Brigitte Deschrevel

Differential effects of hyaluronan and its fragments on fibroblasts: Relation to wound healing

Hyaluronan (HA) is involved in wound healing and its biological properties depend on its molecular size. The effects of native HA and HA‐12 and HA‐880 saccharide fragments on human fibroblast proliferation and expression of matrix‐related genes were studied. The three HA forms promoted cell adhesion and proliferation. Matrix metalloproteinase‐1 and ‐3 mRNA were increased by all HA forms, whereas only HA‐12 stimulated the expression of the tissue inhibitor of metalloproteinase 1. HA‐12 enhanced type I collagen and transforming growth factor‐β (TGF‐β) 1 expression. Interestingly, HA‐12 and native HA stimulated type III collagen and TGF‐β3. HA and its fragments activated Akt and extracellular‐regulated kinases 1/2 and p38. Inhibition of these signaling pathways suggested their implication in most of the effects. Only native HA activated nuclear factor‐κB and activating protein 1. Use of CD44 siRNA suggests that this HA receptor is partly implicated in the effects, although it does not rule out the involvement of other receptors. Depending on its size, HA may exert differential regulation on the wound‐healing process. Furthermore, the HA up‐regulation of type III collagen and TGF‐β3 expression suggests that it may promote a fetal‐like cell environment that favors scarless healing.

Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and Akt

OBJECTIVE To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS). METHODS The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1beta (IL-1beta). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by real-time polymerase chain reaction analysis. The effect of CS and IL-1beta on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography. RESULTS CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1beta increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1beta-induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1beta-induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1beta-treated FLS than in FLS treated with IL-1beta alone. CONCLUSION CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1beta-enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint.

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase.

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.

Electrostatic interactions between hyaluronan and proteins at pH 4: How do they modulate hyaluronidase activity

Hyaluronan (HA) hydrolysis catalyzed by hyaluronidase (HAase) is inhibited at low HAase over HA ratio and low ionic strength, because HA forms electrostatic complexes with HAase, which is unable to catalyze hydrolysis. Bovine serum albumin (BSA) was used as a model to study the HA-protein electrostatic complexes at pH 4. At low ionic strength, there is formation of (i) neutral insoluble complexes at the phase separation and (ii) small positively-charged or large negatively-charged soluble complexes whether BSA or HA is in excess. According to the ionic strength, different types of complex are formed. Assays for HA and BSA led to the determination of the stoichiometry of these complexes. HAase was also shown to form the various types of complex with HA at low ionic strength. Finally, we showed that at 0 and 150 mmol L(-1) NaCl, BSA competes with HAase in forming complexes with HA and thus induces HAase release resulting in a large increase in the hydrolysis rate. These results, in addition to data in the literature, show that HA-protein complexes, which can exist under numerous and varied conditions of pH, ionic strength and protein over HA ratio, might control the in vivo HAase activity.

The hyaluronan-protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at low HAase over HA concentration ratio and under low ionic strength conditions. The reason is the ability of long HA chains to form electrostatic and non-catalytic complexes with HAase. For a given HA concentration, low HAase concentrations lead to very low hydrolysis rates because all the HAase molecules are sequestered by HA, whilst high HAase concentrations lead to high hydrolysis rates because the excess of HAase molecules remains free and active. At pH 4, non-catalytic proteins like bovine serum albumin (BSA) are able to compete with HAase to form electrostatic complexes with HA, liberating HAase which recovers its catalytic activity. The general scheme for the BSA-dependency is thus characterised by four domains delimited by three noticeable points corresponding to constant BSA over HA concentration ratios. The existence of HA-protein complexes explains the atypical kinetic behaviour of the HA / HAase system. We also show that HAase recovers the Michaelis-Menten type behaviour when the HA molecule complexed with BSA in a constant complexion state, i.e. with the same BSA over HA ratio, is considered for substrate. When the ternary HA / HAase / BSA system is concerned, the stoichiometries of the HA-HAase and HA-BSA complexes are close to 10 protein molecules per HA molecule for a native HA of 1 MDa molar mass. Finally, we show that the behaviour of the system is similar at pH 5.25, although the efficiency of BSA is less.

INFLUENCE OF SUBSTRATE AND ENZYME CONCENTRATIONS ON HYALURONAN HYDROLYSIS KINETICS CATALYSED BY HYALURONIDASE

ABSTRACT It has been shown that both hyaluronan (HA) and hyaluronidase (HAase) are present at high levels in the extracellular matrix (ECM) of cancer tumours. As high molecular weight HA (h-HA) is anti-angiogenic and low molecular weight HA (1-HA) is angiogenic, HAase may play an important role in regulating the h-HA/1-HA balance. A detailed study of the HA hydrolysis kinetics catalysed by HAase may thus be useful to understand its implication in cancer development and should be examined in vitro through a model system. Kinetics was monitored by the Reissig method (improved in our laboratory) which estimates the number of reducing ends formed by hydrolysis of β(1–4) glycosidic bonds. We carried out the effects of both substrate and enzyme concentrations and of ionic strength on the kinetics. The most original results concern the non-linear shape of the time course and the atypical behaviour of the initial rate versus both substrate and enzyme concentrations. The substrate dependence curve seems to be of the Michaelian type only for low concentrations. A significant decrease in the initial rate is observed for higher concentrations, which suggests inhibition phenomena. In order to explain our experimental results, we have to consider that enzymatic degradation of a polysaccharide is a particular case of enzymatic reactions because of the polymeric nature of the substrate. Elaboration of a kinetic modelling, in agreement with the experimental results, allows us to suggest a few assumptions about the reaction mechanism.

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence.

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.

Biological processes in organised media

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.

Shift of a Peptide Bond Equilibrium towards Synthesis Catalyzed by α-Chymotrypsin

Under physiological conditions, reactions catalyzed by proteases or other hydrolases, usually shift far over towards hydrolysis. Suitable conditions, such as reduction of the concentration of water (more exactly its activity) in the reaction medium, are thus required to shift the equilibrium towards synthesis. Reduction of the water concentration may be ensured by addition of organic solvents in the reaction medium1–3. This possibility has been examined to synthesize highly valuable compounds such as biologically active peptides3–5. It may also be used to build a new artificial model of active transport and such models have been worked out in our laboratory 6–8. They were based on reversible enzyme reactions occurring in gel slabs in which the asymmetrical distribution of enzyme activities was ensured by maintaining a difference in pH between the two faces of the barrier. The same kind of functional asymmetry may be induced by a difference in water concentration between the two faces if water participates in the reversible reaction.