In order to study the role of Ankrd2 in cell signaling pathways we silenced endogenous Ankrd2 in human myotubes and monitored gene expression by microarray analysis. Silencing Ankrd2 expression affected genes involved in intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-β and Wnt signaling). Using protein arrays we identified several interacting partners of Ankrd2; PDZ- and SH3-containing proteins and transcription factors. Interestingly, the TF proteins MeCP2, Pax6, NFIL3 and LHX2 bind both to the Ankrd2 protein and Ankrd2 promoter DNA. Another novel finding was that Nkx2.5 and p53 can act as upstream effectors of the Ankrd2 gene and that Ankrd1/CARP can affect the transcriptional ability of MyoD on the Ankrd2 promoter. From the information obatined we can assert that Ankrd2 can act as a powerful regulator in skeletal muscle cells, affecting a multitude of pathways and processes including myogenesis, regulation of gene expression, as well as intra- and intercellular signaling. It exerts its function through interaction with transcription regulators, structural and signaling proteins. Our data are in favor of the proposed function for Ankrd2 in transmitting and transforming mechanical signals into cellular response.
From microarray profiling results, it is evident that alteration in Ankrd2 expression can cause changes in the expression of genes involved in several pathways identified using the KEGG database. Most of the affected genes belong to metabolic pathways, which is not surprising as muscle remodeling process in which Ankrd2 take a part, demands also changes in supporting energy metabolism. Apart from the collection of diverse metabolic pathways that had no particular pathway affected, there are basically three main groups of pathways with differentially expressed genes. The first group is involved in intracellular communication and affects the following signaling pathways: calcium, insulin, MAPK, p53, TGF-β and Wnt signaling. The second group is that of intercellular communication pathways affecting: cytokine-cytokine receptor interaction, endocytosis, focal adhesion (FA), tight junction (TJ), gap junction and regulation of the actin cytoskeleton. The third group is that of disease pathways including Cancer, chronic myeloid leukemia, Hungtington's disease, DCM and HCM cardiomyopathies.
In intracellular communication the majority of external signals move into the cell via ion channels, G-proteins or enyzme linked receptors. Silencing Ankrd2 affects genes in the Calcium pathway: calcium behaves as a second messenger transmitting neuromuscular activity into changes in transcription via calcineurin, calcium-dependent or calcium–calmodulin-dependent protein kinases. Interestingly, in Ankrd2 silenced myotubes FATZ-1/myozenin-1/calsarcin-2 
, a calcineurin/NFAT regulator 
is down regulated ( and S1
) whereas FATZ-2/calsarcin-1/myozenin-2 that affects fiber type composition by blocking calcineurin/NFAT activity is upregulated ( and S2
Another important pathway affected by Ankrd2 silencing is the MAPK pathway which is activated by exercise, environmental stress as well as implicated in muscle growth and differentiation 
. The majority of the detected differentially expressed genes of the MAPK pathway are upregulated upon Ankrd2 silencing ( and S2
). Also several TF proteins that interact with the Ankrd2 protein () are associated with the MAPK pathway: CRK, JUN, p53, MEF2C, PAX6 and MeCP2. It is noteworthy that PAX6 and MEPC2 can also bind the Ankrd2
promoter DNA indicating the presence of control by a feedback loop mechanism.
It is interesting that one of the pathways affected by Ankrd2 silencing is the Insulin signaling pathway especially since DARP, a MARP family member, is up regulated in type 2 diabetes and thought to have a role in glucose uptake in muscle 
. The insulin receptor substrate 1 (IRS-1) plays a key role in transmitting signals from the insulin and insulin-like growth factor-I receptor (IGF-IR) to the PI3K/Akt and Erk/MAPK pathways. Cullin7, one of the genes down regulated on silencing Ankrd2, is an E3 ubiquitin ligase that targets IRS-1 for degradation by the proteasome 
and an increase in the IGF-IR was found to up-regulate Pax6 and glucagon which in turn activated the IRS-2/MAPK pathway that could lead to dysregulation associated with type 2 diabetes 
The intercellular pathways involving cell junctions are linked to the regulation of the actin cytoskeleton and cell signaling pathways. Focal adhesions act as multi-protein signaling complexes as well as having the structural role of linking membrane receptors and the actin cytoskeleton. Gap junctions are an important component of intercalated discs in cardiac muscle 
and are necessary for skeletal muscle differentiation 
. Tight junctions, also known as zonula occludens, are important for signaling 
. TJ proteins ZO-1, ZO-2 and ZO-3 have PDZ and SH3 domains and link the TJ transmembrane proteins to the actin cytoskeleton 
. Here we show that Ankrd2 can bind ZO-1 ( and ) and that ZO-2 (TJP2) is downregulated on Ankrd2 silencing ( and S1
). It is interesting that both ZO-1 
and Ankrd2 () can also bind the Src tyrosine-kinase substrate, Cortactin.
