The Specific Role of Dermatan Sulfate as an Instructive Glycosaminoglycan in Tissue Development

The crucial roles of dermatan sulfate (DS) have been demonstrated in tissue development of the cutis, blood vessels, and bone through construction of the extracellular matrix and cell signaling. Although DS classically exerts physiological functions via interaction with collagens, growth factors, and heparin cofactor-II, new functions have been revealed through analyses of human genetic disorders as well as of knockout mice with loss of DS-synthesizing enzymes. Mutations in human genes encoding the epimerase and sulfotransferase responsible for the biosynthesis of DS chains cause connective tissue disorders including spondylodysplastic type Ehlers–Danlos syndrome, characterized by skin hyperextensibility, joint hypermobility, and tissue fragility. DS-deficient mice show perinatal lethality, skin fragility, vascular abnormalities, thoracic kyphosis, myopathy-related phenotypes, acceleration of nerve regeneration, and impairments in self-renewal and proliferation of neural stem cells. These findings suggest that DS is essential for tissue development in addition to the assembly of collagen fibrils in the skin, and that DS-deficient knockout mice can be utilized as models of human genetic disorders that involve impairment of DS biosynthesis. This review highlights a novel role of DS in tissue development studies from the past decade.


Classical Functions of DS
Although classical functions of DS have been described in the literature [4,10,46], they are briefly introduced in this section. The DS side chain of decorin binds to collagen to assemble the extracellular matrix [47,48] (Table 1). A focused ion beam scanning electron microscope revealed that the DS side chain of decorin forms a ring-mesh like structure, with each ring surrounding a collagen fibril [49,50].
Heparin cofactor II, like antithrombin III, inhibits proteolytic enzymes involved in blood coagulation via interaction with DS as well as heparin [51] ( Table 1) 3 , from porcine skin has been identified as the smallest fragment of DS binding to heparin cofactor II with high affinity [52].
DS and/or DS-PGs are up-regulated in tumor cells as well as in the stroma [62][63][64][65], which is consistent with up-regulations of glycosyltransferases, epimerases, and sulfotransferases responsible for the biosynthesis of DS [65]. Furthermore, IdoA-deficient human esophagus squamous cell carcinoma by shRNA showed decreased migration and invasion capabilities in vitro, which was associated with reduced cellular interaction with HGF, inhibition of pERK-1/2 signaling, and deregulated actin cytoskeleton dynamics and focal adhesion formation [65]. These findings suggest that DS and/or DS-PGs may contribute to proliferation, invasion, and metastasis via binding with effector proteins. However, what remains unclear is the ratio of CS/DS, the content of IdoA, chain length, sulfation pattern, binding molecules, and cell signaling. Further studies are required in order to clarify the molecular mechanisms involving DS-PGs through the use of model animals as well as clinical specimens. Anti-coagulation, cell growth, assembly of extracellular matrix [2,47,51,66] Ascidian (A. nigra) --~100% Heparin cofactor II Anti-coagulation, neurite outgrowth-promoting activity [57,67] Ascidian (S. plicata) --~70% Heparin cofactor II Anti-coagulation, neurite outgrowth-promoting activity [57,68] Embryonic sea urcin --~100% --Neurite outgrowth-promoting activity [57,69] Hagfish --, not reported.

