Myeloid Zinc Finger Research Articles

LDOC1 Gene Expression in Men With Klinefelter Syndrome.

Klinefelter syndrome (KS) results from an extra chromosome X, which is due to the failure of normal chromosomal segregation during meiosis. Patients with KS have gynecomastia, small testes, and azoospermia. Apoptosis is a mechanism responsible for the normal regulation of spermatogenesis. LDOC1 gene is a known regulator of nuclear factor mediated pathway to apoptosis through inhibition of nuclear factor kappa B (NF-kappaB). Furthermore, the transcription factor myeloid zinc finger gene 1 (MZF-1) has been shown to interact with LDOC1 and to enhance LDOC1 activity favoring apoptosis. We investigated the expression of LDOC1 gene mRNA, by quantitative reverse transcription polymerase chain reaction (qRT-PCR), in peripheral blood leukocytes of 13 patients with KS compared to 13 healthy men chosen as controls. LDOC1 expression was higher in 9 of the 13 KS patient compared to normal controls. These finding led us to hypothesize that LDOC1 gene upregulation may play a role in the spermatogenesis derangement observed in patients with KS.

Abstract WP268: Leukemia Inhibitory Factor Upregulates Superoxide Dismutase 3 in Neurons via Activation of Myeloid Zinc Finger-1

Objective: To determine whether leukemia inhibitory factor (LIF) upregulates superoxide dismutase 3 (SOD3), a neuroprotective antioxidant enzyme, through the transcription factor myeloid zinc finger-1 (MZF-1). Hypothesis: MZF-1 facilitates LIF-mediated neuroprotection by increasing transcription of the SOD3 gene in neurons. Methods: After middle cerebral artery occlusion or sham surgery, male Sprague-Dawley rats were injected with PBS or LIF (125 μg/kg) (n=4 per group). Rats were euthanized 72 h after injury and western blotting was used to measure MZF-1 protein expression in brain tissue. For in vitro studies, rat neurons were transfected with scrambled or MZF-1 siRNA (n=3 per group). Neurons were treated with PBS or 200 ng/mL LIF prior to 24 h in vitro ischemia induced by oxygen glucose deprivation. Lactate dehydrogenase (LDH) release was measured to assess neuronal death. MZF-1 levels were quantified in cultured neurons with immunocytochemistry. SOD3 and MZF-1 mRNA levels were measured with real-time PCR. Results: LIF (0.938 OD units ± 0.170), but not PBS (0.562 OD units ± 0.223), significantly increased MZF-1 protein expression in ipsilateral tissue 72 h after stroke compared to sham surgery (0.411 OD units ± 0.039, p<0.05). LIF treatment prior to 24 h in vitro ischemia significantly increased the percentage of MZF-1-positive neurons (52.17 % ± 0.93) compared to PBS 44.84 % ± 1.11, p<0.01). PCR results confirmed the increase in MZF-1 mRNA (1.89 fold change ± 0.33) in LIF-treated neurons compared to PBS-treated neurons (1.00 fold change ± 0.23, p<0.05). Moreover, LIF increased SOD3 mRNA after 24 h ischemia (1.35 fold change ± 0.03) compared to PBS (1.00 fold change ± 0.13, p<0.05). LIF decreased LDH release (363,967 ± 68,557 neuronal units) compared to PBS (559,856 ± 60,555 neuronal units, p<0.05) among neurons transfected with scrambled siRNA. However, MZF-1 siRNA attenuated the neuroprotective effect of LIF (690,633 neuronal units ± 19,167; p<0.05). Conclusions: MZF-1 plays a fundamental role in a novel neuroprotective pathway by enhancing SOD3 expression and provides insight regarding protection by antioxidant enzymes during stroke.

Genetic effect of an A/G polymorphism in the HSP70 gene on thermotolerance in chicken.

