-
Frontiers in Immunology 2023Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy...
INTRODUCTION
Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less.
METHODS
Three potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h.
RESULTS
Suppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h.
DISCUSSION
Takeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.
Topics: Humans; T-Lymphocytes; Leukocytes, Mononuclear; Tumor Necrosis Factor-alpha; Mitogens; Immunosuppression Therapy; Mesenchymal Stem Cells; Intercellular Signaling Peptides and Proteins; Caspases
PubMed: 37822938
DOI: 10.3389/fimmu.2023.1225047 -
Developmental Dynamics : An Official... Nov 2008The cytoplasmic serine/threonine kinases transduce extracellular signals into regulatory events that impact cellular responses. The induction of one kinase triggers the... (Review)
Review
The cytoplasmic serine/threonine kinases transduce extracellular signals into regulatory events that impact cellular responses. The induction of one kinase triggers the activation of several downstream kinases, leading to the regulation of transcription factors to affect gene function. This arrangement allows for the kinase cascade to be amplified, and integrated according to the cellular context. An upstream mitogen or growth factor signal initiates a module of three kinases: a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK; e.g., Raf) that phosphorylates and activates a MAP kinase kinase (MAPKK; e.g., MEK) and finally activation of MAP kinase (MAPK; e.g., ERK). Thus, this MAP3K-MAP2K-MAPK module represents critical effectors that regulate extracellular stimuli into cellular responses, such as differentiation, proliferation, and apoptosis all of which function during development. There are 21 characterized MAP3Ks that activate known MAP2Ks, and they function in many aspects of developmental biology. This review summarizes known transduction routes linked to each MAP3K and highlights mouse models that provide clues to their physiological functions. This perspective reveals that some of these MAP3K effectors may have redundant functions, and also serve as unique nexus depending on the context of the signaling pathway.
Topics: Animals; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mitogens; Models, Biological
PubMed: 18855897
DOI: 10.1002/dvdy.21750 -
European Journal of Medicinal Chemistry Aug 2022A series of novel pyrrolidinedione-thiazolidinones was synthesized and subjected to physico-chemical characteristics. They were screened on a panel of cell lines...
A series of novel pyrrolidinedione-thiazolidinones was synthesized and subjected to physico-chemical characteristics. They were screened on a panel of cell lines representing different types of cancer, as well as normal human keratynocytes and lymphocytes of peripheral human blood. High antiproliferative activity of 1-(4-chlorophenyl)- and 1-(4-hydroxyphenyl)-3-{5-[(Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene]-4-oxo-2-thioxothiazolidin-3-yl}-1-(4-hydroxyphenyl)-pyrrolidine-2,5-diones 2a and 2b was revealed along with satisfactory cytotoxicity characteristics. Human T-leukemia cells of Jurkat line were the most sensitive to the action of 2a, 2b and 5-(2-allyloxybenzylidene) derivative 2f. At the same time, synthesized compounds demonstrated low toxicity towards normal human keratinocytes of HaCaT line and mitogen-activated lymphocytes of peripheral blood of healthy human donor. The compounds 2а and 2b demonstrated high selectivity (SI >9.2) towards studied leukemia, lung, breast, cervical, colon carcinoma and glioblastoma cells. Compounds 2a, 2b induced mitochondria-dependent apoptosis in treated Jurkat T-cells via increasing the level of proapoptotic Bax and EndoG proteins, and decreasing the level of antiapoptotic Bcl-2 protein. The cytotoxic action of compounds 2a, 2b towards Jurkat T-cells was associated with the single-strand brakes in DNA and its inter-nucleosomal fragmentation, without significant intercalation of these compounds into the DNA molecule. Compounds 2a, 2b did not induce significant DNA damage and changes in morphology of mitogen-activated lymphocytes of peripheral blood of healthy donor. Altogether, these data demonstrated anticancer potential of novel hybrid pyrrolidinedione-thiazolidinones which were relatively non-toxic for normal human cells.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Leukemia; Mitogens; Succinimides
PubMed: 35533562
DOI: 10.1016/j.ejmech.2022.114422 -
Blood Apr 2023Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that...
Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.
