Biomedical subjects

Nan-Shan Chang

Publications and source records attributed to Nan-Shan Chang.

15 recordsLinked to original sources

PubMed2007

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells.

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.

Cell Line, Tumor↗

PubMed2006

WW domain-containing oxidoreductase: a candidate tumor suppressor.

Common fragile site gene WWOX encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Alteration of this gene, along with dramatic downregulation of WWOX protein, is shown in the majority of invasive cancer cells. Ectopic WWOX exhibits proapoptotic and tumor inhibitory functions in vitro and in vivo, probably interacting with growth regulatory proteins p53, p73 and others. Hyaluronidases regulate WWOX expression, increase cancer invasiveness and seem to be involved in the development of hormone-independent growth of invasive cancer cells. Estrogen and androgen stimulate phosphorylation and nuclear translocation of WWOX, although binding of WWOX to these sex hormones is unknown. We propose that suppression of WWOX expression by overexpressed hyaluronidases might contribute in part to the development of hormone independence in invasive cancer.

Biomarkers↗

PubMed2005

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.

Active Transport, Cell Nucleus↗

PubMed2005

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.

Adult↗

PubMed2005

Cloning and characterization of a small-size peptide Zfra that regulates the cytotoxic function of tumor necrosis factor by interacting with JNK1.

By cDNA library screening, here we isolated an unusual gene transcript encoding a 31-amino-acid zinc finger-like peptide that regulates apoptosis (named Zfra). Northern blotting and RT/PCR showed the transcript is abundant in spleen but absent in several prostate and breast cancer cells. When stably expressed in L929 fibroblasts, Zfra conferred resistance to the cytotoxic effects of TNF and FasL. In contrast, transiently expressed Zfra could enhance or inhibit the cytotoxicity of overexpressed death domain proteins TRADD, FADD, and RIP of the TNF signaling pathway. By GST pull-down assay and co-immunoprecipitation, TNF and UV light were shown to induce Zfra to rapidly self-associate and bind JNK1. While JNK1 is a downstream effector of the TNF signaling, Zfra regulation of the TNF cytotoxic function is likely due to its interaction, in part, with JNK1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

17beta-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 17beta-estradiol (E(2)) 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 E(2) 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.

Animals↗

PubMed2004

Down-regulation of WW domain-containing oxidoreductase induces Tau phosphorylation in vitro. A potential role in Alzheimer's disease.

Numerous enzymes hyperphosphorylate Tau in vivo, leading to the formation of neurofibrillary tangles (NFTs) in the neurons of Alzheimer's disease (AD). Compared with age-matched normal controls, we demonstrated here that the protein levels of WW domain-containing oxidoreductase WOX1 (also known as WWOX or FOR), its Tyr33-phosphorylated form, and WOX2 were significantly down-regulated in the neurons of AD hippocampi. Remarkably knock-down of WOX1 expression by small interfering RNA in neuroblastoma SK-N-SH cells spontaneously induced Tau phosphorylation at Thr212/Thr231 and Ser515/Ser516, enhanced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and ERK, and enhanced NFT formation. Also an increased binding of phospho-GSK-3beta with phospho-Tau was observed in these WOX1 knock-down cells. In comparison, increased phosphorylation of Tau, GSK-3beta, and ERK, as well as NFT formation, was observed in the AD hippocampi. Activation of JNK1 by anisomycin further increased Tau phosphorylation, and SP600125 (a JNK inhibitor) and PD-98059 (an MEK1/2 inhibitor) blocked Tau phosphorylation and NFT formation in these WOX1 knock-down cells. Ectopic or endogenous WOX1 colocalized with Tau, JNK1, and GSK-3beta in neurons and cultured cells. 17Beta-estradiol, a neuronal protective hormone, increased the binding of WOX1 and GSK-3beta with Tau. Mapping analysis showed that WOX1 bound Tau via its COOH-terminal short-chain alcohol dehydrogenase/reductase domain. Together WOX1 binds Tau via its short-chain alcohol dehydrogenase/reductase domain and is likely to play a critical role in regulating Tau hyperphosphorylation and NFT formation in vivo.

Alzheimer Disease↗

PubMed2004

TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53.

