfer were compared with those of LacZ-treated animals. We confirmed selected microarray results by comparison with mRNA levels obtained by Northern blot analysis or real-time quantitative RT-PCR. As shown in Table II of the online data supplement, similar fold changes in mRNA levels, as measured by both microarray and Northern/RT-PCR, were observed. The microarray analysis identified 264 transcripts that were upregulated more than 2-fold in the LV overexpressing p38 MAPK compared with LacZ-injected hearts according to our selection criteria (supplemental Table III). Upregulated genes were further organized into groups representing their known biological functions: apoptosis, cell adhesion, cell division, cell signaling/communication (subgrouped into hormones and growth factors, intracellular transducers, protein modification, and receptors), cell structure/motility, cell/organism defense, channel/transport proteins, inflammatory and immune response, metabolism (subgrouped into amino acid, carbohydrate, lipid, nucleotide, and nucleic acid metabolism), protein synthesis/turnover/posttranslational modification, TFs, translation factors, unclassified genes and expressed-sequence tags (ESTs), as well as other unknown transcripts (Figure 2 and supplemental Table III). Of note, only 2 genes (pyruvate dehydrogenase kinase isoenzyme-4 and gap junction membrane channel protein-1) were downregulated more than 2-fold. When DNA microarray analysis was extended to include genes that were upregulated 1.5- to 2.0-fold in hearts with p38 MAPK overexpression, 651 additional genes (521 upregulated, 130 downregulated) were identified, the majority of which belonged to the groups of unknown function and ESTs (data not shown).
The major functional groups of known genes affected by p38 MAPK overexpression were genes related to cell division (31 genes) and cell signaling/communication (28 genes) as well as genes related to inflammatory and immune response (23 genes). Yet, the most transcripts to be upregulated in the p38 MAPK overexpressing hearts were genes with unknown function or ESTs and related sequences (Figure 2 and supplemental Table III). Strikingly, the individual genes upregulated by p38 MAPK included critical regulators of cell cycle and many fundamental inflammation-associated genes like interleukin-6 (IL-6) and Toll-like receptor-2. Furthermore, the group of cell adhesion genes included well-known fibrosis related factors, such as selectin E, connective tissue growth factor (CTGF), tenascin C, and osteopontin (supplemental Table III). Most of the genes we uncovered have not previously been known to be regulated by p38 MAPK in any tissue types. These novel p38 MAPK target genes include genes involved in the regulation of cell cycle, such as cyclins A2, B1, and B2, kinesin family member-23, and polo-like kinases. Interestingly, a number of them belong to transcription and cell signaling/communication-related functional groups, including TFs ATF-3, Runx1, and Krox20. However, our microarray analysis also identified several genes that have previously been suggested as cardiac p38 MAPK target genes, such as COX-2, ANP,11,13 and cell cycle target histone H3.15
Finally, the complete list of 266 differentially expressed genes was loaded into PathwayAssist. Of those genes, 73 were recognized by the software, and 39 of them (printed in bold in supplemental Table III) were found to have pathway relationships in published research literature. The interaction map showing potential connections among genes regulated by p38 MAPK overexpression in the heart is shown in supplemental Figure I. A central signaling molecule in this pathway is IL-6, which has been implicated in the regulation of 20 genes found to be affected by p38 MAPK. In addition, chemokine (C-C motif) ligand-2 (CCL2), myelocytomatosis oncogene (myc), and cyclin-dependent kinase inhibitor 1A (CDKN1A) also figure prominently in this network.
Even though the microarray analysis identified increased gene expression of several TFs, such as Egr-1 and Egr-2, the expression levels of many well-characterized cardiac TF genes remained unaffected. Because these factors are known to be subject to posttranscriptional and posttranslational modification by p38 MAPK,1 and the cardiac-restricted TF GATA-4 is regulated by p38 MAPK in vitro,9 we first measured LV GATA-4 DNA binding from the MKK3bE+WTp38-injected hearts by gel mobility-shift assay using a 30-bp oligonucleotide probe containing the –90 GATA sites of the rat BNP promoter. Consistently with previous in vitro results, the gene transfer resulted in a 1.6-fold increase (Figure 3A) in BNP GATA-4 DNA binding. However, the transcriptional effects of p38 MAPK were not restricted to GATA-4, because significant increases were also observed in AP-1 (Figure 3B; 4.5-fold versus LacZ), NF-B (Figure 3C; 1.7-fold versus LacZ), and SRF (Figure 3D; 1.4-fold versus LacZ) DNA binding in MKK3bE+WTp38-treated hearts 3 day after injection. In contrast, MKK3bE+WTp38 gene transfer did not affect MEF-2 binding (data not shown).