Recent Additional Functions of DS
Recently, it was shown that molecules longer than tetrasaccharides derived from DS enhance the activation of anaplastic lymphoma kinase, a receptor tyrosine kinase, by clustering of anaplastic lymphoma kinase [62].
Furthermore, it was demonstrated that the expressions of Dsel and D4st1 increased during formation of the embryonic body from mouse embryonic stem cells, and that an addition of DS to the culture medium promoted neuronal differentiation by activation of extracellular signal-regulated kinase 1/2, and also accelerated neurite outgrowth in mouse embryonic stem cells [71]. On the other hand, knockdown or overexpression of D4ST1 in mouse embryonic stem cells led to the promotion or suppression of endodermal differentiation, respectively [72]. These opposite effects of the addition of DS as well as knockdown or overexpression of D4ST1 on differentiation of mouse embryonic stem cells remain unclear; further study is required to explain these phenomena. In addition, DS promoted neuronal differentiation and neuronal migration, but not neurite outgrowth in human neuronal stem cells [71]. These findings indicate that DS may modulate neuronal differentiation in both mouse and human stem cells.
Although knockdown of C4ST1 by antisense morpholino oligonucreotide accelerated regeneration of axons after spinal cord injury in zebrafish, knockdown of D4ST1 did not [73], indicating that 4-O-sulfation of CS, but not DS, inhibit axonal regrowth after spinal cord injury.

Dse
In 2009, Maccarana and coworkers generated Dse knockout mice. Their studies showed that the knockout mice of Dse were smaller, with a 20-30% reduced body weight at birth compared with that of wild-type mice [28] (Table 2). All Dse -/pups had kinked tails until 4 weeks of age, and Dse -/mice showed reduced fertility. Histological analysis of the skin from Dse -/mice exhibited sparser loose connective tissue in the hypodermal layer compared with that of wild-type mice. However, the distributions and amounts of core proteins, decorin and biglycan, that are DS-PGs, did not change in the skin of Dse -/mice. The mean diameters of collagen fibrils from skin were 60 and 85 nm in wild-type and Dse -/mice, respectively. Accordingly, the stress at failure was reduced in the skin of Dse -/mice. In decorin-deficient mice, which were generated by Iozzo's group, the collagen fibrils showed irregular outlines with larger diameters than wild-type skin [74]. Thus, DS as well as DS-PGs play a role in skin development. On the other hand, the histological phenotypes of adrenal glands, brains, intestines, kidneys, and lungs in Dse -/mice were not detected. Further analyses are required to expand the findings on DS functions.
Epimerase activity of the skin, spleen, lung, and kidney in Dse -/mice was markedly lower compared with that of wild-types, whereas its activity in the brain was comparable between Dse +/+ and Dse -/mice [28]; this was consistent with their mRNA levels [75,76]. The remaining activity can most likely be attributed to DSE2 encoded by Dsel. Furthermore, IdoA-containing structures in the whole body as well as in the skin of 10-day-old pups were markedly reduced in the Dse -/mice compared with that of wild-type mice. These findings indicate that DSE and the DS side chains of PGs contribute to the DS biosynthesis and formation of collagen bundles, respectively, in the skin.

Chst14
Increased skin fragility, disorganized collagen fibers, thoracic kyphosis, reduced fertility, kinked tail, myopathy-related phenotypes such as variation in fiber size and spread of the muscle interstitium, smaller body mass, alterations in the vascular structure of the placenta, an abnormal structure of the basement membrane of capillaries in the placental villus, better recovery after femoral nerve injury.

Dsel
In 2015, Maccarana and coworkers generated Dsel knockout mice [77,78]. Their studies showed that the Dsel -/mice were fertile, and that there were no significant differences in the weight or length at weaning and at 9 weeks of age between wild-type and Dsel -/mice [78] (Table 2). Furthermore, Dsel -/mice had no anatomical or histological defects in the brain, heart, kidney, liver, lungs, lymph nodes, muscles, pancreas, skin, small intestines, spleen, stomach, or thymus compared with wild-type mice; however, total DS-epimerase activity in the brain, kidney, spleen, liver, lung, and skin was reduced to 11,45,56,62,66, and 76%, respectively, compared with wild-type mice. Consistently, IdoA contents in CS/DS of brain and kidney from 3-day-old Dsel -/were reduced to 62 and 87%, respectively, compared with wild-type mice. Normal extracellular matrix features were detected in the brain section from Dsel -/mice compared with wild type. Thus, further behavioral analyses are needed, such as an open-field test, a separation-reunion test, resident-intruder test, and social dominant tube test, in order to explore the functions of DS in the brain, since it has been reported that various mutations in DSEL cause bipolar disorder [75].