TIAF1 is a TGF-beta 1-induced factor that protects L929 fibroblasts from TNF-mediated apoptosis. In contrast, overexpressed TIAF1 induces growth inhibition and apoptosis of monocytic U937 and various nonfibroblast cells. TIAF1-mediated apoptosis of U937 cells involves upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. To determine whether p53 and TIAF1 functionally interact in regulating cell death, ectopic TIAF1 and p53 were shown to induce apoptosis of U937 cells in both synergistic and antagonistic manners. At optimal levels both TIAF1 and p53 mediated apoptosis cooperatively. Also, both proteins suppressed adherence-independent growth of L929 cells. In contrast, initiation of apoptosis by overexpressed TIAF1 was blocked by low doses of p53, and vice versa. Furthermore, ectopic p53 blocked an ongoing apoptosis in U937 cells stably expressing TIAF1. Yeast two-hybrid analyses failed to demonstrate the binding of p53 with TIAF1, suggesting an unidentified protein that links the p53/TIFA1 interaction. Suppression of TIAF1 expression by siRNA could not inhibit Ser15 phosphorylation in p53 in response to UV and etoposide. However, nuclear translocation of these Ser15-phosphorylated p53 was significantly reduced in TIAF1-silenced cells. Taken together, TIAF1 and p53 functionally interact in regulating apoptosis, and TIAF1 is likely to participate in the nuclear translocation of activated p53.

Animals↗

PubMed2003

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.

Apoptosis↗

PubMed2003

High expression of TIAF-1 in chronic kidney and liver allograft rejection and in activated T-helper cells.

BACKGROUND: T helper cells are important modulators of the allograft immune response. A small number of genes are already known to be differentially expressed in T helper 1 (Th1) and T helper 2 (Th2) cells, but it is likely that many other genes are functionally important. To study gene expression in Th1 and Th2 cells, we used serial analysis of gene expression. One of the differentially expressed genes was TIAF-1, which is a TGF-beta 1-induced antiapoptotic factor, known to inhibit the cytotoxic effects of tumor necrosis factor-alpha on mouse fibroblasts. We hypothesized that TIAF-1 plays a protective role against apoptosis during allograft rejection. METHODS: We examined TIAF-1 mRNA and protein expression in kidney and liver allograft biopsy specimens from patients with chronic or acute rejection by reverse transcriptase-polymerase chain reaction and immunohistochemistry. RESULTS: TIAF-1 mRNA and protein were not detectable in normal kidney and liver; however, the expression of TIAF-1 was up-regulated in most biopsy specimens with chronic and a few with acute allograft rejection. Immunohistochemistry for TIAF-1 revealed expression in the inflammatory infiltrate and in tubular epithelial cells. CONCLUSIONS: TIAF-1 mRNA and protein are predominantly up-regulated in kidney and liver allografts with chronic rejection. This does not seem to be related to the cyclosporine A therapy. Expression of TIAF-1 in the lymphocytes during chronic allograft rejection may be related to the predominance of a Th2 response in this condition. The expression in the transplanted tissue may protect these cells from apoptosis.

Apoptosis Regulatory Proteins↗

PubMed2003

JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis.

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

Animals↗

PubMed2003

TIAF1 participates in the transforming growth factor beta1--mediated growth regulation.

TGF-beta induces growth suppression and apoptosis of various types of cells, but supports fibroblast growth. We previously isolated TIAF1 (TGF-beta1-induced antiapoptotic factor 1), which protects murine L929 fibroblasts from TNF cytotoxicity. Here, we show that TIAF1 induced growth inhibition and apoptosis of monocytic U937 and other types of cells. In contrast, like TGF-beta1, TIAF1 supported transforming growth of L929 fibroblasts. TIAF1 increased the expression of p53, Cip1/p21, and Smad proteins; suppressed ERK phosphorylation; and altered TGF-beta1-mediated Smad2/3 phosphorylation in U937 cells. Antisense TIAF1 mRNA significantly enhanced the proliferation of mink lung Mv1Lu epithelial cells. Together, these observations indicate that TIAF1 participates in the TGF-beta-mediated growth regulation.

Animals↗

PubMed2002

Transforming growth factor-beta1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts.

