Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism

Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency(Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized.     

Effect of hyaluronan to inhibit caspase activation in porcine granulosa cells

We studied the ability of hyaluronan (HA) to inhibit apoptosis in porcine granulosa cells. The granulosa layer with cumulus-oocyte complex is cultured in media supplemented with follicle stimulating hormone (FSH) and 4-MU an inhibitor of hyaluronan synthases. The concentration of HA significantly increased after supplemented with FSH, but significantly decreased with 4-MU. CD44, receptor of HA, expressed after cultured with FSH, decreased in addition low concentration of 4-MU, whereas not detected in high concentration of 4-MU, indicating parallel relation between the amount of HA and CD44 expression. The 4-MU treatment also decreased the expression of procaspase-3, -8, -9 suggesting that inhibition of HA synthesis leads to activation of these caspases. Moreover, addition of anti-CD44 antibody decreased the expression of procaspases suggesting that perturbation of HA-CD44 binding leads activation of caspases. Hence, HA has ability to inhibit apoptosis and HA-CD44 binding is important on apoptosis inhibitory mechanism in porcine granulosa cells.     

PKC-induced stiffening of hyaluronan/CD44 linkage; local force measurements on glioma cells

Interaction of cells with hyaluronan (HA) rich extracellular matrix involves the membrane receptor CD44. HA-CD44 interactions are particularly important in the development of glioma pathogenesis for its implication in tumor cells spreading. Highly motile states rely on the spaciotemporal regulation of HA-CD44 interactions occurring in specific cytoskeletal-supported membrane organization such as microvilli or the leading edge observed in migrating cell. We used AFM-based force measurement to probe the HA-CD44 interaction at localized regions at the surface of living glioma cells expressing high level of the CD44 standard isoform. We show that unstimulated cells interact with HA over their entire surfaces and are highly deformable when force is exerted on individual HA molecules bound to membrane CD44 receptors. Conversely, in PKC-activated cells the probed interactions are concentrated at the leading edge of the cells with reduced membrane deformability. Taken together, our results show that PKC-enhanced motility in glioma cells is associated with a redistribution of CD44 receptors at the leading edges concomitant with a stiffer anchoring of CD44 to the cell surface involving the actin cytoskeleton.     

Up-regulation of hyaluronan receptors in Toxoplasma gondii-infected monocytic cells

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.     

Evidences for correlation between the reduced VCAM-1 expression and hyaluronan synthesis during cellular senescence of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) undergo cellular senescence during in vitro expansion culture, which accompanies the loss of migration and homing abilities. In this study, we analyzed expression levels of several surface markers of human MSCs at different passages of expansion culture. It has been shown that expression of vascular cell adhesion molecule-1 (VCAM-1) was most markedly decreased among the tested markers in the senescent MSCs. Interestingly the reduced VCAM-1 expression could be restored by applying hyaluronan, a major glycosaminoglycan ligand of CD44, to the culture. It was found that the hyaluronan level in extracellular and pericellular matrices was greatly reduced in the senescent MSCs, mainly due to the decreased expression of hyaluronan synthases, suggesting a correlation between the reduced VCAM-1 expression and hyaluronan synthesis. In fact, when hyaluronan synthases were knock-downed by siRNA transfection, the VCAM-1 expression was also reduced. Our results indicate that VCAM-1 expression in the senescent MSCs was down-regulated because of the reduced synthesis of hyaluronan. Thus, we suggest that hyaluronan supplementation in expansion culture of MSCs would compensate adverse effects induced by its decreased synthesis and subsequently enhance cell adhesion and migration abilities.     

Immunolocalization of the hyaluronan receptor CD44 in the reproductive tract of the mare

Hyaluronan (HA), a glycosaminoglycan, is a major component of the pericellular matrix which envelopes mammalian cells. Binding of hyaluronan to one of its specific receptors, CD44, modulates transduction of intracellular signals which direct a variety of processes, including embryogenesis, wound healing, inflammation, and neoplasia. Since regulation of these processes is critical to equine reproductive success, localization of constitutive CD44 expression was evaluated by immunohistochemical methods in ovarian, oviductal, and uterine tissues from healthy mares. Ovarian stroma contained thecal cells with varying CD44 immunopositivity. Follicular and granulosa cells of some antral and atretic follicles were positive for CD44. In the oviduct, the luminal epithelium was variably positive for CD44, with overall decreasing intensity of immunostaining from the infundibulum to the isthmus. The CD44 molecule was expressed strongly by surface epithelial cells of the uterine endometrium, but was present only rarely among cells of uterine glands. In addition, CD44 was expressed by smooth muscle cells of vascular walls, oviduct, and uterus. Since CD44 is known to modulate cell movement and differentiation, and was present at multiple sites in the reproductive tract of normal mares, we inferred there may be an important role for the HA-CD44 signaling pathway in reproductive function and inflammation.     

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.     

Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action

Texas red-labeled hyaluronan (TR-HA) is rapidly taken up in a CD44 independent manner into ras-transformed 10T1/2 fibroblasts, where it accumulates in both cell ruffles/lamellae, the perinuclear area, and the nucleus. HA does not accumulate in the cell ruffles/lamellae of parental 10T1/2 cells. Addition of HA to ras-transformed cells promotes their random motility but has no effect on 10T1/2 cell motility. 10T1/2 cells can be modified to take up HA into cell ruffles by exposure to phorbol ester or direct microinjection of HA into cells. Both treatments significantly stimulate 10T1/2 cell motility.     

