A biological guide to glycosaminoglycans: current perspectives and pending questions.

Mammalian glycosaminoglycans (GAGs), except hyaluronan (HA), are sulfated polysaccharides that are covalently attached to core proteins to form proteoglycans (PGs). This article summarizes key biological findings for the most widespread GAGs, namely HA, chondroitin sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), and heparan sulfate (HS). It focuses on the major processes that remain to be deciphered to get a comprehensive view of the mechanisms mediating GAG biological functions. They include the regulation of GAG biosynthesis and postsynthetic modifications in heparin (HP) and HS, the composition, heterogeneity, and function of the tetrasaccharide linkage region and its role in disease, the functional characterization of the new PGs recently identified by glycoproteomics, the selectivity of interactions mediated by GAG chains, the display of GAG chains and PGs at the cell surface and their impact on the availability and activity of soluble ligands, and on their move through the glycocalyx layer to reach their receptors, the human GAG profile in health and disease, the roles of GAGs and particular PGs (syndecans, decorin, and biglycan) involved in cancer, inflammation, and fibrosis, the possible use of GAGs and PGs as disease biomarkers, and the design of inhibitors targeting GAG biosynthetic enzymes and GAG-protein interactions to develop novel therapeutic approaches.

Hyaluronan-Induced CD44-iASPP Interaction Affects Fibroblast Migration and Survival.

In the present study, we show that the inhibitor of the apoptosis-stimulating protein of p53 (iASPP) physically interacts with the hyaluronan receptor CD44 in normal and transformed cells. We noticed that the CD44 standard isoform (CD44s), but not the variant isoform (CD44v), bound to iASPP via the ankyrin-binding domain in CD44s. The formation of iASPP-CD44s complexes was promoted by hyaluronan stimulation in fibroblasts but not in epithelial cells. The cellular level of p53 affected the amount of the iASPP-CD44 complex. iASPP was required for hyaluronan-induced CD44-dependent migration and adhesion of fibroblasts. Of note, CD44 altered the sub-cellular localization of the iASPP-p53 complex; thus, ablation of CD44 promoted translocation of iASPP from the nucleus to the cytoplasm, resulting in increased formation of a cytoplasmic iASPP-p53 complex in fibroblasts. Overexpression of iASPP decreased, but CD44 increased the level of intracellular reactive oxygen species (ROS). Knock-down of CD44s, in the presence of p53, led to increased cell growth and cell density of fibroblasts by suppression of p27 and p53. Our observations suggest that the balance of iASPP-CD44 and iASPP-p53 complexes affect the survival and migration of fibroblasts.

In Vitro Investigation of Hyaluronan/CD44 Network.

Hyaluronan is one of the most influential components of the extracellular matrix. It is involved in the regulation of normal tissue function and architecture, while its metabolism is perturbed in a multitude of human diseases like inflammation, cancer, and viral infection. Given the implication of hyaluronan in a vast array of diseases, we describe here assays that can be utilized to study the quantity, size, subcellular localization, and binding capacity of hyaluronan by cells as well as its interactions with its major cellular receptor, CD44. Hopefully, these protocols will provide researchers with useful tools to study the complex hyaluronan biology.

Identification of a Small Molecule Inhibitor of Hyaluronan Synthesis, DDIT, Targeting Breast Cancer Cells.

Breast cancer is a common cancer in women. Breast cancer cells synthesize large amounts of hyaluronan to assist their proliferation, survival, migration and invasion. Accumulation of hyaluronan and overexpression of its receptor CD44 and hyaluronidase TMEM2 in breast tumors correlate with tumor progression and reduced overall survival of patients. Currently, the only known small molecule inhibitor of hyaluronan synthesis is 4-methyl-umbelliferone (4-MU). Due to the importance of hyaluronan for breast cancer progression, our aim was to identify new, potent and chemically distinct inhibitors of its synthesis. Here, we report a new small molecule inhibitor of hyaluronan synthesis, the thymidine analog 5'-Deoxy-5'-(1,3-Diphenyl-2-Imidazolidinyl)-Thymidine (DDIT). This compound is more potent than 4-MU and displays significant anti-tumorigenic properties. Specifically, DDIT inhibits breast cancer cell proliferation, migration, invasion and cancer stem cell self-renewal by suppressing HAS-synthesized hyaluronan. DDIT appears as a promising lead compound for the development of inhibitors of hyaluronan synthesis with potential usefulness in breast cancer treatment.

