Nan-Shan Chang

Latex allergy can induce clinical reactions to specific foods.

Objective The purpose of this study was to investigate crossreactivity between latex and foods, to identify crossreacting IgE binding proteins, and to assess the clinical significance.

WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses.

Human WWOX gene encodes a putative tumor suppressor WW domain-containing oxidoreductase WOX1 (also known as WWOX or FOR). A high frequency of loss of heterozygosity (LOH) of this gene has been shown in prostate, lung, breast and other cancers. In addition, numerous aberrant WWOX mRNA transcripts have been found in cancer cells. WOX1 is a proapoptotic protein. In response to stress or apoptotic stimuli, WOX1 became phosphorylated at Tyr33, which enabled its complex formation with activated p53 and JNK1. The p53/WOX1 complex translocated to the mitochondria and further to the nuclei to mediate apoptosis. WOX1 mutants, which were inactivated for nuclear translocation or Tyr33 phosphorylation, failed to induce apoptosis, indicating that activation of WOX1 via Tyr33 phosphorylation, followed by nuclear translocation, is essential for inducing cell death. WOX1 induced apoptosis synergistically with p53. In contrast, transiently activated JNK1 induced anti-apoptotic response, and this protective activity inhibited WOX1-induced apoptosis. Taken together, WOX1 is involved in stress and apoptotic responses, and is likely to regulate the activation of both p53 and JNK1.

17β-estradiol upregulates and activates WOX1/WWOXv1 and WOX2/WWOXv2 in vitro: Potential role in cancerous progression of breast and prostate to a premetastatic state in vivo

Human WWOX gene encodes a proapoptotic WW domain-containing oxidoreductase WOX1 (also named WWOX, FOR2 or WWOXv1). Apoptotic and stress stimuli activate WOX1 via Tyr33 phosphorylation and nuclear translocation. WOX1 possesses a tetrad NSYK motif in the C-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain, which may bind estrogen and androgen. Here, we determined that 17β-estradiol (E2) activated WOX1, p53 and ERK in COS7 fibroblasts, primary lung epithelial cells, and androgen receptor (AR)-negative prostate DU145 cells, but not in estrogen receptor (ER)-positive breast MCF7 cells. Androgen also activated WOX1 in the AR-negative DU145 cells. These observations suggest that sex hormone-mediated Tyr33 phosphorylation and nuclear translocation of WOX1 is independent of ER and AR. Stress stimuli increase physical binding of p53 with WOX1 in vivo. We determined here that E2 increased the formation of p53/WOX1 complex and their nuclear translocation in COS7 cells; however, nuclear translocation of this complex could not occur in MCF7 cells. By immunohistochemistry, we determined that progression of prostate from normal to hyperplasia, cancerous and metastatic stages positively correlate with upregulation and activation of WOX1 and WOX2 (FOR1/WWOXv2). In contrast, breast cancer development to a premetastatic state is associated with upregulation and Tyr33 phosphorylation of cytosolic WOX1 and WOX2, followed by significant downregulation or absent expression during metastasis. These Tyr33-phosphorylated proteins are mostly located in the mitochondria without translocating to the nuclei, which is comparable to those findings in cultured breast cancer cells. Together, sex steroid hormone-induced activation of WOX1 and WOX2 is independent of ER and AR, and this activation positively correlates with cancerous progression of prostate and breast to a premetastatic state.

Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

Background Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells. Methodology/Principal Findings DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues. Conclusions/Significance We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.

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.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 μJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 μm² at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

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.

Light-induced retinal damage involves tyrosine 33 phosphorylation, mitochondrial and nuclear translocation of WW domain-containing oxidoreductase in vivo

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, is a known proapoptotic protein and a candidate tumor suppressor. Stress stimuli activate WOX1 via tyrosine 33 (Tyr33) phosphorylation and translocation to the mitochondria and nuclei in vitro. Here, the potential role of WOX1 in light-induced retinal degeneration in vivo was investigated. WOX1 is expressed primarily in the inner retina at perinatal stages, whereas an enhanced expression of WOX1, along with its Tyr33 phosphorylation (p-WOX1), is shown specifically in the retinal ganglion cells in adults. Prolonged exposure of mature rats to constant, low-intensity light (500 lux) for 1-2 months resulted in substantial death of photoreceptors and the presence of activated microglia, astrocytes and Muller glial in the outer retina. However, the inner retina was not or barely affected. In the damaged inner and outer nuclear layers of rat retina, WOX1 and p-WOX1 were overly expressed. Also, WOX1 colocalized with fragments of opsin-positive cones. In rd mice with an inherited retinal deficiency, upregulation of WOX1 and p-WOX1 in degenerated retina was observed with age. By electron microscopy, a large number of immunogold particles of WOX1 and p-WOX1 were found in the damaged mitochondria and condensed nuclei of degenerating photoreceptors, indicating that WOX1 undergoes activation and translocation to these organelles. In contrast, little or no WOX1-positive particles were found in the Golgi apparatus. In conclusion, activated WOX1 is likely to exert apoptosis of neuronal cells in the outer retina during the light-induced injury and in mice with an inherited retinal defect.