Manuela Viola

miR-145-dependent targeting of junctional adhesion molecule A and modulation of fascin expression are associated with reduced breast cancer cell motility and invasiveness.

Micro RNAs are small non-coding RNAs, which regulate fundamental cellular and developmental processes at the transcriptional and translational level. In breast cancer, miR-145 expression is downregulated compared with healthy control tissue. As several predicted targets of miR-145 potentially regulate cell motility, we aimed at investigating a potential role for miR-145 in breast cancer cell motility and invasiveness. Assisted by Affymetrix array technology, we demonstrate that overexpression of miR-145 in MDA-MB-231, MCF-7, MDA-MB-468 and SK-BR-3 breast cancer cells and in Ishikawa endometrial carcinoma cells leads to a downregulation of the cell–cell adhesion protein JAM-A and of the actin bundling protein fascin. Moreover, podocalyxin and Serpin E1 mRNA levels were downregulated, and gamma-actin, transgelin and MYL9 were upregulated upon miR-145 overexpression. These miR-145-dependent expression changes drastically decreased cancer cell motility, as revealed by time-lapse video microscopy, scratch wound closure assays and matrigel invasion assays. Immunofluorescence microscopy demonstrated restructuring of the actin cytoskeleton and a change in cell morphology by miR-145 overexpression, resulting in a more cortical actin distribution, and reduced actin stress fiber and filopodia formation. Nuclear rotation was observed in 10% of the pre-miR-145 transfected MDA-MB-231 cells, accompanied by a reduction of perinuclear actin. Luciferase activation assays confirmed direct miR-145-dependent regulation of the 3′UTR of JAM-A, whereas siRNA-mediated knockdown of JAM-A expression resulted in decreased motility and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. Our data identify JAM-A and fascin as novel targets of miR-145, firmly establishing a role for miR-145 in modulating breast cancer cell motility. Our data provide a rationale for future miR-145-targeted approaches of antimetastatic cancer therapy.

Modifications of hyaluronan influence the interaction with human bone morphogenetic protein-4 (hBMP-4).

In this study, we have demonstrated that the modification of hyaluronan (hyaluronic acid; Hya) with sulfate groups led to different binding affinities for recombinant human bone morphogenetic protein-4 (rhBMP-4). The high-sulfated sHya2.8 (average degree of sulfation (D.S.) 2.8) exhibited the tightest interaction with rhBMP-4, followed by the low-sulfated sHya1.0, as determined with surface plasmon resonance (SPR), ELISA, and competition ELISA. Unmodified Hya, chondroitin-sulfate (CS), and heparan sulfate (HS) showed significantly less binding affinity. SPR data could be fitted to an A + B = AB Langmuir model and binding constants were evaluated ranging from 13 pM to 5.45 microM. The interaction characteristics of the differentially sulfated Hyas are promising for the incorporation of these modified polysaccharides in bioengineered coatings of biomaterials for medical applications.

Hyaluronan: biosynthesis and signaling.

BACKGROUND Hyaluronan is a critical component of extracellular matrix with several different roles. Besides the contribution to the tissue hydration, mechanical properties and correct architecture, hyaluronan plays important biological functions interacting with different molecules and receptors. SCOPE OF REVIEW The review addresses the control of hyaluronan synthesis highlighting the critical role of hyaluronan synthase 2 in this context as well as discussing the recent findings related to covalent modifications which influence the enzyme activity. Moreover, the interactions with specific receptors and hyaluronan are described focusing on the importance of polymer size in the modulation of hyaluronan signaling. MAJOR CONCLUSIONS Due to its biological effects on cells recently described, it is evident how hyaluronan is to be considered not only a passive component of extracellular matrix but also an actor involved in several scenarios of cell behavior. GENERAL SIGNIFICANCE The effects of metabolism on the control of hyaluronan synthesis both in healthy and pathologic conditions are critical and still not completely understood. The hyaluronan capacity to bind several receptors triggering specific pathways may represent a valid target for new approach in several therapeutic strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Molecular cloning and characterization of UDP-glucose dehydrogenase from the amphibian Xenopus laevis and its involvement in hyaluronan synthesis.

