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 (T␤RII), 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...

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.

WW domain-containing oxidoreductase (named WWOX, FOR or WOX1) is a pro-apoptotic protein and tumor suppressor. Animals treated with dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+) develop Parkinson's disease (PD)-like symptoms. Here we investigated whether WOX1 is involved in MPP+-induced neurodegeneration. Upon insult with MPP+ in rat brains, WOX1 protein was upregulated and phosphorylated at Tyr33 (or activated) in the injured neurons in the striatum and cortex ipsilaterally to intoxication, as determined by immunohistochemistry and Western blotting. Also, WOX1 was present in the condensed nuclei and damaged mitochondria of degenerative neurons, as revealed by transmission immunoelectron microscopy. Time-lapse microscopy revealed that MPP+ induced membrane blebbing and shrinkage of neuroblastoma SK-N-SH cells. Dominant-negative WOX1, a potent inhibitor of Tyr33 phosphorylation, abolished this event, indicating a critical role of the phosphorylation in apoptosis. c-Jun N-terminal kinase (JNK1) is known to bind and counteract the apoptotic function of WOX1. Suppression of JNK1 function by a dominant-negative spontaneously induced WOX1 activation. WOX1 physically interacted with JNK1 in SK-N-SH cells and rat brain extracts. MPP+ rapidly increased the binding, followed by dissociation, which is probably needed for WOX1 to exert apoptosis. We synthesized a short Tyr33-phosphorylated WOX1 peptide (11 amino acid residues). Interestingly, this peptide blocked MPP+-induced neuronal death in the rat brains, whereas non-phospho-WOX1 peptide had no effect. Together, activated WOX1 plays an essential role in the MPP+-induced neuronal death. Our synthetic phospho-WOX1 peptide prevents neuronal death, suggestive of its therapeutic potential in mitigating the symptoms of PD.

SummaryProtein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, we identified one role that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. We further identified Rab4 GTPase as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wingblade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. We found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, our findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration.

It has been suggested that the concept of gliomas originate from tumor stem cell (TSC) may provide clues to our understanding of gliomas pathogenesis. Here, we are using immunohistochemistry (IHC) and immunofluorescence (IF) methods to examine 16 glioblastomas (GBM), 2 anaplastic astrocytomas, 3 astrocytomas, and 4 normal brains with stem cell markers to test the hypothesis that TSCs and stem cell markers are differentially expressed in the gliomas. IHC revealed that there were relatively low number of CD133 and nestin antibodies positive cells in normal brain, grade II and grade III astrocytomas. CD133 and nestin were strongly expressed and co‐expressed in the smooth muscle cells of proliferating glomeruloid vessels in GBMs but not in the small cortical or larger meningeal vessels. CD133+ cells either weakly or not co‐express with vimentin. Nestin+ cells co‐express vimentin and GFAP. The IF density and number of CD133+or nestin+ cells decreased inversely to the distance of the glomeruloid vessels in GBMs. IF also revealed that nestin co‐express with vimentin, and GFAP. MAP2 and tubulin III antibodies highlight entrapped neurofilaments in the tumors. Given these three intermediate filaments present in different stage of glial cell development, and nestin and vimentin differentially co‐expressed in CD133+ cells, our findings suggest that intermediate filaments may play important role in stem cell derived GBMs.