Original articleInduction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection
Introduction
CCN1 (also known as Cyr61) was first identified as an immediate early gene upregulated by growth factor stimulation and subsequently shown to be induced in response to a wide range of extracellular stimuli [1], [2]. CCN1, classified as a matricellular protein, is secreted from cells and serves to regulate diverse responses including cell migration, proliferation, angiogenesis, senescence and cell survival. CCN1 is a multi-domain protein which includes a number of distinct integrin binding sites [2], [3]. CCN1 binding to integrins mediates the majority of its diverse, and at times opposing cellular effects [2], [4], [5], [6], [7]. We have demonstrated that activation of GPCRs by lysophospholipids (S1P and LPA) or thrombin leads to robust induction of CCN1 expression in glioblastoma cells and that this is mediated through the activation of RhoA [6], [8]. RhoA involvement in S1P induced CCN1 induction has also been demonstrated in other glioma cells lines [9], [10] and in stretch-induced responses of smooth muscle cells [11], [12].
RhoA is best recognized as transducer of signals for actin cytoskeletal rearrangement. A critical, albeit less appreciated role, for RhoA is in transcriptional regulation, as first discovered through the effects of RhoA activation on serum response factor (SRF) target gene expression [13]. SRF, a widely expressed member of the MADS (MCM-1, Agamous, and Deficients, SRF) box superfamily, is constitutively localized to the nucleus and bound to SRE sequences [14], [15]. Transcriptional activity of SRF is regulated through its association with other transcriptional co-activators which provide combinatorial control of SRF target genes [16], [17]. To date, two major families of coactivators are known to activate SRF, the ternary complex factors (TCFs) and the myocardin-related transcription factors (MRTFs; also known as MAL or MKL). The effect of RhoA on SRF dependent genes is mediated through a TCF-independent mechanism [15]. Recent seminal studies demonstrated that the myocardin family proteins MRTF-A/B provide the link between RhoA-dependent cytoskeletal regulation and SRF-dependent gene expression [16], [18], [19], [20]. Mechanistically, MRTF-A associates with G-actin and is thus sequestrated in the cytoplasm under resting conditions. Serum stimulation and signals that activate RhoA to promote actin polymerization lead to MRTF-A dissociation from G-actin, whereupon it translocates into the nucleus and triggers activation of SRF target genes. MRTF-A activation was recently implicated in the ability of mechanical stretch to induce RhoA-mediated CCN1 gene expression in smooth muscle cells [11]. In the heart, deletion of MRTF-A has been shown to decrease cardiac hypertrophic responses induced by pressure overload or angiotensin II (Ang-II) [21], consistent with our early findings on RhoA involvement in hypertrophic ANF gene expression in cardiomyocytes [22], [23].
Relatively little is known about the regulation or functional role of CCN1 in cardiomyocytes. Global CCN1 gene deletion results in embryonic lethality associated with altered cardiac development [24] and Drexler's laboratory reported that CCN1 expression is highly upregulated in the myocardium of patients with heart failure or ischemic myopathy [25]. CCN1 appears to serve as a survival signal for cardiomyocytes by activating kinases such as Akt and ERK that protect against oxidative stress [7]. Conversely CCN1 has been shown to sensitize to apoptosis induced by TNFα or Fas ligand [4], [5] but this depends on specific integrin binding sites [4], [5] and is context dependent [2], [5], [7], [26].
We and others have demonstrated that S1P and RhoA signaling confers cardioprotection [27], [28], [29], [30]. It is not known whether CCN1 signaling contributes to this response. In the present study we demonstrate that CCN1 is induced in cardiomyocytes by S1P and other agonists that activate RhoA, that this occurs through MRTF-A signaling, and that CCN1 confers cardioprotection against ischemia/reperfusion injury both in cardiomyocytes and the isolated perfused heart based on findings using mice in which cardiac CCN1 is genetically deleted.
