If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Gene expression profiling of mouse skin wounds has led to the discovery of numerous target genes that may have therapeutic or diagnostic value. Among these, cardiac ankyrin repeat protein (CARP, ankrd1) expression was markedly and persistently elevated in several cutaneous compartments. This review summarizes the current state of knowledge of CARP and its regulation in biological systems. In addition to its role as a nuclear transcription cofactor in many cell types including vascular endothelium, CARP is also a structural component of the sarcomere. CARP transcripts are prominent in cardiogenesis and muscle injury, and they are under complex regulation by cytokines, hypoxia, doxorubicin, and other forms of stress. CARP overexpression in wounds by adenoviral gene transfer leads to a high vascular density, and CARP exerts effects on endothelial behavior. The unusual cellular distribution and actions of CARP make it a novel candidate gene in tissue repair.
atrial natriuretic factor
cardiac ankyrin repeat protein
epidermal growth factor
human microvascular endothelial cell
muscle ankyrin repeat protein
transforming growth factor-beta
vascular endothelial cell
vascular smooth muscle cell
3′ untranslated region
Healing of wounds involves an orchestrated cascade of events, and it requires a complex interplay between multiple cell types that must respond to the initial insult with a progressive change in their genetic programming. Beginning with the activation of platelets and their release of clotting factors and cytokines and ending with re-epithelization and the final maturation and remodeling of the wound area, gene expression profiles of all involved cell types are dynamic and responsive to the ever-changing wound milieu. To “fish” for novel genes involved in this process, cDNA subtractive hybridization was performed in collaboration with Switch Biotech AG, comparing intact skin and 1 day full-thickness wounds in the mouse (
). Using this approach, the expression of ankrd1, a gene which encodes the transcriptional cofactor, cardiac ankyrin repeat protein (CARP) was among several hundred genes that were found to be highly induced in wounds. The time course of CARP mRNA and protein expression, determined by quantitative, real-time RT-PCR and Western blot analysis, showed that they were significantly increased within hours, reaching peak levels by 15 hours and remaining elevated for 2 weeks during the wound healing process (Figure 1). Furthermore, immunohistochemistry and in situ hybridization for CARP protein and mRNA in day 1 wounds revealed that monocytes, cells of the epidermis and the vasculature, and skeletal muscle had increased expression (
) (Figure 2). Thus, changes in the abundance of this transcriptional cofactor may modulate the gene expression profiles in these cells. This review will focus on what is known about CARP and its actions, and how it affects its role in neovascularization.
The gene encoding CARP was initially identified in a screen for cytokine-inducible factors in microvascular endothelial cells (
). Found on chromosome 10 in humans and 19 in mouse, the ankrd1 gene structure is highly conserved, with nine exons and a canonical TATA box in the proximal promoter which, to date, appears to produce a single transcript (
). No alternative splicing of the RNA has been described. Other canonical response elements identified in the 5′ flanking sequence of CARP include GATA sites, E-box elements, a CCAC box, a CAGA box, and M-CAT, activator protein-1 and SP-1 binding sites (
) (Figure 3). These sites exert both positive and negative regulation of CARP. A short 5′ untranslated region (UTR) and a longer 3′ UTR, which contains a conserved degradation motif, characterize the 10.6 kb CARP pre-mRNA. The primary transcript containing nine exons is processed to encode a 319 amino-acid protein with a molecular weight of 36 kDa. CARP protein sequence and domain organization is highly conserved among mammalian species. Analysis of the peptide sequence identifies a bipartite nuclear localization signal, a PEST-like sequence (a four amino acid sequence associated with rapidly degraded proteins), four highly conserved ankyrin-like repeats and another less conserved half repeat, and numerous potential modification sites for phosphorylation, glycosylation, and myristilation (
) (Figure 3). Ankyrin-repeats have been found in proteins that are involved in protein–protein interactions, including those characterized as cyclin-dependent kinase inhibitors (p16, p18, and p19), developmental regulators (Notch and IκBα), and transcription factors (Swi6 and GABPα, β) (reviewed in
), and the potential for multiple forms of protein modification suggest that the expression and activity of this protein is highly regulated at the post-transcriptional level. Indeed, the steady-state levels of both CARP protein and mRNA are significantly increased by β-adrenergic agonists (
). Lower, constitutive CARP expression in adult mice is most prominent in the heart, whereas lung and skeletal muscle transcripts are also detected by Northern blot analysis. CARP mRNA is also found in the highly vascularized placenta. Using a 2.5 kb CARP promoter-driven lacZ transgene, embryonic expression appears to be primarily localized in the developing heart, where CARP expression has been studied most extensively. Removal of the cardiogenic homeobox gene Nkx2.5 using knockout strategies results in selectively reduced CARP expression (
). This result suggests that, at least in cardiac muscle, CARP is downstream of the Nkx2.5 pathway and that Nkx2.5 either directly or indirectly regulates CARP expression. GATA 4, a direct target of Nkx2.5, is also involved in cardiac development and has been shown to regulate CARP expression through direct binding to canonical response elements in the CARP proximal promoter (
CARP expression is increased in several cardiomyopathies and in cardiovascular injury. Left ventricular dilated cardiomyopathy and ischemic cardiomyopathy in humans exhibit increased CARP expression as compared to non-affected human donor hearts (
). In a well-characterized, in vitro model of cardiac hypertrophy that involves activation of fetal genes in cardiac myocytes by α1-adrenergic stimulation, α1-adrenergic agonists increase endogenous CARP expression whereas antagonists lower expression in myocytes. Analysis of the CARP proximal promoter has identified TEF-1 and GATA 4 binding to MCAT and GATA elements, respectively, as the transcriptional mediators of the α1-adrenergic stimulation (
). Vascular smooth muscle cells (VSMCs) in rat atherosclerotic lesions exhibit increased CARP expression, and similar induction is observed in both VSMCs and vascular endothelial cells (VECs) of collateral arteries that form following femoral artery occlusion (
). However, our own data employing selective inhibitors in the MS-1 vascular endothelial cell line suggest dominant signaling through the p38 mitogen-activated protein kinase pathway and a lesser contribution of Smad signaling (Y Shi, unpublished).
