Shur Tzu Chen

Transforming Growth Factor β1 Signaling via Interaction with Cell Surface Hyal-2 and Recruitment of WWOX/WOX1

Transforming growth factor β (TGF-β) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-β1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF-β 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-2·WOX1 complexes for relocation to the nuclei. TGF-β1 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 two-hybrid 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-β1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-β1-induced apoptosis. Together, TGF-β1 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.

WOX1 Is Essential for UVB Irradiation–Induced Apoptosis and Down-Regulated via Translational Blockade in UVB-Induced Cutaneous Squamous Cell Carcinoma In vivo

Purpose: We investigated the role of candidate tumor suppressor and proapoptotic WOX1 (also named WWOX, FOR, or WWOXv1) in UVB-induced apoptosis and formation of cutaneous squamous cell carcinomas (SCC). Experimental Design: Expression of WOX1 and family proteins (WWOX) in human primary cutaneous SCCs was examined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. UVB irradiation–induced WOX1 activation (Tyr33 phosphorylation and nuclear translocation), apoptosis, and cutaneous SCC formation were examined both in vitro and in vivo. Results: Up-regulation of human WOX1, isoform WOX2, and Tyr33 phosphorylation occurred during normal keratinocyte differentiation before cornification and death. Interestingly, significant reduction of these proteins and Tyr33 phosphorylation was observed in nonmetastatic and metastatic cutaneous SCCs (P < 0.001), but without down-regulation of WWOX mRNA (P > 0.05 versus normal controls), indicating a translational blockade of WWOX mRNA to protein. During acute exposure of hairless mice to UVB, WOX1 was up-regulated and activated in epidermal cells in 24 hours. In parallel with the clinical findings in humans, chronic UVB-treated mice developed cutaneous SCCs in 3 months, with significant reduction of WOX1 and Tyr33 phosphorylation and, again, without down-regulation of WWOX mRNA. Human SCC-25 and HaCaT cells were transfected with small interfering RNA–targeting WOX1 and shown to resist UVB-induced WOX1 expression, activation, and apoptosis. Conclusions: WOX1 is essential for UVB-induced apoptosis and likely to be involved in the terminal differentiation of normal keratinocytes. During UVB-induced cutaneous SCC, epidermal cells have apparently prevented the apoptotic pressure from overexpressed WOX1 by shutting down the translation machinery for WWOX mRNA.

Melatonin attenuates MPP+-induced neurodegeneration and glutathione impairment in the nigrostriatal dopaminergic pathway

In this study we selected a rat model of Parkinsons disease (PD) by using intrastriatal infusion of the 1‐methyl‐4‐phenyl‐pyridinium ion (MPP+) to investigate the neuroprotective action of melatonin and its inhibitory activity on MPP+‐impaired glutathione (GSH) system in the nigrostriatal system. Results show that MPP+ caused not only a severe neuronal injury in the striatum and in the ipsilateral substantia nigra (SN), but it also induced a significant decrease in GSH levels and an increase in the GSSG/GSH ratio 3u2003days after intrastriatal MPP+ infusion. Intraperitoneal co‐administration of melatonin (10u2003mg/kg, five times) significantly attenuated MPP+‐induced nigrostriatal neurotoxicity and GSH impairment. Depletion of cytosolic GSH by L‐buthionine sulfoximine (BSO) did not cause neuronal damage by itself. It, however, when co‐administrated with MPP+, potentiated the GSH reduction in the striatum, without aggravating nigrostriatal neurodegeneration induced by MPP+. Moreover, the MPP+‐caused neuronal damage was positively correlated with a rising ratio of GSSG/GSH, but not with a drop of GSH. These results suggest that the MPP+‐triggered oxidative stress may play a more important role than the loss of the antioxidant GSH in determining neuronal injury. Interestingly, the neuronal damage and oxidative stress elicited by co‐treatment of BSO with MPP+ were effectively reduced by melatonin. Our results hence provide direct evidence showing that melatonin attenuates MPP+‐induced nigrostriatal dopaminergic injury by its ability to impede the increase of GSSG/GSH ratio; therefore melatonin may have therapeutic implications in PD.

Dramatic co-activation of WWOX/WOX1 with CREB and NF-κB in delayed loss of small dorsal root ganglion neurons upon sciatic nerve transection in rats

Background Tumor 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 Findings Sciatic 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/Significance Rapid 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.

Brain region-dependent increases in β-amyloid and apolipoprotein E levels in hypercholesterolemic rabbits

Summary. Recent studies indicate a possible link between serum cholesterol level, β-amyloid (Aβ) peptide concentrations, and the incidence of Alzheimers disease (AD). In the present report, the effects of dietary cholesterol on Aβ and apolipoprotein E (APOE) levels in several brain regions were examined using diet-induced hypercholesterolemic rabbits as the animal model. Increased dietary cholesterol levels increased Aβ concentrations in temporal cortex (p = 0.02). A similar trend was observed in the frontal cortex (p = 0.06), yet not in the cerebellum. Interestingly, the regional levels of Aβ in the hypercholesterolemic rabbit paralleled the amyloid pathology observed in AD brain. Elevated APOE levels were also noticed in temporal (p < 0.01) and frontal (p < 0.01) cortices, but not in cerebellum, in the rabbit fed with cholesterol-abundant diet. These results suggest that high serum cholesterol levels could induce the elevation of brain APOE, which may play a role in aggravating the Aβ accumulation.