To confirm the specificity of the DNA-binding reactions, we performed supershift and competitor analysis using specific antibodies and competitor DNAs. An antibody-induced supershift was seen for GATA-4 but not for GATA-5 or GATA-6 complexes, and GATA-4 DNA binding was effectively inhibited by unlabeled self DNA but unaffected by nonrelated competitor DNA Oct-1 or competitor DNA-containing mutated GATA site (Figure 4A). Similarly AP-1, NF-B, and SRF binding was attenuated by unlabeled self DNAs but not by mutated or nonrelated competitor DNAs. Antibody induced supershifts were observed for AP-1, NF-B, and SRF, demonstrating that the complexes bound by oligonucleotide probe contained AP-1, NF-B, and SRF proteins (Figure 4B through 4D).
Functional Effects of p38 MAPK Overexpression in the Adult Heart
To determine the functional significance of changes in cell cycle regulatory and inflammatory genes identified by microarray analysis, we studied the morphological changes in histological analysis and hemodynamic consequences by echocardiography 1 week after MKK3bE+WTp38 gene transfer. Because microarray analysis revealed several genes that are associated with cell division and proliferation, we first performed immunohistochemical staining against Ki-67 antigen to identify cells undergoing proliferation. Ki-67 is considered as a specific marker of cellular proliferation, as all cells that express Ki-67 go on to divide.16 Ki-67 immunostaining 1 week after gene transfer showed large numbers of Ki-67–positive nuclei in the anterior wall of the MKK3bE+WTp38-treated hearts. In contrast, only few Ki-67–positive cells were observed in the LacZ-treated hearts (Figure 5A). Next, histological sections from both experimental groups were stained with hematoxylin/eosin, Sirius Red, and Masson’s trichrome. Remarkably, the histological sections showed a massive infiltration of inflammatory cells in the MKK3bE+WTp38-treated hearts. As shown in Figure 5A, large numbers of mononuclear inflammatory cells were present in the interstitium of the p38 MAPK overexpressing myocardium. Furthermore, Masson’s trichrome and Sirius Red stainings showed large stained areas in the p38 MAPK overexpressing hearts, indicating massive cardiac fibrosis. The area covered by collagen as assessed from Masson’s trichrome-stained sections was significantly higher in p38 MAPK overexpressing than control hearts (11.1% versus 4.3%; P<0.05; n=5 to 6). Histological sections also revealed a notable thickening of the anterior wall (Figure 5B). At day 3 after gene transfer, only minor fibrotic lesions and inflammatory cell infiltration were observed, suggesting that upregulation of gene expression of inflammatory and fibrosis promoting factors preceded the functional changes (data not shown).
To determine the effects of p38 MAPK overexpression on cardiac function, we performed echocardiographic analysis 1 week after gene transfer. These measurements showed preserved cardiac function in the MKK3bE+WTp38-treated animals, as no statistically significant changes in LV diameter, fractional shortening, or ejection fraction were observed (Figure 6). Consistently with the anterior wall thickening observed in histological analysis, the diameter of the interventricular septum was significantly increased. Moreover, the E/A-ratio in MKK3bE+WTp38-treated animals was raised suggesting development of diastolic dysfunction. The LV weight versus body weight (LVW/BW) ratio was also increased in the MKK3bE+WTp38-treated animals in comparison to that of LacZ-injected group 1 week after gene transfer (Figure 6).
Identification of Cell Types Affected by p38 MAPK Overexpression
To illuminate the cell types affected by p38 MAPK overexpression, immunohistochemistry with specific antibodies was performed. p38 MAPK, GATA-4, and IL-6 immunostaining was localized on cardiac myocytes, whereas inflammatory cells and endothelial cells were stained with Ki-67 (Figure 7).