Studying thermotolerance is important for the prevention of thermostress in chickens. This study aimed to analyze the effect of mutations in the heat shock protein 70 (HSP70) gene on chicken thermotolerance. The C.-69A>G SNP in the 5'-flanking region of the HSP70 gene was genotyped in Lingshan and White Recessive Rock (WRR) chickens. Association of this SNP with thermotolerance traits revealed it to be significantly associated with CD4+/CD8+, and potentially associated with heterophil-to-lymphocyte ratio in WRR chickens exposed to thermoneutral temperature (15°C). Online prediction detected a putative myeloid zinc finger protein 1 binding factor in the C.-69A>G mutation. Under acute thermostress, mRNA levels of HSP70 in individuals with different C.-69A>G genotypes varied in the heart, leg muscle, and liver tissues. The HSP70 protein was expressed at higher levels in individuals with the GG genotype than in those with the AA genotype. In heart and liver, protein expression of HSP70 in individuals with the GG genotype was significantly higher than in those with the AA genotype. In leg muscle, protein expression was higher in birds with the GG genotype than in those with the AA and AG genotypes. Luciferase activity of the GG genotype was significantly higher than that of the AA genotype, suggesting that the C.-69A>G SNP regulates HSP70 gene expression. These results indicate that the C.-69A>G SNP in the 5'-flanking region of the HSP70 gene might affect chicken thermotolerance and that the GG genotype might be advantageous for the prevention of thermostress.

Bioinformatic Evaluation of Transcriptional Regulation of WNT Pathway Genes with reference to Diabetic Nephropathy.

Objective. WNT/β-catenin pathway members have been implicated in interstitial fibrosis and glomerular sclerosis disease processes characteristic of diabetic nephropathy (DN), processes partly controlled by transcription factors (TFs) that bind to gene promoter regions attenuating regulation. We sought to identify predicted cis-acting transcription factor binding sites (TFBSs) overrepresented within WNT pathway members. Methods. We assessed 62 TFBS motif frequencies from the JASPAR databases in 65 WNT pathway genes. P values were estimated on the hypergeometric distribution for each TF. Gene expression profiles of enriched motifs were examined in DN-related datasets to assess clinical significance. Results. Transcription factor AP-2 alpha (TFAP2A), myeloid zinc finger 1 (MZF1), and specificity protein 1 (SP1) were significantly enriched within WNT pathway genes (P values < 6.83 × 10−29, 1.34 × 10−11, and 3.01 × 10−6, resp.). MZF1 expression was significantly increased in DN in a whole kidney dataset (fold change = 1.16; 16% increase; P = 0.03). TFAP2A expression was decreased in an independent dataset (fold change = −1.02; P = 0.03). No differential expression of SP1 was detected. Conclusions. Three TFBS profiles are significantly enriched within WNT pathway genes highlighting the potential of in silico analyses for identification of pathway regulators. Modification of TF binding may possibly limit DN progression, offering potential therapeutic benefit.

Abstract A36: Decreased levels of the transcription factors Ik-1 and MZF1 contribute to upregulation of IGF-IR expression in NPM-ALK+ T-cell anaplastic large-cell lymphoma.

