Abstract
Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.
Highlights
Hyaluronidases Hyal-1 and Hyal-2 and hyaluronan (HA) are associated with embryonic development, morphogenesis, wound healing, neurodegeneration, and cancer progression [1,2,3,4]
We determined that exposure of malignant prostate DU145 cells to high-molecular-weight hyaluronan (105–106 kDa) for 1 hr resulted in nuclear translocation of full-length WWOX (46 kDa), isoform WWOX2 (42 kDa), p-FAK, p-Smad2/3, Smad4, p-ERK, and pS46-p53 (Figure 1A; ~100-300% increased in nuclear accumulation)
We have previously determined that TGF-β1 induces the signaling of the Hyal-2/WWOX/Smad4 complex for transcriptional activation of SMADresponsive element [21]
Summary
Hyaluronidases Hyal-1 and Hyal-2 and hyaluronan (HA) are associated with embryonic development, morphogenesis, wound healing, neurodegeneration, and cancer progression [1,2,3,4]. Hyal-1 is a lysosomal enzyme that is secreted from cells, and is considered as a tumor suppressor [5]. It enhances extravasation www.impactjournals.com/oncotarget and metastasis of many types of cancer cells [6,7,8]. Hyal-2 is a lysosomal protein and a candidate tumor suppressor [9], and is anchored on cell surface via glycosylphosphatidylinositol (GPI) [10]. PH-20 induces the expression of proapoptotic p53 and WW domain-containing oxidoreductase (WWOX, WOX1, or FOR) [16,17,18,19], which contributes in part to the increased TNF sensitivity in murine L929 fibroblasts
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