Several potential new interactions were discovered by probing arrays of PDZ, SH3 and transcription factor proteins with Ankrd2 (, , , ) corroborating its regulatory role. As can be seen in some of these proteins have roles in cell junction (MUPP1, ZO1, cortactin) and signaling pathways such as TGF-β (HDC1, MADH3), Wnt (MADH3), MAPK (JUN, JUNB, MECP2, MEF2C, PAX6, p53, CRK) and NFAT (HAND2, SDB2). RIL and hCLIM are members of the Enigma family of PDZ LIM proteins that have been shown to interact with the members of both the FATZ (calsarcin/myozenin) and myotilin families of Z-disc proteins 
. Also of note is the fact that Ankrd2 can bind to MUPP1, a multiple PDZ domain protein, known to bind the tight junction claudins 
Apart from the role of Ankrd2 in intracellular signaling, our results indicate a new role for Ankrd2 in intercellular signaling, in transmitting and transforming mechanical signals into cellular response. It could be hypothesized that Ankrd2 is implicated in spreading stress signals through a strictly intracellular route as well as an inside/outside path to the sarcolemma and back to the nucleus through cell-surface receptor pathways. The results obtained by DNA and protein arrays are in accordance and strongly implicate Ankrd2 role in regulatory and signaling processes.
It was demonstrated that tumor suppressor p53 has complex and multilevel interaction with MARP family members Ankrd1/CARP and Ankrd2. It behaves as an important regulator of their expression and MARPs are able to modulate the activity of p53. We have already shown their physical interaction on protein level, ability of Ankrd1/CARP to modulate p53 transcriptional activity on different promoters and potential p53 dependant regulation of Ankrd1/CARP expression through upregulation of Ankrd1 promoter activity 
. Here we show that Ankrd2
gene expression can be regulated by p53 since it significantly increased Ankrd2
promoter activity in luciferase assays (). Since in adult muscle both p53 and Ankrd2 levels increase in response to stress 
it could be suggested that p53 probably acts on Ankrd2
gene expression in differentiated muscle cells that are exposed to stress stimuli such as stretch. Our results implicate a novel role for p53 in up-regulation of Ankrd2
gene expression and as common regulator of MARP expression.
