Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Significance of TLR2 signaling during megakaryocyte development: regulatory cross-talk of miR-125b, cytokine induction, and MAPK pathway during dengue infection.

OBJECTIVE: Dengue is a viral infection endemic in more than 100 countries as per the WHO reports with approximately 5.2 million patients worldwide that spreads from mosquitoes to humans. Severe form of dengue fever can cause serious bleeding (low platelets) and death. Megakaryocytes are the immune cells responsible for the production of platelets. The molecular drivers behind platelet defects are mostly ambiguous. Here, we attempted to understand the distinct pathogen-elicited toll-like receptors (TLRs) functions in megakaryocyte biology. To understand the TLR induction and the molecular events that are governed in the mammalian system during dengue infection and to study TLR2-mediated cellular signaling-associated mechanisms with respect to their dimerization partners during dengue infection. METHODS: In this study, we used the human Megakaryoblastic cells, DAMI, and treated them with TLR agonists (LPS and Zymosan) and Dengue virus (DNV-II). RESULTS AND DISCUSSION: TLR2 could play an important role by dimerizing with TLR1, TLR4, and TLR6, which we induced for functional characterization. We observed that megakaryocyte maturation markers CD-41 and CD-61 were elevated. This augmentation under the LPS and Zymosan system along with DNV Infection was further confirmed. Our analysis also suggested that activation of miR-125b and MAPK signaling led to lipid droplet elevation. This led us to analyze TLR-mediated consequences and their impact on megakaryocyte development under diverse pathogen-elicited conditions. CONCLUSION: Pathogenic challenges associated with toll-like receptor system activation could further our understanding of the platelet biogenesis mechanistic pathways under various pathogenic circumstances.

Endocannabinoid system: Role in blood cell development, neuroimmune interactions and associated disorders.

The endocannabinoid system (ECS) is a complex physiological network involved in creating homeostasis and maintaining human health. Studies of the last 40 years have shown that endocannabinoids (ECs), a group of bioactive lipids, together with their set of receptors, function as one of the most important physiologic systems in human body. ECs and cannabinoid receptors (CBRs) are found throughout the body: in the brain tissues, immune cells, and in the peripheral organs and tissues as well. In recent years, ECs have emerged as key modulators of affect, neurotransmitter release, immune function, and several other physiological functions. This modulatory homoeostatic system operates in the regulation of brain activity and states of physical health and disease. In several research studies and patents the ECS has been recognised with neuro-protective properties thus it might be a target in neurodegenerative diseases. Most immune cells express these bioactive lipids and their receptors, recent data also highlight the immunomodulatory effects of endocannabinoids. Interplay of immune and nervous system has been recognized in past, recent studies suggest that ECS function as a bridge between neuronal and immune system. In several ongoing clinical trial studies, the ECS has also been placed in the anti-cancer drugs spotlight. This review summarizes the literature of cannabinoid ligands and their biosynthesis, cannabinoid receptors and their distribution, and the signaling pathways initiated by the binding of cannabinoid ligands to cannabinoid receptors. Further, this review highlights the functional role of cannabinoids and ECS in blood cell development, neuroimmune interactions and associated disorders. Moreover, we highlight the current state of knowledge of cannabinoid ligands as the mediators of neuroimmune interactions, which can be therapeutically effective for neuro-immune disorders and several diseases associated with neuroinflammation.

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop.

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.

Virodhamine, an endocannabinoid, induces megakaryocyte differentiation by regulating MAPK activity and function of mitochondria.

Endocannabinoids are well-known regulators of neurotransmission by activating the cannabinoid (CB) receptors. Endocannabinoids are being used extensively for the treatment of various neurological disorders such as Alzheimer's and Parkinson's diseases. Although endocannabinoids are well studied in cell survival, proliferation, and differentiation in various neurological disorders and several cancers, the functional role in the regulation of blood cell development is less examined. In the present study, virodhamine, which is an agonist of CB receptor-2, was used to examine its effect on megakaryocytic development from a megakaryoblastic cell. We observed that virodhamine increases cell adherence, cell size, and cytoplasmic protrusions. Interestingly, we have also observed large nucleus and increased expression of megakaryocytic marker (CD61), which are the typical hallmarks of megakaryocytic differentiation. Furthermore, the increased expression of CB2 receptor was noticed in virodhamine-induced megakaryocytic cells. The effect of virodhamine on megakaryocytic differentiation could be mediated through CB2 receptor. Therefore, we have studied virodhamine induced molecular regulation of megakaryocytic differentiation; mitogen-activated protein kinase (MAPK) activity, mitochondrial function, and reactive oxygen species (ROS) production were majorly affected. The altered mitochondrial functions and ROS production is the crucial event associated with megakaryocytic differentiation and maturation. In the present study, we report that virodhamine induces megakaryocytic differentiation by triggering MAPK signaling and ROS production either through MAPK effects on ROS-generating enzymes or by the target vanilloid receptor 1-mediated regulation of mitochondrial function.

RUNX1 and TGF-ß signaling cross talk regulates Ca2+ ion channels expression and activity during megakaryocyte development.