BACKGROUND: Functional antagonism between transforming growth factor beta (TGF-beta) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-beta1-mediated growth inhibition of epithelial cells. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-beta1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-beta1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined. RESULTS: Murine L929 fibroblasts were engineered to stably express green-fluorescent protein (GFP)-tagged hyaluronidase (GFP-Hyal-1 or GFP-Hyal-2) or GFP alone. Compared to control cells, Hyal-2-expressing cells had a significantly increased sensitivity to TNF cytotoxicity (approximately 60-110% increase), while Hyal-1-expressing cells were less sensitive to TNF (approximately 20-90% increase). TNF activated NF-kappaB, along with IkappaBalpha degradation, occurred at 20 to 60 min in Hyal-2 cells post stimulation, but at the 20 min time point in both control and Hyal-1 cells. Hyal-2 cells, but not Hyal-1 and control cells, constitutively expressed WOX1, and transiently expressed Hyal-2 enhanced WOX1-mediated cell death. Unlike PH-20, Hyal-1 and Hyal-2 did not induce p53 expression. Hyal-2 translocated from the lysosome to the mitochondria during staurosporine-mediated apoptosis, suggesting that Hyal-2 may damage mitochondria. Finally, Hyal-1 and Hyal-2 blocked TGF-beta1-enhanced L929 cell growth. In contrast, TGF-beta1 inhibited Hyal-1- and Hyal-2-increased TNF cytotoxicity in L929 cells by 30-50%. CONCLUSIONS: TGF-beta1 limits the ability of Hyal-2 to induce TNF cytotoxicity in L929 cells. Hyal-2-increased TNF cytotoxicity in L929 cells appears to be correlated with upregulation of WOX1, a prolonged NF-kappaB activation, and Hyal-2 translocation to the mitochondria during apoptosis.

Animals↗

PubMed2002

The non-ankyrin C terminus of Ikappa Balpha physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-beta 1-mediated growth suppression.

Transforming growth factor beta (TGF-beta1) suppresses the growth of mink lung Mv1Lu epithelial cells, whereas testicular hyaluronidase abolishes the growth inhibition. Exposure of Mv1Lu cells to TGF-beta1 rapidly resulted in down-regulation of cytosolic IkappaBalpha and hyaluronidase prevented this effect, suggesting a possible role of IkappaBalpha in the growth regulation. Ectopic expression of wild-type and dominant negative IkappaBalpha prevented TGF-beta1-mediated growth suppression. Nonetheless, the blocking effect of IkappaBalpha is not related to regulation of NF-kappaB function by its N-terminal ankyrin-repeat region (amino acids 1-243). Removal of the PEST (proline-glutamic acid-serine-threonine) domain-containing C terminus (amino acids 244-314) abolished the IkappaBalpha function, and the C terminus alone blocked the TGF-beta1 growth-inhibitory effect. Co-immunoprecipitation by anti-p53 antibody using Mv1Lu and other types of cells, as well as rat liver and spleen, revealed that a portion of cytosolic IkappaBalpha physically interacted with p53. In contrast, Mdm2, an inhibitor of p53, was barely detectable in the immunoprecipitates. The cytosolic p53 x IkappaBalpha complex rapidly dissociated in response to apoptotic stress, etoposide- and UV-mediated DNA damage, hypoxia, and TGF-beta1-mediated growth suppression. Also, a rapid increase in the formation of the nuclear p53 x IkappaBalpha complex was observed during exposure to etoposide and UV. In contrast, TGF-beta1-mediated promotion of fibroblast growth failed to mediate p53 x IkappaBalpha dissociation. Mapping by yeast two-hybrid showed that the non-ankyrin C terminus of IkappaBalpha physically interacted with the proline-rich region and a phosphorylation site, serine 46, in p53. Deletion of serine 46 or alteration of serine 46 to glycine abolished the p53 x IkappaBalpha interaction. Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53 x IkappaBalpha complex formation. Functionally, enhancement of p53 apoptosis was observed when p53 and IkappaBalpha were transiently co-expressed in cells. Together, the IkappaBalpha x p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress.

Animals↗

PubMed2002

A potential role of p53 and WOX1 in mitochondrial apoptosis (review).

A large number of p53-transcribed proteins have been shown to mediate growth arrest and/or apoptosis in vitro, whereas their in vivo roles remain largely unclear. p53 is capable of initiating apoptosis without transcription of apoptosis inducer genes, although the underlying mechanism is unknown. p53 is present in the mitochondria and appears to contribute to the biogenesis, function and apoptosis of this organelle. We have recently cloned a p53-binding mitochondrial WW domain-containing oxidoreductase (WOX1). Suppression of WOX1 expression abolishes p53 apoptotic function, indicating that WOX1 is a likely partner of p53 in cell death. In this review article, the potential role of WOX1/p53 as a signaling complex in the mitochondrial apoptosis is discussed.

Apoptosis↗