Subunit Gene Promoter Research Articles

Hypermethylation of the sodium channel beta subunit gene promoter is associated with colorectal cancer

AimsTo better understand the role of sodium channel beta subunit (SCNN1B) in the initiation and progression of colorectal cancer (CRC) and to identify potential biomarkers for the early detection and prognosis of CRC.MethodsA total of 74 pairs of CRC tissues and their adjacent normal tissues were collected between October 2016 and November 2017. The methylation levels of the SCNN1B promoter region in CRC tissues and their adjacent normal tissues were investigated by pyrosequencing. The expression of both SCNN1B mRNA and protein were detected by RT‒qPCR and immunohistochemistry, respectively.ResultsThe results showed that the methylation levels of the SCNN1B promoter region were significantly higher in CRC tissues than in adjacent normal tissues. The expression levels of SCNN1B mRNA and protein were significantly lower in the CRC tissues than in their adjacent normal tissues. Moreover, Pearson’s correlation analysis showed that the methylation levels of the SCNN1B promoter were negatively correlated with the SCNN1B mRNA levels in CRC tissues. In addition, the high methylation levels and low mRNA expression of SCNN1B showed a significant association with advanced tumour stage, increased risk of lymph node metastasis and poor prognosis of CRC patients.ConclusionThis study suggested that the decreased expression of SCNN1B due to its promoter hypermethylation may play an important role in the progression and prognosis of CRC, and the methylation levels of the SCNN1B promoter may serve as an effective molecular marker for predicting the progression and prognosis of CRC.

Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT

BackgroundFructans are water-soluble carbohydrates that accumulate in wheat and are thought to contribute to a pool of stored carbon reserves used in grain filling and tolerance to abiotic stress.ResultsIn this study, transgenic wheat plants were engineered to overexpress a fusion of two fructan biosynthesis pathway genes, wheat sucrose: sucrose 1-fructosyltransferase (Ta1SST) and wheat sucrose: fructan 6-fructosyltransferase (Ta6SFT), regulated by a wheat ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (TaRbcS) gene promoter. We have shown that T4 generation transgene-homozygous single-copy events accumulated more fructan polymers in leaf, stem and grain when compared in the same tissues from transgene null lines. Under water-deficit (WD) conditions, transgenic wheat plants showed an increased accumulation of fructan polymers with a high degree of polymerisation (DP) when compared to non-transgenic plants. In wheat grain of a transgenic event, increased deposition of particular fructan polymers such as, DP4 was observed.ConclusionsThis study demonstrated that the tissue-regulated expression of a gene fusion between Ta1SST and Ta6SFT resulted in modified fructan accumulation in transgenic wheat plants and was influenced by water-deficit stress conditions.

High-Dose Thyroid Hormone Replacement in Bexarotene-Induced Central Hypothyroidism

Background: Central hypothyroidism is a rare disorder characterized by a defect in thyroid hormone production by an otherwise normal thyroid gland due to decreased stimulation by TSH. Medications are an uncommon cause. Case Presentation: A 72-year-old man was referred for evaluation of a low TSH. He had a long history of hypothyroidism, euthyroid on levothyroxine, and was diagnosed with cutaneous T cell lymphoma (CTCL). Due to disease progression on dapsone, PUVA and TAR baths, he was started on bexarotene. Soon after, he developed recurrent symptoms of hypothyroidism including fatigue, cold intolerance, dry skin, and myalgias. Workup revealed a TSH of <0.01 ulU/mL (0.27-4.20) with a free T4 of 0.6 ng/dl (0.9-1.7). After evaluation, his levothyroxine dose was increased. Repeat labs 3 months later showed a TSH of <:0.01 with a free T4 0.8 ng/dl and T3 43 ng/dl (80-200). Over several months, levothyroxine titration to a supraphysiologic dose of 800 mcg daily was required, despite optimal administration, to normalize FT4. Given persistent hypothyroid symptoms and a low T3 level, liothyronine 5 mcg BID was added and resulted in clinical and biochemical euthyroidism. Clinical Lessons: Unlike in primary thyroid disorders, the TSH assay is unreliable in central hypothyroidism since values can be low, normal, or even mildly elevated; regardless, TSH has subnormal bioactivity. Ineffectual TSH leads to a low T4, which is a required for diagnosis. Bexarotene, a derivative of Vitamin A, is a retinoid X receptor (RXR) selective ligand approved for the treatment of CTCL. The exact mechanism of bexarotene-induced thyroid dysfunction is not clear, although it involves both central and peripheral effects. Bexarotene inhibits TSH gene expression by decreasing the activity of the thyrotropin β subunit gene promoter in a dose-dependent and thyroxine-independent manner; TSH levels drop as early as 4-8 hours after exposure. Bexarotene also directly lowers T4 and T3 levels, even in athyreotic patients, by negatively impacting deiodinase activity and hepatic conjugation. Stopping bexarotene is often not possible given its effectiveness in CTCL. Therefore, thyroid hormone should be initiated with the goal of achieving a normal FT4, although as the current case demonstrates, massively supraphysiologic doses and/or the addition of liothyronine may be necessary to achieve clinical euthyroidism.

Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera

A major pest of chickpea, Helicoverpa armigera, can be controlled by expressing genes from the bacterium Bacillus thuringiensis as an environmentally compatible option. Here we show that transgenic chickpeas containing a cry1Ac gene conferred a high degree of resistance to H. armigera. The Agrobacterium binary vector contained the nptII gene as the selectable marker and cry1Ac gene driven by the Arabidopsis rubisco small subunit gene (ats1A) promoter. We generated 54 and 47 independent transgenic lines using truncated (trcry1Ac) and full-length versions of the cry1Ac (flcry1Ac) gene, respectively. Of these lines, twelve transmitted the trcry1Ac transgene to the next generation at a 3:1 ratio, while only 8 flcry1Ac lines segregated in a 3:1 ratio. Five lines expressed trCry1Ac protein > 50 μg/g fresh weight, however, only one line accumulated about 30 μg/g flCry1Ac protein. Such high levels of trCry1Ac protein have not been reported before in chickpea. When trCry1Ac lines were challenged to whole plant bioassays in the greenhouse, lowest pod damage was observed in BS100B (1.4%) followed by BS81P (4.4%), and BS100E (6.2%) compared to the parental line (49.9%). The phenotypes of the lines expressing high levels of Cry1Ac protein were indistinguishable from their null segregants and controls. Thus, trCry1Ac lines could be suitable for crossing with our existing Cry2Aa lines for generation of a pyramided Bt chickpea for enhanced insect resistance management in the field.

The base pair-scale diffusion of nucleosomes modulates binding of transcription factors

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone β subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF's binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.

SIN3B promotes integrin αV subunit gene transcription and cell migration of hepatocellular carcinoma.

Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed β-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.

A Transgenic Mouse Model to Selectively Identify α3 Na,K-ATPase Expressing Cells in the Nervous System

The α3 Na+,K+-ATPase (α3NKA) is one of four known α isoforms of the mammalian transporter. A deficiency in α3NKA is linked to severe movement control disorders. Understanding the pathogenesis of these disorders is limited by an incomplete knowledge of α3NKA expression in the brain as well as the challenges associated with identifying living cells that express the isoform for subsequent electrophysiological studies. To address this problem, transgenic mice were generated on the C57BL/6 genetic background, which utilize the mouse α3 subunit gene (Atp1a3) promoter to drive the expression of ZsGreen1 fluorescent protein. Consistent with published results on α3NKA distribution, a ZsGreen1 signal was detected in the brain, but not in the liver, with Atp1a3-ZsGreen1 transgenic mice. The intensity of ZsGreen1 fluorescence in neuronal cell bodies varied considerably in the brain, being highest in the brainstem, deep cerebellar and select thalamic nuclei, and relatively weak in cortical regions. Fluorescence was not detected in astrocytes or white matter areas. ZsGreen1-positive neurons were readily observed in fresh (unfixed) brain sections, which were amenable to patch-clamp recordings. Thus, the α3NKA-ZsGreen1 mouse model provides a powerful tool for studying the distribution and functional properties of α3NKA-expressing neurons in the brain.

Expression of Pinellia pedatisecta Lectin Gene in Transgenic Wheat Enhances Resistance to Wheat Aphids.

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat.

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Synvip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Synvip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (synbar) gene. Marker gene excision was achieved in hybrids with up to 41.18% recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII+cre-synbar), were established in subsequent generation through genetic segregation.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvβ6 integrin expression in vitro. We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvβ6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Association between GRIN2A promoter polymorphism and recovery from concussion

Objective: To determine genetic variability within the N-methyl-D-aspartate receptor 2A sub-unit (GRIN2A) gene promoter and its association with concussion recovery time. The hypothesis tested was that there would be a difference in allele and/or genotype distribution between two groups of athletes with normal and prolonged recovery.Methods: DNA was extracted from saliva collected from a total of 87 athletes with a physician-diagnosed concussion. The (GT) variable number tandem repeats (VNTR) within the promoter region of GRIN2A was genotyped. The long (L) allele was an allele with ≥25 repeats and the short (S) allele was an allele with <25 repeats in the GT tract. Participants’ recovery time was followed prospectively and was categorized as normal (≤60 days) or prolonged (>60 days).Results: LL carriers were 6-times more likely to recover longer than 60 days following the concussive event (p = 0.0433) when compared to SS carriers. Additionally, L allele carriers were found more frequently in the prolonged recovery group (p = 0.048).Conclusion: Determining genetic influence on concussion recovery will aid in future development of genetic counselling. The clinical relevance of genotyping athletes could improve management of athletes who experience concussion injuries.

Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.

Isolation and characterization of rubisco small subunit gene promoter from common wheat (Triticum aestivum L.)

Choice of an appropriate promoter is critical to express target genes in intended tissues and developmental stages. However, promoters capable of directing gene expression in specific tissues and stages are not well characterized in monocot species. To identify such a promoter in wheat, this study isolated a partial sequence of the wheat small subunit of RuBisCO (TarbcS) promoter. In silico analysis revealed the presence of elements that are characteristic to rbcS promoters of other, mainly dicot, species. Transient expression of the TarbcS:GUS in immature wheat embryos and tobacco leaves but not in the wheat roots indicate the functionality of the TarbcS promoter fragment in directing the expression of target genes in green plant tissues.

The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon

The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for β-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.

Structure, variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> −700bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.

Functional analysis of a Lemna gibba rbcS promoter regulated by abscisic acid and sugar

Photosynthesis-associated nuclear genes (PhANGs) are able to respond to multiple environmental and developmental signals, including light, sugar and abscisic acid (ABA). PhANGs have been extensively studied at the level of transcriptional regulation, and several cis-acting elements important for light responsiveness have been identified in their promoter sequences. However, the regulatory elements involved in sugar and ABA regulation of PhANGs have not been completely characterized. A ribulose-1,5-bisphosphate carboxylase small subunit gene (rbcS) promoter (SSU5C promoter) was isolated from duckweed (Lemna gibba). A series of SSU5C promoter 5' deletion fragments were fused to an intron-gus gene, and transgenic tobacco suspension cell lines were generated. Assay of tobacco suspension cell line harbouring the complete promoter in the fusion construct indicated that SSU5C promoter was negatively regulated by sugar and ABA under the condition of regular photoperiod. 5' deletion analysis of SSU5C promoter in transgenic tobacco suspension cell lines confirmed that a region between positions -310 and -152 included the ABA-response region, and that sugar-response cis-acting elements might be located in the region between -152 and -117. Taken together, our results confirmed that the cis-regulatory region responsible for repression by ABA and sugar in the SSU5C promoter was located between -310 and -117.

Isolation and characterization of the 5´-upstream region of the human voltage-gated Ca2+ channel α2δ-1 auxiliary subunit gene: promoter analysis and regulation by transcription factor Sp1

The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.

Gene therapy using the human telomerase catalytic subunit gene promoter enables targeting of the therapeutic effects of vesicular stomatitis virus matrix protein against human lung adenocarcinoma

The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), is highly active in immortalized cells and more than 90% of human cancer cells, but is quiescent in the majority of normal somatic cells. Thus, the hTERT promoter has been extensively used in targeted cancer gene therapy. Vesicular stomatitis virus (VSV) matrix protein (MP) induces the apoptosis of tumor cells in the absence of other viral components. In our previous studies, we successfully constructed the pVAX-M plasmid from the pVAX plasmid, which expressed wild-type VSV MP (VSV MP is under the control of the CMV promoter) and demonstrated that pVAX-M efficiently suppresses the growth of malignant tumors via the induction of apoptosis in vitro and in vivo. The present study was designed to construct the plasmid phTERTM (VSV MP is under the control of the hTERT promoter) and investigate whether it had a targeted antitumor effect in nude mice bearing human lung adenocarcinoma. In vitro, A549 human lung adenocarcinoma cells were treated with NS, Lip-null, etoposide, Lip-pVAX-M or Lip-phTERT-M, and examined for cell viability through MTT assays or for apoptosis by flow cytometry and TUNEL assays. In vivo, A549 human lung carcinoma models in nude mice were established. Mice were treated with 10 4-weekly intravenous administrations of NS, Lip-null, etoposide (2 mg/kg), Lip-pVAX-M or Lip-phTERT-M. Subsequently, Lip-phTERT-M was found to be the most efficient inhibitor of tumor growth and inducer of tumor cell apoptosis when compared with the other groups in vivo and in vitro (P<0.05). Notably, immunohistochemical staining showed that Lip-phTERT-M significantly limited the overexpression of VSV MP to the tumor tissues and reduced VSV MP expression in other organs in comparison with Lip-pVAX-M (P<0.05). Therefore, it can be concluded that phTERT-M demonstrates a targeted antitumor effect on A549 human lung adenocarcinoma cells. These observations suggest that phTERT-M gene therapy may be a novel and potent strategy for targeting human lung adenocarcinoma.

The LIM Domain Protein FHL2 Interacts with the NR5A Family of Nuclear Receptors and CREB to Activate the Inhibin-α Subunit Gene in Ovarian Granulosa Cells

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.