Transforming growth factor-beta1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts.
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.
Chang
Nan-Shan
NS
Guthrie Research Institute, Laboratory of Molecular Immunology, Guthrie Medical Center, 1 Guthrie Square, Sayre, Pennsylvania, 18840, USA. nschang@inet.guthrie.org
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001483:Base Sequence; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058851:GPI-Linked Proteins; D006821:Hyaluronoglucosaminidase / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis; D021381:Protein Transport; D019311:Staurosporine / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000633:toxicity; D000074584:WW Domain-Containing Oxidoreductase
D000970:Antineoplastic Agents; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D016328:NF-kappa B; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D010088:Oxidoreductases; D000074584:WW Domain-Containing Oxidoreductase; C428130:Wwox protein, mouse; C469220:Hyal1 protein, mouse; C469221:Hyal2 protein, mouse; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20; D019311:Staurosporine