Background: Functional antagonism between transforming growth factor beta (TGF-beta) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-beta1-mediated growth inhibition of epithelial cells. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-beta1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-beta1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.
Results: Murine L929 fibroblasts were engineered to stably express green-fluorescent protein (GFP)-tagged hyaluronidase (GFP-Hyal-1 or GFP-Hyal-2) or GFP alone. Compared to control cells, Hyal-2-expressing cells had a significantly increased sensitivity to TNF cytotoxicity (approximately 60-110% increase), while Hyal-1-expressing cells were less sensitive to TNF (approximately 20-90% increase). TNF activated NF-kappaB, along with IkappaBalpha degradation, occurred at 20 to 60 min in Hyal-2 cells post stimulation, but at the 20 min time point in both control and Hyal-1 cells. Hyal-2 cells, but not Hyal-1 and control cells, constitutively expressed WOX1, and transiently expressed Hyal-2 enhanced WOX1-mediated cell death. Unlike PH-20, Hyal-1 and Hyal-2 did not induce p53 expression. Hyal-2 translocated from the lysosome to the mitochondria during staurosporine-mediated apoptosis, suggesting that Hyal-2 may damage mitochondria. Finally, Hyal-1 and Hyal-2 blocked TGF-beta1-enhanced L929 cell growth. In contrast, TGF-beta1 inhibited Hyal-1- and Hyal-2-increased TNF cytotoxicity in L929 cells by 30-50%.
Conclusions: TGF-beta1 limits the ability of Hyal-2 to induce TNF cytotoxicity in L929 cells. Hyal-2-increased TNF cytotoxicity in L929 cells appears to be correlated with upregulation of WOX1, a prolonged NF-kappaB activation, and Hyal-2 translocation to the mitochondria during apoptosis.