**1. Introduction**

The cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (CI-M6P/IGF2R, hereafter IGF2R) is a type-1 transmembrane glycoprotein consisting of a large N-terminal extracytoplasmic domain, which allows it to bind to a wide variety of ligands [1,2]. The IGF2 and M6P ligands [3–5] of IGF2R have distinct but important roles in normal development and mesoderm differentiation [6]. Many studies have demonstrated the suppression action of IGF2R on insulin-like growth factor 1 receptor (IGF1R) signaling by scavenging extracellular IGF2 [7]. Furthermore, several lines of evidence demonstrate that IGF2 is highly expressed in rodent embryos, where it functions as an embryonic growth factor, while its amount is diminished at birth [8]. Smith et al. recently showed that a transgene-induced overexpression of IGF2 blocked programmed cell death, one of the main pathological features of cancer [9]. Furthermore, in some cancers such as mammary tumors, IGF2R behaves as a tumor suppressor gene [10], whereas in other cancers such as cervical tumors or glioblastomas, IGF2R acts as an oncogene [11,12]. Thus, these two traits of IGF2R might depend on cell type. Interestingly, cervical tumors and glioblastomas have common mesenchymal founders, namely myofibroblasts [13], which are also involved in muscle disease. Muscle repair is a complex and tightly regulated event that recruits different cell types, starting from macrophage and lymphocyte consecutive involvement and terminating with satellite cell (SC) activation and differentiation [14]. Among the common hallmarks of muscular dystrophy are the infiltration of immune cells into skeletal muscle fibers, and fibrotic cell proliferation [15–18].

Impaired muscle regeneration with SC pool exhaustion is considered an additional pathological feature of Duchenne muscular dystrophy (DMD) [19]. The main biological function of IGF2R is the suppression of IGF1R signaling via the deprivation of extracellular IGF2 ligands. Some studies have explained the tumor suppressive functions of IGF2R by its negative regulation of the oncogenic IGF2–IGF1R signal axis [2]. However, in muscle tissues, the IGFs bind to the IGF1R leading to conformational changes and activation of its tyrosine kinase activity, promoting muscle regeneration [20]. In injured tissues, IGF1 is secreted by SCs and mediates muscle-derived stem cell proliferation and differentiation into myoblasts, which contribute to myofiber formation for restoring normal tissue structures [21]. It has been demonstrated that prolonged expression of IGF1 causes an exaggerated protein synthesis and is responsible for muscular hypertrophy, by increasing myofiber diameter [22–25], and also preventing muscle atrophy in cases of cachexia or chronic inflammation [26]. Thus, tissue-specific IGF1 upregulation rises to the challenge of counteracting the development of muscular dystrophy.

Similar to IGF1, autocrine IGF2 is fundamental to mediate the differentiation of SCs in vitro, but little is known about its role in skeletal muscle development and regeneration in vivo [27]. The expression profile of *IGF2* is quite complicated as it depends on multiple-promoter activation, alternative translation initiation and messenger RNA (mRNA) stability. IGF2R functions as a negative regulator of IGF2 in embryonic skeletal muscles and modulates the amount of systemic IGF2 by inducing its degradation through lysosomes and clearance from the circulation [28,29].

Even if the signaling cascade that regulates the activation of IGF2 at muscular level is not determined, it has been shown that the phosphatidylinositol 3-kinase (PI3K)–the serine/threonine protein kinase B (AKT) pathway is the IGF2 downstream pathway contributing to mammalian target of rapamycin (mTOR) functions [30]. A study by Ge et al. [31] showed that the synergic activity of mTOR with miR-125b regulated *IGF2* production both transcriptionally and post-transcriptionally, and that these events positively influence myogenesis.

In muscle pathology and ageing contexts, where there is a predominant switch of the fiber phenotype from fast to slow [32,33], IGF2 was also able to orchestrate the development of fast myofibers by acting as a twitch motor unit during secondary myogenesis. The modulation of IGF2 expression had a dramatic impact on the amount of fast myofibers in the respiratory (intercostal and diaphragmatic) muscles, likely lessening cardio-respiratory dysfunction related to DMD. IGF2 targeting was suggested as a feasible therapeutic strategy, since IGF2 has a small size and consequently it could be easily distributed to a skeletal muscle target [34]. However, the IGF2R signaling pathways involved in muscle repair and disease remain to be identified.

#### **2. Structure, Genomic Organization and Gene Imprinting of IGF2R**

Imprinting genes are those whose expression is determined by one's parents. They occur in discrete clusters that are regulated by DNA elements called imprinting control regions (ICR). The two copies of one imprinted gene are characterized by methylation of cytosine–guanine base pairs. This modification originates in the paternal germ cells—after adding a methyl group, the chromatin becomes inaccessible to transcription machinery, so the gene is silenced. The IGF2/H19 locus is one of the imprinted gene clusters in human chromosome 11p15.5 or mouse distal chromosome 7 and plays a primary role in muscle cell development [35]. The expression of this cluster is regulated by a distant enhancer downstream of the H19 coding region. A recent study presents paxillin (PXN), a focal adhesion protein, as a transcriptional regulator of the IGF2 and H19 genes; in particular, it has the opposite effect on the activity of the IGF2 and H19 promoters [36]. The knockdown of PXN in human HepG2 cells allows for an increase in the activity of the H19 promoter and at the same time a decrease in the activity of the IGF2 promoter [36]. In a recent study, it was demonstrated that the loss of imprinting (LOI) in a mouse model of Beckwith–Wiedemann syndrome (BWS) results in impaired muscle differentiation and hypertrophy. It was also proposed that there is a signaling pathway in which IGF2 overexpression allows for an overactivation of mitogen-activated protein kinase (MAPK) signaling, while a loss of H19 long non-coding RNA (lncRNA) prevents the regulation of p53 levels, resulting in reduced AKT/mTOR signaling [35].