Topics: Adult; Humans; Animals; Mice; Integrin alpha6; Mitogens; Hematopoietic Stem Cells; Antigens, CD34; Cell Proliferation; Cell Cycle Checkpoints; Cell Cycle; Intercellular Signaling Peptides and Proteins
PubMed: 36652668
DOI: 10.1182/blood.2022017174 -
The Journal of Biological Chemistry Nov 2014The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and...
The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases.
Topics: Animals; Cell Proliferation; Endoribonucleases; Gene Expression Regulation; HEK293 Cells; HeLa Cells; Humans; Mice; Mitogens; Models, Biological; RNA Stability; Transcription, Genetic; Tristetraprolin
PubMed: 25301952
DOI: 10.1074/jbc.M114.589556 -
Journal of Veterinary Science Jul 2022In lipopolysaccharide-induced RAW264.7 cells, (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways...
BACKGROUND
In lipopolysaccharide-induced RAW264.7 cells, (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways of osteoclast development and bone resorption.
OBJECTIVES
This study examined how aster saponin A2 (AS-A2) isolated from AT affects the processes and function of osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264.7 cells and bone marrow macrophages (BMMs).
METHODS
The cell viability, tartrate-resistant acid phosphatase staining, pit formation assay, polymerase chain reaction, and western blot were carried out to determine the effects of AS-A2 on osteoclastogenesis.
RESULTS
In RAW264.7 and BMMs, AS-A2 decreased RANKL-initiated osteoclast differentiation in a concentration-dependent manner. In AS-A2-treated cells, the phosphorylation of ERK1/2, JNK, and p38 protein expression were reduced considerably compared to the control cells. In RAW264.7 cells, AS-A2 suppressed the RANKL-induced activation of osteoclast-related genes. During osteoclast differentiation, AS-A2 suppressed the transcriptional and translational expression of NFATc1 and c-Fos. AS-A2 inhibited osteoclast development, reducing the size of the bone resorption pit area.
CONCLUSION
AS-A2 isolated from AT appears to be a viable therapeutic therapy for osteolytic illnesses, such as osteoporosis, Paget's disease, and osteogenesis imperfecta.
Topics: Animals; Bone Marrow Cells; Bone Resorption; Cell Differentiation; Mitogen-Activated Protein Kinases; Mitogens; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Osteogenesis; RANK Ligand; Saponins; Signal Transduction
PubMed: 35698806
DOI: 10.4142/jvs.21246 -
Scientific Reports Nov 2022The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms...
The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms an inactive ternary cytoplasmatic complex with the MAPKK Wis1 and the MAPKAP kinase Srk1. Wis1 phosphorylates and activates Sty1, inducing the nuclear translocation of the complex. Once in the nucleus, Sty1 phosphorylates and activates Srk1, which in turns inhibits Cdc25 and cell cycle progression, before being degraded in a proteasome-dependent manner. In parallel, active nuclear Sty1 activates the transcription factor Atf1, which results in the expression of stress response genes including pyp2 (a MAPK phosphatase) and srk1. Despite its essentiality in response to stress, persistent activation of the MAPK pathway can be deleterious and induces cell death. Thus, timely pathway inactivation is essential to ensure an appropriate response and cell viability. Here, uncover a role for the MAPKAP kinase Srk1 as an essential component of a negative feedback loop regulating the Sty1 pathway through phosphorylation and inhibition of the Wis1 MAPKK. This feedback regulation by a downstream kinase in the pathway highlights an additional mechanism for fine-tuning of MAPK signaling. Thus, our results indicate that Srk1 not only facilitates the adaptation to stress conditions by preventing cell cycle progression, but also plays an instrumental role regulating the upstream kinases in the stress MAPK pathway.