Abstract Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase is a homodimeric protein that is composed of 2 extracellular α; and 2 transmembranous β; subunits connected by disulfide bonds. IGF-IR belongs to the insulin receptor family whose members exhibit a common structural characteristic in the form of an amino acid motif (YXXXYY) within the activation loop of their respective kinase domains. Ligand stimulation of IGF-IR causes its dimerization, phosphorylation, and the subsequent activation of downstream signaling systems. It has been shown that IGF-IR is aberrantly overexpressed in and contributes to the survival of a variety of human solid tumors and hematological neoplasms and, therefore, it may represent an important therapeutic target. Pathological activation of IGF-IR induces cellular transformation and protection from apoptosis-- prerequisites for the establishment and growth of malignant tumors. Of important note is that the mechanisms underlying increased expression of IGF-IR in cancer cells are not completely understood. For instance, only a few transcription factors are known to bind the IGF-IR gene promoter (15q26.3) and modulate its activity through stimulation or inhibition. These transcription factors include Sp1, WT1, E2F1, STAT1, and EGR-1. Recently, we identified IGF-IR as a major survival molecule that interacts reciprocally with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in a subtype of T-cell anaplastic large-cell lymphoma. NPM-ALK+ T-cell lymphoma is an aggressive type of cancer that is frequently encountered in children and young adults. Compared with its expression in normal human T lymphocytes and reactive lymphoid tissues, the expression of IGF-IR mRNA and protein is remarkably upregulated in NPM-ALK+ T-cell lymphoma cell lines and primary human tumors. Similar to other types of cancer, the mechanisms leading to increased expression of IGF-IR in NPM-ALK+ T-cell lymphoma have not been identified. In this study, we hypothesized that increased IGF-IR expression can be explained by transcriptional aberrancies that exist inherently in NPM-ALK+ T-cell lymphoma. To identify transcription factors that may potentially bind to the human IGF-IR gene promoter, 3 web-based transcription factor search algorithms were used: TFSearch (www.cbrc.jp/research/db/TFSEARCH.html), Genomatix (www.genomatix.de), and MATCH (www.gene-regulation.com/pub/programs.html). These algorithms identified the transcription factors Ikaros (Ik) and myeloid zinc finger 1 (MZF1) to potentially bind to IGF-IR gene promoter. Western blotting (WB) studies demonstrated lower levels of Ik-1 and MZF1 in 5 NPM-ALK+ T-cell lymphoma cell lines than in T lymphocytes. WB also showed that Ik-1 and MZF1 are decreased in 85% and 100%, respectively, of 15 ALK+ T-cell lymphoma patient specimens. A luciferase assay supported that Ik-1 and MZF1 regulate the IGF-IR gene promoter. Furthermore, ChIP assay showed that these transcription factors bind specific sites located within the IGF-IR gene promoter. Forced expression of Ik-1 and MZF1 in NPM-ALK+ T-cell lymphoma cells significantly decreased IGF-IR mRNA and protein expression levels. This decrease in IGF-IR was associated with downregulation of pIGF-IR and the phosphorylation of its interacting molecules, including IRS-1, AKT, and NPM-ALK. In addition, overexpression of Ik-1 and MZF1 decreased the viability, proliferation, migration, and anchorage-independent colony formation of NPM-ALK+ T-cell lymphoma cells. Collectively, our findings provide novel evidence that aberrant decreases in the transcription factors Ik-1 and MZF1 play important roles in the pathogenesis of NPM-ALK+ T-cell lymphoma through upregulation of IGF-IR expression. These findings could be exploited to devise more effective strategies to treat this lymphoma. Citation Format: Deeksha Vishwamitra, Choladda V. Curry, Serhan Alkan, Ping Shi, Hesham M. Amin. Decreased levels of the transcription factors Ik-1 and MZF1 contribute to upregulation of IGF-IR expression in NPM-ALK+ T-cell anaplastic large-cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A36.

Role and Regulation of Myeloid Zinc Finger Protein 1 in Cancer.

Myeloid zinc finger 1 (MZF1) belongs to the SCAN-Zinc Finger (SCAN-ZF) transcription factor family that has recently been implicated in a number of types of cancer. Although the initial studies concentrated on the role of MZF1 in myeloid differentiation and leukemia, the factor now appears to be involved in the etiology of major solid tumors such as lung, cervical, breast, and colorectal cancer. Here we discuss the regulation of MZF1 that mediated its recruitment and activation in cancer, concentrating on posttranslational modification by phosphorylation, and sumoylation, formation of promyelocytic leukemia nuclear bodies and its association with co-activators and co-repressors.

Abstract 1975: Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion

Abstract ErbB2 overexpression gives rise to a highly invasive, aggressive phenotype associated with an increased activity of lysosomal cysteine cathepsins B and L and with a pericellular accumulation of lysosomes at the close vicinity of the cell membrane, enabling lysosomal exocytosis and secretion of lysosomal contents to the extracellular space. We recently showed that ErbB2 expression level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. We found that the increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and that it is mediated by a novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, Myeloid Zinc Finger 1 (MZF1) and ETS1, which regulate the ErbB2-induced expression of cathepsins B and L. We currently work on understanding the mechanisms that increase the transcriptional activity of MZF1 in breast cancer. Thus far we have identified two basic mechanisms that can increase MZF1 target gene expression: cancer-induced, increased levels of MZF1 and ErbB2 signaling-induced, increased transcriptional activity of MZF1. Our results thus far show that the level of MZF1 can be directly downregulated by specific, non-coding RNAs. The cancer-induced increase in the MZF1 level is most likely due to changes in the expression of these non-coding RNAs. Preliminary data employing two-dimensional SDS-PAGE as well as the factual involvement of several kinases downstream of ErbB2 for its transcriptional activity, suggests that MZF1, a prominent phosphoprotein, is activated by phosphorylation in response to ErbB2 signaling. Towards this end we have set up various experiments to verify the phosphorylation of MZF1 in response to ErbB2 signaling. We now aim at the identification of the ErbB2-regulated phosphorylation sites. With this work we expect to collect more information about the molecular mechanisms underlying ErbB2-induced invasion. Increased knowledge of the mechanisms is expected to enable the development of novel therapies and biomarkers for aggressive, ErbB2-positive breast cancer. Citation Format: Tuula Kallunki, Ditte M. Brix, Knut Kristoffer B. Clemmensen, Siri A. Tvingsholm, Bo Rafn, José Moreira, Irina Gromova, Marja Jäättelä. Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1975. doi:10.1158/1538-7445.AM2015-1975