Alterations in interaction between Ankrd2 and p53, as well as other players in p53 pathway could be involved in pathogenesis of some tumors. In fact, the expression of Ankrd2 is elevated in a very high percentage of rhabdomyosarcomas and its use as a potential tumor marker for differential diagnosis of this soft tissue sarcoma has been suggested 
. Although there is overexpression of Wnt in embryonal rhabdomyosarcomas the canonical Wnt/B-catenin signaling pathway was down-regulated possibly due to altered AP-1 
. Since both Wnt 
and Ankrd2 
are up regulated on skeletal muscle injury it is not surprising that several genes of the Wnt pathway are affected by Ankrd2 silencing. Apart from tumors, Ankrd2 could be linked to dystrophies and cardiac diseases since some proteins from the FATZ (myozenin/calsarcin), myotilin and Enigma families are differentially expressed in Ankrd2 silenced myotubes (, S1
Ankrd2 has an active role in the processes that coordinate proliferation and differentiation in muscle 
. Our results support the indispensable role of Ankrd2 in myogenesis by demonstrating that Ankrd2 silencing alters genes involved in cell to cell communication, which is very important in myogenesis. The changes in gene expression and cell morphology that occur during myogenic differentiation must be coordinated in a spatiotemporal fashion and one of the ways to achieve this is through regulation of these processes by cell-cell adhesion and resultant signaling 
. Also, primary and secondary myoblast fusion processes require cell-cell contact 
Ankrd2 interacts with a variety of proteins that have diverse function (structural and regulatory) and contain ankyrin repeats, modules for protein-protein interaction. Our results revealed that Ankrd2 has distinct binding patterns for its interacting partners. It uses exclusively ankyrin repeats for interaction with sarcomeric proteins (titin and telethonin), whereas N terminal domain that maps to aa 98–121 is also needed for its interaction with TFs (PML, YB-1 and p53). There are several SH3 and PDZ binding sites predicted by ELM 
within the Ankrd2 N-terminus and although PDZ domains predominantly bind short C-terminal peptides they can also bind internal peptide sequences 
. It is possible that binding motif(s) located in the N-terminus stabilize the interaction between Ankrd2 and regulatory proteins. On the other hand, calpain 3 could be also involved in regulation of Ankrd2 protein-protein interactions and its intracellular localization. Both Ankrd2 and Ankrd1/CARP are the substrates of this modulator protease as well as titin 
. Since the cleavage site of Ankrd2 by calpains is Arg 77 which is situated proximally from NLS, there is also a possibility that calpain 3 mediated proteolysis, apart from regulation of Ankrd2 and titin interaction, could also introduce conformational changes into Ankrd2 protein that allow differential binding of Ankrd2 to sarcomeric or regulatory proteins.
These results and observations should be analyzed in a light of the most recent result that Ankrd2 is found to be a downstream target in Akt pathway as demonstrated by Cenni and colleagues 
. Akt-mediated signaling pathways are important in differentiation, regeneration and hypertrophy of muscle 
. It was found that Ankrd2 is a novel substrate specific for Akt2 and that oxidative stress triggers phosphorylation of Ankrd2 Ser 99 which in turn induced nuclear translocation of Ankrd2. In fact, the site of Ankrd2 phosphorylation Ser99 corresponds to Ser72 in the Ankrd2 primary sequence reported under accession number CAI14194.1 in which Arg77 is the site of calpain 3 proteolysis. This finding sheds a completely different light on these results since the sites are very close. Phosphorylation of Ankrd2 by Akt2 induces nuclear translocation of Ankrd2. The proteolysed Ankrd2 could bind more strongly to the N2A region of titin in a similar way as demonstrated for Ankrd1/CARP 
. As phosphorylation and cleavage sites are separated by only 5 amino acids, it is possible that phosphorylation and proteolysis are competitive processes that can alter the inter-cellular distribution of Ankrd2. We hypothesize that the phosphorylated pool of Ankrd2 is predominantly located in the nuclei and that the proteolysed Ankrd2 is sequestered by the titin N2A region located at the I-band. In muscle cells that are in early phase of differentiation (binucleated cells), as well as in normal muscle tissue, both nuclear and cytoplasmic localization of Ankrd2 can be observed. Since it is known that Ankrd2 expression in the nucleus increases with stress, a possible mechanism could be that calpain 3 is not able to proteolyse Ankrd2 when Ser72 is phosphorylated, therefore Ankrd2 is not sequestered by the titin N2A region but is free to move to the nucleus. Rationalization of these separate observations on Ankrd2 selective interactions, calpain proteolysis and phosphorylation by Akt 2 kinase has yet to occur, but an association with coordination of stress response could be a possible link. The interrelation and interdependence between these three phenomena is another open question.
