The membrane receptor CD44: novel insights into metabolism
Authors: Xiong Weng, Samuel Maxwell-Warburton, Annie Hasib, Lifeng Ma, Li Kang
Journal: Trends in Endocrinology and Metabolism (2022)

CD44 contributes to obesity-associated insulin resistance in muscle, liver, and adipose tissue via binding to its ligands HA and osteopontin.CD44 regulates glucose and lipid homeostasis in metabolic tissues, contributing to the pathogenesis of chronic metabolic diseases, including obesity and diabetes.Intracellular signalling of CD44 in metabolic regulation is not fully understood and requires further investigation.Despite limited success of anti-CD44 therapies in cancer, their repurposing to treat metabolic diseases may prove to be promising. CD44, a cell-surface glycoprotein, has long been studied as a cancer molecule due to its essential role in physiological activities in normal cells and pathological activities in cancer cells, such as cell proliferation, adhesion, and migration; angiogenesis; inflammation; and cytoskeleton rearrangement. Yet, recent evidence suggests a role of CD44 in metabolism, especially insulin resistance in obesity and diabetes. In line with the current concept of fibroinflammation in obesity and insulin resistance, CD44 as the main receptor of the extracellular matrix component, hyaluronan (HA), has been shown to regulate diet-induced insulin resistance in muscle and other insulin-sensitive tissues. In this review, we integrate current evidence for a role of CD44 in regulating glucose and lipid homeostasis and speculate about its involvement in the pathogenesis of chronic metabolic diseases, including obesity and diabetes. We summarize the current development of CD44-targeted therapies and discuss its potential for the use in treating metabolic diseases. CD44, a cell-surface glycoprotein, has long been studied as a cancer molecule due to its essential role in physiological activities in normal cells and pathological activities in cancer cells, such as cell proliferation, adhesion, and migration; angiogenesis; inflammation; and cytoskeleton rearrangement. Yet, recent evidence suggests a role of CD44 in metabolism, especially insulin resistance in obesity and diabetes. In line with the current concept of fibroinflammation in obesity and insulin resistance, CD44 as the main receptor of the extracellular matrix component, hyaluronan (HA), has been shown to regulate diet-induced insulin resistance in muscle and other insulin-sensitive tissues. In this review, we integrate current evidence for a role of CD44 in regulating glucose and lipid homeostasis and speculate about its involvement in the pathogenesis of chronic metabolic diseases, including obesity and diabetes. We summarize the current development of CD44-targeted therapies and discuss its potential for the use in treating metabolic diseases. CD44 was originally described as a lymphocyte-homing receptor and later identified and cloned as a member of the cartilage link protein family during 1980s [1.Stamenkovic I. et al.A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family.Cell. 1989; 56: 1057-1062Abstract Full Text PDF PubMed Scopus (561) Google Scholar]. The human CD44 gene was mapped to chromosome locus 11p13, and has 19 exons, while the mouse cd44 has 20 exons, including an extra exon, V1, which is not found in humans (Figure 1). Exons 1–5 encode the extracellular common region, while exons 16 and 17, 18, and 19 and 20 encode the stalk region, transmembrane domain, and intracellular cytoplasmic domain, respectively. Exons 6–15 undergo alternative splicing, and are inserted between the extracellular common region and stalk region, resulting in multiple CD44 variant isoforms. CD44 is widely expressed in lymphocytes, fibroblasts, and smooth muscle cells in humans and mice. CD44 variants have been reported to express in various normal epithelial tissues, most expressing variant (v)9 and fewer expressing v6 or v4 [2.Mackay C.R. et al.Expression and modulation of CD44 variant isoforms in humans.J. Cell Biol. 1994; 124: 71-82Crossref PubMed Scopus (407) Google Scholar]. Aberrant expression of CD44 splice variants has been associated with tumour progression [3.Naor D. et al.CD44 in cancer.Crit. Rev. Clin. Lab. Sci. 2002; 39: 527-579Crossref PubMed Scopus (433) Google Scholar]. CD44 that is only coded by exons 1–5 and 16–20 is the standard form (CD44s). CD44s, the smallest isoform, with 365 amino acids, is expressed on the membrane of most vertebrate cells. CD44v8-10 (CD44E) is predominantly expressed on epithelial cells, while the isoform of CD44v3-10 is the largest CD44 isoform, mainly present in keratinocytes [4.Ouhtit A. et al.Novel CD44-downstream signaling pathways mediating breast tumor invasion.Int. J. Biol. Sci. 2018; 14: 1782-1790Crossref PubMed Scopus (33) Google Scholar]. The expression and distribution of CD44 isoforms have been previously reviewed elsewhere [5.Zoller M. CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?.Nat. Rev. Cancer. 