Topics: Feedback; Gene Expression Regulation, Fungal; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mitogens; Phosphorylation; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 36376357
DOI: 10.1038/s41598-022-23970-8 -
American Journal of Physiology. Renal... Jul 2004Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an imperative role in tubular repair and regeneration after acute renal injury. Growing evidence... (Review)
Review
Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an imperative role in tubular repair and regeneration after acute renal injury. Growing evidence indicates that HGF is also an endogenous renoprotective factor that possesses a potent antifibrotic ability. HGF prevents the initiation and progression of chronic renal fibrosis and inhibits transforming growth factor (TGF)-beta(1) expression in a wide variety of animal models. In vitro, HGF counteracts the action of TGF-beta(1) in different types of kidney cells, resulting in blockade of the myofibroblastic activation from interstitial fibroblasts and glomerular mesangial cells, as well as inhibition of the mesenchymal transition from tubular epithelial cells. Recent studies reveal that HGF antagonizes the profibrotic actions of TGF-beta(1) by intercepting Smad signal transduction through diverse mechanisms. In interstitial fibroblasts, HGF blocks activated Smad-2/3 nuclear translocation, whereas it specifically upregulates the expression of the Smad transcriptional corepressor SnoN in tubular epithelial cells. In glomerular mesangial cells, HGF stabilizes another Smad corepressor, TGIF, by preventing it from degradation. Smad corepressors bind to activated Smad-2/3 and sequester their ability to transcriptionally activate TGF-beta target genes. This article reviews recent advances in our understanding of the cellular and molecular mechanisms underlying HGF inhibition of renal fibrosis.
Topics: Disease Progression; Fibrosis; Hepatocyte Growth Factor; Humans; Kidney; Mitogens; Proto-Oncogene Proteins c-met; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1
PubMed: 15180923
DOI: 10.1152/ajprenal.00451.2003 -
Molecular Oncology Aug 2021In psychotherapy, paradoxical interventions are characterized by a deliberate reinforcement of the pathological behavior to improve the clinical condition. Such a... (Review)
Review
In psychotherapy, paradoxical interventions are characterized by a deliberate reinforcement of the pathological behavior to improve the clinical condition. Such a counter-intuitive approach can be considered when more conventional interventions fail. The development of targeted cancer therapies has enabled the selective inhibition of activated oncogenic signaling pathways. However, in advanced cancers, such therapies, on average, deliver modest benefits due to the development of resistance. Here, we review the perspective of a 'paradoxical intervention' in cancer therapy: rather than attempting to inhibit oncogenic signaling, the proposed therapy would further activate mitogenic signaling to disrupt the labile homeostasis of cancer cells and overload stress response pathways. Such overactivation can potentially be combined with stress-targeted drugs to kill overstressed cancer cells. Although counter-intuitive, such an approach exploits intrinsic and ubiquitous differences between normal and cancer cells. We discuss the background underlying this unconventional approach and how such intervention might address some current challenges in cancer therapy.
Topics: Cell Proliferation; Drug Resistance, Neoplasm; Homeostasis; Humans; Mitogens; Neoplasms; Signal Transduction; Tumor Microenvironment
PubMed: 33955157
DOI: 10.1002/1878-0261.12979 -
Cellular and Molecular Life Sciences :... Oct 2008Mitogenic signals stimulate cell division by activating cyclin/cyclin-dependent kinase (CDK) complexes. Their timely regulation ensures proper cell cycle progression. It... (Review)
Review
Mitogenic signals stimulate cell division by activating cyclin/cyclin-dependent kinase (CDK) complexes. Their timely regulation ensures proper cell cycle progression. It is therefore not surprising that cyclin/CDK complexes are integrators of multiple signals from both the extracellular environment and intracellular cues. Important regulators of cyclin/CDKs are the CDK inhibitors that have attracted attention due to their association with disease. p27(KIP1) is a CDK inhibitor that controls CDK activity throughout the cell cycle. As a CDK inhibitor, p27(KIP1) has tumor suppressor activity. Besides CDKs, p27(KIP1) regulates additional cellular processes, including cell motility, some of which seem to mediate oncogenic activities of p27(KIP1). These activities of p27(KIP1) are regulated through multiple phosphorylation sites, targeted by several signal transduction pathways. Understanding functions and regulation of p27(KIP1) will be important to determine which isoform of p27(KIP1) has anti- or pro-tumorigenic activities. Such knowledge might be of prognostic value and may offer novel therapeutic windows.
Topics: Animals; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Humans; Mitogens; Neoplasms; Protein Processing, Post-Translational
PubMed: 18636226
DOI: 10.1007/s00018-008-8296-7