UDP-glucose dehydrogenase (UGDH) supplies the cell with UDP-glucuronic acid (UDP-GlcUA), a precursor of glycosaminoglycan and proteoglycan synthesis. Here we reported the cloning and the characterization of the UGDH from the amphibian Xenopus laevis that is one of the model organisms for developmental biology. We found that X. laevis UGDH (xUGDH) maintained a very high degree of similarity with other known UGDH sequences both at the genomic and the protein levels. Also its kinetic parameters are similar to those of UGDH from other species. During X. laevis development, UDGH is always expressed but clearly increases its mRNA levels at the tail bud stage (i.e. 30 h post-fertilization). This result fits well with our previous observation that hyaluronan, a glycosaminoglycan that is synthesized using UDP-GlcUA and UDP-N-acetylglucosamine, is abundantly detected at this developmental stage. The expression of UGDH was found to be related to hyaluronan synthesis. In human smooth muscle cells the overexpression of xUGDH or endogenous abrogation of UGDH modulated hyaluronan synthesis specifically. Our findings were confirmed by in vivo experiments where the silencing of xUGDH in X. laevis embryos decreased glycosaminoglycan synthesis causing severe embryonic malformations because of a defective gastrulation process.

Hyaluronan-CD44-ERK1/2 Regulate Human Aortic Smooth Muscle Cell Motility during Aging

The glycosaminoglycan hyaluronan (HA) modulates cell proliferation and migration, and it is involved in several human vascular pathologies including atherosclerosis and vascular restenosis. During intima layer thickening, HA increases dramatically in the neointima extracellular matrix. Aging is one of the major risk factors for the insurgence of vascular diseases, in which smooth muscle cells (SMCs) play a role by determining neointima formation through their migration and proliferation. Therefore, we established an in vitro aging model consisting of sequential passages of human aortic smooth muscle cells (AoSMCs). Comparing young and aged cells, we found that, during the aging process in vitro,HA synthesis significantly increases, as do HA synthetic enzymes (i.e. HAS2 and HAS3), the precursor synthetic enzyme (UDP-glucose dehydrogenase), and the HA receptor CD44. In aged cells, we also observed increased CD44 signaling that consisted of higher levels of phosphorylated MAP kinase ERK1/2. Further, aged AoSMCs migrated faster than young cells, and such migration could be modulated by HA, which alters the ERK1/2 phosphorylation. HA oligosaccharides of 6.8 kDa and an anti-CD44 blocking antibody prevented ERK1/2 phosphorylation and inhibited AoSMCs migration. These results indicate that, during aging, HA can modulate cell migration involving CD44-mediated signaling through ERK1/2. These data suggest that age-related HA accumulation could promote SMC migration and intima thickening during vascular neointima formation.

Proinflammatory Cytokines Induce Hyaluronan Synthesis and Monocyte Adhesion in Human Endothelial Cells through Hyaluronan Synthase 2 (HAS2) and the Nuclear Factor-κB (NF-κB) Pathway

Chronic inflammation is now accepted to have a critical role in the onset of several diseases as well as in vascular pathology, where macrophage transformation into foam cells contributes in atherosclerotic plaque formation. Endothelial cells (EC) have a critical function in recruitment of immune cells, and proinflammatory cytokines drive the specific expression of several adhesion proteins. During inflammatory responses several cells produce hyaluronan matrices that promote monocyte/macrophage adhesion through interactions with the hyaluronan receptor CD44 present on inflammatory cell surfaces. In this study, we used human umbilical chord vein endothelial cells (HUVECs) as a model to study the mechanism that regulates hyaluronan synthesis after treatment with proinflammatory cytokines. We found that interleukin 1β and tumor necrosis factors α and β, but not transforming growth factors α and β, strongly induced HA synthesis by NF-κB pathway. This signaling pathway mediated hyaluronan synthase 2 (HAS2) mRNA expression without altering other glycosaminoglycan metabolism. Moreover, we verified that U937 monocyte adhesion on stimulated HUVECs depends strongly on hyaluronan, and transfection with short interference RNA of HAS2 abrogates hyaluronan synthesis revealing the critical role of HAS2 in this process.