Section snippets
Materials
S1P and LPA were purchased from Avanti Polar Lipids (Alabaster, AL, USA), prepared according to manufactory instruction. C3 exoenzyme was obtained from Cytoskeleton, Inc. Antibodies against CCN-1, RhoA, MRTF-A and α-actinin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-RhoGDI and anti-Lamin A/C were purchased from Cell Signaling Technology (Danvers, MA, USA). CCG-1423 and CCG-203,971 were kindly provided by Dr. Scott Larsen (University of Michigan). All other chemicals and
Differential effects of GPCR agonists on CCN1 expression in neonatal rat ventricular myocytes
The cardioprotective lysophospholipid S1P was tested for its ability to induce CCN1 expression in neonatal rat ventricular myocytes (NRVMs). CCN1 mRNA assessed by quantitative RT–PCR increased within 15 min after S1P treatment, reached a peak at 30 min and declined to basal levels by 2 h (Fig. 1A). S1P treatment also lead to robust increases in CCN1 protein in whole cell lysates which were significant as early as 30 min and sustained over 24 h (Fig. 1B). We then compared the ability of other GPCR
Discussion
CCN1 is a pleiotropic molecule, expression of which is highly induced in response to diverse stimuli. Cellular functions including cell migration, proliferation, differentiation, survival/apoptosis and senescence can be regulated through CCN1 signaling [2]. CCN1 is highly expressed in the myocardium of patients with heart failure or ischemic myopathy, and it has also been shown to be increased in the mouse heart in response to pressure overload and myocardial infarction [25]. The molecular
Funding
This work was supported by National Institutes of Health grant HL028143 and GM036927 to Joan Heller Brown and SM by HL097037 to Shigeki Miyamoto. Sunny Xiang was supported by an AHA Postdoctoral Fellowship (11POST7580130) and Olivia Yu by Pharmacological Sciences Training Grant (T32-GM007752).
Acknowledgments
We thank Dr. Scott Larsen for kindly providing CCG-1423 and CCG-203,971 and Dr. David Roberts for excellent technical assistance.
Conflict of interest
None declared.
Disclosure
None.
References (66)
- et al.
CCN1 protects cardiac myocytes from oxidative stress via beta1 integrin-Akt pathway
Biochem Biophys Res Commun
(Apr 13 2007) - et al.
Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness
Exp Cell Res
(May 1 2007) - et al.
Mechanical regulation of the proangiogenic factor CCN1/CYR61 gene requires the combined activities of MRTF-A and CREB-binding protein histone acetyltransferase
J Biol Chem
(Aug 21 2009) - et al.
The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF
Cell
(Jun 30 1995) - et al.
Transcriptional regulation by extracellular signals: mechanisms and specificity
Cell
(Jan 27 1995) - et al.
Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors
J Biol Chem
(Oct 8 2004) - et al.
Actin dynamics control SRF activity by regulation of its coactivator MAL
Cell
(May 2 2003) - et al.
Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes. Dissociation of Ras and Rho pathways
J Biol Chem
(Dec 6 1996) - et al.
Focal adhesion kinase as a RhoA-activable signaling scaffold mediating Akt activation and cardiomyocyte protection
J Biol Chem
(Dec 19 2008) - et al.
Physical and functional interactions of Galphaq with Rho and its exchange factors
J Biol Chem
(May 4 2001)
Galphaq expression activates EGFR and induces Akt mediated cardiomyocyte survival: dissociation from Galphaq mediated hypertrophy
J Mol Cell Cardiol
In vitro ADP-ribosylation of Rho by bacterial ADP-ribosyltransferases
Methods Enzymol
Design, synthesis and prostate cancer cell-based studies of analogs of the Rho/MKL1 transcriptional pathway inhibitor, CCG-1423
Bioorg Med Chem Lett
Redox modification of nuclear actin by MICAL-2 regulates SRF signaling
Cell
Signalling via the reperfusion injury signalling kinase (RISK) pathway links closure of the mitochondrial permeability transition pore to cardioprotection
Int J Biochem Cell Biol
Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins
J Biol Chem
Lysophospholipids control integrin-dependent adhesion in splenic B cells through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11)
J Biol Chem
RhoA/Rho kinase up-regulate Bax to activate a mitochondrial death pathway and induce cardiomyocyte apoptosis
J Biol Chem
Lysophosphatidic acid induces hypertrophy of neonatal cardiac myocytes via activation of Gi and Rho
J Mol Cell Cardiol
