Transforming growth factor  (TGF-) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF- receptor-deficient HCT116 cells, as determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2⅐WOX1 complexes for relocation to the nuclei. TGF-1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast twohybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8 -9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-1-induced apoptosis. Together, TGF-1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death. Transforming growth factor  (TGF-)4 plays a dual role in cell growth and tumorigenesis (1, 2). TGF- inhibits mammary epithelial cell growth. In contrast, invasive cancer cells frequently overproduce TGF- to promote growth and metastasis (1, 2). The underlying mechanism is largely unknown. TGF- induces the development of metastatic phenotypes, i.e. stimulation of epithelial-mesenchymal transitions in cancerous mammary epithelial cells (1, 2). These cells are normally devoid of functional type II TGF- receptor (TRII), suggesting that TGF- binds to an alternative receptor for signaling.Hyaluronan is the major components of pericellular coat and plays a key role in affecting cell morphology, communication, and behavior (3-5). Up-regulation of hyaluronan and hyaluronidases Hyal-1, Hyal-2, and PH-20 is associated with cancer metastasis (3-5). Hyaluronidases counteract the activity of TGF-1 (6 -8). TGF-1 suppresses the proliferation of normal epithelial cells, whereas PH-20 blocks the TGF-1 effect (6). Hyal-1 and Hyal-2 enhance the cytotoxic function of TNF and block TGF-1-mediated protection of murine L929 fibroblasts from TNF cytotoxicity (6 -8).Hyaluronidases PH-20, Hyal-1, and Hyal-2 induce the expression of tumor suppressor WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1 (8 -11). Human WWOX gene is located on a chromosomal fragile site 16q23 and encodes WWOX/FOR/WOX1 and isoforms (9, 10, 12-16). The full-length 46-kDa WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence between the WW domains, and a C-terminal short chain alcohol dehydrogenase/reductase domain. Numerous exogenous stimuli, including sex stero...
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.
BackgroundTumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats.Methodology/Principal FindingsSciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-κB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-κB (40–65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-κB. WOX1 directly activated NF-κB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons.Conclusions/SignificanceRapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-κB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-κB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury.
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