The *IGF2R* is an example of differential imprinting in the human and mouse genomes; the *IGF2R* is repressed on the paternal chromosome in the murine genome, but the same gene is expressed from both alleles in humans. For humans, the study was conducted on several tissues—adult liver, placenta, fetus, kidney, adrenal, brain, intestine, heart, tongue, skin and muscle: in all these samples both IGF2R alleles were expressed more or less at the same level. Accordingly, it was established that this character is subject to Mendelian segregation, escaping imprinting. As a plausible explanation for this phenomenon, Kalscheuer et al. suggested that the stages of initiation and maintenance of imprinting could be under the control of trans-acting factors that could act differently in mice compared to in humans [37]. They also hypothesized an alternative explanation based on the structural difference of the mouse and human *IGF2R* genes, as an "imprinting box" was previously discovered that could be modified by methylation in the female gamete and allowed maternal expression [37]. The *IGF2R* gene is located on mouse chromosome 17: it is composed of 48 exons and encoded for a 2482-amino acid protein. Exons 1–46 comprise the extracellular part of the receptor, while the transmembrane portion and the cytoplasmic region are located, respectively, on exon 46 and 46–48 [38].

### **3. IGF2R-Dependent Pathway**

M6P/IGF2-R lacks intrinsic kinase activity, and the role of G-proteins in its downstream pathway has been investigated. Functionally, G-proteins are a class of proteins that interact with multi-spanning receptors (seven transmembrane receptors or heptahelical receptors). Some studies have speculated that M6P/IGF2-R, although characterized by a single-spanning structure, might initiate signaling cascades through G-proteins in a direct or indirect manner. It is well known that the pertussis toxin exerts its activity by binding and blocking the activation of the α subunit of the Gq/11-protein [39]. El-Shewy et al. showed that the use of this toxin is able to inhibit the function of M6P/IGF2-R, therefore suggesting the involvement of G-proteins in the downstream pathway of M6P/IGF2-R. Pre-treatment of Human embryonic kidney 293 cells (HEK-293) cells with the pertussis toxin can significantly reduce the level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation resulting from the interaction of IGFs with their receptors. The indirect activity of these receptors is carried on through parallel activation of G-protein-coupled receptors (GPCR). They also observed that the administration of IGF1 and IGF2 ligands activates sphingosine kinase (SK) 4, which is translocated from the cytosol to the plasma membrane. There, it promotes an increase in extra- and intracytoplasmic levels of sphingosine-1-phosphate (S1P). S1P's interaction with its G-protein-coupled receptor represents a general mechanism for indirect G-protein-dependent signaling of M6P/IGF2-R resulting in ERK1/2 phosphorylation [40]. Conversely, the direct activity of IGF2R and G-proteins was hypothesized by Nishimoto et al. [41,42]: based on their observations, a region of the cytoplasmatic domain of M6P/IGF2-R may contain aminoacidic residues (2410–2423) that directly bind and activate G-proteins, in particular, Gi-2. This is supported by evidence that the use of both human and rat antibodies targeting aminoacidic residues is able to inhibit the activation of Gi-2 resulting from M6P/IGF2-R stimulation.

#### **4. Functions of IGF2R**

#### *4.1. IGF2R Expression Levels Regulate Cardiac Development and Remodeling*

The expression of the *IGF2R* gene is particularly abundant in embryo hearts. IGF2R-deficient mice display dramatic cardiac dysfunction and heart failure development. In adults, low levels of IGF2R expression lead to heart disease and apoptosis in cardiac myocytes [43], while upregulation determines myocardial infarction, remodeling [44] and hypertrophy [45]. In particular, the IGF2R activates phospholipase C (PLC) through the heterotrimeric G-protein-coupled receptor: this interaction is mediated by αq G subunits (Gαq) that in turn allow the function of different enzymes such as the protein kinase C-α (PKC-α), Ca2<sup>+</sup>-calmodulin-dependent protein kinase II (CaMKII) and ERK kinase—all upregulated in cardiac hypertrophy [46]. Alternatively, the modulation of IGF2R can enhance cardiomyocyte apoptosis and cardiac contractility by inhibiting protein kinase A (PKA) phosphorylation [47]. A recent study showed that IGF2R expression is negatively controlled by the cardioprotective heat shock transcription factor 1 (HSF1). Antitumor drugs such as doxorubicin (DOX), meanwhile, lead to high levels of IGF2R expression in cardiomyocytes, through a decrease in HSF1, and trigger cardiac apoptotic processes [48]. IGF2R expression in the heart may also mediate increased sizes of cardiomyocytes, in a manner dependent on PKA activation, and mediate atrial natriuretic peptide (ANP), calcium-dependent channels (SERCA) and phospho-troponin I signaling, as described recently by Wang et al. [49].