Thrombocytopenia is characterized by low platelet count and is typically observed among all preterm and low birthweight neonates admitted to the neonatal intensive care unit. Although the underlying cause for this predisposition is unclear, recent studies have proposed that the intrinsic inability of neonatal hematopoietic stem/progenitor cells to produce mature polyploid megakaryocytes (MKs) may result in delayed platelet engraftment. The developmental and molecular differences between neonatal and adult MKs are not yet fully understood. Previously, we had reported that the key MK transcription factor RUNX1, which is crucial for the regulation of MK specification and maturation, is down-regulated in neonatal MKs when compared with adult MKs. In humans, loss-of-function mutations in RUNX1 cause familial platelet disorder, which is characterized by thrombocytopenia, indicating its crucial role in MK development. However, information about its cross talk with developmentally regulated signaling pathways in MKs is lacking. In this study, we performed a differential gene expression analysis in MKs derived from human cord blood (CB) and adult peripheral blood (PB) CD34+ cells. Further, validation and correlation studies between RUNX1 and transforming growth factor beta (TGF-ß) were performed in a differentiating megakaryocytic cell line model. The analysis revealed that TGF-ß pathway was the main pathway affected between CB- and PB-MKs. RUNX1 is reported to be a modulator of TGF-ß signaling in several studies. The correlation between RUNX1 and TGF-ß pathway was analyzed in the PMA-induced megakaryocytic differentiating K562 cells, which exhibit mature megakaryocytic features. The RT2 profiler PCR array analysis revealed that TGF-ß pathway components were up-regulated in the PMA-induced megakaryocytic differentiating cells. Furthermore, our study indicated that human TGF-ß1 promotes cytosolic calcium (Ca2+ ) activity and MK maturation. We noticed that TGF-ß1 increased intracellular free Ca2+ ([Ca2+ ]i) via reactive oxygen species-mediated activation of transient receptor potential (TRP) ion channels. Moreover, we observed that decreased cytosolic Ca2+ activity in the siRUNX1-transfected cells was associated with down-regulation of TRP ion channel expression. Finally, we demonstrated that TGF-ß/SMAD signaling augments the development of MKs derived from CB-CD34+ . Present data suggest that RUNX1/TGF-ß pathway cross talk is crucial for MK maturation.

Endoplasmic reticulum stress induced apoptosis and caspase activation is mediated through mitochondria during megakaryocyte differentiation.

Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs, many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence about the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We used PMA (Phorbol 12-myristate 13 acetate) to induce megakaryocytic differentiation to understand the involvement of ER stress and caspase activation during MK maturation. Further, we used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as a positive control to induce ER stress. We observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage. The increased expression of megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.

Exosome mediated differentiation of megakaryocytes: a study on TLR mediated effects.

Megakaryocytes are large polyploid bone marrow cells whose function is to produce circulatory platelets. Megakaryocytes are also known to release extracellular vesicles (EVs) of varying sizes. Toll like receptors (TLRs), present on the sentinel cells are essential components of the innate immune response, these receptors are also expressed by platelets and megakaryocytes. Our data provide the evidence that TLR-2 induced MKEVs are able to recapitulate TLR-2 signalling in megakaryocytic cell line (Dami cells) and that likely induces megakaryocytic maturation by increasing the production of cytokines involved in MK maturation. TLR-2 induced MKEVs may be involved in replenishment of the immune effector platelets in circulation and its progenitor megakaryocyte in bone marrow for the physiological need of the platelets by inducing the maturation of megakaryocyte.

Current Updates on Role of Lipids in Hematopoiesis.

Hematopoiesis is the process which generates all the mature blood cells from the rare pool of Hematopoietic stem cells (HSCs). Asymmetric cell division of HSCs provide it dual capacity for self-renewal and multi-potent differentiation. Hematopoiesis is a steady state process in which mature blood cells are produced at the same rate at which they are lost, establishing a homeostasis. HSCs are regulated through their environmental niche, cytokine signalling, and the orchestrated activities of various transcription factors. However, there is very little information available about the signal transduction events that regulate HSC function; in particular, the effects of bioactive lipids and lipid mediators are not well understood. Recent studies have added an important aspect of this process, introducing the role of lipids in cell fate decisions during hematopoiesis. The mechanisms of bioactive lipids and their derivatives have been studied extensively in signal transduction and various other cellular processes. This review focuses on various categories of lipids and their regulatory mechanisms in HSCs and their comment into different blood cells. Moreover, we also discuss the role of lipid signalling specifically in megakaryocyte and platelets.

Epigenetic Mechanisms: Role in Hematopoietic Stem Cell Lineage Commitment and Differentiation.

Major breakthroughs in the last several decades have contributed to our knowledge of the genetic regulation in development. Although epigenetics is not a new concept, unfortunately, the role of epigenetics has not come to fruition in the past. But the field of epigenetics has exploded within the past decade. Now, growing evidences show a complex network of epigenetic regulation in development. The epigenetic makeup of a cell, tissue or individual is much more complex than their genetic complement. Epigenetic modifications are more important for normal development by maintaining the gene expression pattern in tissue- and context-specific manner. Deregulation of epigenetic mechanism can lead to altered gene expression and its function, which result in altered tissue specific function of cells and malignant transformation. Epigenetic modifications directly shape Hematopoietic Stem Cell (HSC) developmental cascades, including their maintenance of self-renewal and multilineage potential, lineage commitment, and aging. Hence, there is a growing admiration for epigenetic players and their regulatory function in haematopoiesis. Epigenetic mechanisms underlying these modifications in mammalian genome are still not completely understood. This review mainly explains 3 key epigenetics mechanisms including DNA methylation, histone modifications and non-coding RNAs inference in hematopoietic lineage commitment and differentiation.