HDAC7 Is a Repressor of Myeloid Genes Whose Downregulation Is Required for Transdifferentiation of Pre-B Cells into Macrophages

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes

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The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction

The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1

The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion

From Springer Nature via Jisc Publications RouterHistory: received 2020-04-21, rev-recd 2021-03-23, accepted 2021-04-14, registration 2021-04-15, pub-electronic 2021-05-12, online 2021-05-12, pub-print 2021-06-10Publication status: PublishedFunder: Worldwide Cancer Research; doi: https://doi.org/10.13039/100011713; Grant(s): 15-1283Funder: RCUK | MRC | Medical Research Foundation; doi: https://doi.org/10.13039/501100009187; Grant(s): MC_PC_18056Abstract: There is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior

The transcriptional repressor HDAC7 promotes apoptosis and c-Myc downregulation in particular types of leukemia and lymphoma

The generation of B cells is a complex process requiring several cellular transitions, including cell commitment and differentiation. Proper transcriptional control to establish the genetic programs characteristic of each cellular stage is essential for the correct development of B lymphocytes. Deregulation of these particular transcriptional programs may result in a block in B-cell maturation, contributing to the development of hematological malignancies such as leukemia and lymphoma. However, very little is currently known about the role of transcriptional repressors in normal and aberrant B lymphopoiesis. Here we report that histone deacetylase 7 (HDAC7) is underexpressed in pro-B acute lymphoblastic leukemia (pro-B-ALL) and Burkitt lymphoma. Ectopic expression of HDAC7 induces apoptosis, leads to the downregulation of c-Myc and inhibits the oncogenic potential of cells in vivo, in a xenograft model. Most significantly, we have observed low levels of HDAC7 expression in B-ALL patient samples, which is correlated with the increased levels of c-Myc. From a mechanistic angle, we show that ectopically expressed HDAC7 localizes to the nucleus and interacts with the transcription factor myocyte enhancer factor C (MEF2C) and the corepressors HDAC3 and SMRT. Accordingly, both the HDAC7-MEF2C interaction domain as well as its catalytic domain are involved in the reduced cell viability induced by HDAC7. We conclude that HDAC7 has a potent anti-oncogenic effect on specific B-cell malignancies, indicating that its deregulation may contribute to the pathogenesis of the disease.This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-28290; to MP). MP was supported by a Ramón y Cajal contract from the Spanish Ministry of Science and Innovation (MICIIN). LR-G was supported by an IDIBELL PhD fellowship. NL-B acknowledges the funding from MICIIN (SAF2012-36199)

Aberrant expression of extracellular signal-regulated kinase 5 in human prostate cancer

Abnormal intracellular signaling contributes to carcinogenesis and may represent novel therapeutic targets. mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) overexpression is associated with aggressive prostate cancer. In this study, we examined the role of extracellular signal-regulated kinase (ERK5, an MAPK and specific substrate for MEK5) in prostate cancer. ERK5 immunoreactivity was significantly upregulated in high-grade prostate cancer when compared to benign prostatic hyperplasia (<i>P</i><0.0001). Increased ERK5 cytoplasmic signals correlated closely with Gleason sum score (<i>P</i><0.0001), bony metastases (<i>P</i>=0.0044) and locally advanced disease at diagnosis (<i>P</i>=0.0023), with a weak association with shorter disease-specific survival (<i>P</i>=0.036). A subgroup of patients showed strong nuclear ERK5 localization, which correlated with poor disease-specific survival and, on multivariant analysis, was an independent prognostic factor (<i>P</i><0.0001). Analysis of ERK5 expression in matched tumor pairs (before and after hormone relapse, n=26) revealed ERK5 nuclear expression was significantly associated with hormone-insensitive disease (<i>P</i>=0.0078). Similarly, ERK5 protein expression was increased in an androgen-independent LNCaP subline. We obtained the following <i>in vitro</i> and <i>in vivo</i> evidence to support the above expression data: (1) cotransfection of ERK5wt and MEK5D constructs in PC3 cells results in predominant ERK5 nuclear localization, similar to that observed in aggressive clinical disease; (2) ERK5-overexpressing PC3 cells have enhanced proliferative, migrative and invasive capabilities in vitro (<i>P</i><0.0001), and were dramatically more efficient in forming tumors, with a shorter mean time for tumors to reach a critical volume of 1000 mm3, <i>in vivo</i> (<i>P</i><0.0001); (3) the MEK1 inhibitor, PD184352, blocking ERK1/2 activation at low dose, did not suppress proliferation but did significantly decrease proliferation at a higher dose required to inhibit ERK5 activation. Taken together, our results establish the potential importance of ERK5 in aggressive prostate cancer