Dse/Dsel
Double knockout mice of Dse and Dsel, which were generated by Maccarana's group in 2015, showed complete loss of the IdoA residue of CS/DS in 2-day-old pups [77]. Total levels of CS/DS disaccharides from various organs on embryonic (E) day 19.5 in Dse -/-/Dsel -/were reduced to 30% compared with Dse +/+ /Dsel +/+ . Dse -/-/Dsel -/embryos showed Mendelian ratios on E13.5-E19.5, and exhibited the defects of abdominal wall closure, a kinked tail, and exencephaly in varying ratios with each phenotype, ranging macroscopically from normal to severely affected. The major organs including the brain, spinal cord, liver, and lung from Dse -/-/Dsel -/embryos appeared normal when compared with control embryos, Dse +/+ /Dsel -/-. Nonetheless, all newborns died in the 48 h after birth, even in the absence of a phenotype, which may be due to multiple fragility and multiple organ failure. Further investigation using this double knockout mouse model is needed to reveal the mechanism of neonatal lethality.

Chst14
In 2011, Schachner and coworkers generated Chst14 knockout mice [29,79]. Their studies showed that the Chst14 -/mice were fertile, but that one-third of the knockout mice died between E16.5-E18.5 and/or within a few days after birth [29] ( Table 2). The body weight as well as weight of the tibia, heart, liver, and kidney, but not brain, were decreased in the Chst14 -/mice compared with their wild-type littermates. These observations are partially similar to those of humans with mutations in CHST14 [22], and indicate that Chst14 and/or DS may be indispensable for early tissue development. DS binds to FGFs as well as HGF [55,56]. Thus, defects in forms of signaling may lead to tissue development in Chst14 -/mice.
In 2018, Yoshizawa et al. demonstrated that the placenta derived from Chst14 -/mice exhibited an alteration in the vascular structure with ischemic and/or necrotic-like change, an abnormal structure of the basement membrane of capillaries in the placental villus, a reduced weight of the placenta, and significantly decreased DS [80]. These findings offer further evidence to support the perinatal lethality caused by a defect in CHST14 [22], and suggest why DS may be essential for placental vascular development and/or assembly of collagen fibrils by DS.
Chst14 -/mice exhibited elephant teeth, a kinked tail, increased skin fragility, and thoracic kyphosis, compared with Chst14 +/+ mice [29,31]. Moreover, Nitahara-Kasahara et al. demonstrated that decreases in DS in muscle in addition to myopathy-related phenotypes such as variation in fiber size and spread of the muscle interstitium which caused lower grip strength and decreased exercise capacity, were observed in Chst14 -/mice compared with Chst14 +/+ as well as Chst14 +/mice [31,32].
In 2021, Hirose et al. demonstrated that the skin tensile strength of Chst14 -/mice was lower than that of Chst14 +/+ mice, which may be most likely caused by abnormal collagen fibrils in the reticular layer [30]. A DS-PG, decorin-PG, regulates collagen fiber formation [74]. Although DS with a round conformation wrapped the collagen fibrils in the wild-type mice, rod-shaped linear DS was detected at one end of collagen fibrils and protruded outside the fibrils in Chst14 -/mice [30]. These observations suggest that the DS side chain of decorin is essential for the assembly of collagen and for supporting skin strength, indicating that Chst14 -/mice is a good model for musculocontractural Ehlers-Danlos syndrome caused by mutations in CHST14 [22][23][24]87,88].
Bian et al. demonstrated that neurospheres from Chst14 -/mice showed larger diameters and fewer total numbers than those from Chst14 +/+ mice [79]. This was caused by dysfunctions in proliferation and self-renewal of neural stem cells in vivo [79], indicating that DS and/or DS-PGs play important roles in the proliferation and differentiation of neural stem cells. Chst14 -/mice exhibited longer cell processes as well as a higher proliferation rate of cultured Schwann cells from dorsal roots and nerves, in addition to longer neurites of cultured neurons from cerebella compared with those of Chst14 +/+ mice [29]. After femoral nerve injury, Chst14-deficiency accelerates the recovery of motor functions, whereas there were no significant differences in the motor reinnervation pattern, degree of myelination, or Schwann cell proliferation between Chst14 +/+ and Chst14 -/mice. Therefore, further analysis may be needed to understand the molecular mechanism underlying motor recovery by Chst14-ablation.

