Dual inhibition of P38 MAPK and JNK pathways preserves stemness markers and alleviates premature activation of Muscle Stem Cells during isolation
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ABSTRACT: Dual inhibition of P38 MAPK and JNK pathways preserves stemness markers and alleviates premature activation of Muscle Stem Cells during isolation
Project description:Adult skeletal muscle contains resident Muscle Stem Cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored. We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources. We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC. Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.
Project description:p38 and JNK are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators GIGYF1 and 2 by p38-dependent MK2 phosphorylation and 14-3-3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.
Project description:In order to identify the effects of JNK inhibitor or p38 Inhibitor on the transcriptome of ATP7B H1069Q-overexpressing liver cells, we performed RNAseq experiments
Project description:Facioscapulohumoral muscular dystrophy (FSHD) is a progressive muscle wasting disease caused by misexpression of the DUX4 transcription factor. DUX4 expression in skeletal muscle cells results in a dramatic alteration in their transcriptional profile and cellular phenotype and subsequently, cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology such as DNA damage and altered mRNA splicing before detectable changes in protein levels. Implementation of phosphoproteomics uncovered that widespread changes in protein phosphorylation preceded changes in protein levels indicating that alterations in kinase signaling may play a role in DUX4-mediated stress and cell death. Here we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Pharmacological inhibition of either of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover JNK as a novel pathway involved in DUX4-mediated cell death, as well as provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.
Project description:Facioscapulohumoral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes, and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered widespread changes in protein phosphorylation rapidly following DUX4 induction indicating that alterations in kinase signaling may play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover JNK as a novel pathway involved in DUX4-mediated cell death as well as provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.
Project description:In primary melanoma, the amount of VEGF-C expression and lymphangiogenesis predict the probability of metastasis to sentinel nodes, but conditions boosting VEGF-C expression in melanoma are poorly characterized. By comparative mRNA expression analysis of a set of 22 human melanoma cell lines, we found a striking negative correlation between VEGF-C and MITF expression, which was confirmed by data mining in GEO databases of human melanoma Affymetrix arrays. Moreover, in human patients, high VEGF-C, and low MITF levels in primary melanoma significantly correlated with the chance of metastasis. Pathway analysis disclosed the respective JNK- and p38/MAPK activities as being responsible for the inverse regulation of VEGF-C and MITF. Predominant JNK signaling results in a VEGF-Clow/MITFhigh phenotype, these melanoma cells are highly proliferative, show low mobility and are poorly lymphangiogenic. Predominant p38 signaling results in a VEGF-Chigh/MITFlow phenotype, corresponding to a slowly cycling, highly mobile, lymphangiogenic and metastatic melanoma.
Project description:The gene expression pattern of PAM3 treated monocytes was compared to that of M-CSF, the conventional method of generating immunosuppressive macrophage. Pathways regulated by NF-KB and Akt played a central role in the general process of monocyte to macrophage differentiation. p38 MAPK and PTGS2 signaling biased this process towards the generation of immunosuppressive rather than pro-inflammatory macrophage whereas ERK and JNK were essential for Pam3 but not M-CSF driven maturation
Project description:Bis-2-chloroethyl sulfide (sulfur mustard, SM) is a potent alkylating agent and vesicant. Exposure to SM results in activation of numerous signaling cascades, including mitogen-activated protein kinase (MAPK) signaling pathways. These pathways include the Erk, p38, and JNK pathways, which are involved in cell growth, inflammation, and stress signaling. However, the precise roles of these pathways in SM toxicity have not been fully elucidated. We used Western blotting and microarray analysis to examine the activation and role of each pathway following SM exposure in primary human epidermal keratinocytes. Western blotting revealed increased phosphorylation of p38 and JNK following SM exposure; however, phosphorylation of Erk was equivocal, suggesting that growth conditions may impact activation of Erk by SM. We used pharmacologic inhibitors to target each MAPK and then compared the gene expression profiles to identify SM-induced gene networks regulated by each MAPK. Cells were pretreated with 10 µM SB 203580 (p38 inhibitor), PD 98059 (Erk inhibitor), or SP 600125 (JNK inhibitor) 60 minutes before exposure to 200 µM SM. Cells were harvested at 1h, 4h, and 8h post-exposure, and RNA was extracted for synthesis of microarray probes. Probes were hybridized to Affymetrix U133 Plus 2.0 arrays for gene expression profiling. Analysis of variance was performed to identify genes significantly modulated due to pharmacologic inhibition in SM-exposed cells. Pathway mapping confirmed alterations in SM-induced Erk, JNK, and p38 MAPK signaling due to pharmacologic inhibition. SM-induced expression of IL-8, IL-6, and TNF-alpha was decreased by p38 MAPK inhibition, but not by inhibition of other MAPKs. Based on the number of significant pathways mapped to each MAPK in the presence and absence of inhibitors, the p38 MAPK pathway appeared to be the MAPK pathway most responsive to SM exposure. Interestingly, pathway mapping of the microarray data identified potential cross-talk between MAPK signaling pathways and other pathways involved in SM-induced signaling. Mining of these results will increase our understanding of the role of MAPK pathways in SM-induced signal transduction and may identify potential therapeutic targets for medical countermeasure development.
Project description:Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38α in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion and transcription. Several previously unknown p38 target genes were found. Using gel mobility shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression induced gene expression results in cell proliferation, myocardial inflammation and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart. Keywords: Gene transfer