The caudal homeodomain protein activates Drosophila E2F gene expression

The Drosophila caudal homeobox gene is required for definition of the anteroposterior axis and for gut development, and CDX1 and CDX2, human homologs, play crucial roles in the regulation of cell proliferation and differentiation in the intestine. Most studies have indicated tumor suppressor functions of Cdx2, with inhibition of proliferation, while the effects of Cdx1 are more controversial. The influence of Drosophila Caudal on cell proliferation is unknown. In this study, we found three potential Caudal binding sequences in the 5′-flanking region of the Drosophila E2F (DE2F) gene and showed by transient transfection assays that they are involved in Caudal transactivation of the dE2F gene promoter. Analyses with transgenic flies carrying an E2F-lacZ fusion gene, with and without mutation in the Caudal binding site, indicated that the Caudal binding sites are required for expression of dE2F in living flies. Caudal-induced E2F expression was also confirmed with a GAL4-UAS system in living flies. In addition, ectopic expression of Caudal with heat-shock promotion induced melanotic tumors in larvae. These results suggest that Caudal is involved in regulation of proliferation through transactivation of the E2F gene in Drosophila.     

Genetic screening for modifiers of the DREF pathway in Drosophila melanogaster: identification and characterization of HP6 as a novel target of DREF

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5′-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5′-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.     

Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.     

The Transcription Factor C/EBP-β Mediates Constitutive and LPS-Inducible Transcription of Murine SerpinB2

SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by ∼1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the ∼5 kb 5''-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5′ flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-β containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-β-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-β-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-β-dependent regulation of inflammation-associated SerpinB2 expression.     

前列腺细胞REGⅣ基因功能的初步研究

目的:探讨人再生基因Ⅳ(REGIV)在前列腺细胞中的表达及意义.方法:构建REGIV基因的全序列过表达质粒.将全序列过表达质粒采用脂质体转染的方式转入前列腺细胞系PC-3中.应用Real-time-PCR方法检测REGIV基因mRNA表达,Westembloting检测REGIV基因蛋白质表达,MTT法分析细胞增殖活性.结果:通过荧光显微镜观察计数,细胞转染成功.REGIV基因的全序列过表达使REGIV基因mRNA的表达,蛋白表达提高,细胞增殖能力增强.结论:应用全序列过表达技术可以使前列腺癌REGIV表达水平特异性增高.前列腺癌增值能力的增强说明REGW可能与肿瘤快速增长有关.     

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

Expression Profile of the REG Gene Family in Colorectal Carcinoma

Regenerating (REG) gene family belongs to the calcium-dependent lectin gene superfamily and encodes small multifunctional secretory proteins, which might be involved in cell proliferation, differentiation, and carcinogenesis. To clarify REG expression profile in colorectal carcinoma (CRC), the authors examined the expression of REG Iα, Iβ, III, HIP/PAP, and REG IV by immunohistochemistry on tissue microarray. The expression of REG Iα, III, and HIP/PAP was more frequently observed in the CRCs than adjacent non-neoplastic mucosa (p < 0.001), whereas it was the converse for REG Iβ and IV (p < 0.001). The expression of REG Iα, Iβ, III, and HIP/PAP was negatively correlated with the depth of invasion of CRCs (p < 0.05). The REG Iβ and HIP/PAP were less expressed in CRCs with than without venous invasion (p < 0.05). The positive rates of REG Iα and HIP/PAP were significantly higher in CRCs without than with lymph node metastasis (p < 0.05). Mucinous carcinoma more frequently expressed REG IV protein than well- and moderately differentiated ones (p < 0.05). There was a positive relationship between REG Iα, Iβ, III, and HIP/PAP expression (p < 0.05). Survival analysis indicated the REG Iβ or HIP/PAP expression was positively linked to favorable prognosis of carcinoma patients (p < 0.05). This study indicated that aberrant REG expression might be closely linked to the pathogenesis, invasion, or lymph node metastasis of CRCs. REG Iβ and HIP/PAP could be considered reliable markers of favorable prognosis of CRC patients.     

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the −0.4- to −0.2-kb 5′-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the −0.4/−0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.     

CDX2 can be regulated through the signalling pathways activated by IL-6 in gastric cells

The inflammatory infiltrate of the gastric mucosa associated with Helicobacter pylori infection increases the presence of the pro-inflammatory cytokine IL-6 that activates both the SHP-2/ERK/MAPK and the JAK/STAT signalling pathways. Furthermore, the ectopic expression of CDX2 is detected in pre-neoplasic lesions associated with decreased levels of SOX2, and we found that in gastric adenocarcinomas their expression is inversely correlated. To determine the role of IL-6 in the regulation of CDX2, MKN45 that constitutively expresses p-STAT3, and NUGC-4 gastric cancer cell lines were treated with IL-6, which induced the CDX2 up-regulation and SOX2 down-regulation. ChIP assays determined that in IL-6-treated cells, c-JUN and p-STAT3 bound to CDX2 promoter in MKN45 cells whereas in NUGC-4 cells, p-STAT3 binds to and c-JUN releases from the CDX2 promoter. Specific inhibition of STAT3 and ERK1/2 phosphorylation through AG490 and U0126, respectively, and STAT3 down-regulation using shRNA verified that the SHP-2/ERK/MAPK pathway regulates the expression of CDX2 in basal conditions, and the CDX2 up-regulation by IL-6 is through the JAK/STAT pathway in NUGC-4 cells whereas in MKN45 cells both pathways contribute to the CDX2 up-regulation. In conclusion, the signalling pathways activated by IL-6 have a crucial role in the regulation of CDX2 that is a key factor in the process of gastric carcinogenesis, suggesting that the inflammatory infiltrate in the gastric mucosa is relevant in this process and a potential target for new therapeutic approaches.     

Vitamin A Metabolite,All-trans-retinoic Acid,Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cδ (PKCδ), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCδ, PKCδVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCδVIII via utilization of a downstream 5′ splice site of exon 10 on PKCδ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCδVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCδVIII alternative splicing. To identify the cis-elements involved in 5′ splice site selection we cloned a minigene, which included PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5′ splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCδ minigene promoted selection of 5′ splice site II. Mutation of the SC35 binding site in the PKCδ minigene abolished RA-mediated utilization of 5′ splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCδ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCδ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCδVIII.