Overexpression Of PBX1 Research Articles

PBX1-promoted SFRP4 transcription inhibits cell proliferation and epithelial-mesenchymal transition in endometrial carcinoma

ObjectiveTo explore the effects and mechanisms of action of the PBX1/secreted frizzled-related protein 4 (SFRP4) axis in endometrial carcinoma (EC). MethodsThe expression of PBX1 and SFRP4 was analyzed using bioinformatics prediction, followed by validation in EC cells using quantitative reverse transcription-polymerase chain reaction and western blotting. After transduction with overexpression vectors for PBX1 and SFRP4, migration, proliferation, and invasion of EC cells were measured, accompanied by the detection of E-cadherin, Snail, N-cadherin, Vimentin, β-catenin, GSK-3β, and C-myc expression. The association between PBX1 and SFRP4 was validated using dual luciferase reporter gene and chromatin immunoprecipitation assays. ResultsPBX1 and SFRP4 were downregulated in EC cells. Overexpression of PBX1 or SFRP4 resulted in weakened cell proliferation, migration, and invasion, as well as decreased expression of Snail, N-cadherin, Vimentin, β-catenin, GSK-3β, and C-myc and increased expression of E-cadherin. PBX1 bound to the SFRP4 promoter and promoted its transcription. Knockdown of SFRP4 reversed the repression of overexpressed PBX1 in the malignant phenotypes and EMT of EC cells, and PBX1 repressed Wnt/β-catenin pathway activation by upregulating SFRP4 transcription. ConclusionPBX1 inhibited activation of the Wnt/β-catenin pathway by promoting SFRP4 transcription, thereby suppressing malignant phenotypes in EC cells and the EMT process.

Spindle function and Wnt pathway inhibition by PBX1 to suppress tumor progression via downregulating DCDC2 in colorectal cancer

PBX1 is a transcription factor that regulates a variety of genes, involved in intracellular lipid metabolism, cell proliferation, and other pathways. The promoting and inhibiting function of PBX1 in various cancer types was extensively discussed, however, there have been no studies on PBX1 proteins in colorectal cancer (CRC). This study aimed to reveal the anti-tumor function of PBX1 in CRC and the underlying molecular mechanism. Bioinformatics analysis revealed that PBX1 is downregulated in CRC, indicating that is a potential antioncogene in CRC. Overexpression of PBX1 suppresses tumor growth and metastasis in vitro and in vivo. Mechanistically, we found that PBX1 acted as a transcription factor that suppressed DCDC2 expression and inhibited spindle function. Moreover, the PBX1-DCDC2 axis controlled the Wnt pathway in CRC cells. Overexpression of DCDC2 restored CRC proliferation, metastasis abilities and Wnt pathway. In conclusion, this study suggests that PBX1 acts as a transcription factor to suppress DCDC2 expression and inhibit cell proliferation and metastasis by disrupting spindle function and the Wnt pathway in CRC.

Functional Assessment of a New PBX1 Variant in a 46,XY Fetus with Severe Syndromic Difference of Sexual Development through CRISPR-Cas9 Gene Editing.

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

PBX1 Participates in Estrogen-mediated Bladder Cancer Progression and Chemo-resistance Affecting Estrogen Receptors.

Bladder cancer (BCa) is a common cancer associated with high morbidity and mortality worldwide. Pre-B-cell leukemia transcription factor 1 (PBX1) has been reported to be involved in tumor progression. The aim of the study was to explore the specific role of PBX1 in BCa and its underlying mechanisms. The relative expressions of PBX1 in muscle-invasive BCa tissues and cell lines were analyzed through RT-qPCR and western blotting. Kaplan-Meier analysis was used to analyze the relationship between PBX1 levels and survival status. Co-immunoprecipitation (CO-IP) and chromatin immunoprecipitation (ChIP)-qPCR assays were adopted to verify the interaction between PBX1 and Estrogen receptors (ERs) and explore the estrogen receptors (ERs)-dependent genes transcription. PBX1 was upregulated in invasive BCa patients and BCa cells, positively associated with tumor size, lymph node metastasis, distant metastasis and poorer survival status. The overexpression of PBX1 promoted cell growth, invasion, epithelial-mesenchymal transition (EMT) process and cisplatin resistance in BCa cells, while the silence of PBX1 showed opposite effects. Furthermore, PBX1 interacted with ERs and was required for ER function. PBX1 overexpression aggravated the tumorpromoting effect of estrogen on BCa cells, while it partially suppressed the inhibitory effects of ER antagonist AZD9496 on BCa cells. This study revealed that PBX1 participated in estrogen mediated BCa progression and chemo-resistance through binding and activating estrogen receptors. Hence, PBX1 may serve as a potential prognostic and therapeutic target for BCa treatment.

