Advances in research of biological functions of Isthmin-1

ISM1 in growth and development

Current reports on the role of ISM1 focus on its dynamic expression during different periods of embryonic development and the fact that it affects the embryonic body axis, organs, craniofacial morphology, and hematopoietic cell formation.

Dynamic expression of ISM1

Several studies have shown that ISM1 is expressed early in the embryonic development of frogs and zebrafish (Pera et al. 2002; Xiang et al. 2011). Pera et al. (Pera et al. 2002) observed that ISM1 was expressed in several sites in frog embryos, including the ventral ectodermal lip, proximal mesoderm, neural folds, notochord, MHB, isthmus, and hind mesencephalon.Additionally, Ism1 is expressed in the branchial arches. A correlation study of the timing and location of ISM1 expression in zebrafish found that it was expressed in the MHB and hind trunk regions during the late prometaphase/early segmentation stage and thereafter only in the tail bud region and notochord; the expression level in the notochord increased at 22 h post fertilization (hpf), decreased in the tail bud at 30 hpf, decreased in the notochord at 48 hpf, and disappeared at 72 hpf (Kesavan et al. 2021). Interestingly, deletion of ISM1 did not affect embryonic morphological changes during early development of zebrafish embryos, which may be triggered by genetic compensation phenomena through mutations in other genes (El-Brolosy et al. 2019; Kesavan et al. 2021).

The expression of ISM1 in chicks is dynamic. In 10-somites embryos, ISM1 expression is spatially restricted to the anterior region (Osório et al. 2014). At 21–22 somites, ISM1 transcripts are strongly expressed in the epithelium of the ventral ear sac and begin to be weakly expressed in the neural tube of the anterior trunk region (Osório et al. 2014). In embryos of 31–33 somites, ISM1 is expressed in the epithelium of the optic cup and ventral midbrain, as well as the ear vesicle, the neural tube, the dermatomyotome, and the lateral plate mesoderm (Osório et al. 2014). The absence of ISM1 expression in the MHB and precursor mesoderm or somites of chick embryos may be related to species differences and varying differentiation mechanisms of somites and dermomyotomes (Osório et al. 2014).

ISM1 also starts to be expressed early in mouse embryonic development, and the regions where ISM1 transcripts exist differ depending on the stage of development; for example, low levels of ISM1 can be observed at embryonic day 6.75, ISM1 transcripts are expressed in the anterior mesoderm at embryonic day 7.5, and the expression level gradually increases as ISM1 transcripts become present in the somites, MHB, anterior somites, anterior and lateral plate mesoderm, ear vesicle, and neural tube (Osório et al. 2014). The expression of ISM1 in the dorsal aspect of the trunk neural tube in mice starts at 5–6 somites and adapts in a gradient according to he anterior–posterior axis of the embryo, suggesting that ISM1 may be associated with spine formation (Osório, et al. 2014). Analysis against human gene expression databases revealed that skin, mucosal tissue, and lymphoid tissue cells express ISM1 and have barrier functions; however, the human nervous system has low levels of ISM1 expression (Valle-Rios, et al. 2014). The identification of ISM1-expressing lymphocytes revealed that human and mouse CD4+ T cells express ISM1 upon activation, which suggests that ISM1 may be associated with the body's innate and adaptive immunity (Valle-Rios, et al. 2014). Furthermore, although ISMI is present in the genomes of a wide range of vertebrates, its expression in mammals differs from that of other animals. ISM1 is virtually absent from the mammalian nervous system, but it is present in CD4+ T cells of peripheral blood. Thus, ISM1 is expressed in organs of multiple animals at different times and may affect the growth and development of the sites in which it is expressed.

Studies have shown that ISM1 is directly and indirectly controlled by β-catenin. Injection of the nodal signaling gene, sqt, and the homologous frame transcription factor, bozozok (boz), mRNA during zebrafish gastrula embryogenesis resulted in ectopic expression of ISM1 in the embryonic marginal region, as well as upregulation of ISM1 expression, suggesting that both genes may be upstream of ISM1 (Kesavan et al. 2021). Notably, sqt and boz upregulate ISM1 expression even when β-catenin is blocked, which, combined with previous studies reporting that β-catenin is a transient signaling molecule, suggests that the Wnt/β-catenin pathway may not be required for ISM1 activation (Kesavan et al. 2021). However, in contrast to these early observations, late ISM1 expression appears to be independent of nodal signaling (Kesavan et al. 2021), suggesting that ISM1 expression is temporally controlled by a different signaling system.