Abstract 4768: Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer

Abstract Objective: PAX2 is recognized as a significant factor in the development of urogenital system. It is expressed in multiple tumors and is essential in tumor cell survival. This study aimed to investigate the effect, expression, methylation of PAX2 in endometrial cancer, and the possible regulative mechanism of DNA methylation related PAX2 expression. Methods: PAX2 expression in endometrial cancer, hyperplasia and normal tissues was detected by immunohistochemistry, and its expression in cell lines was measured by real-time PCR, western blot and immunocytochemistry. The role of PAX2 on cell viability and mobility was measured by CCK-8, invasion and migration assays. Bisulfite sequencing and MassARRAY were used to quantify the DNA methylation level of PAX2 promoter in endometrial cells and tissues. Deletion analysis of PAX2 promoter was performed by luciferase reporter gene to analyze its promoter activity. Transcription factors (TFs) combined with PAX2 promoter were forecasted by TF search database and the regulatory function of these TFs were measured by detecting the expression of PAX2 mRNA and protein following the knock down of these TFs. Results: PAX2 was over-expressed in endometrial cancer tissues compared to hyperplasia and normal endometrium. Compare to normal endometrial epithelial cells, endometrial cancer cell lines exhibited moderate to high PAX2 expression. Knocking down PAX2 by siRNA reduced cell viability, invasion and migration, while PAX2 over-expression exhibited opposite role. The methylation level of PAX2 promoter was higher than normal counterparts in both cancer tissues and cells, which was positively related to PAX2 expression. After 5-Aza-CdR treatment, the PAX2 mRNA and protein was down-regulated along with the methylation level of PAX2 promoter. Deletion analysis confirmed that a repressive transcriptional regulatory region of PAX2 promoter coincided with the hypermethylated region found in MassARRAY analysis. The binding sites of myeloid zinc finger 1 (MZF1) and the early growth responsive gene 1(EGR-1) were found in the repressive transcriptional regulatory region of PAX2 promoter. Knocking down MZF1 or EGR-1 upregulated both the mRNA and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 and EGR-1 may act as repressive transcription factors in the DNA methylation related PAX2 transcription. Conclusion: PAX2 is involved in carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. Overexpression of PAX2 in endometrial cancer may be regulated by promoter hypermethylation via repressive transcription factors such as MZF1 or EGR-1. Citation Format: Nan Jia, Jieyu Wang, Qing Li, Xiang Tao, Keqin Hua, Yinhua Yu, Jan P. Baak, Kwong Kwok Wong, Weiwei Feng. Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2015-4768

Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single-nucleotide polymorphism (SNP) and crucial single-nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele-specific mutations functionally reassembled wild-type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa-B), MZF1 (myeloid zinc finger 1), and NRF-2 (nuclear factor erythroid 2-related factor 2). In vitro, a mutant allele that gained novel NFKB-binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome-wide sequencing (GWS) of numerous non-coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild-type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells.

Myeloid Zinc Finger 1 (MZF-1) Regulates Expression of the CCN2/CTGF and CCN3/NOV Genes in the Hematopoietic Compartment.

Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25-dihydroxyvitamin D3 (calcitriol) and all-trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger-1 (MZF-1), a hematopoietic transcription factor. We show the interaction of MZF-1 with the CTGF and NOV promoters in several cell types. Up-regulation of MZF-1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF-1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma.

Myeloid zinc finger 1 (MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin (FS) is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from −1 800 to +16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs (−302/+16 bp)-FS and (−180/+16 bp)-FS (P<0.01). Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 (MZF1) binding sequence had significantly decreased luciferase activity (P<0.05). Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence “TCCCCACC” (the recognition site of transcription factor MZF1) was the most important for FS transcription activation in the porcine.

MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma.

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (∆DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1∆DBD or Elk-1∆DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1∆DBD or Elk-1∆DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter.

Expression of myeloid zinc finger 1 and the correlation to clinical aspects of oral squamous cell carcinoma.