Hyaluronan nanoscale clustering and Hyaluronan synthase 2 expression are linked to the invasion of child fibroblasts and infantile fibrosarcoma in vitro and in vivo.

Infantile fibrosarcoma is a rare childhood tumour that originates in the fibrous connective tissue of the long bones for which there is an urgent need to identify novel therapeutic targets. This study aims to clarify the role of the extracellular matrix component hyaluronan in the invasion of child fibroblasts and Infantile fibrosarcoma into the surrounding environment. Using nanoscale super-resolution STED (Stimulated emission depletion) microscopy followed by computational image analysis, we observed, for the first time, that invasive child fibroblasts showed increased nanoscale clustering of hyaluronan at the cell periphery, as compared to control cells. Hyaluronan was not observed within focal adhesions. Bioinformatic analyses further revealed that the increased nanoscale hyaluronan clustering was accompanied by increased gene expression of Hyaluronan synthase 2, reduced expression of Hyaluronidase 2 and CD44, and no change of Hyaluronan synthase 1 and Hyaluronidases 1, 3, 4 or 5. We further observed that the expression of the Hyaluronan synthase 1, 2 and 3, and the Hyaluronidase 3 and 5 genes was linked to reduced life expectancy of fibrosarcoma patients. The invasive front of infantile fibrosarcoma tumours further showed increased levels of hyaluronan, as compared to the tumour centre. Taken together, our findings are consistent with the possibility that while Hyaluronan synthase 2 increases the levels, the Hyaluronidases 3 and 5 reduce the weight of hyaluronan, resulting in the nanoscale clustering of hyaluronan at the leading edge of cells, cell invasion and the spread of Infantile fibrosarcoma.

CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence.

Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism.

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness.

Plasma hyaluronan, hyaluronidase activity and endogenous hyaluronidase inhibition in sepsis: an experimental and clinical cohort study.

BACKGROUND: Plasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase. METHODS: Plasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 × 108 CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 × 108 CFU/h of 6 h followed by 1 × 109 CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6). RESULTS: In experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock. CONCLUSION: Elevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreatitis.

TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation.

The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFß) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.

Salicylate suppresses the oncogenic hyaluronan network in metastatic breast cancer cells.

The oncogenic role of hyaluronan in several aspects of tumor biology has been well established. Recent studies by us and others suggest that inhibition of hyaluronan synthesis could represent an emerging therapeutic approach with significant clinical relevance in controlling different breast cancer subtypes, including triple-negative breast cancer. Epidemiological and preclinical studies have revealed the therapeutic potential of aspirin (acetyl salicylate), a classical anti-inflammatory drug, in patients with cancer. However, the underlying molecular mechanisms remain unknown. The present study demonstrates that salicylate, a break down product of aspirin in vivo, alters the organization of hyaluronan matrices by affecting the expression levels of hyaluronan synthesizing (HAS1, 2, 3) and degrading (HYAL-1, -2) enzymes, and that of hyaluronan receptor CD44. In particular, salicylate was found to potently activate AMPK, a kinase known to inhibit HAS2 activity, and caused a dose-dependent decrease of cell associated (intracellular and membrane-bound) as well as secreted hyaluronan, followed by the down-regulation of HAS2 and the induction of HYAL-2 and CD44 in metastatic breast cancer cells. These salicylate-mediated effects were associated with the redistribution of CD44 and actin cytoskeleton that resulted in a less motile cell phenotype. Interestingly, salicylate inhibited metastatic breast cancer cell proliferation and growth by inducing cell growth arrest without signs of apoptosis as evidenced by the substantial decrease of cyclin D1 protein and the absence of cleaved caspase-3, respectively. Collectively, our study offers a possible direction for the development of new matrix-based targeted treatments of metastatic breast cancer subtypes via inhibition of hyaluronan, a pro-angiogenic, pro-inflammatory and tumor promoting glycosaminoglycan.