ISM1 and the growth and development process

Orofacial cleft is a common congenital malformation with a complex etiology that is influenced by both genetic and environmental factors (Martinelli, Palmieri, Carinci, and Scapoli 2020; Nasreddine, El Hajj, and Ghassibe-Sabbagh 2021). FGFs, Sprouty Homolog 1 (Spry1), and Spry2 in Fgf-8 are also involved in craniofacial development and associated with cleft lip or palate (Conte et al. 2016; Porntaveetus et al. 2010; Reynolds et al. 2020). Since the ISM1 expression pattern is similar to and belongs to the same expression group as Fgf-8, it is expressed in MHB, ear substrate, maxilla, mandible, periorbital, and other sites important for craniofacial development,  including branchial arch 1 (BA1) which gives rise to the maxilla, the structure that is associated with cleft lip and/or palate (Osório et al. 2014; Pera et al. 2002). Craniofacial defects occur in cases with heterozygous deletions of ISM1; therefore, Lansdon et al. (Lansdon et al. 2018) identified ISM1 as an orofacial clefting candidate for study and concluded that it was haploinsufficient. Inhibition of ISM1 in African clawed frog embryos showed shortened embryonic body axes, missing or defective eyes, abnormal tail, and craniofacial malformations including median facial clefts analogous to those found in humans, where increasing inhibition caused whole embryo abnormalities or even headlessness (Lansdon et al. 2018). Furthermore, the expression of LIM homeobox protein 8 (Lhx8), which is associated with craniofacial morphology, decreased with the knockdown of ISM1 (Lansdon et al. 2018). This suggests that ISM1 is involved in craniofacial development (Table 1, Fig. 4).

Table 1 Expression and biological functions of ISM1 in different sitesFig. 1figure 1

Effect of ISM1 on organ morphology and hematopoiesis during growth and development

ISM1 deficiency in Xenopus laevis embryos may cause craniofacial malformations via Lhx8, causing whole-embryo abnormalities as the degree of inhibition increases. In chick embryos, ISM1 inhibits NODAL-SMAD2 and downstream targets, leading to left–right asymmetry and abnormal heart localization in chick embryos. Wnt is both an important factor mediating HSC production and an upstream signal for ISM1, and ISM1 knockdown leads to reduced HSPC production and fewer mature erythrocytes and bone marrow cells, so Wnt may affect hematopoiesis through ISM1 cell production. (Paired-liked homeodomain transcription factor 2, PITX2)、CER1 (Cerberus 1).

The TGF-β superfamily includes several subfamilies of TGF-β, NODAL, growth differentiation factors (GDF), and activating and bone morphogenetic proteins (BMP), which play important roles in embryonic development, body immunity, and cancer (Hayes et al. 2021; Magro-Lopez and Muñoz-Fernández 2021; Stuelten and Zhang 2021). ISM1 contains a TSR1 structural domain, which typically mediates TGF-β family signaling, suggesting that ISM1 may be involved in regulating TGF-β signaling (Adams and Lawler 2011; Rossi et al. 2004). Osório et al. (Osório et al. 2019) found that ISM1 caused a significant decrease in phosphorylation of the ligand SMAD2 of NODAL by examining the effect of ISM1 on the main ligands of the TGF-β family, while the effect on the expression of several other ligands was not significant. Interestingly, it was shown that the loss of inhibitory function of ISM1 on NODAL in the absence of the AMOP structural domain was not significantly affected when the TSR1 structural domain was deleted, suggesting that the effect of ISM1 on NODAL may not be dependent on TSR1, but on the AMOP structural domain (Adams and Lawler 2011; Osório et al. 2019; Pera et al. 2002). In addition, NODAL is important for the development of the embryonic mesoderm and endoderm, as well as for the formation of the anterior–posterior and left–right body axes (Schier 2009). ISM1 is highly expressed in the anterior mesoderm of chick and mouse embryos, and mesoderm and endoderm formation is associated with NODAL signaling, which is consistent with the fact that ISM1 is thought to be a regulatory gene for NODAL signaling in zebrafish embryos (Bennett et al. 2007; Montague and Schier 2017). In vitro, the AMOP structural domain of ISM1 inhibits NODAL during development by interacting with the activin A receptor type 1B ligand, resulting in left–right asymmetry and abnormal heart positioning in chick embryos. (Table 1, Fig. 1).