MPP+-induced neuronal death in rats involves tyrosine 33 phosphorylation of WW domain-containing oxidoreductase WOX1.

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 Parkinsons 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.

Mechanisms Underlying Benzyl Alcohol Cytotoxicity (Triamcinolone Acetonide Preservative) in Human Retinal Pigment Epithelial Cells

PURPOSEnBenzyl alcohol (BA) is the preservative in triamcinolone acetonide (TA) suspensions, which are used in treating vitreoretinal diseases and during surgery. This paper investigates the molecular mechanisms and signaling pathways underlying BA toxicity in human retinal pigment epithelial (RPE) cells.nnnMETHODSnCultured human RPE cells from the ARPE-19 cell line were exposed to culture medium alone (control) or with BA (0.0225, 0.225, 0.9, 3, or 9 mg/mL) for up to 6 hours. BA toxicity was assessed by TUNEL assay, propidium iodide/annexin V-FITC staining and flow cytometry, caspase activation assay, caspase and apoptosis inhibition assays, mitochondrial transmembrane potential by rhodamine staining and flow cytometry, reactive oxygen species by chemiluminescence, and apoptosis-inducing factor staining.nnnRESULTSnBA caused RPE cell death not only by necrosis but also by apoptosis, evidenced by exposure to 9 mg/mL BA for 6 hours leading to 19.0% early apoptotic cells and 64.2% apoptotic necrotic cells. Apoptotic signaling involved the immediate production of reactive oxygen species, activation of caspase-8, impairment of the mitochondrial transmembrane potential, and further activation of caspase-9 and -3. In addition, BA induced translocation of apoptosis-inducing factor into the nucleus, indicating caspase-independent apoptosis.nnnCONCLUSIONSnBA leads to necrosis of RPE cells and triggers mitochondrial apoptosis through both caspase-dependent and - independent pathways. Extreme caution is suggested in the intraocular use of TA suspensions and meticulous evaluation before adoption of BA as a preservative in the future development of ophthalmic formulations.

Differential expression of Bcl-2 and APP immunoreactivity after intrastriatal injection of MPP+ in the rat.

While there is growing evidence that Bcl-2 proto-oncogene and beta-amyloid precursor proteins (APP) are neuroprotective in function, our recent studies have demonstrated that Bcl-2 and APP may be co-expressed and co-regulated in retinal neurons or glia under normal or experimental conditions. Whether Bcl-2 and APP are functionally coupled in other neuronal systems is not clear. This issue was investigated further in the present experiments by examining the expression pattern of two molecules after unilateral intrastriatal injection of 1-methyl-4-phenyl-pyridinium (MPP(+)), a neurotoxic metabolite that selectively damages dopaminergic neurons. One hour to 2 months after MPP(+) injection into rat striatum, a significant increase in Bcl-2 expression was observed in distinct populations of neurons, astrocyte-like and OX-42-positive cells not only in traumatic regions but also in remote areas including the ipsilateral cortex and substantia nigra (SN). No detectable change was observed in the striatum, cortex or SN on the contralateral side of the brain. The immunoreactive pattern and time-dependent APP increase was similar to that of Bcl-2 in the severely injured striatum and cortex. However, an up-regulation of Bcl-2 expression, but not APP, appears in dopaminergic neurons in the ipsilateral SN pars compacta where there was retrograde degeneration. In contrast, APP immunoreactivity was decreased in the hippocampus following intrastriatal injury, whereas, no alteration in Bcl-2 expression was detected. The differential changes in Bcl-2 and APP expression in nigral neurons and some other brain tissues suggest that these proteins may not be co-regulated by a common mechanism, at least in certain neuronal pathways.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

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.

Expression of WW domain-containing oxidoreductase WOX1 in human nervous system tumors

Background and ObjectiveS: We aimed to evaluate the expression levels of the tumor suppressor WOX1 in nervous system tumors and its co-expression with p53 and neurofibromatosis type 2/merlin (NF2) tumor suppressor gene products. Methods: Immunohistochemistry, western blotting and in situ hybridization were used for WOX1 protein and WWOX mRNA expression. Immunofluorescence and electron microscopical immunohistochemistry were performed for colocalization of gene products. Results: WOX1 expression is low in normal cortical neurons, mainly on the axon fibers, whereas there is moderate to high immunoreactivity in the cytosol and nuclei of certain tumor cells. In the microcystic (WHO grade I) and malignant (WHO grade III) meningiomas, WOX1 expression is intense, but various in transitional (WHO grade I) and atypical (WHO grade II) subtypes. WOX1 levels are moderate to high in the menigiotheliomatous area, but relatively low in the fibroblastic area. WOX1 and NF2/merlin, but not p53, colocalized in certain tumor cells, primarily at the borders of nuclei. Schwannoma and astrocytoma specimens stained moderately to strongly positive for the WOX1 protein. Interestingly, the expression of WOX1, NF2/merlin and mutant p53 is intense in high grade glioblastoma, but WOX1 expression is low in metastatic carcinoma or adenocarcinoma. Conclusions: The expression of WOX1 on different types of nervous system tumors, including primary and metastatic tumors, is differential.