Intracellular hyaluronan: Importance for cellular functions.

Hyaluronan-rich matrices are abundant in ECM and are involved in biological processes, such as cell growth and migration. Hyaluronan is synthesized by the hyaluronan synthase family of enzymes, HAS1, HAS2 and HAS3; the HAS1 and HAS3 genes give rise to different transcripts through alternative splicing, and the HAS2 gene to a non-coding RNA antisense transcript in addition to the protein-coding transcript. Biosynthesis of hyaluronan increases during inflammation and cancer and is regulated by cytokines and growth factors. In addition to extracellular hyaluronan-rich matrices, cytoplasmic and nuclear forms of hyaluronan have been detected in normal and pathological processes. Extra- and intra-cellular hyaluronan binds to hyaluronan binding proteins, such as CD44, RHAMM, CDC37 and USP17, affecting cellular behavior. Although neither the exact mechanisms by which hyaluronan is present in the intracellular compartments, nor its function at these sites are currently understood, there are evidence that intracellular hyaluronan has important regulatory roles during cell cycle, cell motility, RNA translation and splicing, and autophagy.

Involvement of hyaluronan and CD44 in cancer and viral infections.

Hyaluronan and its major receptor CD44 are ubiquitously distributed. They have important structural as well as signaling roles, regulating tissue homeostasis, and their expression levels are tightly regulated. In addition to signaling initiated by the interaction of the intracellular domain of CD44 with cytoplasmic signaling molecules, CD44 has important roles as a co-receptor for different types of receptors of growth factors and cytokines. Dysregulation of hyaluronan-CD44 interactions is seen in diseases, such as inflammation and cancer. In the present communication, we discuss the mechanism of hyaluronan-induced signaling via CD44, as well as the involvement of hyaluronan-engaged CD44 in malignancies and in viral infections.

High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity.

BACKGROUND: A main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known. METHODS: A cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Serum hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability. FINDINGS: Elevated serum hyaluronan levels (≥70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity. INTERPRETATION: Pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection. FUND: This work was supported in part by grants from the Swedish Cancer Society (2018/337; 2016/445), the Swedish Research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106-2314-B-037-088- and 106-2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3 T05) and Academy of Finland. The funders played no role in the design, interpretation or writing of the manuscript.

Transforming growth factor ß (TGFß) induces NUAK kinase expression to fine-tune its signaling output.

TGFß signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and thus affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis. We here report that NUAK family kinase 1 (NUAK1) and NUAK2 are two TGFß target genes. NUAK1/2 belong to the AMP-activated protein kinase (AMPK) family, whose members control central and protein metabolism, polarity, and overall cellular homeostasis. We found that TGFß-mediated transcriptional induction of NUAK1 and NUAK2 requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases. Genomic mapping identified an enhancer element within the first intron of the NUAK2 gene that can recruit SMAD proteins, which, when cloned, could confer induction by TGFß. Furthermore, NUAK2 formed protein complexes with SMAD3 and the TGFß type I receptor. Functionally, NUAK1 suppressed and NUAK2 induced TGFß signaling. This was evident during TGFß-induced epithelial cytostasis, mesenchymal differentiation, and myofibroblast contractility, in which NUAK1 or NUAK2 silencing enhanced or inhibited these responses, respectively. In conclusion, we have identified a bifurcating loop during TGFß signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFß activity.

Tumor-suppressive functions of 4-MU on breast cancer cells of different ER status: Regulation of hyaluronan/HAS2/CD44 and specific matrix effectors.