PBX1-SIRT1 Positive Feedback Loop Attenuates ROS-Mediated HF-MSC Senescence and Apoptosis

Stem cell senescence and depletion are major causes of aging and aging-related diseases. The NAD (Nicotinamide adenine dinucleotide) – SIRT1 (Silent Information Regulator 1) – PARP1 (Poly (ADP-ribose) polymerase-1) axis has gained interest owing to its significant role in regulating stem cell senescence and organismal aging. A recent study from our lab showed that pre-B-cell leukemia transcription factor1 (PBX1) overexpression attenuates hair follicle-derived mesenchymal stem cells (HF-MSCs) senescence and apoptosis by regulating ROS-mediated DNA damage via PARP1 downregulation; thus, suggesting that PARP1 downregulation is a common manifestation of the roles of both PBX1 and SIRT1 in HF-MSCs senescence attenuation, and implying a potential link between PBX1 and SIRT1. To this end, HF-MSCs overexpressing PBX1, overexpressing both PBX1 and PARP1, downregulating SIRT1, and overexpressing PBX1 as well as downregulating SIRT1 were generated, and senescence, apoptosis, DNA damage, and repair biomarkers were analyzed. Our results showed that (1) PBX1 overexpression alleviated HF-MSCs senescence and apoptosis accompanied by SIRT1 upregulation, PARP1 downregulation, and increased intracellular NAD and ATP levels. (2) SIRT1 knockdown enhanced cellular senescence and apoptosis, accompanied by increased ROS accumulation, DNA damage aggravation, and decreased intracellular NAD and ATP levels. (3) PBX1 overexpression rescued HF-MSCs senescence and apoptosis induced by SIRT1 knockdown. (4) PBX1 rescued PARP1 overexpression-mediated ATP and NAD depletion, accompanied by increased SIRT1 expression. Collectively, our results revealed that a positive interaction feedback loop exists between PBX1 and SIRT1. To the best of our knowledge we are the first to report that there is a PBX1-SIRT1-PARP1 axis that plays a critical role in alleviating HF-MSCs senescence and apoptosis. We provide a new perspective on the mechanisms underlying stem cell senescence as well as age-related disease prevention and treatment.Graphical

Rs6426881 in the 3'-UTR of PBX1 is involved in breast and gastric cancers via altering the binding potential of miR-522-3p.

Breast and gastric cancers are the most important diseases that lead to cancer death and socialhealthcare challenge. Overexpression of PBX1, a proto-oncogene, is correlated with the progression and metastasis of various cancers. For the first time, in this study the researchers evaluated the relationship between rs6426881, affecting miR-522-3p binding to the PBX1, with breast and gastric cancers. The Microarray analysis was performed for finding the relative expression level of PBX1 and hsa-miR-522-3p, based on high throughput experiments. The GSE54397, GSE112369, GSE10810, GSE241585.ER, GSE24185.PR, GSE68373, and GSE38167 datasets were analyzed. A case-control study was carried out in 123 Iranian suffering from breast cancer and 132 participants as control samples as well as 130 people suffering from gastric cancer and 54 people as control group members. SNP rs6426881 in the 3'-UTR of PBX1 was genotyped by the High-Resolution Melting (HRM) method. Association analysis revealed that rs6426881 is correlated with Estrogen and Progesterone receptors, grade, and stage of breast cancer. Furthermore, a significant relationship was observed between the genotypes and blood groups in gastric cancer, while the distribution of alleles was significantly related to smoking, status of the primary tumor, and metastasis (Chi-Square P < 0.05). Finally, Bioinformatics analyses suggested that rs6426881 contains binding sites for miR-522-3p in the 3'-UTR of PBX1 transcript. The finding suggested that TT genotype is associated with poor prognosis in breast and gastric cancer. The rs6426881 T allele at PBX1 3'-UT is significantly related to breast and gastric cancers by altering the regulatory affinity of miR-522-3p to PBX1 3'-UTR and may be suggested as a novel prognostic biomarker for the diseases.

PBX1: a key character of the hallmarks of cancer.