In spinal animals, hematopoietic stem cells (HSC) are constantly self-renewing and differentiating to maintain blood homeostasis throughout development (Montazersaheb et al. 2022). HSCs differentiate into populations of progenitor cells of various types of blood cells, which together maintain the body's blood cell population (Manz et al. 2002; Weinreb, Rodriguez-Fraticelli, Camargo, & Klein, 2020). The zebrafish is a perfect model for studying the biology of hematopoietic stem and progenitor cells (HSPCs) due to its similarity of circulatory and hematopoietic systems to humans and its transparent coloration (Carroll and North 2014; Gore, Pillay, Venero Galanternik, and Weinstein 2018). Berrun et al. (Berrun, Harris, & Stachura 2018) showed that ISM1 expression levels were high in all three cell lines (zebrafish embryonic stromal stem cell line, embryonic caudal hematopoietic stromal tissue cell line, and zebrafish renal stromal cell line), supporting the idea of different time points of hematopoiesis in zebrafish, and that knockdown of ISM1 results in a decrease in bone marrow cells, such as erythrocytes, neutrophils, and macrophages, in the organism; furthermore, ISM1 deletion resulted in a decrease in HSPC production. Previous studies have shown that Wnt, an important factor mediating HSC production, can in turn regulate ISM1 expression, which may explain the involvement of ISM1 in hematopoietic cell formation during zebrafish development (Campbell et al. 2015; Stachura et al. 2009; Wolf et al. 2017). In mice, the lungs are one of the active hematopoietic organs (Lefrançais et al. 2017). ISM1, expressed mainly in lung NK and NKT-like cells in mice (Osório et al. 2014; Valle-Rios et al. 2014). Rivera et al. (Rivera-Torruco et al. 2022) recently showed that mice ISM1+ cells have a progenitor phenotype associated with endothelial cells (EC), mesenchymal cells, and hematopoietic cells in the lungs, and that ISM1+ LSK cells may represent a subset of hematopoietic progenitors. These results suggest that ISM1 is closely associated with the hematopoietic function (Fig. 1) In addition, recent studies have shown that gene enrichment analysis of ISM1 is associated with TGF-β signaling, which has an important role in embryonic development, hematopoiesis, and spinal cord disease (Bataller et al. 2019; Wu et al. 2021). However, whether ISM1 is involved in hematopoiesis via TGF-β needs further investigation.

ISM1 deficiency in zebrafish embryos may cause craniofacial malformations via Lhx8, causing whole-embryo abnormalities as the degree of inhibition increases. In chick embryos, ISM1 inhibits NODAL-SMAD2 and downstream targets, leading to left–right asymmetry and abnormal heart localization in chick embryos. Wnt is both an important factor mediating HSC production and an upstream signal for ISM1, and ISM1 knockdown leads to reduced HSPC production and fewer mature erythrocytes and bone marrow cells, so Wnt may affect hematopoiesis through ISM1 cell production. (Paired-liked homeodomain transcription factor 2, PITX2)、CER1 (Cerberus 1).

ISM1 and metabolism

Adipose tissue, the liver, and skeletal muscle play a vital role in maintaining systemic energy homeostasis (Korenblat et al. 2008). In the state of obesity, excessive energy intake leads to subcutaneous fat spillage, which causes ectopic lipid accumulation in skeletal muscle, liver, and other tissues, decreased glucose tolerance, and inflammatory reactions, resulting in a variety of metabolic diseases. Insulin is the only hypoglycemic hormone in the body, and it not only promotes glucose uptake, but also promotes lipid synthesis, which may even lead to obesity or the development of alcoholic fatty liver disease in the case of hyperinsulinemia (Kolb et al. 2020). Therefore, finding cytokines that can both increase glucose uptake and inhibit lipogenesis may be a crucial direction for current glucose uptake research. Recent studies have defined ISM1 as an adipokine that has important roles in promoting glucose uptake, inhibiting lipogenesis, and stimulating protein synthesis.

ISM1 and glucose metabolism

ISM1 positively correlates with obesity in human and mouse adipocytes, and in the plasma of females (Jiang et al. 2021). However, circulating ISM1 levels were lower in the middle-aged overweight population with type 2 diabetes than in the non-type-2-diabetic overweight group; this suggests that elevated ISM1 may reduce the risk of developing diabetes (J. Wang et al. 2022).

In 2021, by sequencing RNA from brown and white mature adipocytes in mice and performing bioinformatics analysis, Jiang et al. (Jiang et al. 2021) found that ISM1 was highly expressed in mature adipocytes, especially brown adipocytes, suggesting that ISM1 may be closely related to the function of mature adipose tissue. This study used recombinant ISM1 or insulin to treat human SGBS adipocytes, primary mouse adipocytes, 3T3-L1 adipocytes, and human skeletal muscle cells, which are closely related to glucose metabolism (Jiang et al. 2021). The study found that ISM1 mediated glucose uptake in a variety of cells in a non-insulin-dependent pathway, and that ISM1-mediated glucose uptake capacity differed between different cell types and species, which may be related to ISM1-dependent cell type-specific receptors or glucose transporter proteins (Jiang et al. 2021). Glucose transporter 4 (GLUT4) is a glucose transporter protein regulated mainly by adenosine triphosphate (ATP) or insulin in adipose and skeletal muscle cells (Fazakerley et al. 2022). In vitro experiments have demonstrated that ISM1 promotes GLUT4 translocation from the cytoplasm to the plasma membrane, while endogenous phosphorylation of the energy metabolism factor AKTS473 increases glucose uptake (Jiang et al. 2021). ISM1 was able to induce pAKTS473 phosphorylation levels in a variety of mature adipocytes and human primary skeletal muscle cells, and treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors revealed that ISM1-induced glucose uptake was completely blocked, suggesting that that ISM1 requires PI3K to induce glucose uptake in adipocytes (Jiang et al. 2021).