The malignant phenotype of various cancers is linked to enhanced expression of hyaluronan, a pro-angiogenic glycosaminoglycan whose expression is suppressed by 4-methylumbelliferone (4-MU), a non-toxic oral agent used as a dietary supplement to improve health and combat prostate cancer. In this study, we investigated the role of 4-MU in mammary carcinoma cells with distinct malignant phenotypes and estrogen receptor (ER) status, a major prognostic factor in the clinical management of breast cancers. We focused on two breast cancer cell lines, the low metastatic and ERα+ MCF-7 cells, and the highly-aggressive and ERα- MDA-MB-231 cells. Treatment with 4-MU caused a dose-dependent decrease of hyaluronan accumulation in the extracellular matrix as well as within the breast cancer cells, most prevalent in cells lacking ERα. This decrease in hyaluronan was accompanied by suppression of Hyaluronan Synthase 2 (HAS2), the major enzyme responsible for the synthesis of hyaluronan, and by induction of hyaluronidases (HYALs) -1 and -2. Moreover, 4-MU induced intense phenotypic changes and substantial loss of CD44, a major hyaluronan receptor, from cell protrusions. Importantly, 4-MU evoked differential effects depending on the absence or presence of ERα. Only the ERα+ cells showed signs of apoptosis, as determined by cleaved PARP-1, and anoikis as shown by concurrent loss of E-cadherin and ß-catenin. Interestingly, 4-MU significantly reduced migration, adhesion and invasion of ERα- breast cancer cells, and concurrently reduced the expression and activity of several matrix degrading enzymes and pro-inflammatory molecules with tumor-promoting functions. Collectively, our findings suggest that 4-MU could represent a novel therapeutic for specific breast cancer subtypes with regard to their ER status via suppression of hyaluronan synthesis and regulation of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators.

Regulation of hyaluronan biosynthesis and clinical impact of excessive hyaluronan production.

The tightly regulated biosynthesis and catabolism of the glycosaminoglycan hyaluronan, as well as its role in organizing tissues and cell signaling, is crucial for the homeostasis of tissues. Overexpression of hyaluronan plays pivotal roles in inflammation and cancer, and markedly high serum and tissue levels of hyaluronan are noted under such pathological conditions. This review focuses on the complexity of the regulation at transcriptional and posttranslational level of hyaluronan synthetic enzymes, and the outcome of their aberrant expression and accumulation of hyaluronan in clinical conditions, such as systemic B-cell cancers, aggressive breast carcinomas, metabolic diseases and virus infection.

Has2 natural antisense RNA and Hmga2 promote Has2 expression during TGFß-induced EMT in breast cancer.

The glycosaminoglycan hyaluronan has a crucial role in tissue organization and cell signaling. Hyaluronan accumulates in conjunction with rapid tissue remodeling during embryogenesis, as well as in inflammatory conditions and cancer. We report a negative correlation between the expression of genes encoding hyaluronan synthase HAS2, its natural antisense transcript HAS2-AS, the chromatin modulating factor HMGA2 and transforming growth factor-ß (TGFß), and survival of patients with invasive breast carcinomas. In mouse mammary epithelial cells, TGFß activates Smad and non-Smad signaling pathways, resulting in the transcriptional induction of Has2, Has2as (the mouse ortholog of HAS2-AS) and Hmga2, as well as epithelial-mesenchymal transition (EMT)-promoting transcription factors, such as Snail. Importantly, Has2as abrogation suppressed the TGFß induction of EMT markers, including Snai1, Hmga2, Fn1, and suppressed the mesenchymal phenotype. TGFß induction of Hmga2, Has2as and Has2, and synthesis of hyaluronan were accompanied with activation of Akt and Erk1/2 MAP-kinase signaling and were required for breast cancer cell motility. Importantly, the hyaluronan receptor Cd44, but not Hmmr, was required for TGFß-mediated EMT phenotype. Interestingly, Has2as was found to contribute to the maintenance of stem cell factors and breast cancer stemness. Our findings show that Has2as has a key role in TGFß- and HAS2-induced breast cancer EMT, migration and acquisition of stemness.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

HAS2 and CD44 in breast tumorigenesis.

Metastatic spread of breast cancer cells, facilitated by the epithelial-mesenchymal transition (EMT) process, is responsible for the majority of breast cancer mortality. Increased levels of hyaluronan due to deregulation of hyaluronan-synthesizing enzymes, like HAS2, and expression of CD44, the key receptor for hyaluronan, are correlated to poor outcome of patients with basal-like breast cancer. TGFß induces HAS2 and CD44, both of which are required in the course of efficient TGFß-induced EMT processes by mammary epithelial cells. Elucidation of the molecular mechanisms underlying tumor-stroma interactions in breast cancer including the regulation of HAS2 and CD44 expression may contribute to the development of better strategies to treat breast cancer patients.