Pre-B-cell leukemia homeobox transcription factor 1 (PBX1) was first identified as part of a fusion protein resulting from the chromosomal translocation t(1;19) in pre-B cell acute lymphoblastic leukemias. Since then, PBX1 has been associated with important developmental programs, and its expression dysregulation has been related to multifactorial disorders, including cancer. As PBX1 overexpression in many cancers is correlated to poor prognosis, we sought to understand how this transcription factor contributes to carcinogenesis, and to organize PBX1's roles in the hallmarks of cancer. There is enough evidence to associate PBX1 with at least five hallmarks: sustaining proliferative signaling, activating invasion and metastasis, inducing angiogenesis, resisting cell death, and deregulating cellular energetics. The lack of studies investigating a possible role for PBX1 on the remaining hallmarks made it impossible to defend or refute its contribution on them. However, the functions of some of the PBX1's transcription targets indicate a potential engagement of PBX1 in the avoidance of immune destruction and in the tumor-promoting inflammation hallmarks. Interestingly, PBX1 might be a player in tumor suppression by activating the transcription of some DNA damage response genes. This is the first review organizing PBX1 roles into the hallmarks of cancer. Thus, we encourage future studies to uncover the PBX1's underlying mechanisms to promote carcinogenesis, for it is a promising diagnostic and prognostic biomarker, as well as a potential target in cancer treatment.

Upregulation of microRNA-650 by PBX1 is correlated with the development of Helicobacter pylori-associated gastric carcinoma.

Helicobacter pylori (HP) infection induces the development of gastric carcinoma (GC), one of the most frequent and fatal cancers worldwide, via a progressive cascade. The roles of microRNAs (miRNAs) involved in the cascade and the behind mechanisms, however, are still unclear. This study was designed to investigate the expression of miR-650, a well-recognized oncogenic miRNA in GC samples and to analyze the associations between this miRNA and HP infection, and the molecular mechanism. Following miRNA- and mRNA-based microarray analyses, miR-650, pre-B-cell leukemia transcription factor 1 (PBX1), and LATS2 were filtered as targets. After that, function assays were implemented to assess their function in GC cells. miR-650 was upregulated in HP+ tissues and cells, and inhibition of miR-650 attenuated cell proliferation, invasion, migration, yet enhanced apoptosis. PBX1 was overexpressed in HP+ tissues and cells and promoted miR-650 transcription. Overexpression of PBX1 abrogated the effect of the miR-650 inhibitor on GC cells. miR-650 targeted LATS2, and LATS2 was poorly expressed in HP+ tissues and cell lines. Simultaneous knockdown of miR-650 and LATS2 reduced GC cell apoptosis. These results display that upregulation of miR-650 induced by HP infection and PBX1 dampens LATS2 in GC cells, potentially offering novel intervention targets for GC.

PBX1 is a valuable prognostic biomarker for patients with breast cancer.

Pre-B-cell leukemia transcription factor (PBX) proteins have important roles in the development of numerous organs. To date, four members of the PBX family have been identified to be involved in human cancer but little is known about their expression patterns and precise functions in breast cancer (BC) progression. The aim of the present study was to determine whether they have the potential to be prognostic biomarkers in patients with BC. The expression patterns of PBXs were evaluated using Oncomine, Cancer Cell Line Encyclopedia and Gene expression-based Outcome for Breast cancer Online algorithm analyses. The prognostic value of PBX1 was determined by Kaplan-Meier plotter analysis. It was observed that, among all PBX family members, only PBX1 was significantly upregulated in BC vs. normal tissues. Meta-analysis in the Oncomine database revealed that PBX1 was significantly upregulated in invasive breast carcinoma stroma, ductal breast carcinoma, invasive lobular breast carcinoma, invasive mixed breast carcinoma and male breast carcinoma compared with normal tissues. In addition, PBX1 was significantly correlated with forkhead box protein A1. Subtype analysis indicated that PBX1 overexpression was associated with luminal-like and hormone receptor-sensitive subtypes. In the survival analysis, a high expression level of PBX1 was associated with poor prognosis of patients with estrogen receptor (ER)-positive, luminal A and luminal B subtypes of BC. The results of the present study indicate that PBX1 may serve as a specific biomarker and essential prognostic factor for ER-positive, luminal A and luminal B subtypes of BC.

NANOG Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence by Upregulating PBX1 and Activating AKT Signaling.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG—a well-known core transcription factor—plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-β-galactosidase- (SA-β-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-β-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.

Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families.

The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome‐Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0‐fold (P = 0.036). Pre‐B‐cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3‐fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild‐type homozygote genotype had overexpression of PBX1. There is a 3‐fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

PBX1 attributes as a determinant of connexin 32 downregulation in Helicobacter pylori-related gastric carcinogenesis.

