Transforming growth factor-β1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts

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 (~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%.TGF-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.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]. Hyaluronidase is an extracellular matrix-degrading enzyme. Interestingly, malignant and metastatic breast and prostate cancer cells frequently overexpress both hyaluronidase and TGF-β proteins [3-7]. Previously we have shown that bovine testicular hyaluronidase, also 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 doma