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.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

Modulation of matrix metalloproteinase-9 activity by hyaluronan is dependent on NF-kappaB activity in lymphoma cell lines with dissimilar invasive behavior

Expression and activity of matrix metalloproteinase-9 (MMP-9) as well as its relationship with hyaluronan (HA) and NF-kappaB activity were analyzed in two murine lymphoma cell lines with dissimilar migration and invasive behavior. MMP activity was evaluated by zymograms in supernatants, membrane extracts of tumor cells, and in the organs invaded by these cells. The more aggressive LBLa cell line showed MMP-9 activity in vitro, which increased after HA treatment and was blocked by anti-CD44 mAb. Such activity was not found in the less aggressive LBLc. MMP-9 and MMP-2 activity was found in organs invaded by both cell lines, although differential MMP-9 activity was observed in lung infiltrated only by LBLa cell line. NF-kappaB activation was evaluated to determine whether differential activity of MMP-9 was dependent on downstream signaling pathway, showing higher NF-kappaB activity in the more aggressive LBLa cell line. Our results showed that MMP-9 activity modulated by HA through NF-kappaB signaling pathway may be involved in the aggressive behavior of LBLa.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

The decreased secretion of hyaluronan by older human fibroblasts under physiological conditions is mainly associated with the down-regulated expression of hyaluronan synthases but not with the expression levels of hyaluronidases

Although it has been reported that levels of hyaluronan are decreased in the dermis of aged skin, little is known about the cellular mechanism(s) underlying that hyaluronan deficiency. Since hyaluronan is produced by dermal fibroblasts and is secreted into the surrounding dermal tissues, we examined the secretion of hyaluronan by dermal fibroblasts and characterized its cellular mechanism using real-time RT-PCR and western blotting for its synthesizing and degrading enzymes, hyaluronan synthase and hyaluronidase, respectively. The secretion of hyaluronan by dermal fibroblasts derived from differently aged human donors, was higher in the younger human fibroblasts tested (0 and 19 years old) compared to the older human fibroblasts tested (39, 56 and 77 years old). The relative secretion levels of hyaluronan by the different human fibroblasts tested were attributable to the relative expression of hyaluronan synthases 1, 2, 3 but not hyaluronidases 1, 2 enzymes at the gene and protein levels among those fibroblasts. These findings indicate that the deficiency of hyaluronan in the aged dermis might result from the down-regulation in the potential of older human fibroblasts to secrete hyaluronan and that decrease in secretory potential is mainly associated with the down-regulated expression of hyaluronan synthases, especially hyaluronan synthase 2, but not with the expression levels of hyaluronidases.     

Lactate stimulates fibroblast expression of hyaluronan and CD44: the Warburg effect revisited

Hyaluronan, a high-molecular-weight glycosaminoglycan of the extracellular matrix, is prominent during rapid tissue growth and repair. It stimulates cell motility and hydrates tissue, providing an environment that facilitates cell movement. Markedly enhanced levels of hyaluronan also occur in the stroma surrounding human cancers, thus providing an environment that promotes spread of cancer cells. The ability of malignant tumors to generate lactate, even in the presence of adequate oxygen, is known as the Warburg effect. Early in wound healing as blood and oxygen supply decrease, lactate levels increase, as does stromal hyaluronan, suggesting a cause-and-effect relationship. Similarly, peritumor stromal fibroblast hyaluronan may be a response to cancer cell lactate. To test this, fibroblasts were cultured in the presence of lactate. With increasing lactate, higher levels of hyaluronan were observed, as were levels of CD44 expression, the predominant receptor for hyaluronan. The ability of tumor cells to utilize anaerobic metabolism and to generate lactate, even in the presence of adequate supplies of oxygen, may be one of the mechanisms used to recruit host fibroblasts to deposit hyaluronan and to express CD44, thereby participating in the process of cancer invasion and metastasis.     

The effect of hydroxyl radicals on the rheological performance of hylan and hyaluronan

Shear flow, dynamic oscillation and extensional viscosity measurements were used to compare the rheological performance of several hylan samples (M_v 1.6, 3.2, 3.7, 4.7 and 5.6×10⁶) and hyaluronan (M_v 1.4 and 1.8×10⁶) before and after hydroxyl radicals (√OH) induced degradation. It was found that the higher molecular weight cross-linked structure of hylan was more resistant to degradation than hyaluronan and that this superior stability was reflected in various rheological parameters. The √OH degradation of the initial hylan and hyaluronan samples produced a range of polysaccharides based on hylan and hyaluronan with molecular weight covering a range from 0.5–5.6×10⁶. The rheological parameters associated with the polysaccharides could then also be studied. Zero shear values of the complex viscosity (η*), dynamic viscosity (η′) and shear viscosity (η) were calculated using the method of Morris¹ and shown to approach the same value at zero shear or frequency. An adaptation of the method of Gibbs et al.² gave a ‘master curve’ for the storage and loss modulus of hyaluronan and hylan, which encompasses a 10-fold molecular weight and a 5-fold concentration variation. In all instances for hylan, the storage modulus predominates over the loss modulus, whereas for hyaluronan, the reverse is true, demonstrating the greater elasticity of hylan throughout the whole experimental range of molecular weights and concentrations.     

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

Background

Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes.

Methods

mRNA and related protein levels were measured for CD44, PKCδ, PKC?, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed.

Results

CD44, PKCδ, and PKC? mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKC? levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKC?, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKC?, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation.

General Significance

These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.