The myeloid zinc finger 1 (MZF1) is a zinc finger transcription factor which regulates myeloid differentiation and oncogenesis. However, little information is available concerning the MZF1 expression in oral squamous cell carcinoma (OSCC) and its correlation with patients' prognosis. We detected the expression of MZF1 in 274 patients with OSCC using tissue microarrays (TMAs) and evaluated the associations between nuclear MZF1 expression and the clinical parameters of OSCC patients. We found that nuclear MZF1 expression was present in 190/274 (69.3 %) cases, and loss of nuclear expression of MZF1 was associated with more advanced clinical stages (p = 0.011) and larger tumor size (p = 0.002), but not associated with positive lymph node metastasis and distal metastasis. Importantly, tongue squamous cell carcinomas (SCC) patients with negative nuclear MZF1 expression had significantly worse overall survival rates (log-rank test, p = 0.028). In conclusion, our results revealed that the loss of nuclear expression of MZF1 in OSCC samples can predict the progression of OSCC and the survival of OSCC patients in Taiwan.

Alcohol Inhibits Osteopontin-dependent Transforming Growth Factor-β1 Expression in Human Mesenchymal Stem Cells

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor β1 (TGF-β1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-β1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-β1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-β1 protein expression, decreases MZF1-dependent TGF-β1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-β1 expression. Last, we showed that EtOH exposure reduces the TGF-β1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-β1 signaling pathway in MSC.

Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma.

Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense (AS) RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naive rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 AS RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder.

The transcription factors Ik-1 and MZF1 downregulate IGF-IR expression in NPM-ALK⁺ T-cell lymphoma.

BackgroundThe type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase promotes the survival of an aggressive subtype of T-cell lymphoma by interacting with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein. NPM-ALK+ T-cell lymphoma exhibits much higher levels of IGF-IR than normal human T lymphocytes. The mechanisms underlying increased expression of IGF-IR in this lymphoma are not known. We hypothesized that upregulation of IGF-IR could be attributed to previously unrecognized defects that inherently exist in the transcriptional machinery in NPM-ALK+ T-cell lymphoma.Methods and resultsScreening studies showed substantially lower levels of the transcription factors Ikaros isoform 1 (Ik-1) and myeloid zinc finger 1 (MZF1) in NPM-ALK+ T-cell lymphoma cell lines and primary tumor tissues from patients than in human T lymphocytes. A luciferase assay supported that Ik-1 and MZF1 suppress IGF-IR gene promoter. Furthermore, ChIP assay showed that these transcription factors bind specific sites located within the IGF-IR gene promoter. Forced expression of Ik-1 or MZF1 in the lymphoma cells decreased IGF-IR mRNA and protein. This decrease was associated with downregulation of pIGF-IR, and the phosphorylation of its interacting proteins IRS-1, AKT, and NPM-ALK. In addition, overexpression of Ik-1 and MZF1 decreased the viability, proliferation, migration, and anchorage-independent colony formation of the lymphoma cells.ConclusionsOur results provide novel evidence that the aberrant decreases in Ik-1 and MZF1 contribute significantly to the pathogenesis of NPM-ALK+ T-cell lymphoma through the upregulation of IGF-IR expression. These findings could be exploited to devise new strategies to eradicate this lymphoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0324-2) contains supplementary material, which is available to authorized users.

Abstract T P248: Leukemia Inhibitory Factor Induced-Akt Activation Protects Neurons from Ischemic Damage by Increasing Superoxide Dismutase Activity and Expression

Objective: To determine the molecular mechanisms by which leukemia inhibitory factor (LIF) protects neurons during permanent middle cerebral artery occlusion (MCAO). Hypothesis: LIF protects neurons from ischemic oxidative injury through Akt-mediated increases in superoxide dismutase 3 (SOD3) expression and total SOD activity. Methods: Male Sprague-Dawley rats injected with vehicle (PBS) or LIF (125 μg/kg) were euthanized 24, 48, and 72 h post-MCAO or sham surgery for assessment of SOD activity and SOD3 expression. Rat cortical neurons were subjected to 24 h oxygen glucose deprivation or normoxia following treatment with vehicle , 50, 200, or 1000 ng/mL LIF (n=3 per group). In a third experiment, neurons were treated with vehicle, 50 ng/mL LIF,10 μM Akt inhibitor, or 50 ng/mL LIF+10 μM Akt inhibitor (n=3 per group) prior to oxygen glucose deprivation. Lactate dehydrogenase levels in media were measured to assess neuronal death. We used immunocytochemistry to assess expression of SOD3, phospho-Akt (Ser473), and myeloid zinc finger-1 in neurons. Results: LIF significantly increased brain SOD activity (2.085 ± 0.476 U/mg lysate; n=5) compared to vehicle (0.919 ± 0.285 U/mg lysate; n=7) 72 h post-MCAO (p&lt;0.05). LIF significantly increased SOD3 protein expression in the brain (3.707 ± 0.541 units; n=3) compared to vehicle (1.401 ± 0.825 units; n=3) at 72 h post-MCAO (p&lt;0.05). Co-localization of phospho-Akt and SOD3 occurred 24 h post-MCAO. Additionally, 50 ng/mL LIF (0.789 ± 0.018 U/mL media; n=3) significantly decreased lactate dehydrogenase levels in vitro compared to vehicle (1.000 ± 0.023 U/mL media; n=3) (p&lt;0.0001). Co-incubation of 50/mL ng LIF with 10 μM Akt inhibitor (1.070 ±0.106 U/mL media; n=3) reversed the protective effect of LIF (0.705 ± 0.050 U/mL media; n=3) (p&lt;0.01). Increases in SOD3, phospho-Akt, and myeloid zinc finger-1 staining after oxygen glucose deprivation and LIF were abolished upon Akt inhibition. Conclusion: Our data demonstrates that LIF-mediated protection against ischemia in vivo and in vitro results from Akt-dependent increases in SOD3 expression and SOD activity.

Osteopontin mediates an MZF1-TGF-β1-dependent transformation of mesenchymal stem cells into cancer-associated fibroblasts in breast cancer.

Interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TMEN) significantly influence cancer growth and metastasis. Transforming growth factor-β (TGF-β) is known to be a critical mediator of the CAF phenotype, and osteopontin (OPN) expression in tumors is associated with more aggressive phenotypes and poor patient outcomes. The potential link between these two pathways has not been previously addressed. Utilizing in vitro studies using human mesenchymal stem cells (MSCs) and MDA-MB231 (OPN+) and MCF7 (OPN−) human breast cancer cell lines, we demonstrate that OPN induces integrin-dependent MSC expression of TGF-β1 to mediate adoption of the CAF phenotype. This OPN-TGF-β1 pathway requires the transcription factor, myeloid zinc finger 1 (MZF1). In vivo studies with xenotransplant models in NOD-scid mice showed that OPN expression increases cancer growth and metastasis by mediating MSC-to-CAF transformation in a process that is MZF1- and TGF-β1-dependent. We conclude that tumor-derived OPN engenders MSC-to-CAF transformation in the microenvironment to promote tumor growth and metastasis via the OPN-MZF1-TGF-β1 pathway.

Myeloid zinc finger 1 (Mzf1) differentially modulates murine cardiogenesis by interacting with an Nkx2.5 cardiac enhancer.

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.

Disordered signaling governing ferroportin transcription favors breast cancer growth

Iron is a necessary chemical element needed by all organisms. Iron metabolism is finely tuned in mammals, and the hepcidin–ferroportin (FPN) axis is the central signaling in governing systemic iron homeostasis. Deregulation of this signaling would lead to iron disorders and likely other diseases including cancers. Reduced FPN was previously found to correlate with poor prognosis in breast cancer patients. Nonetheless, the biological effects of abnormal FPN expression in tumor cells remain largely unexplored, and the mechanisms underlying misregulated expression of FPN in cancers keep elusive. In the current study, we scrutinized the effects of abnormal FPN on tumor growth and the molecular mechanisms of diminished tumor FPN. Downregulation of FPN significantly promoted breast cancer growth, whereas FPN upregulation impeded tumor growth. We demonstrated that the transcription factors Nrf2 (nuclear factor erythroid 2-like 2) and MZF-1 (myeloid zinc finger-1) synergistically transactivated FPN expression in breast cancer cells. Moreover, CpG island methylation at the FPN promoter was the reason of attenuated FPN expression. Downregulation of Nrf2 and MZF-1 and hypermethylation of the FPN promoter were concurrently associated with decreased FPN concentration in breast tumors. Taken together, our study highlighted the contribution of disordered iron metabolism to breast cancer growth, and also signified an oncogenic effect of misregulated ferroportin in breast cancers. This work represents a promising starting point to the possibility of restraining breast cancer through targeting FPN or its upstream regulatory factors.