Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

BACKGROUND: Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. METHODS: We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). RESULTS: Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1 was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. CONCLUSIONS: Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

Using Omics to Study Leprosy, Tuberculosis, and Other Mycobacterial Diseases.

Mycobacteria are members of the Actinomycetales order, and they are classified into one family, Mycobacteriaceae. More than 20 mycobacterial species cause disease in humans. The Mycobacterium group, called the Mycobacterium tuberculosis complex (MTBC), has nine closely related species that cause tuberculosis in animals and humans. TB can be detected worldwide and one-fourth of the world's population is contaminated with tuberculosis. According to the WHO, about two million dies from it, and more than nine million people are newly infected with TB each year. Mycobacterium tuberculosis (M. tuberculosis) is the most potential causative agent of tuberculosis and prompts enormous mortality and morbidity worldwide due to the incompletely understood pathogenesis of human tuberculosis. Moreover, modern diagnostic approaches for human tuberculosis are inefficient and have many lacks, while MTBC species can modulate host immune response and escape host immune attacks to sustain in the human body. "Multi-omics" strategies such as genomics, transcriptomics, proteomics, metabolomics, and deep sequencing technologies could be a comprehensive strategy to investigate the pathogenesis of mycobacterial species in humans and offer significant discovery to find out biomarkers at the early stage of disease in the host. Thus, in this review, we attempt to understand an overview of the mission of "omics" approaches in mycobacterial pathogenesis, including tuberculosis, leprosy, and other mycobacterial diseases.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration.

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Increasing cytosolic Ca2+ levels restore cell proliferation and stem cell potency in aged MSCs.

Aging is an inescapable complex physiological but extendable process, and all cells, including stem cells, are altered over time. Diverse mechanism(s) could modulate stem cell number, their proliferation rate, and promote tissue repair during aging that leads to longevity. However, the factors that could restore aging stem cell potency and would lead to healthy aging are not fully identified. Here we show that maintaining cytosolic Ca2+ levels was essential for modulating stem cells function in aged mesenchymal stem cells (MSCs). Increasing external Ca2+ induced spindle shape stem cell morphology and maintained stem cell surface marker expression in aged bone marrow-derived MSCs. Similarly, stem cell survival and proliferation of aged MSCs was dependent on cytosolic Ca2+ levels. Importantly, Ca2+ entry potentiated cell cycle progression, and stem cell potential was increased in cells incubated with higher external Ca2+. Moreover, blocking Ca2+ entry using SKF 96365, decreased stem cell survival and its proliferation but, treatment with 2-APB did not significantly affected cell proliferation, rather only modulated cell viability. Evaluation of Ca2+ entry channels, showed that TRPC1/Orai1/Orai3 and their regulator STIM1 was essential for MSCs proliferation/viability as gene silencing of Orai1/Orai3/TRPC1/STIM1 significantly inhibited stem cell viability. Finally, MSCs isolated from aged mice that were subjected to higher Ca2+ levels, were able to rescue age-induced loss of MSCs function. Together these results suggest that Ca2+ entry is essential for preventing the loss of aged stem cell function and supplementing Ca2+ not only restored their proliferative potential but, allowed them to develop into younger stem cell lineages that could be critical for regenerative medicine.

Calcium Signaling Regulates Autophagy and Apoptosis.

Calcium (Ca2+) functions as a second messenger that is critical in regulating fundamental physiological functions such as cell growth/development, cell survival, neuronal development and/or the maintenance of cellular functions. The coordination among various proteins/pumps/Ca2+ channels and Ca2+ storage in various organelles is critical in maintaining cytosolic Ca2+ levels that provide the spatial resolution needed for cellular homeostasis. An important regulatory aspect of Ca2+ homeostasis is a store operated Ca2+ entry (SOCE) mechanism that is activated by the depletion of Ca2+ from internal ER stores and has gained much attention for influencing functions in both excitable and non-excitable cells. Ca2+ has been shown to regulate opposing functions such as autophagy, that promote cell survival; on the other hand, Ca2+ also regulates programmed cell death processes such as apoptosis. The functional significance of the TRP/Orai channels has been elaborately studied; however, information on how they can modulate opposing functions and modulate function in excitable and non-excitable cells is limited. Importantly, perturbations in SOCE have been implicated in a spectrum of pathological neurodegenerative conditions. The critical role of autophagy machinery in the pathogenesis of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, would presumably unveil avenues for plausible therapeutic interventions for these diseases. We thus review the role of SOCE-regulated Ca2+ signaling in modulating these diverse functions in stem cell, immune regulation and neuromodulation.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Expression of interferon regulatory factors (IRF-1 and IRF-2) during radiation-induced damage and regeneration of bone marrow by transplantation in mouse.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors of IRF-family that regulate expression of genes for cytokines, chemokines and growth factors in mammalian cells. IRF-1 and IRF-2 play crucial roles in the differentiation of bone marrow cells for immune response. Bone marrow (BM) is the soft lymphoid organ that contains many types of stem cells and produces different types of cells of the blood and immune system. Genetic alterations and damage of the bone marrow cells can lead to different types of blood and immune system-related diseases including anemia and cancer. We have studied the expression of IRF-1 and IRF-2 during radiation-induced damage and regeneration of bone marrow cells after transplantation of freshly isolated bone marrow cells in the mouse. Cell cycle analysis, colony forming unit-fibroblast (CFU-F) assay and bone marrow histology showed that after radiation-induced damage, the bone marrow transplantation resulted in regeneration of the bone marrow up to 24-35% recovery. Real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the mRNA expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (4.34× fold for IRF-1, and 3.87× fold for IRF-2) compared to control and transplanted (1.13× fold for IRF-1, and 1.12× fold IRF-2) mice and immuno-fluorescence analysis for the protein expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (2.12× fold for IRF-1 and 1.71× fold for IRF-2) compared to control and transplanted (1.73× fold for IRF-1 and 1.21× fold for IRF-2) mice. Thus, IRF-1 and IRF-2 are sensitive and responsive to radiation-induced damage in the bone marrow cells and may also be involved in the bone marrow regeneration process.