Advanced colorectal cancers are often associated with elevated amounts of hyaluronan. To investigate the importance of hyaluronan in colon carcinoma tumor progression, we have expressed by stable transfection hyaluronan synthase 2 (Has2) and hyaluronidase 1 (Hyal1) in the rat colon carcinoma cell line, PROb. We found that hyaluronan overproduction led to a higher growth rate of tumor cells in vitro, and to a faster development of transplantable tumors in syngeneic rats, compared to the mock-transfectants. Has2 transfected PROb cells gave rise to tumors that were significantly less vascularized, but had a significantly larger viable tumor fraction compared to tumors generated from mocktransfectants. In contrast, Hyal1 overexpression suppressed the growth rate of tumor cells both in vitro and in vivo. Moreover, tumors derived from Hyal1-transfected cells had a significantly larger necrotic area than tumors derived from mock-and Has2-transfectants. Our study demonstrates that Has2 overproduction promotes tumorigenicity, whereas Hyal1 overexpression suppresses tumorigenicity in an experimental model for colon carcinoma.

Hyaluronan (HA) is a linear glycosaminoglycan that accumulates in the interstitium of injured lung and inhibits gas exchange between air and blood. In the present study we investigated the molecular mechanisms behind the local turnover of HA during the early phase of irradiation-evoked lung fibrosis in rats. Irradiation with a single dose of 30 Gy to the lower part of the right lung of rats induced an accumulation of HA in bronchoalveolar lavage fluid 6 wk after irradiation, followed by return to almost normal levels at 10 wk after irradiation. This was parallelled with a transient downregulation of HA receptors on alveolar macrophages (AMs); 4 and 6 wk after irradiation the binding of [(3)H]HA to AMs was decreased to about 50% of that of AMs from nonirradiated control rats, returning to almost normal level at 10 wk after irradiation. Analysis of the expression of rat HA synthase (HAS) isoforms (rHAS1, rHAS2, and rHAS3) and rat hyaluronidases (rHYAL1 and rHYAL2) by Northern blotting revealed an upregulation of rHAS2 messenger RNA at 4, 6, and 10 wk after irradiation, but a progressive decrease in the constitutive expression of rHYAL2 at 6 and 10 wk after irradiation; rHAS1 was undetectable, whereas rHAS3 and rHYAL1 were faintly detectable. Although transforming growth factor-beta1 stimulated HA production by normal lung fibroblasts, it inhibited HYAL activity in lysosomes and HYAL activity released into the culture media. Another interesting observation was that HA fragments, which likely result from the action of HYAL, induced expression of types I and III collagen genes. Our results indicate that rHAS2 and rHYAL2 are involved in the turnover of HA during the early phase of lung injury and that rHAS2 and rHYAL2 as well as HA fragments may play important roles in the pathogenesis of lung fibrosis.

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Cultured brain capillary endothelial cells grown in a 3-dimensional collagen gel can form tubular structures after stimulation by angiogenic factors. We found that treatment of such cultures with testicular hyaluronidase led to formation of tubular structures and cell survival. Anion-exchange chromatography of the enzyme preparation on a MonoQ column revealed the presence of the angiogenic factor basic fibroblast growth factor (bFGF) in the flow-through fraction, as determined by immunoblotting; part of the effect on endothelial cell morphogenesis could thus be ascribed to bFGF. However, adsorbed fractions eluted with increasing concentrations of NaCl, which exhibited hyaluronan-degrading activity at neutral pH, did not contain bFGF but were still able to induce tube-like structures of the endothelial cells. Streptomyces hyaluronidase failed to evoke the same effect. Interestingly, blocking of hyaluronan binding to CD44 receptors by the monoclonal antibody KM114 inhibited the effect of hyaluronidase, but not of bFGF, on endothelial cell tube formation. Our data suggest a CD44-mediated specific role for certain populations of testicular hyaluronidase in the induction of angiogenesis.