Discussion
The family members of the MAPKs play a vital role in numerous biological processes in response to extracellular stimuli. p38 MAPK was originally identified as a protein kinase that underwent tyrosine phosphorylation in response to osmotic shock and endotoxin treatment.1 Subsequent studies demonstrated that p38 MAPK responds strongly to proinflammatory cytokines, tumor necrosis factor (TNF)-, heat stress, and growth factors.1 Within the cardiomyocytes, p38 MAPK has been documented to be activated in response to G protein-coupled receptor (GPCR) agonists, such as phenylephrine, endothelin-1, and angiotensin II as well as ischemia-reperfusion injury, mechanical stretch and pressure overload.12,17–19 Considering its central position in signal transduction, it is surprising to note that the p38 MAPK-dependent gene-expression pattern has remained fundamentally unknown. This study provides a comprehensive analysis regarding the target genes and TFs of p38 MAPK in the myocardium as well as the functional effects of p38 MAPK activation in the adult heart.
Our findings demonstrate that p38 MAPK controls a wide array of genes at transcriptional level in the heart, the majority of which are related to cell division, inflammation, cell signaling, cell adhesion, and transcription. All of these categories include individual genes that have been previously suggested to be regulated by p38 MAPK in other cell types but also a number of new, previously nondescribed p38 MAPK targets. Although this study was aimed to characterize the gene-expression profile controlled by p38 MAPK in the heart, it is likely that the significance of our findings can be extended beyond the cardiovascular system. So far, p38 MAPK-related genome profiling has been performed by using pharmacological p38 MAPK inhibitors in transformed follicular lymphoma cells and in cultured cardiomyocytes.20,21 Given the diverse effects of pharmacological inhibitors, these studies do not provide direct evidence concerning genes that are controlled by p38 MAPK. In addition, gene-expression profiles in transgenic adult mouse hearts have been determined at 4- to 7-day and 2- to 4-week time points following MKK3b transgene induction by tamoxifen.22 In the current experimental design, the gene-expression profile was analyzed at a time point when no or only minor functional or structural changes were observed, suggesting that the present results represent targets that are directly regulated by p38 MAPK. Furthermore, the profile of the genes that were upregulated by p38 MAPK in our data were not cardiac restricted, as it included a number of conserved genes that are widely expressed in different tissue types. It is also noteworthy that our results, like those following MKK3b transgene induction by tamoxifen,22 represent p38 MAPK targets in several types of cells, such as cardiac myocytes, fibroblasts, and endothelial cells, because adenovirus-mediated gene transfer into the LV was performed as a local intramyocardial injection. On the other hand, the effects of chronic and potent activation of the p38 MAPK pathway achieved with adenovirus-mediated gene transfer may differ from those of more transient activation observed in many experiment models.1–4
A key finding of the present study is the role of p38 MAPK in the cell cycle control in the heart. Previous studies suggest that p38 MAPK may play either a positive or a negative role in this process, depending on cell types and stimuli.1,21 Our microarray data identified important regulators of diverse cell cycle events ranging from DNA replication (DNA topoisomerase ), the control of the cell cycle at the G2/mitosis transition (cyclins A2, B1, and B2 and CDC2) to centrosome function and chromosome segregation in mitosis (kinesins, pituitary tumor-transforming 1, Aurora kinase B). Specifically, p38 MAPK seems to play a major role in the regulation of cell division through transcriptional upregulation of central molecules needed in G2/mitosis cell cycle checkpoint activation. Functionally, the upregulation of the cell cycle regulatory genes was associated with cellular proliferation, as the Ki-67 immunostaining revealed a large number of replicating cells in the myocardium overexpressing p38 MAPK. Based on histological stainings, the majority of the proliferating cells belong to other cell types than cardiomyocytes, such as inflammatory and endothelial cells. However, a recent study showed that constitutively expressed cyclin A2 is able to induce cardiomyocyte mitosis in postmitotic myocardium,23 and downregulation of polo-like kinase has been reported to correlate with loss of proliferative ability of cardiac myocytes.24 In view of these findings, it is tempting to speculate that p38 MAPK-induced gene expression may also promote cardiomyocyte proliferation. Even though p38 was originally reported as a proapoptotic protein kinase in cultured cardiomyocytes, our microarray analysis identified virtually no apoptosis-related genes.25 The only gene upregulated in this category was Bcl-related protein A2, suggesting that the in vitro observations regarding proapoptotic effects of p38 MAPK may not be extended to the adult heart. Taken together, cardiac p38 MAPK is a growth promoting molecule, likely playing a key role in G2/mitosis checkpoint activation. Interestingly, microarray analysis further revealed activation of several genes related to amino acid and nucleotide metabolism, probably also reflecting the vigorous cell growth in the heart.