AIMTo clarify the mechanisms of connexin 32 (Cx32) downregulation by potential transcriptional factors (TFs) in Helicobacter pylori (H. pylori)-associated gastric carcinogenesis.METHODSApproximately 25 specimens at each developmental stage of gastric carcinogenesis [non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma (GC)] with H. pylori infection [H. pylori (+)] and 25 normal gastric mucosa (NGM) without H. pylori infection [H. pylori (-)] were collected. After transcriptional factor array analysis, the Cx32 and PBX1 expression levels of H. pylori-infected tissues from the developmental stages of GC and NGM with no H. pylori infection were measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Regarding H. pylori-infected animal models, the Cx32 and PBX1 mRNA expression levels and correlation between the gastric mucosa from 10 Mongolian gerbils with long-term H. pylori colonization and 10 controls were analyzed. PBX1 and Cx32 mRNA and protein levels were further studied under the H. pylori-infected condition as well as PBX1 overexpression and knockdown conditions in vitro.RESULTSIncremental PBX1 was first detected by TF microarray in H. pylori-related gastric carcinogenesis. The identical trend of PBX1 and Cx32 expression was confirmed in the developmental stages of H. pylori-related clinical specimens. The negative correlation of PBX1 and Cx32 was confirmed in H. pylori-infected Mongolian gerbils. Furthermore, decreased PBX1 expression was detected in the normal gastric epithelial cell line GES-1 with H. pylori infection. Enforced overexpression or RNAi-mediated knockdown of PBX1 contributed to the diminished or restored Cx32 expression in GES-1 and the gastric carcinoma cell line BGC823, respectively. Finally, dual-luciferase reporter assay in HEK293T cells showed that Cx32 promoter activity decreased by 30% after PBX1 vector co-transfection, indicating PBX1 as a transcriptional downregulator of Cx32 by directly binding to its promoters.CONCLUSIONPBX1 is one of the determinants in the Cx32 promoter targeting site, preventing further damage of gap junction protein in H. pylori-associated gastric carcinogenesis.

Abstract A32: PBX1, a transcriptional regulator, promotes stemness and chemoresistance in ovarian cancer.

Abstract Background: Cancer recurrence and chemoresistance contribute greatly to the high mortality of ovarian cancers; however, molecular players involved in these processes remain under-determined. Several theories have been proposed to account for the development of resistance to chemotherapeutic agents. One of the emerging concepts is tumor heterogeneity, which argues for the presence of different cell subpopulations in bulk tumor with varying degrees of tumor initiating potential and drug sensitivity. The fractions of cells with enhanced tumor initiating potential are referred to as cancer stem-like cells (CSC). These cells have defining “stemness” phenotypes, including unlimited cell division, repopulation by a minimum cell number and resistance to cytotoxic agents and irradiation. New Findings: To identify embryonic stem cell factors that are potentially involved in tumor recurrence and chemoresistance, we have screened expression levels of four of these factors by immunohistochemistry in pairs of matched recurrent and primary tumor tissues from 41 patients. Our data demonstrated that PBX1 is significantly up-regulated in recurrent/chemoresistant ovarian tumors. Moreover, PBX1 overexpression in ascites tumors correlates with shorter overall survival in post-chemotherapy ovarian cancer patients. When PBX1 is ectopically expressed, it promotes cancer stem cell-like phenotypes, including increased side population and ALDH1 activity, enhanced tumorigenicity at low cell density, and increased resistance to platinum-based therapy. In platinum-resistant cell lines that overexpress PBX1, silencing PBX1 expression using RNA interference sensitizes cells to platinum treatment and reduces their stem cell-like phenotypes. Gene expression and chromatin immunoprecipitation analyses identified PBX1 direct target genes involved in multi-drug resistance, stem cell maintenance, immunomodulation, and DNA damage response. In the TCGA ovarian cancer recurrent series, expression of several PBX1 direct target genes, including ABCA1, Nanog, BMP3, and ATM, is tightly associated with PBX1 expression, further supporting the tissue-level transcriptional regulation of these genes by PBX1. Conclusion: The above findings establish PBX1 as an upstream regulator of key functional networks that mediate cancer stem-like and drug resistant phenotypes. Studies on the hematopoietic system have indicated that PBX1 maintains a viable pool of quiescent stem cells. Recently, in a renal cell carcinoma system, chemotherapy has been shown to induce damage signaling and to stimulate cell division and repopulation of quiescent CSCs. We speculate that chemotherapy in ovarian cancer may induce comparable injury responses and trigger PBX1 signaling, which in turn may activate downstream networks that support CSC survival and create a microenvironment niche that is essential for CSC repopulation at the tissue level. Collectively, the association between PBX1 and a cascade of stemness pathways points to a potential Achilles's heel critical to responding to chemotherapy and developing chemoresistance and argues for the development of antagonists of PBX1 signaling as anticancer agents. Citation Format: Jin-Gyoung Jung, Tae-Hoen Kim, Emily Gerry, Jen-Chun Kuan, Ayse Ayhan, Ben Davidson, Ie-Ming Shih, Tian-Li Wang. PBX1, a transcriptional regulator, promotes stemness and chemoresistance in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A32.

