Jeannine Mertens-Strijthagen

Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na+/H+ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2.

Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase

The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles.

Endocytosis of hyaluronidase-1 by the liver

It has been suggested that intracellular Hyal-1 (hyaluronidase-1), which is considered a lysosomal enzyme, originates via endocytosis of the serum enzyme. To test this proposal we have investigated the uptake and intracellular distribution of rhHyal-1 (recombinant human Hyal-1) by mouse liver, making use of centrifugation methods. Experiments were performed on wild-type mice injected with 125I-labelled rhHyal-1 and on Hyal-1-/- mice injected with the unlabelled enzyme, which were killed at various times after injection. Activity of the unlabelled enzyme was determined by zymography. Intracellular distribution of Hyal-1 was investigated by differential and isopycnic centrifugation. The results of the study indicated that rhHyal-1 is endocytosed by the liver, mainly by sinusoidal cells, and follows the intracellular pathway described for many endocytosed proteins that are eventually located in lysosomes. However, Hyal-1 endocytosis has some particular features. First, endocytosed rhHyal-1 is quickly degraded. Secondly, its distribution, as analysed by differential centrifugation, differs from the distribution of beta-galactosidase, taken as the reference lysosomal enzyme. Further analysis by isopycnic centrifugation in a sucrose gradient shows endocytosed rhHyal-1 behaves like beta-galactosidase shortly after injection. However the Hyal-1 distribution is markedly less affected than beta-galactosidase, following a prior injection of Triton WR-1339, which is a specific density perturbant of lysosomes. The behaviour in centrifugation of endogenous liver Hyal-1, identified by hyaluronan zymography, exhibits some similarity with the behaviour of the endocytosed enzyme, suggesting that it could originate from endocytosis of the serum enzyme. Overall, these results can be explained by supposing that active endocytosed Hyal-1 is mainly present in early lysosomes. Although its degradation half-time is short, Hyal-1 could exert its activity due to a constant supply of active molecules from the blood.

Permeability of mitochondria to sucrose induced by hydrostatic pressure.

When subjected to increasing pressure at 0 degree C, rat liver mitochondria become permeable to sucrose, causing them to swell and their outer membrane to rupture. Afterwards they are lysed and their matrix content is released into the medium. This permeation to sucrose may be prevented to some extent by increasing the temperature at which compression is carried out. 0.75 mM imipramine protects mitochondria against lysis caused by hydrostatic pressure, but does not oppose their permeation to sucrose nor the swelling resulting from the compression. At this concentration, the drug does not exhibit a significant effect on the lateral phase separations which take place in the inner mitochondrial membrane under pressure. The mitochondria of rat fetal liver (21 days), kidney and Morris hepatoma 16 become permeable to sucrose when they are subjected to compression; under these conditions, lateral phase separations occur in their inner membrane. Contrary to liver mitochondria, the former do not undergo lysis. Taking into account both present and previous results, events leading to mitochondrial membrane deterioration by hydrostatic pressure may be summarized in the following way. Pressure first leads to a phase transition of the membrane lipids, thus causing a permeation to sucrose; as a result the mitochondria swell because they have absorbed osmotic water. The membrane lipids freeze increasingly as the pressure increases; the inner membrane becomes fragile and finally, in the case of the adult liver organelles, can no longer resist the swelling. All these events can be avoided by increasing the temperature; imipramine only prevents inner membrane lysis.

Role of the endothelin axis in the proliferation of human thyroid cancer cells

Objective  Endothelin‐1 (ET‐1) may play a role in carcinogenesis. ET‐1 axis is overexpressed in thyroid carcinoma. We investigated the expression and the production of ET‐1 by thyroid cancer cells as well as the effect of ET‐1 receptor antagonism on cell proliferation.

Coronary circulation response to hyperoxia after vagotomy and combined alpha and beta adrenergic receptors blockade in the anesthetized intact dog

SummaryIn closed-chest vagotomized dogs with alpha and beta adrenergic receptors blockade, ventilation with 100 per cent oxygen at atmospheric pressure did not modify the tension-time index nor the myocardial oxygen consumption. However, coronary blood flow decreased and coronary resistance increased significantly. A rise of the myocardialpO2 was not likely to be primarily responsible for the elevation of the coronary resistance. Since in the presence of a fixed oxygen consumption the myocardialpO2-elevation would occur following the rise of the arterialpO2 whose direct effect on the vascular smooth muscle tone has been demonstratedin vitro by other workers, it may be concluded that elevation of arterialpO2 exerted a direct constrictive action on the coronary vessels.However, oxygen transport to the left ventricle remained commensurate to the myocardial oxygen consumption. It is suggested that an additional mechanism adjusted the elevated coronary resistance. Shifts of the myocardialpO2, resulting from transient imbalances between oxygen supply and demand, may be the stimulus initiating the adjustment through changing release of vasodilator or vasoconstrictor substances.Results of this paper and those previously published indicated that autonomic influences normally play a dominant role in the hyperoxia-induced reduction in cardiac work and metabolism.

Biochemical characterisation of mitochondrial and lysosomal particles in foetal and neonatal rats

1. The sedimentation coefficients of the mitochondria are larger in the foetal as compared to the adult rats. For cytochrome oxidase, the values are respectively 18.700 x 10(3) and 13,550 x 10(3) S. 2. Lysosomal sedimentation coefficients are smaller in the foetal as compared to the adults. For beta galactosidase, the values are respectively 5.090 x 10(3) and 10.430 x 10(3) S. 3. It is concluded that the mitochondria are larger and the lysosomes smaller in size in the foetus as compared to the adults. 4. The external membrane of neonatal mitochondria and the membrane of foetal and neonatal lysosomes are more resistant to osmotic swelling.

Biochemical characterization of mitochondrial and lysosomal particles in old rat liver

Sedimentation rates, equilibrium densities, and membrane fragility of liver mitochondrial and lysosomal particles were estimated in adult and 36-month-old rats. The sedimentation coefficient and the size of particles were also calculated. The fractionation experiments indicated a similar enzymatic distribution for mitochondrial and lysosomal tracer enzymes in both types of animals. The liver mitochondria of senescent and mature rats were identical in sedimentation rate, sedimentation constants, equilibrium densities, fragility under isotonic conditions, and oxidative phosphorylation. Only in hypotonic media was there a decreased cohesiveness of the external mitochondrial membrane in older animals. In old rats several lysosomal tracer enzymes had lower sedimentation rates and sedimentation coefficients. The equilibrium densities were higher in these animals too. The lysosomal latency in old and mature rats was identical. It can be concluded that in very old age liver lysosomes are smaller in size than those in mature animals.

The effect of pressure on fetal and neonatal liver mitochondrial membranes

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.

The effect of sodium valproate on the subcellular particles of the rat liver

Sodium valproate, 200 mg/kg, i.p., for three weeks produced an increase in the mean sedimentation coefficient of liver mitochondria, while no changes were observed in lysosomes and peroxisomes. As the mean density of the mitochondria, the lysosomes and the peroxisomes of the control and treated animals were not different, it was concluded that the size of the mitochondria had increased in the liver of the treated rats. The mean sedimentation coefficient of the mitochondria returned to normal after the administration of the drug was interrupted for 8 days, indicating a reversible phenomenon. The mechanical and osmotical fragility of the mitochondria and the peroxisomes were not affected by sodium valproate. The osmotic stability of the lysosomes was increased in the in vivo experimental conditions.