Molecular mechanisms that regulate Ankrd2
gene expression and its role in the heart are completely unknown. Here we demonstrate that the cardiac specific transcription factor Nkx2.5 up-regulates the activity of Ankrd2
promoter and that Ankrd1/CARP, a cardiac specific MARP family member, could regulate Ankrd2 expression through activation of MyoD. Apart from the well established critical role of the transcriptional activator Nkx2.5 in cardiac morphogenesis 
, it also has a role in the regulation of cardiac-specific gene expression in the adult heart. Its expression is upregulated in response to hypertrophic stimulation which may have implications in the transcriptional regulation of the cardiac gene program in hypertrophied hearts 
. In the adult heart, Nkx2.5 also plays an important role in protecting the myocardium against cytotoxic damage 
. Nkx2.5 mediated regulation of Ankrd2 expression in the heart could be the mechanism underlying its role in cardiac signaling pathways activated upon stress.
Although Ankrd1/CARP acts as negative co-factor in the regulation of cardiac specific gene expression 
, we recently showed that Ankrd1/CARP could behave as a positive regulator of gene expression and modulate p53 activity on the p21
. Here we demonstrate that Ankrd1/CARP also acts as positive regulator of MyoD activity on the Ankrd2
promoter (). Therefore, apart from p53 
, we have identified MyoD as another transcription factor whose activity can be modulated by Ankrd1/CARP. Although MyoD is known as a key regulator of skeletal muscle differentiation it was only recently detected in cardiac muscle, in periarterial Purkinje fibers 
. Purkinje fibers are conduction cells located in the inner ventricular walls and since Ankrd2 is expressed in the ventricles 
it is possible that the expression of Ankrd2 in cardiac muscle cells is under the control of MyoD and that Ankrd1/CARP could up-regulate MyoD dependant Ankrd2 expression in the heart. The emerging role of Ankrd2 in cardiac muscle is further supported by our finding that the HCM and DCM pathways are both affected when Ankrd2 is silenced in myotubes. One of the promising lines of future studies on Ankrd2 could be to identify mutations in Ankrd2 gene that are linked to these cardiomyopathies as has been done for Ankrd1/CARP 
It is interesting that both the Ankrd2
promoter DNA and the Ankrd2 protein can bind transcription factors MECP2, LHX2, NFIL3 and PAX6 indicating the existence of a regulatory feedback loop mechanism ( and ). Transcriptional regulators HOXA5, KLF12 and LHX2 participate in developmental processes and their interaction with Ankrd2 could be important for its function in myogenesis. MECP2 is particularly interesting as a nuclear protein with a role in gene regulation. Recently it has been proposed to act not only as a transcriptional repressor but also as an activator; in fact most genes appear to be activated rather than repressed by MECP2 
. It should be noted that the DNA of the Ankrd2
promoter that bound MECP2 was not methylated, however MECP2 is also capable of binding non-methylated DNA 
. MECP2 is upregulated in differentiated cardiomyocytes with a concomitant increase in global methylation and condensed chromatin 
. The finding that Ankrd2 binds MECP2 suggests that Ankrd2 could affect not only transcription but also chromatin remodeling. Therefore, the final target of signaling cascades involving Ankrd2 could be the structural modification of chromatin.
Our data support a multi-tasking role of Ankrd2 in many cellular processes regulating skeletal muscle differentiation, growth and remodeling. The results obtained from both the DNA- and protein arrays give a strong indication that Ankrd2 represents a central node within regulatory networks involved in the determination of muscle cells (MRF4), the regulation of trunk (SIX4, MEF2C) and head (PITX2, LBD1) skeletal muscle formation, control of muscle phenotype (MEF2, NFAT, JUNB, HDACs), regulation of calcineurin activity (FATZs) as well as control of muscle protein turnover (FOXO3A, PIK3C2B, NBR1, AKT signaling, FATZs). As mechano-transcriptional links in the myoblasts are found at distinct sarcomeric regions and activate different transcriptional programmes it raises the question of whether crosstalk between these pathways exists. Our data suggest that the Ankrd2 protein, itself, represents a possible link between distinct mechano-transcriptional connections. In fact, previous and current results demonstrate its functional interaction with proteins localized in the Z-disc (FATZ-1/myozenin-1/calsarcin-2, FATZ-2/myozenin-2/calsacin-1, telethonin) and M-band (NBR1 and MURFs) mechanosensing complexes. The functional significance of crosstalk between different mechanosensors and synergistic or antagonistic activation of transcriptional programmes that regulate muscle remodeling remain to be elucidated.