Inhibition of non-small-cell lung cancer cell proliferation by Pbx1.

Lung cancer is one of the most deadly human cancers and continues to be a major unsolved health problem worldwide. Here, we evaluate the function of Pbx1 in the proliferation of non-small-cell lung cancer (NSCLC). In contrast with its known proliferative function, we found that Pbx1 inhibits the proliferation of lung cancer cells. In particular, Pbx1-specific RNA interference resulted in increased proliferation in lung cancer cells. In addition, histone H3 phosphorylation was also increased following inhibition of Pbx1 expression. In contrast, Pbx1 overexpression repressed the proliferation of lung cancer cells and inhibited DNA synthesis. Collectively, our data indicate that Pbx1 inhibits proliferation in lung cancer cells, suggesting a complex role for Pbx1 in modulating the proliferation of cancer cells and making this protein a potential new target for lung cancer therapy.

Endocrine pancreatic tissue plasticity in obese humans is associated with cytoplasmic expression of PBX-1 in pancreatic ductal cells

In vivo lineage tracing experiments in mice have recently cast doubt on the potential islet neogenesis from ductal precursors in adult mammals. We examined, in human obesity, a model for pancreatic endocrine tissue plasticity, the gene and protein expression of PBX-1—a transcription factor expressed in regenerating rat ductules and potentially implicated in the pancreatic development, alone or in association with PDX-1. When comparing gene expression, by quantitative real-time RT-PCR, in pancreatic exocrine tissue from obese non-diabetic subjects with increased islet mass, we found that Pbx-1 and Pdx-1 were up-regulated (5.9 ± 1.2 and 2.4 ± 0.6 versus non-obese). Immunohistochemistry confirmed PBX-1 over-expression and its cytoplasmic sequestration in ductal cells of obese subjects, associated with pronounced islet neogenesis (cytokeratin 19/chromogranin A double labeling). cDNA microarray analysis also showed up-regulation of other genes implicated in islet regeneration, including betacellulin, laminin, TGFa, NeuroD1, Pax6, substantiating the role of the islet neogenesis pathway in human obesity.

Identification of homeodomain proteins, PBX1 and PREP1, involved in the transcription of murine leukemia virus.

Cyclin-dependent kinase inhibitors (CDKIs) have been shown to block human immunodeficiency virus and herpes simplex virus. It is hypothesized that CDKIs block viral replication by inhibiting transcription of specific cellular genes. Here we find that three CDKIs, flavopiridol, purvalanol A, and methoxy-roscovitine, block Moloney murine leukemia virus (MLV) transcription events. Using gene expression microarray technology to examine the inhibitory effects of CDKIs, we observed a cellular gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, down-regulated by CDKI treatment. The PBX consensus element (PCE), TGATTGAC, is conserved in the long terminal repeats of several murine retroviruses, including Moloney MLV. Mutations in the PCE completely inhibited viral transcription whereas overexpression of PBX1 and a PBX1-associated protein, PREP1, enhanced viral transcription. The interaction between the PCE and PBX1-PREP1 proteins was confirmed by gel shift experiments. Blocking PBX1 protein synthesis resulted in a significant decrease in viral transcription. Collectively, our results represent the first work demonstrating that the homeodomain proteins PBX1 and PREP1 are cellular factors involved in Moloney MLV transcription regulation.

CAMP-Dependent Transactivation Involving the Homeodomain Protein Pbx1

Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acute lymphoblastic leukemia. Previously we reported a cAMP-regulatory sequence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17α-hydroxylase cytochrome P450 (P450c17) to be the first endogenous Pbx1 binding site and that overexpression of Pbx1 in mouse adrenal Y1 tumor cells enhances cAMP-dependent transcription mediated by this element. Here we report further characterization of Pbx1 binding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated transcription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/G)GA(T/C)A which represents the 5′ portion of CRS1. An artificial Pbx-binding sequence (PRS), previously determined by random PCR analysis, is similar to the Pbx1-binding sequence in CRS1 and by both gel shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhancing CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and nonsteroidogenic (HepG2 and S194) cells when coexpressed with the catalytic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of participating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors.

A cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the homeodomain protein Pbx1.

Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.