Hirotaka Nishi

Hypoxia-Inducible Factor 1 Mediates Upregulation of Telomerase (hTERT)

ABSTRACT Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1α (HIF-1α) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between −165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.

HIF-1-mediated activation of telomerase in cervical cancer cells

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of O2 homeostasis, which regulates the expression of several genes linked to angiogenesis and energy metabolism. Tumor hypoxia has been shown to be associated with poor prognosis in a variety of tumors, and HIF-1 induced by hypoxia plays pivotal roles in tumor progression. The presence of putative HIF-1-binding sites on the promoter of human telomerase reverse transcriptase gene (hTERT) prompted us to examine the involvement of HIF-1 in the regulation of hTERT and telomerase in tumor hypoxia. The telomeric repeat amplification protocol (TRAP) assay revealed that hypoxia activated telomerase in cervical cancer ME180 cells, with peak induction at 24–48 h of hypoxia. Notably, hTERT mRNA expression was upregulated at 6–12 h of hypoxia, concordant with the elevation of HIF-1 protein levels at 6 h. hTERT protein levels were subsequently upregulated at 24 h and later. Luciferase assays using reporter plasmids containing hTERT core promoter revealed that hTERT transcription was significantly activated in hypoxia and by HIF-1 overexpression, and that the two putative binding sites within the core promoter are responsible for this activation. Chromatin immunoprecipitation assay identified the specific binding of HIF-1 to these sites (competing with c-Myc), which was enhanced in hypoxia. The present findings suggest that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays a critical role as a transcription factor. They also suggest the existence of novel mechanisms of telomerase activation in cancers, and have implications for the molecular basis of hypoxia-induced tumor progression and HIF-1-based cancer gene therapy.

Expression of a Novel Human Gene, Human Wings Apart-Like (hWAPL), Is Associated with Cervical Carcinogenesis and Tumor Progression

In Drosophila melanogaster, the wings apart-like (wapl) gene encodes a protein that regulates heterochromatin structure. Here, we characterize a novel human homologue of wapl (termed human WAPL; hWAPL). The hWAPL mRNA was predominantly expressed in uterine cervical cancer, with weak expression in all other normal and tumor tissues examined. hWAPL expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse hWAPL expression was found in all invasive squamous cell carcinomas examined. In addition, NIH3T3 cells overexpressing hWAPL developed into tumors on injection into nude mice. Furthermore, repression of hWAPL expression by RNA interference induced cell death in SiHa cells. These results demonstrate that hWAPL is associated with cell growth, and the hWAPL expression may play a significant role in cervical carcinogenesis and tumor progression.

Matrix metalloproteinase‐26 is expressed in human endometrium but not in endometrial carcinoma

The human matrix metalloproteinase (MMP)‐26, also called matrilysin‐2 or endometase, has been isolated as a matrilysin (MMP‐7) homolog. Matrix metalloproteinase‐26 was expressed in tissue samples from the placenta and endometrial tumors and its expression may be related to the development of endometrial carcinomas.

USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis.

Paclitaxel (also known as Taxol) is a well-known anticancer agent that blocks cell mitosis and kills tumor cells, and is often used in clinic to treat cancers. Despite the success of Paclitaxel, the development of drug resistance prevents its clinical applicability. Here, we screened an siRNA library against the entire human genomes using HeLa cells, and have find that lack of USP15 (ubiquitin-specific protease 15) causes Paclitaxel resistance. We also observed the decreased expression of USP15 in Paclitaxel-resistant human ovarian cancer samples. In addition, we have demonstrated that USP15 plays an essential role for stability and activity of caspase-3 during Paclitaxel-induced apoptosis. Thus, USP15 may be a candidate diagnostic marker and therapeutic target for Paclitaxel-resistant cancers.

The correlation of TERT expression with c-myc expression in cervical cancer

Recently putative catalytic telomerase subunit was identified as telomerase reverse transcriptase (TERT). Several reports showed that TERT expression correlated with telomerase activity. It has also been found that c-myc can induce telomerase activation through TERT expression. We examined expression of TERT and c-myc and their correlation in cervical cancers by reverse transcription-polymerase chain reaction (RT-PCR). It was found that TERT and c-myc expression was observed mostly in malignancies and expression of c-myc was concordant for positivity and negativity with TERT. These results support recent studies that c-myc expression is closely associated with TERT and telomerase activity. c-myc up-regulation may play an important role in activation of TERT and telomerase.

Possible role of the exchange protein directly activated by cyclic AMP (Epac) in the cyclic AMP-dependent functional differentiation and syncytialization of human placental BeWo cells

BACKGROUND The mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells. METHODS Epac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N(6)-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated. RESULTS Epac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCG alpha/beta mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion. CONCLUSIONS The Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.

Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac)

INTRODUCTION Human endometrial stromal cells (ESCs) undergo differentiation during the decidualization process. Decidualization is characterized by their enhanced production of IGF binding protein-1 (IGFBP-1), prolactin (PRL), and the forkhead transcriptional factor FOXO1, and transformation into more rounded cells, during the secretory phase of the menstrual cycle and subsequent pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is crucial for this process. The present study was undertaken to examine the involvement of a mediator of cAMP signaling, exchange protein directly activated by cAMP (Epac), in decidualization of cultured ESCs. RESULTS Treatment of ESCs with the Epac-selective cAMP analog 8-CPT-2-OMe-cAMP (CPT) had no effect on IGFBP-1, PRL, and FOXO1 mRNA expression. However, CPT potentiated IGFBP-1 and PRL expression stimulated by the PKA-selective cAMP analog N(6)-Phe-cAMP (Phe) and activated Rap1, a downstream regulator of Epac signaling. Knock-down of Epac1, Epac2, or Rap1 significantly inhibited the Phe- or Phe/CPT-induced increase in IGFBP-1 and PRL expression, as well as Rap1 activation. Furthermore, CPT enhanced IGFBP-1 and PRL expression and the morphological differentiation induced by ovarian steroids, whereas Epac1, Epac2, or Rap1 knock-down suppressed these events. CONCLUSION These data provide evidence for the involvement of the Epac/Rap1 signaling pathway in cAMP-mediated decidualization of human ESCs.

Early growth response-1 mediates downregulation of telomerase in cervical cancer

Early growth response (Egr)‐1 is a transcription factor that triggers transcription of downstream genes within 15–30 min of various stimulations. These genes are expressed rapidly through specific promoter activation and mediate cell growth and angiogenesis. Following the previous computational identification of a site that was thought to be an Egr‐1 consensus binding site at –273 to –281 in the human telomerase reverse transcriptase (hTERT) promoter region, the present study was conducted to evaluate the role of Egr‐1 in the regulation of hTERT and telomerase in uterine cervical cancer. First, the expression of Egr‐1 and hTERT at the mRNA level was examined in cervical cancer tissues. Egr‐1 and hTERT were expressed much higher in cervical cancer tissues than in the normal cervix. However, a negative correlation was noted in the expression between Egr‐1 and hTERT. By luciferase assay using hTERT promoter constructs, hTERT transcriptional activation was shown to be inhibited when Egr‐1 was overexpressed. Furthermore, Egr‐1 overexpression decreased hTERT protein production as well as hTERT mRNA as observed by western blotting analysis and real‐time reverse transcription–polymerase chain reaction, respectively. The present study suggests that Egr‐1 plays an important regulatory role in the transcriptional activation of hTERT. (Cancer Sci 2008; 99: 1401–1406)

The role of exchange protein directly activated by cyclic AMP 2-mediated calreticulin expression in the decidualization of human endometrial stromal cells.

Decidualization of human endometrial stromal cells (ESCs) accompanied by the production of prolactin (PRL) and IGF-binding protein (IGFBP) 1 and rounded-cell morphology is indispensable for the establishment and maintenance of pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is known to be crucial for decidualization. We previously reported that activation of a cAMP mediator, called Exchange protein directly activated by cAMP (EPAC) promotes cAMP analog- or ovarian steroid-induced decidualization in cultured human ESCs. In addition, small interfering RNA-mediated knock-down of the EPAC subtypes, EPAC1 or EPAC2, or knock-down of Rap1, a downstream factor of EPAC signaling, blocked functional and morphological decidualization of ESCs. However, factors downstream of EPAC2 other than Rap1 have not been determined. The present study was undertaken to identify additional downstream targets of EPAC2 associated with decidualization. Using proteomic analysis, we identified calreticulin (CRT) as a potential target of EPAC2. Knock-down of CRT expression in cultured ESCs significantly inhibited PKA-selective cAMP analog- or PKA-selective cAMP analog plus EPAC-selective cAMP analog-induced PRL and IGFBP1 expression. Furthermore, CRT knock-down suppressed the ovarian steroid-stimulated PRL and IGFBP1 expression and morphological differentiation, and silencing of EPAC2 or CRT significantly increased senescence-associated β-galactosidase activity with enhanced p21 expression and decreased p53 expression. These results suggest that EPAC2 and CRT are associated with cellular senescence in ESCs. In conclusion, we demonstrate here that EPAC2-mediated CRT expression is essential for the functional and morphological differentiation of ESCs into decidual cells. Furthermore, both EPAC2 and CRT might prevent ESCs from undergoing abnormal cellular senescence during decidualization.