CARP is also an acronym for cardiac adriamycin-responsive protein. Adriamycin (doxorubicin; DOX) is a chemotherapeutic agent that can produce collateral damage to the myocardium of patients undergoing chemotherapy, and extravasation of the drug during administration produces severe chemical injury to the skin and adjacent tissues. The mechanism of DOX toxicity is primarily by the hyper-induction of oxygen radicals, leading to oxidative stress and programmed cell death/apoptosis (
). Paradoxically, DOX treatment can cause both induction (in vivo) and repression (in vitro) of CARP expression. In piglets, DOX treatment results in left ventricular dilated cardiomyopathy that is associated with increased CARP mRNA and protein 20 days after a single intravenous injection of the drug (
). A more recent microarray analysis of DOX-sensitive transcription products in the heart compares the effects of a single, acute intraperitoneal injection of DOX to weekly intraperitoneal injections of smaller doses (
). Acute DOX increases CARP mRNA significantly, and quantitative, real-time RT-PCR confirms a 4.53-fold induction. However, chronic, low-dose DOX exposure does not result in a significant change in cardiac CARP mRNA. In contrast, cultured cardiomyocytes treated with DOX show a sensitive, dose-dependent repression of CARP that is associated with acute oxidative stress. This effect has been attributed to the action of hydrogen peroxide on a H7-sensitive serine/threonine kinase pathway that interacts with a proximal M-CAT element in the human CARP promoter (
). In a separate study, DOX-treated embryonic heart-derived H9c2 cells developed oxidative stress followed by apoptosis that correlated with the level of GADD153 (CHOP-10), a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors (
). The level of CARP mRNA was found to be inversely proportional to that of GADD153. Furthermore, co-transfection of a GADD153 expression vector with a CARP promoter (-206)-driven luciferase reporter construct resulted in a dose-dependent decrease in luciferase activity. These in vitro data suggest direct regulation of CARP transcription by a GADD153 response element in the proximal promoter.
The opposing DOX regulation of myocardial CARP expression in vivo and in vitro could be related to the timing of analysis. Acute downregulation of CARP by oxidative stress and cardiomyocyte apoptosis could lead to increased mechanical stress and a rebound in CARP expression in surviving cardiomyocytes during the reparative phase. In addition, many forms of myocardial injury can elicit expression of TGF-β and tumor necrosis factor alpha-α, both of which induce CARP expression ((
) and the calpain protease, p94 (Figure 4). Passive muscle stretch induces increased immunostaining for the MARPs both in the nucleus and the I-band, with DARP staining also increasing in the intercalated discs (
). The concept that CARP is responsive to mechanical stress is consistent with its cytoplasmic distribution in striated muscle (cardiac and skeletal). There is still much to learn about the role of MARPs in striated muscle, and we speculate that these ankyrin repeat proteins may link myofibrillar stretch signals to the transcriptional regulation of muscle gene expression.
Cytoplasmic CARP may also participate in regulating cardiac muscle contractility by its interaction with cardiac calsequestrin-2 (CASQ-2), a protein that stores calcium for muscle function (
). Five distinct CASQ-2 binding regions have been identified in CARP: three in the NH2-terminus and two within the ankyrin repeat region. None of the CASQ-2 binding regions overlap the CARP–titin interaction domains (see Figure 3). Without more functional data, the significance of this interaction can only be conjectured; however, overexpression of CARP in engineered heart tissue results in a significant decrease in the force of contraction stimulated by increasing doses of calcium (
). This attenuation in calcium-stimulated contraction could involve more extensive CARP binding to CASQ-2, thereby sequestering calcium in the sarcoplasm and making it unavailable for muscle contraction.