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis

Background: UDP-GlcNAc is a precursor of glycoconjugates, including hyaluronan, and induces protein glycosylation to form O-linked GlcNAc (O-GlcNAcylation). Results: UDP-GlcNAc induces hyaluronan synthesis through O-GlcNAcylation of hyaluronan synthase 2, which stabilizes the enzyme and prevents its proteasomal degradation. Conclusion: O-GlcNAcylation of hyaluronan synthase 2 can control synthesis of extracellular matrices with hyaluronan. Significance: UDP-GlcNAc could control cell microenvironments that are altered in many pathologies, including vascular diseases and cancer. Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies.

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.

Hyaluronan synthesis is inhibited by adenosine monophosphate-activated protein kinase through the regulation of HAS2 activity in human aortic smooth muscle cells.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) involved in cell motility, proliferation, tissue remodeling, development, differentiation, inflammation, tumor progression, and invasion and controls vessel thickening in cardiovascular diseases. Therefore, the control of HA synthesis could permit the fine-tuning of cell behavior, but the mechanisms that regulate HA synthesis are largely unknown. Recent studies suggest that the availability of the nucleotide-sugar precursors has a critical role. Because the formation of UDP-sugars is a highly energetically demanding process, we have analyzed whether the energy status of the cell could control GAG production. AMP-activated protein kinase (AMPK) is the main ATP/AMP sensor of mammalian cells, and we mimicked an energy stress by treating human aortic smooth muscle cells (AoSMCs) with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside and metformin. Under these conditions, HA synthesis, but not that of the other GAGs, was greatly reduced. We confirmed the inhibitory effect of AMPK using a specific inhibitor and knock-out cell lines. We found that AMPK phosphorylated Thr-110 of human HAS2, which inhibits its enzymatic activity. In contrast, the other two HAS isoenzymes (HAS1 and HAS3) were not modified by the kinase. The reduction of HA decreased the ability of AoSMCs to proliferate, migrate, and recruit immune cells, thereby reducing the pro-atherosclerotic AoSMC phenotype. Interestingly, such effects were not recovered by treatment with exogenous HA, suggesting that AMPK can block the pro-atherosclerotic signals driven by HA by interaction with its receptors.

The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells

Extracellular matrix remodeling after proatherosclerotic injury involves an increase in hyaluronan (HA) that is coupled with vascular smooth muscle cell (SMC) migration, proliferation, and with neointima formation. As such events are dependent on HA, in this study we assessed the effects on SMC behavior of 4-methylumbelliferone (4-MU). As previously described in other cell types, 4-MU reduced HA in cultures of primary human aortic SMCs (AoSMCs) as well as the cellular content of the HA precursor UDP-glucuronic acid. We found that SMCs increased UDP-glucuronyl transferase 1 enzymes, which can reduce the cellular content of UDP-glucuronic acid confirming that the availability of the UDP-sugar substrates can regulate HA synthesis. Interestingly, we reported that 4-MU reduced the transcripts coding for the three HA synthases as well as UDP glucose pyrophosphorylase and dehydrogenase. As HA synthase transcript reduction is common to other cell types, the 4-MU effect on gene expression may be considered a mechanism for HA synthesis inhibition. Moreover, we showed that 4-MU strongly inhibits AoSMCs migration, which was restored by the addition of exogenous HA indicating that the rescuing depends on the interaction of HA with its receptor CD44. Besides the decrease in HA synthesis and cell migration, 4-MU reduced AoSMCs proliferation, indicating that 4-MU may exert a vasoprotective effect.