Abstract
BackgroundFunctional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-β1-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-β1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-β1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.ResultsMurine 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 (~60–110% increase), while Hyal-1-expressing cells were less sensitive to TNF (~20–90% increase). TNF activated NF-κB, along with IκBα 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-β1-enhanced L929 cell growth. In contrast, TGF-β1 inhibited Hyal-1- and Hyal-2-increased TNF cytotoxicity in L929 cells by 30–50%.ConclusionsTGF-β1 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-κB activation, and Hyal-2 translocation to the mitochondria during apoptosis.
Highlights
Functional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated
PH-20 induces the expression of proapoptotic p53 [9] and WOX1 (WW domain-containing oxidoreductase; known as WWOX or FOR) [10], which contributes to the increased tumor necrosis factor (TNF) cytotoxic effect
These observations are consistent with our previous finding that exogenous bovine testicular hyaluronidase PH-20 enhances TNF cytotoxicity in various cancer cells, and that TGF-β1 blocks the increased TNF cytotoxicity [8,15]
Summary
Functional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domaincontaining oxidoreductase (WOX1). Transforming growth factor beta (TGF-β) family proteins are multifunctional cytokines capable of regulating cell growth, extracellular matrix protein synthesis, and immune cell functions [1,2]. We have shown that bovine testicular hyaluronidase, known as PH-20, increases the susceptibility of various cancer cells to tumor necrosis factor (TNF or TNF-α) cytotoxicity [[8], review]. PH-20 induces the expression of proapoptotic p53 [9] and WOX1 (WW domain-containing oxidoreductase; known as WWOX or FOR) [10], which contributes to the increased TNF cytotoxic effect. PH-20-induced downregulation of the extracellular matrix inter-α-inhibitor is associated with the enhanced TNF cytotoxicity [9]
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