To investigate its actions in the wound-healing process, CARP was transduced into both full-thickness excisional wounds and an experimental sponge granulation tissue model by adenovirus-CARP infection (
). In these models, CARP significantly increased neovascularization, blood perfusion, and the abundance and organization of migrating VEC. In both rabbit ear excisional wounds and ischemic rat skin wounds, exogenous expression of CARP increased neovascularization and blood perfusion, as assessed by histological analysis of vessel size and numbers as well as laser Doppler perfusion imaging. In an experimental granulation tissue model in which polyvinyl alcohol sponges are implanted subcutaneously in rats, sponge injection with adenovirus-CARP resulted in increased vascularization as assessed by CD34 staining of vessels. In flk-1/lacZ knock-in mice 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside staining showed a significant increase in lacZ-positive endothelial cells in the advenovirus-CARP injected sponges relative to those injected with a control virus.
Potential Actions Of Carp
As observed in striated muscle, CARP protein has been identified in both the cytoplasm and the nucleus of VSMC and VEC. Although CARP-protein associations within these cells have not been determined, the identification of titin-like proteins in smooth muscle and non-muscle cell types suggest a possible commonality with the CARP associations seen in striated muscle. Smitin, a 700 kDa titin-like protein, is associated with the contractile apparatus in smooth muscle (
). Overexpression of the z-repeat constructs results in the loss of stress fibers, suggesting the presence of a titin-like protein in non-muscle that acts to organize the stress fibers, similar to the sarcomere organizing function of titin in which CARP appears to play a role (
). As yet, the association of CARP with the non-muscle titin-like proteins or any other proteins and the effects of protein–protein interactions on CARP activity have not been explored.
CARP expression is increased in VSMC and VEC following wounding, but its effects are not certain. CARP overexpression studies in vivo and in vitro have provided some insights into its function. As reported above, overexpression in wounds and experimental granulation tissue increases angiogenesis and vasculogenesis, leading to increased blood perfusion, which appears to be due to the enhanced infiltration of vascular endothelium and endothelial precursor cells (
). Support for this concept is provided by the effects of CARP overexpression in human microvascular endothelial cells (HMVEC). CARP overexpression does not affect proliferation in tissue, as assessed by proliferating cell nuclear antigen staining (Shi, unpublished). Preliminary findings show that overexpression of CARP in vitro results in the same or lower proliferation than HMVEC treated with either a control virus or nothing. Cell migration is CARP-responsive. Electrical cell-substrate impedance sensing was used to examine migration of CARP-transduced cells after ablation of a portion of the cell monolayer with a brief electrical pulse (
). The rate and extent of monolayer restoration (i.e., cell migration) as measured by impedance, was greater in the CARP-overexpressing cells. Additionally, the haptotactic activity of the HMVEC in response to EGF is also increased, indicating that CARP increases the migration of VEC in response to cytokine signaling. Furthermore, CARP is also capable of inducing HMVECs tube formation, a measure of capillary endothelial morphogenesis, in a Matrigel™ assay. This response is partially abrogated with CARP small interfering RNA, further supporting a direct role for CARP in endothelial cell morphogenesis (Shi, unpublished).
Previous studies indicated that CARP is protective against both hypoxia and oxidative stress-induced programmed cell death or apoptosis (
). When human umbilical vein endothelial cells are subjected to serum starvation, cells transduced with CARP are able to survive better than those transduced with either a control virus or nothing. Treatment of human umbilical vein endothelial cells with DOX causes oxidative stress and initiation of the apoptotic pathway by inducing caspase 3. CARP transduction increases cell survival that correlates with a decrease in basal- and DOX-induced caspase 3 activity (Shi et al., unpublished). Thus, CARP may increase vascular cell abundance by amplification of the migratory stimulus while reducing the rate of apoptosis due to oxidative stress or physiologic attrition.
