Expression of erythropoietin messenger ribonucleic acid in wild-type MED12 uterine leiomyomas under estrogenic influence: new insights into related growth disparities.

OBJECTIVE: To determine factors that impact erythropoietin (EPO) production in leiomyomas. We have previously implicated EPO production in promoting the growth of some leiomyomas. DESIGN: The relationship between EPO messenger RNA (mRNA) expression and MED12 gene mutations or mRNA expression levels of high-mobility group AT-hook (HMGA) 1 and HMGA2 were analyzed. Effects of 10-8 M 17ß-E2 on EPO mRNA expression were evaluated using leiomyoma cells grown in primary cultures. SETTING: Graduate school of medicine. PATIENT(S): Patients with leiomyoma. INTERVENTION(S): We used tissue samples and clinical data of 108 patients with leiomyomas to analyze the relation between EPO mRNA expression and MED12 mutation. Tissue samples from another 10 patients with leiomyomas were collected for in vitro experimentation using primary cultures of leiomyoma and myometrial cells. MAIN OUTCOME MEASURE(S): Relations between EPO mRNA expression, MED12 exon 2 mutation, and HMGA1/HMGA2 mRNA expression levels in leiomyoma samplings, in addition to effects of estrogen (E) on EPO mRNA expression in cultures of leiomyoma cells. RESULT(S): The EPO mRNA level was threefold higher in leiomyomas with wild-type (vs. mutated) MED12 genes. There was no correlation between EPO and HMGA1 or HMGA2 mRNA expression levels. In wild-type MED12 leiomyomas only, E2 treatment produced a twofold increase in EPO mRNA expression, whereas mutated MED12 leiomyomas were unaffected. CONCLUSION(S): The EPO mRNA expression increased significantly after E2 treatment only in leiomyomas lacking MED12 mutations. In conjunction with prior evidence linking EPO mRNA expression levels and tumor size, E2-stimulated EPO mRNA expression may explain the marked growth disparities seen in these tumors.

The Mediator complex subunit MED15, a promoter of tumour progression and metastatic spread in renal cell carcinoma.

BACKGROUND/OBJECTIVE: MED15 is a part of the multiprotein Mediator complex which is involved in the transcription of polymerase (Pol) II-dependent genes. Several studies in this field have reported altered expressions of distinct subunits in human malignancy. However, the role of MED15 in renal cell carcinoma (RCC) has not be investigated yet. METHODS: First, we performed an RNA expression and survival analysis of MED15 in RCC by using the database cBioPortal. To confirm these data on the protein level, we executed immunohistochemical (IHC) staining against MED15 on a tissue microarray containing 184 samples of the most common subtypes of the tumour at the various stages. Further, we performed functional analysis including proliferation, migration, and invasion assays on the RCC cell lines A-498 and ACHN following the siRNA-mediated MED15 knockdown. RESULTS: On the mRNA level, higher expression of MED15 was associated with worse patient survival rates. IHC staining validated this tendency, unfortunately the results were not significant. However, supporting this tendency, in vitro-assays showed a significant decrease in proliferation, migration, and invasion after knockdown of MED15. CONCLUSION: The research concludes that MED15 does seem to play a tumour promoting role in the progression and metastatic spread of renal cell carcinoma.

Splicing regulators in endothelial cell differentiation.

AIMS: Alternative splicing represents a key mechanism of gene regulation. Despite its role in regulating cell pluripotency and differentiation being well known, the underlining mechanisms are still poorly studied. Here, we investigated the possible involvement of splicing regulators during the different steps of endothelial cell differentiation through expression studies on human circulating progenitors. METHODS: Total RNAs were extracted from all cells and reverse-transcribed. Semiquantitative and real-time RT-PCR was performed using selective oligonucleotides. Differences between group means were considered significant at P value less than 0.05 and more significant at P value less than 0.01. Protein extracts were incubated with an antibody directed against MED23. Immunoprecipitation of supernatants and pellets was probed with both anti-Muscleblind-like splicing regulator (MBNL)1 and anti-MBNL2 antibodies. RESULTS: Several clinical trials demonstrated the safety and efficacy of progenitor cells in regenerative therapy of the cardiovascular system. Particularly, we analyzed the expression of genes belonging to muscleblind family members and MED complex subunits, which are known to be involved during differentiation in other models. This study shows that MED23, MBNL1 and MBNL2 were all expressed at high levels only in differentiated cells. Moreover, immunoprecipitation assays indicated that MED23 is able to bind MBNLs in endothelial cells. CONCLUSION: Our data suggest that MED23, MBNL1 and MBNL2 could regulate alternative splicing events activated during differentiation through a common mechanism. Hence, these observations corroborate previous evidence that splicing regulators may have an essential role in the basic apparatus required for cell pluripotency and reprogramming, allowing identification of novel biomarkers to use for early diagnosis in cardiovascular diseases.

Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors.

BACKGROUND: Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. METHODS: Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. RESULTS: In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. CONCLUSIONS: In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15 expression in the preinvasive precursor cells, which may provide diagnostic value to distinguish between benign and pre-malignant testicular specimen, and may indicate a role for MED15 in carcinogenesis in TGCT.

Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells.

The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin ß3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts.

A functional portrait of Med7 and the mediator complex in Candida albicans.

Mediator is a multi-subunit protein complex that regulates gene expression in eukaryotes by integrating physiological and developmental signals and transmitting them to the general RNA polymerase II machinery. We examined, in the fungal pathogen Candida albicans, a set of conditional alleles of genes encoding Mediator subunits of the head, middle, and tail modules that were found to be essential in the related ascomycete Saccharomyces cerevisiae. Intriguingly, while the Med4, 8, 10, 11, 14, 17, 21 and 22 subunits were essential in both fungi, the structurally highly conserved Med7 subunit was apparently non-essential in C. albicans. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogen's ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used epitope tagging and location profiling of the Med7 subunit to examine the distribution of the DNA sites bound by Mediator during growth in either the yeast or the hyphal form, two distinct morphologies characterized by different transcription profiles. We observed a core set of 200 genes bound by Med7 under both conditions; this core set is expanded moderately during yeast growth, but is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also within coding regions and at the 3' ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7p-influenced regulons including genes related to glycolysis and the Filamentous Growth Regulator family. In the absence of Med7, the ribosomal regulon is de-repressed, suggesting Med7 is involved in central aspects of growth control.

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Mechanical stretch via transforming growth factor-ß1 activates microRNA-208a to regulate hypertrophy in cultured rat cardiac myocytes.

BACKGROUND/PURPOSE: MicroRNA-208a (miR208a) and mechanical stress play a key role in cardiac hypertrophy. The relationship between miR208a and mechanical stress in cultured cardiomyocytes has not been investigated. The molecular mechanisms underlying miR208a-induced hypertrophy of cardiomyocytes by mechanical stress is poorly understood. This study investigated whether miR208a is a critical regulator in cardiomyocyte hypertrophy under mechanical stretch. METHODS: Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched at 60 cycles/minute. MiR real-time quantitative assays were used to quantify miRs. A quantitative sandwich enzyme immunoassay technique was used to measure transforming growth factor-ß1 (TGF-ß1). A (3)H-proline incorporation assay was used to measure protein synthesis. RESULTS: Mechanical stretch significantly enhanced miR208a expression. Stretch significantly induced cardiomyocyte hypertrophic protein expression such as ß-myosin heavy chain (MHCß), thyroid hormone receptor-associated protein 100, myostatin, connexin 40, GATA4, and brain natriuretic peptide. MHCα was not induced by stretch. Overexpression of miR208a significantly increased MHCß protein expression while pretreatment with antagomir208a significantly attenuated MHCß protein expression induced by stretch and overexpression of miR208a. Mechanical stretch significantly increased the secretion of TGF-ß1 from cultured cardiomyocytes. Exogenous addition of TGF-ß1 recombinant protein significantly increased miR208a expression and pretreatment with TGF-ß1 antibody attenuated miR208a expression induced by stretch. Mechanical stretch and overexpression of miR208a increased protein synthesis while antagomir208a attenuated protein synthesis induced by stretch and overexpression of miR208a. CONCLUSION: Cyclic stretch enhances miR208a expression in cultured rat cardiomyocytes. MiR208a plays a role in stretch-induced cardiac hypertrophy. The stretch-induced miR208a is mediated by TGF-ß1.

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.

Baculovirus expression: tackling the complexity challenge.

Most essential functions in eukaryotic cells are catalyzed by complex molecular machines built of many subunits. To fully understand their biological function in health and disease, it is imperative to study these machines in their entirety. The provision of many essential multiprotein complexes of higher eukaryotes including humans, can be a considerable challenge, as low abundance and heterogeneity often rule out their extraction from native source material. The baculovirus expression vector system (BEVS), specifically tailored for multiprotein complex production, has proven itself to be uniquely suited for overcoming this impeding bottleneck. Here we highlight recent major achievements in multiprotein complex structure research that were catalyzed by this versatile recombinant complex expression tool.

Expression of Med19 in bladder cancer tissues and its role on bladder cancer cell growth.

OBJECTIVES: The human Med19 gene encodes a critical subunit that stabilizes the whole mediator complex. To understand the role of Med19 in bladder cancer, we studied the effects of lentivirus-mediated suppression of Med19 expression on bladder cancer cells in vitro and in vivo. METHODS AND MATERIALS: In this study, immunohistochemical analysis was used to demonstrate the expression of Med19 in human bladder cancer. The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed. After bladder cancer cells (5637 and T24) were infected, RT-PCR and Western blotting were used to measure Med19 expression. The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT, BrdU, colony formation and tumorigenicity experiments. Cell cycle was analyzed with flow cytometric assay. RESULTS: Med19 was up-regulated in human bladder cancers compared with adjacent benign tissues by immunohistochemical analysis, but was strongly inhibited in 5637 and T24 bladder cancer cells infected with lentiviruses delivering shRNA against Med19. The down-regulation of Med19 increased the proportion of cells in G0/G1 phases and attenuated the growth of 5637 and T24 cells in vitro. The tumorigenicity of Med19-suppressed T24 cells was decreased after inoculation into nude mice. CONCLUSIONS: Our results suggested that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy.

Knockdown of MED19 by short hairpin RNA-mediated gene silencing inhibits pancreatic cancer cell proliferation.

Abnormal gene transcription plays an important role in oncogenesis. In cancer cells, the improper activation of specific genes is usually ascribed to aberrant transcription machinery including transcription factors, RNA polymerase II, and Mediator complex. This study reports on short hairpin RNA (shRNA)-mediated gene silencing of MED19, a subunit of Mediator complex, and its effect on the growth of pancreatic cancer cells. RNA interference was performed by lentivirus shRNA system to specifically knockdown MED19 expression in Aspc-1 and Panc-1 cells. The knockdown efficiency of MED19 was confirmed by quantitative RT-PCR and western blot. The effect of MED19 shRNA on Aspc-1 and Panc-1 cell proliferation was evaluated by methylthiazoletetrazolium assay, BrdU incorporation assay, colony formation assay, and flow cytometry assay. This study shows that downregulation of MED19 remarkably reduced cancer cell proliferation and colony formation capacity in two pancreatic cancer cell lines. In addition, downregulated MED19 induced G1-phase cell cycle arrest and apoptosis. This study provides a potent role of MED19 in promoting pancreatic cancer growth and a possible drug target for cancer therapy.