Our finding that cardiac p38 MAPK overexpression significantly increased IL-6, IL-18, COX-2, CCL2, and Toll-like receptor-2 mRNA levels together with massive inflammatory cell infiltration indicates p38 MAPK as a critical regulator of inflammatory responses of the heart. These observations are consistent with the strong link established between the p38 MAPK pathway and inflammatory responses and diseases in other organ systems. Pharmacological inhibition of p38 MAPK has been previously reported to attenuate the production of proinflammatory cytokines, including IL-1 and IL-6, as well as expression of COX-2.26 Consequently, transgenic mice overexpressing MKK6bE have been previously shown to exhibit increased plasma levels of IL-6.27 The importance of p38 MAPK in myocardial inflammation is further highlighted by the interaction analysis of our microarray results. When genes found to be regulated in the microarray study were used to create a network in which several of these genes are involved in or at least associated with in the literature, IL-6 appears to be a central signaling molecule in this network. Importantly, IL-6 is among the main downstream effectors of pharmacological p38 MAPK inhibition involved in cell proliferation and survival associated pathways.20
In addition to cell proliferation and inflammation, p38 MAPK overexpression caused cardiac fibrosis, which also was tightly coupled to the p38 MAPK-dependent gene-expression profile. p38 MAPK overexpression increased the expression levels of several fibrosis-associated genes, including basic fibroblast growth factor (bFGF), osteopontin, transforming growth factor-2 (TGF)-2, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), bone morphogenic protein-2 (BMP)-2, and CTGF. These findings are supported by previous gain-of-function studies, because transgenic mice with targeted overexpression of constitutively active MKK3 or MKK6 have been shown to exhibit increased interstitial fibrosis.4a,27 Moreover, microarray analysis in MKK3bE transgenic animals revealed changes in expression of genes particularly involved in extracellular matrix remodeling.22 Also, conflicting results have been reported, as cardiac-specific dominant-negative p38, MKK3, and MKK6 transgenic mice develop massive fibrosis in response to pressure overload.28
The upregulation of gene expression by p38 MAPK seems to be a result of transcriptional control, because gel mobility-shift assays demonstrated the activation of several cardiac TFs including GATA-4, AP-1, SRF, and NF-B. Regarding GATA-4, the present results extend our previous in vitro findings indicating that pharmacological p38 inhibition attenuates the DNA binding of GATA-4.9 This is of particular interest considering the central role of GATA-4 in the control of cardiac development and growth as well as in cardiac hypertrophy and heart failure.29 Notably, a very strong 4.5-fold increase was observed in AP-1 DNA binding, and NF-B was upregulated as well. Both of these factors are known to be involved in the inflammatory responses and control of cell cycle. Mechanistically, the activation of TFs in most cases is likely a consequence of direct phosphorylation, because p38 MAPK is known to phosphorylate, eg, GATA-4 and AP-1 components.29 Yet, other mechanisms may be involved as well, because the microarray analysis revealed that p38 MAPK overexpression increases the gene expression of several other intracellular signaling molecules (eg, Rho family GTPase-1 and dual-specificity phosphatase-2).
Our data reveal several novel aspects regarding the role of p38 MAPK and cardiac gene expression in cardiac pathophysiology. The present results demonstrate that in the adult heart, the physiological consequence of p38 MAPK overexpression, via distinct upregulation of gene expression, is cardiac cell proliferation and myocardial inflammation associated with fibrosis. Importantly, persistent inflammation is a characteristic of chronic heart failure. It includes increased levels of proinflammatory cytokines, which contribute to cardiac contractility, cardiomyocyte hypertrophy, and death. It is noteworthy that IL-6, which was strongly upregulated by p38 MAPK, is among the best characterized of these factors and particularly active soon after acute myocardial infarction.30 Fibrosis, in turn, is a critical determinant of postinfarction remodeling, diastolic heart failure, and hypertensive end-organ damage. However, p38 MAPK-induced inflammatory responses may not simply be detrimental to the heart, because under certain conditions, proinflammatory cytokines may also act in a cardioprotective manner by affecting extracellular matrix, integrins, and contribute to vascular and cardiac regeneration and myocardial angiogenesis.30 Accordingly, cardiac fibrosis and cell proliferation may be reparative or harmful depending on pathophysiological context. The cardioprotective role of p38 MAPK is indirectly supported by our observation that p38 MAPK-overexpressing hearts showed preserved systolic function despite the development of fibrosis, diastolic dysfunction, and massive anterior wall thickening. Finally, the findings regarding p38 MAPK and cardiac hypertrophy may be of functional significance. The expression of ANP was significantly increased by p38 MAPK overexpression, but genes encoding structural proteins, such as -myosin heavy chain (-MHC) and -skeletal actin (-SkA), were not upregulated. Furthermore, no morphological signs of cardiomyocyte hypertrophy were observed. These findings support the view that p38 MAPK activation is not prerequisite for the development cardiomyocyte hypertrophy but, in turn, may directly regulate the expression of selected hypertrophy-associated genes.