Inasmuch as CARP is implicated in neovascularization, understanding the signaling mechanisms that regulate its expression becomes important. As mentioned above, CARP mRNA and protein are upregulated by TGF-β signaling in VSMC via a pathway that involves Smad factors (
). Smad 3 potently increases CAGA binding element-dependent activity of a CARP promoter (-1828)-driven luciferase reporter whereas Smad 6, an inhibitory Smad, inhibits TGF-β stimulated activity. Our unpublished data suggest that both TGF-β and tumor necrosis factor alpha-α, but not vascular endothelial growth factor or basic fibroblast growth factor induce CARP expression in VEC. Though the tumor necrosis factor alpha-α signal transduction pathway has not yet been determined in these cells, we have found that the TGF-β signaling induces CARP mRNA and protein in VEC via the mitogen-activated protein kinase/p38 pathway rather than the Smad pathway. The p38 inhibitor SB203580 and a dominant-negative form of p38 attenuate the TGF-β induction of CARP mRNA but Smad 7, another inhibitory Smad, has no effect (Shi et al., unpublished). Similarly, CARP expression in cardiac myocytes is reported to be specifically upregulated by p38 and a constitutively active form of RAC 1 (
). Activation by both factors is reportedly dependent on an M-CAT element within the CARP proximal promoter. Thus, CARP appears to be induced in endothelial cells by inflammatory cytokines and not angiogenic factors. Furthermore, the same inducers activate alternative signaling pathways in different cell types (Table 1 and Figure 4).
Another serious gap in our knowledge of CARP is its downstream effects. Although CARPs function in the cytoplasm of VEC and VSMC remains to be determined, analysis of its nuclear function in cardiomyocytes suggests that it acts as a transcriptional cofactor that regulates expression of heart-enriched genes (Figure 4). CARP is reported to interact with the ubiquitous transcription factor YB-1 (
). Studies to date have emphasized the role of this pairing in the downregulation of transcriptional targets such as myosin light chain-2v via the HF-1a element present in its promoter. Other cardiac-enriched genes such as antinuclear factor, have shown regulation by CARP overexpression (
), possibly by a similar mechanism. Thus, CARP-YB-1 interactions may serve as a prototype for targets of the Nkx2.5 homeobox protein in ventricular cardiomyocytes. Additionally, CARP expression is also associated with circumstances of muscle regeneration, such as bupavicaine injury and Duchenne muscular dystrophy (
) suggesting a transcriptional regulatory function in skeletal muscle. Our preliminary gene expression profiling of VSMC and HMVEC after CARP overexpression gives credence to the idea that CARP or its downstream targets are also responsible for upregulation of a number of relevant genes in these cells.
Although the studies that have been discussed herein describe CARP expression in several systems, these approaches do not define the role of endogenous CARP. As the protein is massively upregulated during cardiomyogenesis (
), it may play a central role in the organization of the sarcomere and/or the transcriptome of the developing cardiomyocyte. In this respect, targeted deletion of CARP might be expected to lead to a fatal disruption of the cardiogenic program. Although lethality of global CARP deletion needs to be confirmed, the use of a conditional deletion in selected tissues or at selected sites should greatly improve our understanding of the intrinsic roles of the molecule. Currently, small interfering RNA reagents in this laboratory appear to be able to abrogate CARP-dependent effects such as endothelial morphogenesis. These tools can be used to fill the serious gap in our knowledge of CARPs downstream effects.
Based on associations with elements of the sarcomere and the transcriptional machinery, CARP appears to have both cytoplasmic and nuclear roles. The presence of titin-like proteins in smooth muscle and non-muscle cells suggests that CARP or a related member of the MARP family could have analogous functions in striated, smooth muscle and non-muscle cell types. At the nuclear level, CARP is strongly implicated as a transcriptional cofactor in regulation of cardiovascular development and response to injury in a variety of tissues. A number of studies show that oxidative stress and several stress-induced signaling molecules modulate CARP in the context of its transcriptional activity, and its own expression through promoter elements that are partially characterized. The mode of action of CARP on target genes is not well understood. The relationship between the cytoplasmic and nuclear sites of CARP interaction may have parallels with the dual role of molecules such as β-catenin (
), in which a structural element of the cytoplasm transforms into a regulatory element when shuttled to the nucleus. In the case of striated muscle, CARP is a key element of a complex containing titin, CASQ-2 and other molecules, which could be capable of relaying mechanical signals. It remains to be determined whether nuclear translocation of CARP contributes to mechanical or other stress responses. Overexpression of CARP in cells and tissues suggests that it could significantly contribute to the quality and extent of tissue repair, particularly with respect to neovascularization. It is not known whether these effects of excess CARP are a direct reflection of its intrinsic role, and targeted downregulation or deletion of CARP will be necessary to define its function. Regarding (cutaneous) wound repair, the pharmacologic action of CARP underscores the potential of gene transfer to reveal heretofore unknown activities of genes, the ability of adenoviral gene delivery to expand the wound healing pharmacopeia to nuclear transcription factors, and the possible presence of a novel mechanism for neovascularization.
Conflict Of Interest
The authors state no conflict of interest.
This work was supported by the NIDDK, the Department of Veterans Affairs, and Switch Biotech AG. We thank Susan R. Opalenik for critical reading of the manuscript and R. Michael Slowey for graphic illustration. Birgit Reitmaier, Michaela Bittner, and Andreas Goppelt contributed significantly to the development of the concepts presented.
Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter.