Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has ...not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.
•BMSC undergo G0 arrest in methylcellulose suspension and on soft polyacrylamide substrates•Induction of G0 enhances CFU-f and osteogenic potential of BMSC (in vitro & in vivo) while reducing adipogenic potential.•Transcriptome of G0 and post-G0 BMSC is markedly different from pre-G0 cells.•BMP signaling is upregulated during G0 and post-G0 BMSC.•G0 and post-G0 BMSC showed enhanced expression of paracrine factors.
Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies.
Using chemical biology, we identified a small molecule ...inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy.
In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation.
These protective mechanisms ensure the negation of Parkinson's disease related motor impairments.
This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.
Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the ...chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs-myogenesis and the cell cycle-while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.
Cardiac fibroblast hyperplasia associated with augmented matrix production is central to wound healing following myocardial injury. Regulation of the cardiac fibroblast cell cycle by factors in the ...diseased myocardium that can potentially modify the hyperplastic response of cardiac fibroblasts has, however, not been investigated. We examined the regulation of the cardiac fibroblast cell cycle by hypoxia, a major constituent of myocardial ischemia. Significant reductions in DNA synthesis and cell number, and flow cytometry indicated decreased G1/S progression in hypoxic adult rat cardiac fibroblasts. Western blot analysis showed reduced levels of cyclin D and cyclin A, induction of p27 and hypophosphorylation of Rb under hypoxia. Skp2, which targets p27 for degradation, was significantly lower and inversely related to p27 protein levels in hypoxic cells. Marked p38 MAPK activation was observed under hypoxia and its inhibition using SB203580 reversed the effects of hypoxia on DNA synthesis, cell cycle phase distribution, p27, and cyclin D1 but not cyclin A. Interestingly, a 2-fold increase in p27 mRNA in hypoxic cells, demonstrated by real-time PCR, was unaffected by SB203580, which, however, reversed the hypoxic inhibition of Skp2. In summary, p38 MAPK is an important determinant of hypoxia-induced G0/G1 block in cardiac fibroblasts. p27 induction in hypoxic cardiac fibroblasts may involve direct transcriptional regulation, independent of p38 MAPK, and post-translational regulation via p38 MAPK-dependent suppression of its degradation by Skp2. The study identifies Skp2 as a potential downstream target of p38 MAPK, suggesting a novel mechanism of G1-S regulation in cardiac fibroblasts exposed to stress conditions.
Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse ...cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of ³H-thymidine incorporation into DNA and a modest reduction in ³H-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in ³H-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of ³H-thymidine. Genistein dramatically reduced ³H-thymidine but not ³H-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of ³H-thymidine incorporation.
Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their ...survival under conditions of stress remain unclear. We explored the possibility that NF-κB protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the antiapoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-κB. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-κB in cardiac fibroblasts. Supershift assay indicated that the active NF-κB complex is a p65/p50 heterodimer. An I-κB-super-repressor was constructed that prevented NF-κB activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-κB. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-κB inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-κB is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role.
Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their ...survival under conditions of stress remain unclear. We explored the possibility that NF-kappaB protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the antiapoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-kappaB. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-kappaB in cardiac fibroblasts. Supershift assay indicated that the active NF-kappaB complex is a p65/p50 heterodimer. An I-kappaB-super-repressor was constructed that prevented NF-kappaB activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-kappaB. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-kappaB inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-kappaB is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role. PUBLICATION ABSTRACT
Double-hit (DH) lymphomas with MYC and either BCL2 (DH-BCL2/MYC) or BCL6 (DH-BCL6/MYC) rearrangements are considered very aggressive, many of which are now included in the category B-cell lymphoma, ...unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) (DLBCL/BL). However, data describing the DH cases are largely based on DH-BCL2/MYC cases. To better characterize DH-BCL6/MYC cases, the clinical, morphologic, phenotypic, and cytogenetic features of 6 cases from University of Pittsburgh Medical Center and 17 cases from the Mitelman database were reviewed. In the University of Pittsburgh Medical Center cases, the median age was 83 years (range, 51 to 89 y) with 5/6 DLBCL/BL cases and 1 large B-cell lymphoma, not otherwise specified. Five of 6 had a germinal center phenotype, 1/6 was BCL2(+), and the median Ki-67 score was 98% (35% to 100%). The Mitelman DH-BCL6/MYC cases included 13 aggressive B-cell lymphomas (diagnosed as DLBCL-5, BL-5, BL-like lymphomas-2, and primary effusion lymphoma-1) and 4 other lymphoid/plasmacytic neoplasms. The median cytogenetic complexity score was 2.5 (range, 0 to 14) in 14 evaluable mature aggressive lymphomas with an immunoglobulin gene partner for MYC in 9/14 and for BCL6 in 7/14 cases. Ten of 13 cases involved extranodal extramedullary sites at presentation, and the median survival for the 10 patients with large cell neoplasms or BL and with available follow-up data was 9 months. Thus, DH-BCL6/MYC lymphomas are aggressive, frequently involve extranodal sites, and are often DLBCL/BL with a germinal center phenotype. Unlike DH-BCL2/MYC lymphomas, however, they are more likely to be CD10(-) but IRF4/MUM-1(+) (P=0.03) and, more like BL, only infrequently express BCL2 (P<0.001), and are cytogenetically less complex (P<0.04).
Abstract
STUDY QUESTION
Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in ...androgen excess?
SUMMARY ANSWER
Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women.
WHAT IS KNOWN ALREADY
PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported.
STUDY DESIGN, SIZE, DURATION
This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria.
PARTICIPANTS/MATERIALS, SETTING, METHODS
This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR.
MAIN RESULTS AND THE ROLE OF CHANCE
We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work.
WIDER IMPLICATIONS OF THE FINDINGS
Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests.
TRIAL REGISTRATION NUMBER
N/A.
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