In summary, our transcriptional profiling results revealed a striking impact of p38 MAPK activation on cell cycle regulatory genes. In addition to cell proliferation, activation of p38 MAPK pathway involves inflammatory and profibrotic gene induction as prominent processes for pathological changes in the heart. We also show that p38 MAPK directly activates multiple cardiac TFs, further emphasizing the role of p38 MAPK as a key regulator of cardiac gene transcription. The results provide a novel link among intracellular signal transduction, gene expression, and cardiac pathophysiology in vivo, as well as an important source of information for development of p38-targeted therapies. The downstream target genes regulated by p38 MAPK suggest a wide variety of additional potential indications besides autoimmune disorders.
Acknowledgments
We thank Dr Jussi Vuoristo (Biocenter Oulu) and Sirpa Rutanen, Pirjo Korpi, Kaisa Penttil, Tuulikki Krn, Marja Arbelius, and Kati Viitala for expert technical assistance.
Sources of Funding
This work was financially supported by the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the National Technology Foundation TEKES, the Aarne Koskelo Foundation, the Ida Montin Foundation, and the Research and Science Foundation of Farmos.
Disclosures
None.
Footnotes
Both authors contributed equally to this study.
Original received April 2, 2006; revision received July 11, 2006; accepted July 17, 2006.
References
Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001; 81: 807–869.
Sugden PH, Clerk A. Regulation of mitogen-activated protein kinase cascades in the heart. Adv Enzyme Regul. 1998; 38: 87–98. [Order article via Infotrieve]
Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol. 2004; 4: 372–377. [Order article via Infotrieve]
Ravingerova T, Barancik M, Strniskova M. Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology. Mol Cell Biochem. 2003; 247: 127–138. [Order article via Infotrieve]
Liao P, Georgakopoulos D, Kovacs A, Zheng M, Lerner D, Pu H, Saffitz J, Chien K, Xiao RP, Kass DA, Wang Y. The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy. Proc Natl Acad Sci U S A. 2001; 98: 12283–12288.
Braz JC, Bueno OF, Liang Q, Wilkins BJ, Dai YS, Parsons S, Braunwart J, Glascock BJ, Klevitsky R, Kimball TF, Hewett TE, Molkentin JD. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J Clin Invest. 2003; 111: 1475–1486.
Kaiser RA, Bueno OF, Lips DJ, Doevendans PA, Jones F, Kimball TF, Molkentin JD. Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia-reperfusion in vivo. J Biol Chem. 2004; 279: 15524–15530.
Martindale JJ, Wall JA, Martinez-Longoria DM, Aryal P, Rockman HA, Guo Y, Bolli R, Glembotski CC. Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo. J Biol Chem. 2005; 280: 669–676.
Behr TM, Nerurkar SS, Nelson AH, Coatney RW, Woods TN, Sulpizio A, Chandra S, Brooks DP, Kumar S, Lee JC, Ohlstein H, Angermann CE, Adams JL, Sisko J, Sackner-Bernstein JD, Willette RN. Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction. Circulation. 2001; 104: 1292–1298.
Tenhunen O, Sarman B, Kerkela R, Szokodi I, Papp L, Toth M, Ruskoaho H. Mitogen-activated protein kinases p38 and ERK1/2 mediate the wall stress-induced activation of GATA-4 binding in adult heart. J Biol Chem. 2004; 279: 24852–24860.
Han J, Molkentin JD. Regulation of MEF2 by p38 MAPK and its implication in cardiomyocyte biology. Trends Cardiovasc Med. 2000; 10: 19–22. [Order article via Infotrieve]
Thuerauf DJ, Arnold ND, Zechner D, Hanford DS, DeMartin KM, McDonough PM, Prywes R, Glembotski CC. p38 mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6. J Biol Chem. 1998; 273: 20636–20643.
