Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway

10.1371/journal.pone.0074334PLoS ONE89-POLN

Accelerated apoptotic death and <i>in vivo</i> turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice

The Pim kinases control rapamycin-resistant T cell survival and activation

Although Pim-1 or Pim-2 can contribute to lymphoid transformation when overexpressed, the physiologic role of these kinases in the immune response is uncertain. We now report that T cells from Pim-1−/−Pim-2−/− animals display an unexpected sensitivity to the immunosuppressant rapamycin. Cytokine-induced Pim-1 and Pim-2 promote the rapamycin-resistant survival of lymphocytes. The endogenous function of the Pim kinases was not restricted to the regulation of cell survival. Like the rapamycin target TOR, the Pim kinases also contribute to the regulation of lymphocyte growth and proliferation. Although rapamycin has a minimal effect on wild-type T cell expansion in vitro and in vivo, it completely suppresses the response of Pim-1−/−Pim-2−/− cells. Thus, endogenous levels of the Pim kinases are required for T cells to mount an immune response in the presence of rapamycin. The existence of a rapamycin-insensitive pathway that regulates T cell growth and survival has important implications for understanding how rapamycin functions as an immunomodulatory drug and for the development of complementary immunotherapeutics

Membrane progestin receptor expression, signaling and function in reproductive somatic cells of female vertebrates

textThe goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker.Marine Scienc

The role of histone kinases in controlling transcription in B cell lymphoma and leukaemia

Protein kinases are central mediators of signal transduction pathways and transcriptional regulation. Lymphoid malignancies are characterised by aberrant activation of key signal transduction pathways and specific gene expression programmes. Consequently, targeting kinases involved in these signal transduction pathways is a promising therapeutic strategy. Because gene expression is regulated at the level of chromatin, the aim of this study was to assess the effects of chromatin-modifying kinases on histone phosphorylation and transcriptional regulation in B cell lymphoma and the consequences of kinase inhibition for tumour cell viability and the chromatin structure of target genes. The kinase PIM1, whose mRNA is highly expressed in the aggressive activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), but not germinal centre B cell-like DLBCL (GCB-DLBCL), has been shown to associate with the transcription factor MYC and to regulate the expression of MYC target genes by phosphorylating histone H3S10. Therefore, effects of PIM1 on viability and gene expression were evaluated in ABC-DLBCL and in the MYC-dependent Burkitt lymphoma (BL). However, pan-PIM kinase inhibition or knockdown of PIM1 did not effectively reduce viability of ABC-DLBCL or Burkitt lymphoma cell lines. Further, the expression of the MYC- and PIM1-bound GNL3 gene was largely unaffected by alterations in PIM kinase levels or activity. In conclusion, PIM kinases do not seem to be bona fide therapeutical targets in DLBCL and BL. The second part of this project aimed to understand the effects of Ibrutinib on chromatin structure in chronic lymphocytic leukaemia (CLL) cells. Ibrutinib inhibits Bruton’s tyrosine kinase, and thus B cell receptor (BCR) signalling, and is currently being tested in clinical trials for the treatment of CLL. In vitro, Ibrutinib inhibited BCR-induced gene expression and histone H3T6 and T11 phosphorylation. A possible kinase targeting H3T6 and H3T11 downstream of the BCR might be zipper-interacting protein kinase (ZIPK), a ZIPK inhibitor blocked H3T6p and H3T11p and gene expression. Short-term Ibrutinib treatment appeared to inhibit histone turnover but did not reduce H3K4me3, H3K9ac, H2A.Z or POL II recruitment at target genes, indicating that it inhibits only some aspects of transcription. In contrast, long-term Ibrutinib treatment decreased H3K4me3 and H3K9ac in promoter regions, possibly by an indirect, gene silencing-dependent mechanism. In summary, the results suggest that Ibrutinib blocks progression of CLL by inhibiting only some branches of BCR signalling and interestingly, many transcription-associated changes to the chromatin remain unaltered, while transcription is effectively inhibited

Regulation of malignant cell transformation by the stress-activated kinase p38alpha

Cancer is a dynamic process that requires the stepwise deregulation of mechanisms affecting various cellular traits. During my PhD, I have characterized how the stress-activated p38alpha MAPK signaling pathway regulates the processes of cellular migration, proliferation, and survival in the context of oncogene-induced malignant transformation, which recapitulates the mechanisms of cancer initiation at the cellular level. My studies have been mostly based on the use of human and mouse cultured cells, which I have analyzed using both biochemical and cell biological approaches. In particular, the development of p38alpha-deficient cell lines and the application of retrovirally-based gene expression techniques have been very useful. The implementation of tools to measure the intracellular levels of reactive oxygen species (ROS) within living cells has also been key for my work. I have found that p38alpha regulates the process of malignant transformation at various levels. First, p38alpha negatively regulates cell cycle progression induced by mitogenic signals in both exponentially proliferating and confluent cells. Oncogene-expressing cells proliferate faster in the absence of p38alpha, which may be accounted for by the negative effect of p38alpha on cyclin D1 expression. Similarly, p38alpha controls the process of cell-cell contact-inhibition, which requires p27Kip1 accumulation and triggers G1-phase cell cycle arrest upon cell confluence. The process of contact inhibition is likely to involve uncharacterized membrane-associated signaling events. Accordingly, I have found that p38alpha regulates the membrane composition of oncogene-transformed cells. In addition to its negative role in cell proliferation, I have shown that p38alpha can interfere with the process of malignant transformation by sensing oxidative stress and inducing apoptosis. Thus, p38alpha becomes activated when oncogene-expressing cells accumulate high levels of carcinogenic ROS and, in turn, induces the elimination of the transformed cells by apoptosis. Interestingly, I have found that human cancer cell lines that contain high ROS levels have developed a mechanism to by-pass this p38alpha function. Finally, in contrast to its anti-proliferative and pro-apoptotic roles, I have found that p38alpha is an important mediator of cytokine-induce cell migration, a process that is thought to be important for cancer cell metastasis

P38 Signaling Pathway

p38 Mitogen activated protein kinases (p38MAPK) are a group of evolutionary conserved protein kinases which are central for cell adaptation to environmental changes as well as for immune response, inflammation, tissue regeneration and tumour formation. The interest in this group of protein kinases has grown continually since their discovery. Recent studies using new genetic and pharmacological tools are providing helpful information on the function of these stress-activated protein kinases and show that they have an acute impact on the development of prevalent diseases related to inflammation, diabetes, neurodegeneration, and cancer. In this Special Issue we present novel advances and review the knowledge on the identification of p38MAPK substrates, functions, and regulation; mechanisms underlying the role of p38MAPK in malignant transformation and other pathologies; and therapeutic opportunities associated with regulation of p38MAPK activity