The mammalian target of rapamycin (mTOR) and its complexes mTORC1 and mTORC2 are activated by insulin, which in turn regulates insulin sensitivity (Destefano and Jacinto 2013). Treatment of cells with mTORC1, 2 inhibitors followed by stimulation with ISM1 or insulin revealed that mTORC2 inhibitors blocked ISM1induced AKT signaling, while mTORC1 inhibitors did not, suggesting that ISM1 may be involved in the induction of PI3K-AKT signaling pathway and glucose uptake through mTORC2 (Jiang et al. 2021). However, when both mTOR inhibitors were present, ISM1 induced a complete loss of phosphorylation of S6S235/S236 and unchanged AKT phosphorylation levels, suggesting that ISM1 induces activation of downstream SD S235/S236 via mTOR1 (Jiang, et al. 2021). Notably, although ISM1 and insulin have similar regulatory effects on glucose, ISM1 does not act on the insulin receptor, but rather activates the PI3K-AKT pathway by binding to a unique receptor to promote glucose uptake by adipocytes. (Table 1, Fig. 2).

Fig. 2figure 2

Diagram of the mechanism of ISM1 regulation of glucose metabolism

In metabolism, ISM1 secreted by adipocytes acts in an autocrine manner to regulate glucose uptake via mTORC2-PI3K-AKT in conjunction with insulin-IR/IGF-1R-PI3K-AKT. ISM1 is able to cause ERK phosphorylation but has no significant effect on other signaling pathways such as PKA, PDK1 or GSK3β.

ISM1 and lipid and protein metabolism

In a high-fat diet-induced obesity and nonalcoholic fatty liver mouse model, injection of recombinant ISM1 can effectively reverse hepatic steatosis, which may provide a new direction for clinical treatment of metabolic diseases (Jiang et al. 2021). ISM1 is similar to insulin in function and downstream signaling pathways, thus ISM1 may have similar roles to insulin in regulating glucose uptake and lipogenesis. Interestingly, ISM1 inhibits insulin-induced expression of the pro-lipid synthesis factor sterol regulatory element binding protein-1 (cSrebp1c) and its target genes acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and low density lipoprotein receptor (LDLR) in a dose-dependent manner, thereby attenuating insulin-induced lipid de novo lipogenesis(Jiang, et al. 2021). Since ISM1 also represses the expression of carbohydrate response element binding protein (ChREBPβ) and peroxisome proliferator-activated receptor γ coactivator 1β (PGC1β) (Jiang, et al. 2021), ISM1 may regulate lipogenesis through multiple pathways. Unfortunately, this experiment only targeted mature adipocytes for detection of ISM1 and did not account for the lipolytic effect on adipocytes at different growth stages. Thus, it is worth further exploring whether ISM1 can directly affect adipocytes at different growth stages in the future (Fig. 3).

Fig. 3figure 3

Diagram of the mechanism of ISM1 regulation of lipid metabolism and protein metabolism

In hepatocytes ISM1 inhibits the expression of Srebp1c and its target genes and affects lipid de novo synthesis. In hepatocytes and skeletal muscle cells, it promotes protein synthesis via AKT-mTORC1-S6.

In hepatocytes, ISM1 induces pS6S235/S236 overactivation, and the combined action of ISM1 and insulin maintains pS6S235/S236 and increases protein synthesis 2.9-fold (Jiang et al. 2021). ISM1 inhibits lipogenesis by switching the cellular anabolic state to a protein synthesis state (Jiang et al. 2021). Skeletal muscle not only controls whole-body energy expenditure, but also serves as a reservoir of protein and is highly sensitive to protein anabolism and catabolism. Recent studies have shown that ISM1 can act directly on skeletal muscle cells to induce protein synthesis via pAKT- pS6S235/S236 (Zhao et al. 2022). Skeletal muscle fibers became smaller, protein degradation increased, muscle strength decreased, and muscle atrophy-related FOXO1 target gene levels increased with ISM1 knockdown (Zhao et al.

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