2011; 11: 254-267Crossref PubMed Scopus (783) Google Scholar]. All CD44 isoforms comprise one cytoplasmic domain, which has binding motifs for cytoskeleton-related proteins (e.g., Ankyrin and cytoplasmic tyrosine kinases), one transmembrane domain, and a distal extracellular domain [5.Zoller M. CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?.Nat. Rev. Cancer. 2011; 11: 254-267Crossref PubMed Scopus (783) Google Scholar]. Its extracellular domain binds to extracellular components, such as its classical ligands, HA (polysaccharides; see Glossary), and osteopontin (OPN, phosphoproteins) through which CD44 transduces intracellular signalling and regulates a variety of biological activities. HA, a nonsulfated glycosaminoglycan comprising alternating N-acetyl-glucosamine (GlcNAc) and glucuronic acid (GlcA) units, is one of the major components of the extracellular matrix (ECM) and a ligand for CD44. HA is crucial in tissue homeostasis and biomechanical integrity, resulting from its remarkable physiochemical properties. It binds to water and forms viscous gels, which interact with proteoglycans and extracellular macromolecules, contributing to ECM remodelling. Aberrant expression of HA has been associated with diseases including various cancers [6.Tammi R.H. et al.Hyaluronan in human tumors: pathobiological and prognostic messages from cell-associated and stromal hyaluronan.Semin. Cancer Biol. 2008; 18: 288-295Crossref PubMed Scopus (213) Google Scholar] and metabolic disorders [7.Kang L. et al.Hyaluronan accumulates with high-fat feeding and contributes to insulin resistance.Diabetes. 2013; 62: 1888-1896Crossref PubMed Scopus (77) Google Scholar,8.Bogdani M. et al.Hyaluronan and hyaluronan-binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis.Diabetes. 2014; 63: 2727-2743Crossref PubMed Scopus (71) Google Scholar]. The molecular mass of natural HA varies from dozens to millions of Daltons (Da). HA with a molecular mass above 500 kDa is usually regarded as high-molecular-weight HA (HMW-HA), 20–500 kDa as low-molecular-weight HA (LMW-HA), and less than 20 kDa as HA oligomers [9.Noble P.W. Hyaluronan and its catabolic products in tissue injury and repair.Matrix Biol. 2002; 21: 25-29Crossref PubMed Scopus (455) Google Scholar,10.Kothapalli D. et al.Differential activation of ERK and Rac mediates the proliferative and anti-proliferative effects of hyaluronan and CD44.J. Biol. Chem. 2008; 283: 31823-31829Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar]. CD44 can bind to HA fragments with different molecular weights, resulting in different, sometimes opposing, cellular functions. For example, when CD44 binds to HMW-HAs, it inhibits mitosis by abrogating the Ras-cyclinD1 interaction, which arrests the cell cycle [10.Kothapalli D. et al.Differential activation of ERK and Rac mediates the proliferative and anti-proliferative effects of hyaluronan and CD44.J. Biol. Chem. 2008; 283: 31823-31829Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar]. By contrast, when binding to LMW-HAs, CD44 promotes Ras-cylinD1-mediated mitosis [11.Kothapalli D. et al.Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells.J. Cell Biol. 2007; 176: 535-544Crossref PubMed Scopus (66) Google Scholar]. Thus, the HA-CD44 interaction may function as a signal switch of cell mitosis in response to different extracellular environments. Upon HA binding, CD44 interacts with, and activates, cytoskeletal proteins, such as Ankyrin, and subsequently forms a HA/CD44 and Ankyrin complex on the cell membrane (Figure 2A ). This complex then triggers membrane invagination and the formation of lipid raft-like caveolae, which contain distinct proteins and lipids, such as cholesterol and sphingolipids [12.Bourguignon L.Y. Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.Semin. Cancer Biol. 2008; 18: 251-259Crossref PubMed Scopus (227) Google Scholar]. These distinct lipids and proteins then recruit intracellular signalling molecules to the plasma membrane and initiate signal cascades, thus regulating cell survival, growth, and migration, and tumour cell invasion (Box 1). In addition to Ankyrin, CD44 also activates cytoskeleton rearrangements and participates in cell adhesion by binding to the Ezrin/Radixin/Moesin (ERM) proteins [13.Singleton P.A. Bourguignon L.Y. CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation.Exp. Cell Res. 2004; 295: 102-118Crossref PubMed Scopus (88) Google Scholar,14.Yonemura S. et al.Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2.J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (502) Google Scholar]. CD44 promotes the phosphorylation of ERM proteins by protein kinases, such as PKC-δ, via its cytoplasmic domain. These phosphorylated ERM proteins attract filamentous actin (F-actin), thus associating the intracellular cytoskeleton with the plasma membrane and affecting the distribution of organelles and transport of molecules [14.Yonemura S. et al.Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2.J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (502) Google Scholar]. Interestingly, the interaction of CD44 and ERM proteins might inhibit the CD44-Ankyrin interaction, although the importance of this inhibition remains unknown [15.Nunomura W. et al.Regulation of CD44-protein 4.1 interaction by Ca2+ and calmodulin. Implications for modulation of CD44-ankyrin interaction.J. Biol. Chem. 1997; 272: 30322-30328Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar].Box 1Intracellular cytoskeletal rearrangement mediated by CD44HA-CD44 signallingUpon HA binding to CD44, activated Ankyrin stimulates intracellular Ca2+ release and mobilisation and the subsequent upregulation of Ca2+/calmodulin-dependent kinase II, which then phosphorylates the cytoskeletal protein filamin, participating in cell migration [13.Singleton P.A. Bourguignon L.Y. CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation.Exp. Cell Res. 2004; 295: 102-118Crossref PubMed Scopus (88) Google Scholar] (see Figure 2A in the main text). Ankyrin binds to small GTP-binding proteins, such as Cdc42 and RhoA. RhoA couples with Rho-binding kinase (ROK) and stimulates Gab-1, a member of the insulin receptor substrate family the phosphorylation of which activates PI3K-MAPK signalling pathways and regulates cell survival and growth [73.Kawano Y. et al.Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins.J. Biol. Chem. 2000; 275: 29628-29635Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Cdc42 interacts with, and activates, P21-activated kinase 1 (PAK1), and subsequently forms a Cdc42-PAK1-filamin complex, which leads to cytoskeleton rearrangement for tumour cell migration and invasion [12.Bourguignon L.Y. Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.Semin. Cancer Biol. 2008; 18: 251-259Crossref PubMed Scopus (227) Google Scholar]. Cdc42 is also linked to ERK signalling via the GTPase-activating-like protein 1 (IQGAP1)-ERK2 complex for transcriptional activation in tumour cells [74.Roy M. et al.IQGAP1 binds ERK2 and modulates its activity.J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]. Moreover, HA is reportedly bound to CD44v3 and activates Vav2 (a guanine nucleotide exchange factor). The CD44v3-Vav2 complex interacts with growth factor receptor protein 2 (Grb2), which binds to P185HER, leading to Ras activation and cell migration in ovarian cancer [75.Bourguignon L.Y. et al.Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth.J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar]. The CD44v3 exon is also required for the CD44-MMP7 interaction and formation of the CD44-MMP7 complex. This complex processes the maturation of the heparin-binding epidermal growth factor (HB-EGF) precursor and leads to subsequent HB-EGF interaction with its receptor, ErB4, regulating cell survival, as occurs in the postpartum uterine and lactating gland epithelium [76.Yu W.H. et al.CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling.Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (379) Google Scholar].OPN-CD44 signallingUpon OPN binding to CD44, the interaction of gelsolin and mDia1 enhances gelsolin-related Src activity and its downstream PI3K signalling activation (see Figure 2B in the main text). The activated Src can also promote actin filament formation and the migration of osteoclasts, which participate in bone resorption and healing [20.Denhardt D.T. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (893) Google Scholar].Signalling triggered by CD44 cleavageCD44 cleavage is regulated by an intricate balance between proteases and their inhibitors, such as 1,10-phenanthroline [77.Okamoto I. et al.CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration.Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (208) Google Scholar] (see Figure 2C in the main text). Ligand binding regulates CD44 cleavage, therefore modulating cell migration in CD44-positive tumour cells. HA containing 6–14-mers, enhances CD44 cleavage in a dose-dependent manner in pancreatic carcinoma cells [78.Sugahara K.N. et al.Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells.J. Biol. Chem. 2003; 278: 32259-32265Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar]. Kazuki et al. showed that HA smaller than 36 kDa significantly promoted CD44 cleavage, and 6.9 kDa (36-mers) HA induced tumour cell migration and invasion [78.Sugahara K.N. et al.Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells.J. Biol. Chem. 2003; 278: 32259-32265Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar]. This is significant because tumour cells tend to generate small HA fragments (10-40 kDa) by upregulating hyaluronidases, which promotes CD44 cleavage, thus providing a favourable environment for tumour cell invasion and metastasis in many types of cancers, such as breast carcinoma, nonsmall cell lung carcinoma, glioma, colon carcinoma, and ovarian carcinoma [79.Okamoto I. et al.Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumors.Am. J. Pathol. 2002; 160: 441-447Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar]. HA-CD44 signalling Upon HA binding to CD44, activated Ankyrin stimulates intracellular Ca2+ release and mobilisation and the subsequent upregulation of Ca2+/calmodulin-dependent kinase II, which then phosphorylates the cytoskeletal protein filamin, participating in cell migration [13.Singleton P.A. Bourguignon L.Y. CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation.Exp. Cell Res. 2004; 295: 102-118Crossref PubMed Scopus (88) Google Scholar] (see Figure 2A in the main text). Ankyrin binds to small GTP-binding proteins, such as Cdc42 and RhoA. RhoA couples with Rho-binding kinase (ROK) and stimulates Gab-1, a member of the insulin receptor substrate family the phosphorylation of which activates PI3K-MAPK signalling pathways and regulates cell survival and growth [73.Kawano Y. et al.Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins.J. Biol. Chem. 2000; 275: 29628-29635Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Cdc42 interacts with, and activates, P21-activated kinase 1 (PAK1), and subsequently forms a Cdc42-PAK1-filamin complex, which leads to cytoskeleton rearrangement for tumour cell migration and invasion [12.Bourguignon L.Y. Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.Semin. Cancer Biol. 2008; 18: 251-259Crossref PubMed Scopus (227) Google Scholar]. Cdc42 is also linked to ERK signalling via the GTPase-activating-like protein 1 (IQGAP1)-ERK2 complex for transcriptional activation in tumour cells [74.Roy M. et al.IQGAP1 binds ERK2 and modulates its activity.J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar]. Moreover, HA is reportedly bound to CD44v3 and activates Vav2 (a guanine nucleotide exchange factor). The CD44v3-Vav2 complex interacts with growth factor receptor protein 2 (Grb2), which binds to P185HER, leading to Ras activation and cell migration in ovarian cancer [75.Bourguignon L.Y. et al.Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth.J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar]. The CD44v3 exon is also required for the CD44-MMP7 interaction and formation of the CD44-MMP7 complex. This complex processes the maturation of the heparin-binding epidermal growth factor (HB-EGF) precursor and leads to subsequent HB-EGF interaction with its receptor, ErB4, regulating cell survival, as occurs in the postpartum uterine and lactating gland epithelium [76.Yu W.H. et al.CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling.Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (379) Google Scholar]. OPN-CD44 signalling Upon OPN binding to CD44, the interaction of gelsolin and mDia1 enhances gelsolin-related Src activity and its downstream PI3K signalling activation (see Figure 2B in the main text). The activated Src can also promote actin filament formation and the migration of osteoclasts, which participate in bone resorption and healing [20.Denhardt D.T. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (893) Google Scholar]. Signalling triggered by CD44 cleavage CD44 cleavage is regulated by an intricate balance between proteases and their inhibitors, such as 1,10-phenanthroline [77.Okamoto I. et al.CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration.Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (208) Google Scholar] (see Figure 2C in the main text). Ligand binding regulates CD44 cleavage, therefore modulating cell migration in CD44-positive tumour cells. HA containing 6–14-mers, enhances CD44 cleavage in a dose-dependent manner in pancreatic carcinoma cells [78.Sugahara K.N. et al.Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells.J. Biol. Chem. 2003; 278: 32259-32265Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar]. Kazuki et al. showed that HA smaller than 36 kDa significantly promoted CD44 cleavage, and 6.9 kDa (36-mers) HA induced tumour cell migration and invasion [78.Sugahara K.N. et al.Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells.J. Biol. Chem. 2003; 278: 32259-32265Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar]. This is significant because tumour cells tend to generate small HA fragments (10-40 kDa) by upregulating hyaluronidases, which promotes CD44 cleavage, thus providing a favourable environment for tumour cell invasion and metastasis in many types of cancers, such as breast carcinoma, nonsmall cell lung carcinoma, glioma, colon carcinoma, and ovarian carcinoma [79.Okamoto I. et al.Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumors.Am. J. Pathol. 2002; 160: 441-447Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar]. In the central nervous system, CD44-HA interactions were found to mediate astrocyte migration through binding to, and activating, the N-terminal antiparallel coiled-coil (ACC) domain of PKNγ [16.Bourguignon L.Y. et al.Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes.J. Neurochem. 2007; 101: 1002-1017Crossref PubMed Scopus (74) Google Scholar]. PKNγ is a Rac-1-activated serine/threonine kinase. Upon activation, PKNγ phosphorylates the cytoskeletal protein cortactin, and suppresses the crosslinking of cortactin to F-actin, resulting in astrocyte migration [16.Bourguignon L.Y. et al.Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes.J. Neurochem. 2007; 101: 1002-1017Crossref PubMed Scopus (74) Google Scholar]. OPN, originally identified as a phosphoprotein secreted from T cells, and also known as SPP1, is another classical ligand of CD44 [17.Nau G.J. et al.A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6414-6419Crossref PubMed Scopus (157) Google Scholar]. It naturally exists as two forms: secreted OPN, which is fully modified by post-translational modifications (PTMs; e.g., phosphorylation and O-glycosylation); and intracellular OPN, which is not or only partly modified by PTMs [18.Kazanecki C.C. et al.Control of osteopontin signaling and function by post-translational phosphorylation and protein folding.J. Cell. Biochem. 2007; 102: 912-924Crossref PubMed Scopus (206) Google Scholar]. OPN is widely expressed by immune, odontoblast, brain, and kidney cells [19.Brown L.F. et al.Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.Mol. Biol. Cell. 1992; 3: 1169-1180Crossref PubMed Scopus (390) Google Scholar]. Its interaction with the alternative splicing domain of CD44 prevents pathogen infection, and the recruitment and activation of neutrophils, macrophages, and granulocytes during the immune response [20.Denhardt D.T. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (893) Google Scholar]. It is reported that OPN-CD44 signalling is vital for both OPN-αVβ3 integrin activation and the interaction between the actin-binding protein gelsolin and the transcriptional activator mDia1, promoting actin filament formation and cell migration [20.Denhardt D.T. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (893) Google Scholar] (Figure 2B, Box 1). In addition, OPN associates with CD44/ERM proteins to regulate fibroblast cell migration [20.Denhardt D.T. et al.Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (893) Google Scholar,21.Zohar R. et al.Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration.J. Cell. Physiol. 