DSE
DSE encoded by DSE converts GlcA into IdoA in the chondroitin precursor chain, which results in the formation of dermatan polysaccharide (Figure 2). [25]. EDS musculocontractural type 2 is caused by pathogenic variants in DSE including p.Ser268Leu, p.Arg267Gly, p.Tyr320*, p.Val333Cysfs*4, p.Pro384Trpfs*9, p.His588Arg, and p.Val938Asp [21,[81][82][83][84] (Table 2). The characteristic manifestations of the EDS musculocontractural type 2 were hypermobility of the finger, elbow, and knee joints, contracture of the thumbs and feet, atrophic scars on the skin, and characteristic facial features such as midfacial hypoplasia, blue sclera, and hypertelorism [21,81,83]. Recombinant DSE-p.Ser268Leu exhibited a loss of epimerase activity [21]. Furthermore, skin fibroblasts from a patient with a mutation of p.Ser268Leu in DSE showed markedly decreased epimerase activity, resulting in a lower level of DS compared with a healthy subject (Table 3) [21]. These findings suggest that these pathogenic variants in DSE may cause a defect in DS, which leads to EDS musculocontractural type 2. CHST14 Not detected 189% [23] * Compared with the healthy subjects.

DSEL
DSE2/DSEL encoded by dermatan sulfate epimerase-like (DSEL) converts GlcA into IdoA in chondroitin, resulting in the formation of dermatan polysaccharide (Figure 2). [44]. DSEL was predominantly expressed in the brain [75,78,97]. A bipolar disorder was caused by a variety of homozygous and heterozygous variants in DSEL including the substitution of a nucleotide in the 5 -non-coding region, p.Val287Ile, p.Pro673Ser, p.Tyr730Cys, p.Pro942Ser, and p.Ile1113Met [75] (Table 2). Furthermore, single nucleotide polymorphism (SNP), rs17077540, detected at 75 kbp upstream of DSEL, causes a recurrent early-onset major depressive disorder [85]. These findings indicate that brain DS and/or DS-PGs produced by DSEL may play a role in the development, homeostasis, and/or function of the central nervous system. A model organism(s) with these variants in DSEL might help explore the pathogenic mechanisms of this disorder.
The heterozygous variant, p.Met14Ile, in DSEL causes a late-presenting, anterior diaphragmatic hernia [86] (Table 2). However, it remains unclear how DSEL, DS, or DS-PGs contribute to the development and/or regulation of the diaphragm.

Conclusions and Perspectives
Mice deficient in DS demonstrated replacement with CS and anomalies of the skin, skeleton, nervous system, and early development, which might be mainly caused by disturbances in collagen bundle formation and in the signaling pathway(s). These findings indicate that interaction of DS with collagens as well as growth factors is dependent on the conformation of the IdoA-containing structure in DS, in a manner similar to heparin. Furthermore, the human genetic disorder Ehlers-Danlos syndrome, caused by a defect in DS, has also been revealed. DS-deficient model mice may become powerful tools in the development of new therapeutics such as applications of mimetics of DSoligosaccharide, enzyme-replacement therapy, or adeno-associated virus for Ehlers-Danlos syndrome caused by a defect in DS.