Alberto Passi

Transcriptional and post-translational regulation of hyaluronan synthesis

Hyaluronan, a ubiquitous high‐molecular‐mass glycinoglycan on cell surfaces and in extracellular matrices, has a number of specific signaling functions in cell–cell communication. Changes in its content, molecular mass and turnover rate are crucial for cell proliferation, migration and apoptosis, processes that control tissue remodeling during embryonic development, inflammation, injury and cancer. To maintain tissue homeostasis, the synthesis of hyaluronan must therefore be tightly controlled. In this review, we highlight some recent data on the transcriptional regulation of hyaluronan synthase (Has1–3) expression and on the post‐transcriptional control of hyaluronan synthase activity, which, in close association with the supply of the UDP‐sugar substrates of hyaluronan synthase, adjust the rate of hyaluronan synthesis.

Collagens, Proteoglycans, MMP-2, MMP-9 and TIMPs in Human Achilles Tendon Rupture

Tendon integrity depends on the extracellular matrix (ECM) metabolism which is regulated by proteolytic enzymes. However, it is unclear which enzymes play a role in tendon rupture. We studied the ECM of 19 ruptured human Achilles tendons, comparing the composition of specimens harvested close to the rupture with specimens harvested from an apparently healthy area in the same tendon. We compared gene expression of collagen Type I, decorin, and versican including enzymes involved in their metabolism as matrix metalloproteases (MMP-2 and -9) and tissue inhibitory of metalloproteinase (TIMP-1 and -2) using real-time PCR, zymography and FACE analysis. We found greater gene expression of proteoglycan core protein decorin and versican, collagen Type I, MMPs and TIMPs in the tendon rupture. Zymography analysis, reflecting expression of enzymatic activity, confirmed the gene expression data at protein level. Carbohydrate content was greater in the macroscopically healthy area than in the ruptured area. In the ruptured area, we found increased core protein synthesis but without the normal glycosaminoglycan production. The tissue in the area of rupture undergoes marked rearrangement at molecular levels and supports the role of MMPs in the pathology.

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.

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.

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.

The sensitivity of versican from rabbit lung to gelatinase A (MMP‐2) and B (MMP‐9) and its involvement in the development of hydraulic lung edema

Large chondroitinsulphate‐containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP‐2) and gelatinase B (MMP‐9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP‐9 and proMMP‐2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP‐2 and MMP‐9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.

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.

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.