2000; 184: 118-130Crossref PubMed Scopus (228) Google Scholar]. OPN/CD44/phospholipase C was found to regulate human mammary epithelial cell migration, which might contribute to the remodelling of plasma membrane phospholipids and the activation of downstream signalling (e.g., AKT activation) [22.Rangaswami H. et al.Osteopontin: role in cell signaling and cancer progression.Trends Cell Biol. 2006; 16: 79-87Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar]. OPN also enhances the HA-CD44 interaction to activate PI3K/AKT signalling, thus promoting cell survival and growth and contributing to drug resistance in mesothelioma [23.Tajima K. et al.Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells.Oncogene. 2010; 29: 1941-1951Crossref PubMed Scopus (39) Google Scholar]. In addition, OPN promotes HA synthesis by increasing HA synthase 2 expression, enhancing HA-CD44 signalling, and promoting breast cancer malignancy [24.Cook A.C. et al.Osteopontin induction of hyaluronan synthase 2 expression promotes breast cancer malignancy.J. Biol. Chem. 2006; 281: 24381-24389Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. However, how OPN-CD44 and HA-CD44 signalling are intertwined is still unknown and merits further investigation. The cleavage of CD44 is controlled in a cell type-dependent fashion by various stimulants and molecules. CD44 is cleaved in two steps (Figure 2C, Box 1). First, stimulations, such as Ca2+ influx, PKC activation, and Ras oncoproteins, activate membrane-associated matrix metalloproteinases (MMPs), resulting in the proteolytic release of the CD44 ectodomain from the membrane. Second, presenilin (PS)-dependent ɣ-secretase mediates intramembranous cleavage, which breaks the transmembrane and cytoplasmic domains of CD44 [25.Nagano O. Saya H. Mechanism and biological significance of CD44 cleavage.Cancer Sci. 2004; 95: 930-935Crossref PubMed Scopus (306) Google Scholar]. When cleaved, the released CD44 extracellular domains become soluble, causing the detachment of CD44-expressing cells from the ECM, contributing to cell migration and adhesion. Antibody-induced shedding of CD44 is linked to the assembly of cytoskeleton, possibly by Rho family GTPase RAC1 and Cdc42 activation, because pharmacological activation of RAC1 and Cdc42 induces elevated CD44 ectodomain release and concomitantly distinct assembly of actin in primary mouse synovial fibroblasts [26.Shi M. et al.Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.J. Immunol. 2001; 167: 123-131Crossref PubMed Scopus (55) Google Scholar]. In addition to the ectodomain, CD44 cleavage also produces a 25-kDa soluble cytoplasmic fragment (CD44ICD). These fragments provide positive feedback for CD44 expression by direct transportation into the nucleus, where they cooperate with the transcriptional coactivator CBP/P300 to initiate CD44 transcription. CD44ICD also regulates the expression of other genes, such as hypoxia-inducible factor (HIF)2a, in a CBP/P300-dependent manner, indirectly regulating HIF-targeted genes, such as those encoding phosphoglycerate kinase 1 (PGK1), glucose transporter 1 (GLUT1), and vascular endothelial growth factor (VEGF). Indeed, CD44ICD was reported to enhance the hypoxic response of glioma by potentiating HIF-2α, negatively impacting the treatment of glioma [27.Johansson E. et al.CD44 interacts with HIF-2alpha to modulate the hypoxic phenotype of perinecrotic and perivascular glioma cells.Cell Rep. 2017; 20: 1641-1653Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. The intracellular cytoskeleton rearrangements mediated by CD44 may contribute to metabolic regulation. For example, activation of Ankyrin/Cdc42 downstream signalling Rho-associated protein kinases (ROCKs), regulates glycogen synthase and glucose transport in peripheral tissues by suppressing IRS-1 phosphorylation [28.Tabur S. et al.Association of Rho/Rho-kinase gene polymorphisms and expressions with obesity-related metabolic syndrome.Eur. Rev. Med. Pharmacol. Sci. 2015; 19: 1680-1688PubMed Google Scholar]. Indeed, deletion of ROCK1, an isoform of ROCK, attenuates obesity-associated insulin resistance in adipose tissue [29.Lee S.H. et al.ROCK1 isoform-specific deletion reveals a role for diet-induced insulin resistance.Am. J. Physiol. Endocrinol. Metab. 2014; 306: E332-E343Crossref PubMed Scopus (38) Google Scholar]. In addition, P21-activated kinase 1 (PAK1), another downstream component of HA-CD44-Ankyrin signalling, is required for the maintenance of mitochondrial function and preserving β cell mass in mice and human islets [30.Ahn M. et al.A requirement for PAK1 to support mitochondrial function and maint