Rapid upregulation of CTGF in cardiac myocytes by hypertrophic stimuli: implication for cardiac fibrosis and hypertrophy
J Mol Cell Cardiol
Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats
J Mol Cell Cardiol
HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart
J Pharm Biomed Anal
Expression of Cyr61, a growth factor-inducible immediate-early gene
Mol Cell Biol
CCN1/Cyr61: the very model of a modern matricellular protein
Cell Mol Life Sci
Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets
Nat Rev Drug Discov
Cytotoxicity of TNFalpha is regulated by integrin-mediated matrix signaling
EMBO J
Extracellular matrix protein CCN1 regulates cardiomyocyte apoptosis in mice with stress-induced cardiac injury
Cardiovasc Res
Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61
FASEB J
G protein-coupled receptors go extracellular: RhoA integrates the integrins
Mol Interv
Sphingosine-1-phosphate regulates glioblastoma cell invasiveness through the urokinase plasminogen activator system and CCN1/Cyr61
Mol Cancer Res
Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways
Am J Physiol Cell Physiol
p67SRF is a constitutive nuclear protein implicated in the modulation of genes required throughout the G1 period
Cell Regul
Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL
Science
Cited by (30)
Cellular communication network factor 1 (CCN1) knockdown exerts a protective effect for hepatic ischemia/reperfusion injury by deactivating the MEK/ERK pathway
2021, Clinics and Research in Hepatology and GastroenterologySphingosine-1-phosphate receptor 3 signaling
2021, Clinica Chimica ActaAn inhibitor role of Nrf2 in the regulation of myocardial senescence and dysfunction after myocardial infarction
2020, Life SciencesCitation Excerpt :For example, mice lacking the senescence effector p16INK4a or p53 were more sensitive to pressure overload-induced cardiac fibrosis and dysfunction probably due to the loss of non-age-dependent premature senescence in cardiac fibroblasts [11]. While cardiomyocyte-specific (CR) knockout (KO) of cysteine-rich angiogenic inducer 61 (Cyr61) or CCN family member 1 (CCN1) is capable of exacerbating infarct size of the ischemic/reperfused heart [12], cardiac overexpression of CNN1 via adeno-associated virus serotype 9 gene transfer protects against both pressure overload- and MI-induced cardiac fibrosis and dysfunction associated with downregulated cellular senescent responses in cardiac myocytes and/or fibroblasts [11,13,14]. In addition, global p53 KO inhibits cardiac fibroblast senescence but enhances cardiac fibrosis in pressure overloaded and infarcted hearts [11,15]; however, CR-p53 KO induces age-dependent cardiac hypertrophy and heart failure but protects against pressure overload-induced cardiomyopathy and heart failure [16].
Blockade of Fibroblast YAP Attenuates Cardiac Fibrosis and Dysfunction Through MRTF-A Inhibition
2020, JACC: Basic to Translational ScienceCitation Excerpt :Although the functional importance of each has been documented in isolation, there is also convincing evidence of interpathway communication leading to complex signaling networks that modulate fibrosis (28). RhoA signaling directs the nuclear localization of MRTF-A to control ECM-related genes (29–31), and the Hippo–YAP pathway intersects with MRTF-A and SRF to further modulate gene expression (19,32). However, the regulation of MRTF-A expression itself is largely unexplored.
Protective transcriptional mechanisms in cardiomyocytes and cardiac fibroblasts
2019, Journal of Molecular and Cellular CardiologyCitation Excerpt :Indeed, CFs may function as important modulatory cells to balance pro-fibrotic mechanisms. CF-derived expression of CCN1 can mediate integrin dependent CM proliferation as part of recovery from injury [73,209]. Similarly, RhoA-dependent induction of CCN1 in CMs can provide integrin-dependent, Akt-mediated protection [73].
RhoA regulates Drp1 mediated mitochondrial fission through ROCK to protect cardiomyocytes
2018, Cellular SignallingCitation Excerpt :Thus activation of RhoA in cardiomyocytes does not lead to phosphorylation of Drp1 at serine-637 or phosphorylation of Drp1 through PKD signaling. Our previous work demonstrates that RhoA signaling in cardiomyocytes regulates the kinases ROCK, Akt, and PKD [2, 3, 6, 9]. We considered possible involvement of Akt downstream of ROCK [2, 6], in the regulation of Drp1; however, phosphorylation of Drp1 by S1P was not blocked by an Akt inhibitor or a PKD inhibitor (data not shown).