Liang F, Gardner DG. Mechanical strain activates BNP gene transcription through a p38/NF-kappaB-dependent mechanism. J Clin Invest. 1999; 104: 1603–1612.
Degousee N, Martindale J, Stefanski E, Cieslak M, Lindsay TF, Fish JE, Marsden PA, Thuerauf DJ, Glembotski CC, Rubin BB. MAP kinase kinase 6-p38 MAP kinase signaling cascade regulates cyclooxygenase-2 expression in cardiac myocytes in vitro and in vivo. Circ Res. 2003; 92: 757–764.
Szatkowski ML, Westfall MV, Gomez CA, Wahr PA. Michele DE, DelloRusso C, Turner II, Hong KE, Albayya FE, Metzger JM. In vivo acceleration of heart relaxation performance by parvalbumin gene delivery. J Clin Invest. 2001; 107: 191–198.
Zhong SP, Ma WY, Dong Z. ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10. J Biol Chem. 2000; 275: 20980–20984.
Yuasa S, Fukuda K, Tomita Y, Fujita J, Ieda M, Tahara S, Itabashi Y, Yagi T, Kawaguchi H, Hisaka Y, Ogawa S. Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats. Mol Cell Biochem. 2004; 259: 177–181. [Order article via Infotrieve]
Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998; 83: 345–352.
Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC, Feuerstein GZ, Yue TL. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation. 1999; 99: 1685–1691.
Fischer TA, Ludwig S, Flory E, Gambaryan S, Singh K, Finn P, Pfeffer MA, Kelly RA, Pfeffer JM. Activation of cardiac c-jun NH(2)-terminal kinases and p38-mitogen-activated protein kinases with abrupt changes in hemodynamic load. Hypertension. 2001; 37: 1222–1228.
Lin Z, Crockett DK, Jenson SD, Lim MS, Elenitoba-Johnson KS. Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells. Mol Cell Proteomics. 2004; 3: 820–833.
Engel FB, Schebesta M, Duong MT, Lu G, Ren S, Madwed JB, Jiang H, Wang Y, Keating MT. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev. 2005; 19: 1175–1187.
Mitchell S, Ota A, Foster W, Zhang B, Fang Z, Patel S, Nelson SF, Horvath S, Wang Y. Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation. Physiol Genomics. 2006; 25: 50–59.
Chaudhry HW, Dashoush NH, Tang H, Zhang L, Wang X, Wu EX, Wolgemuth DJ. Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium. J Biol Chem. 2004; 279: 35858–35866.
Georgescu SP, Komuro I, Hiroi Y, Mizuno T, Kudoh S, Yamazaki T, Yazaki Y. Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes. J Mol Cell Cardiol. 1997; 29: 929–937. [Order article via Infotrieve]
Wang Y, Huang S, Sah VP, Ross J, Brown JH, Han J, Chien KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem. 1998; 273: 2161–2168.
Kumar S, Boehm J, Lee JC. p38 MAP kinases: Key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov. 2003; 2: 717–726. [Order article via Infotrieve]
Li M, Georgakopoulos D, Lu G, Hester L, Kass DA, Hasday J, Wang Y. p38 MAP kinase mediates inflammatory cytokine induction in cardiomyocytes and extracellular matrix remodeling in heart. Circulation. 2005; 111: 2494–2502.
Nishida K, Yamaguchi O, Hirotani S, Hikoso S, Higuchi Y, Watanabe T, Takeda T, Osuka S, Morita T, Kondoh G, Uno Y, Kashiwase K, Taniike M, Nakai A, Matsumura Y, Miyazaki J, Sudo T, Hongo K, Kusakari Y, Kurihara S, Chien KR, Takeda J, Hori M, Otsu K. p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload. Mol Cell Biol. 2004; 24: 10611–10620.
Pikkarainen S, Tokola H, Kerkela R, Ruskoaho H. GATA transcription factors in the developing and adult heart. Cardiovasc Res. 2004; 63: 196–207. [Order article via Infotrieve]
Nian M, Lee P, Khaper N, Liu P. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res. 2004; 94: 1543–1553.
您现在查看是摘要介绍页，详见ORG附件。