瘢痕疙瘩中TIEG1的高表达

转化生长因子-β1(TGF-β1/Smads)信号转导通路的持续激活是瘢痕疙瘩形成的重要机制.研究发现这条通路重要的负反馈调节信号分子Smad7表达明显下调,Smad2/3的磷酸化水平和蛋白质量并无明显改变.但是,Smad7下调的机制尚不清楚.采用生物信息学方法对Smad7的启动子进行分析;用RT-PCR和蛋白质印迹分别检测了正常皮肤、正常瘢痕及瘢痕疙瘩组织中的Sp1样转录因子TIEG1mRNA及蛋白质的表达水平;体外培养正常皮肤、正常瘢痕及瘢痕疙瘩成纤维细胞,检测TIEG1 mRNA及蛋白的表达水平.研究结果显示,Smad7启动子上有Sp1的位点,TIEG1 mRNA及蛋白质水平在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中表达明显高于正常瘢痕和正常皮肤(P<0.05).说明瘢痕疙瘩中TIEG1可能是Smad7下调的重要原因,有必要进一步研究TIEG1对Smad7的调控作用机制.     

Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene

Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.     

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.     

Very high incidence of germ cell tumorigenesis (seminomagenesis) in human papillomavirus type 16 transgenic mice.

Human papillomavirus type 16 (HPV16) is frequently found in carcinomas and precancerous lesions of the uterine cervix and is thought to be closely associated with carcinogenesis in these regions. However, the transforming activity of the E6 and E7 genes in vivo has not been characterized. To investigate this function, we produced transgenic mice carrying HPV16 E6 and E7 open reading frames. We obtained five transgenic founders and established three transgenic lineages. We observed testicular tumors of germ cell origin in mice of all three lineages. Morphological studies showed that these tumors were a type of seminoma. Both testes of all tumor-bearing mice were affected with this type of tumor. Strikingly, in one lineage, all of the male mice developed this tumor. On Northern (RNA) analysis, a high level of expression of HPV mRNA was detected in these tumors. These results suggest that transforming genes of HPV16 have transforming activity in vivo and preferential effects on germ cells in the testis.     

Simian virus 40 large T antigen's association with the CUL7 SCF complex contributes to cellular transformation

Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that delta69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not delta69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and delta69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.     

Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.     

USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.     

DYRK1A stabilizes HPV16E7 oncoprotein through phosphorylation of the threonine 5 and threonine 7 residues

HPV16, a high-risk tumorigenic virus, has been identified as one of the causative agents for the development of cervical cancer. Subsequent to viral infection, the constitutive expression of the viral oncoproteins E6 and E7 plays a number of critical roles in maintaining the transformed phenotype. Here we demonstrate that a cellular kinase, dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), interacts with and phosphorylates HPV16E7 in vitro and in vivo. Using substitution mutations, we identified that DYRK1A specifically phosphorylates HPV16E7 at Thr5 and Thr7, which are located within the N-terminal CRI domain. This interaction greatly increases the steady-state level of HPV-16E7 by interfering with the protein's 26S proteosome-dependent degradation. The half-life of E7 was extended significantly by replacing Thr5 and Thr7 with a phosphorylation mimetic residue, aspartic acid. In addition, DYRK1A-induced phosphorylation protected E7 from degradation and influenced E7's function when modulating pRb degradation. We propose a new mechanism whereby DYRK1A phosphorylates Thr5 and Thr7 within HPV16E7. This phosphorylation then interferes with the degradation of HPV16E7, extending the protein half-life of HPV16E7 and increasing the colony-formation efficacy of HPV16E7. Our findings suggest that DYRK1A increases the transforming potential of HPV16-infected cells because of the greater stability of HPV16E7.     

Link of the unique oncogenic properties of adenovirus type 9 E4-ORF1 to a select interaction with the candidate tumor suppressor protein ZO-2

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region-encoded ORF1 (E4-ORF1) protein. We report here that the PDZ domain-containing protein ZO-2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4-ORF1 but not for non-tumorigenic wild-type E4-ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C-terminal PDZ domain-binding motif of Ad9 E4- ORF1 and the first PDZ domain of ZO-2, and in cells this interaction resulted in aberrant sequestration of ZO-2 within the cytoplasm. Furthermore, transformation-defective Ad9 E4-ORF1 mutants exhibited impaired binding to and sequestration of ZO-2 in cells, and overexpression of wild-type ZO-2, but not mutant ZO-2 lacking the second and third PDZ domains, interfered with Ad9 E4-ORF1-induced focus formation. Our results suggest that the select capacity to complex with the candidate tumor suppressor protein ZO-2 is key to defining the unique transforming and tumorigenic properties of the Ad9 E4-ORF1 oncoprotein.     

Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity.

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.