Critical Role for Histone Deacetylase 6 (HDAC6) in the Regulation of IL-6, and the JAK/STA3 Signaling Cascade in Macrophages

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1039-1039
Author(s):  
Maritza Lienlaf ◽  
Patricio Perez-Villarroel ◽  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Danay Marante ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Histone Deacetylase 6 ◽  
Stat3 Pathway ◽  
Cellular Processes ◽  
Stat3 Signaling Pathway ◽  
Antigen Presenting

Abstract Abstract 1039 Background Histone Deacetylases (HDACs) have divergent effects over the production of anti- and pro-inflammatory cytokines in Antigen Presenting Cells (APCs). We have previously shown that modulation of specific HDACs can alter the immunogenicity of APCs, either to an activating or tolerogenic phenotype. We recently identified HDAC6 to positively regulate IL-10 production. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we are presenting evidence of the important role of HDAC6 in the regulation of IL-6 via activation of the JAK/STAT3 pathway. Methods Stable knockdown clones of HDAC6 (KDHDAC6) and Non-target (NT) cells were generated in RAW264.7 murine macrophages using lentiviral shRNA for HDAC6 or non-target (NT) respectively. Two KDHADC6 and two NT clones were treated with LPS or untreated and then analyzed by microarray using the Affymetrix GeneChip Mouse 430 2.0. Significantly down- or up-regulated genes were analyzed by their ontology distribution and selected genes were validated by quantitative real-time RT-PCR, ELISA, or immunoblots. Additionally, changes in the expression of these selected genes were tested in cells treated with selective HDAC6 inhibitors. Results 1542 genes were down-regulated and 775 up-regulated in KDHDAC6 cells. Their ontology distribution revealed significant changes in immune-related (632) and apoptosis/cell cycle control (47) genes. Importantly, IL-6 was one of the most highly down-regulated genes in KDHDAC6 cells. Therefore, we next analyzed the relevance of these findings by studying the tolerogenic JAK/STAT3 signaling pathway which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. We observed a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in KDHDAC6 cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6. Conclusions These results demonstrate the requirement of HDAC6 in the production of IL-6 in response to LPS, and therefore positions this deacetylase as an important regulator of the JAK/STAT3 pathway. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation. Disclosures: No relevant conflicts of interest to declare.

Role Of Histone Deacetylase 6 (HDAC6) In The Modulation Of STAT Pathways and Immune Function Of APCs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1032-1032
Author(s):  
Maritza Lienlaf ◽  
Patricio Perez-Villarroel ◽  
Fengdong Cheng ◽  
Calvin K. Lee ◽  
Jorge Canales ◽  
...  
Keyword(s):  
Cell Lines ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Histone Deacetylase 6 ◽  
Histone Modifiers

Abstract Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation. We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6. Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

scholarly journals c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells †

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 416
Author(s):  
Derek Strassheim ◽  
Vijaya Karoor ◽  
Hala Nijmeh ◽  
Philip Weston ◽  
Martin Lapel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Pulmonary Artery ◽  
Molecular Mechanisms ◽  
Cell Cycle Control ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Vasa Vasorum ◽  
Angiogenic Factor ◽  
Angiogenic Genes

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

scholarly journals Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6

2017 ◽  
Vol 312 (4) ◽  
pp. H711-H720 ◽  
Author(s):  
Thorsten M. Leucker ◽  
Yohei Nomura ◽  
Jae Hyung Kim ◽  
Anil Bhatta ◽  
Victor Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Histone Deacetylase ◽  
Vascular Endothelium ◽  
High Fat Diet ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Oxidized Ldl ◽  
Hdac Inhibitors ◽  
Histone Deacetylase 6 ◽  
High Fat

Endothelial cystathionine γ-lyase (CSEγ) contributes to cardiovascular homeostasis, mainly through production of H2S. However, the molecular mechanisms that control CSEγ gene expression in the endothelium during cardiovascular diseases are unclear. The aim of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial CSEγ. Reduced CSEγ mRNA expression and protein abundance were observed in human aortic endothelial cells (HAEC) exposed to oxidized LDL (OxLDL) and in aortas from atherogenic apolipoprotein E knockout (ApoE−/−) mice fed a high-fat diet compared with controls. Intact murine aortic rings exposed to OxLDL (50 μg/ml) for 24 h exhibited impaired endothelium-dependent vasorelaxation that was blocked by CSEγ overexpression or the H2S donor NaHS. CSEγ expression was upregulated by pan-HDAC inhibitors and by class II-specific HDAC inhibitors, but not by other class-specific inhibitors. The HDAC6 selective inhibitor tubacin and HDAC6-specific siRNA increased CSEγ expression and blocked OxLDL-mediated reductions in endothelial CSEγ expression and CSEγ promoter activity, indicating that HDAC6 is a specific regulator of CSEγ expression. Consistent with this finding, HDAC6 mRNA, protein expression, and activity were upregulated in OxLDL-exposed HAEC, but not in human aortic smooth muscle cells. HDAC6 protein levels in aortas from high-fat diet-fed ApoE−/− mice were comparable to those in controls, whereas HDAC6 activity was robustly upregulated. Together, our findings indicate that HDAC6 is upregulated by atherogenic stimuli via posttranslational modifications and is a critical regulator of CSEγ expression in vascular endothelium. Inhibition of HDAC6 activity may improve endothelial function and prevent or reverse the development of atherosclerosis. NEW & NOTEWORTHY Oxidative injury to endothelial cells by oxidized LDL reduced cystathionine γ-lyase (CSEγ) expression and H2S production, leading to endothelial dysfunction, which was prevented by histone deacetylase 6 (HDAC6) inhibition. Our data suggest HDAC6 as a novel therapeutic target to prevent the development of atherosclerosis.

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 864-864
Author(s):  
Dewen You ◽  
Andrew Volk ◽  
Clare Sun ◽  
Junping Xin ◽  
Geunhyoung Ha ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Phosphatase Activity ◽  
Focal Adhesion ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Polycytidylic Acid

Abstract Abstract 864 Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten's lipid phosphatase activity. However, the role of Pten's protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied. Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Silencing HDAC3 Enhances Apoptosis of Acute B Lymphoblastic Leukemia Cells smurf2 Inducing By Inhibiting JAK/STAT3 Signal Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5128-5128
Author(s):  
Yongling Guo ◽  
Dan Ma ◽  
Zhengchang He ◽  
Jie Xiong ◽  
Xingyi Kuang ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Mrna Level ◽  
Therapeutic Targets ◽  
Signal Pathway ◽  
Lymphocytic Leukemia ◽  
Clinical Prognosis ◽  
Stat3 Pathway ◽  
Healthy Donors

Abstract Purpose Smad ubiquitin regulatory factor 2(Smurf2) as a member of ubiquitinated E3 ligase was certificated to regulate the ubiquitination level of target gene. In this study, smurf2 as tumor suppressor was studied in acute B lymphoblastic leukemia, Histone deacetylases 3(HDAC3) which was connected with smurf2 was related with poor prognosis. It is further proved that smurf2 and HDAC3 were expected to be therapeutic targets for B-ALL cells and improve the prognosis of patients with B-ALL. Methods Realtime-PCR and westernblot were used to detect the expression of smurf2 and HDAC3 in healthy donors and B-ALL patients by us. Sup-B15 and CCRF-SB were treated with MG-132, small interfering RNA of smurf2 or HDAC3. Up-regulating smurf2 plasmid was also transfected to B-ALL cells by lipo3000. Flow cytometry and westernblot were used to validate the difference of apoptosis and cell cycle. Results Smurf2 mRNA in B-ALL patients was significantly lower than that in healthy donors. HDAC3 was higher in B-ALL patients than that in healthy donors. Protein level of smurf2 and HDAC3 was consistent with mRNA level. Up-regulating smurf2 by plasmid or MG-132 and silencing smurf2 could influence HDAC3, which further depressed JAK/STAT3 signal pathway inducing apoptosis of B-ALL cells. Sup-B15 and CCRF-SB were treated with siHDAC3 and MG-132 for 24h, we discovered that silencing HDAC3 enhanced the apoptosis level of B-ALL cells MG-132 inducing by depressing JAK/STAT3 pathway. It was expected to become a therapeutic target for improving the clinical prognosis of B-ALL patients. On the other hand, we found that MG-132 caused G2/M phase arrest in B-ALL cells and successfully inhibited the JAK/STAT3 pathway leading to apoptosis. Conclusions Silencing HDAC3 inhibited the JAK/STAT3 pathway and further enhanced apoptosis in B-ALL cells MG-132 inducing. HDAC3 and smurf2 were expected to become therapeutic targets for clinical treatment of acute B lymphocytic leukemia and improve survival rate of leukemia patients. Key words Smurf2; HDAC3; Apoptosis; Prognosis; Disclosures No relevant conflicts of interest to declare.

scholarly journals Sirtuins as Potential Therapeutic Targets for Hepatitis B Virus Infection

2021 ◽  
Vol 8 ◽  
Author(s):  
Fanyun Kong ◽  
Qi Li ◽  
Fulong Zhang ◽  
Xiaocui Li ◽  
Hongjuan You ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Hbv Infection ◽  
Viral Propagation ◽  
Therapeutic Modalities ◽  
Cellular Processes ◽  
Hbv Replication ◽  
B Virus

Sirtuins (SIRTs) are well-known histone deacetylases that are capable of modulating various cellular processes in numerous diseases, including the infection of hepatitis B virus (HBV), which is one of the primary pathogenic drivers of liver cirrhosis and hepatocellular carcinoma. Mounting evidence reveals that HBV can alter the expression levels of all SIRT proteins. In turn, all SIRTs regulate HBV replication via a cascade of molecular mechanisms. Furthermore, several studies suggest that targeting SIRTs using suitable drugs is a potential treatment strategy for HBV infection. Here, we discuss the molecular mechanisms associated with SIRT-mediated upregulation of viral propagation and the recent advances in SIRT-targeted therapy as potential therapeutic modalities against HBV infection.

Beyond mRNAs and Mirnas: Unraveling the Full-Spectrum of the Normal Human Platelet Transcriptome Through Next-Generation Sequencing

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3298-3298 ◽  
Author(s):  
Eric R. Londin ◽  
Eleftheria Hatzimichael ◽  
Phillipe Loher ◽  
Yue Zhao ◽  
Yi Jing ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Pearson Correlation ◽  
Critical Role ◽  
Ltr Retrotransposons ◽  
Rna Seq ◽  
Ensembl Database ◽  
Protein Coding ◽  
Protein Coding Genes

Abstract Abstract 3298 The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. While the repertoire of platelet transcripts is a reflection of the megakaryocyte at the time of platelet differentiation, post-transcriptional events are known to occur. Furthermore, a strong correlation between the expressed mRNAs and proteome has been identified. Having a complete understanding of the platelet transcriptome is important for generating insights into the genetic basis of platelet disease traits. To capture the complexity of the platelet transcriptome, we performed RNA sequencing (RNA-seq) in leukocyte-depleted platelets from 10 males, with median age of 24.5 yrs and unremarkable medical history. Their short and long RNA platelet transcriptomes were analyzed on the SOLiD 5500xl sequencing platform. We generated ∼3.5 billion sequence reads ∼40% of which could be mapped uniquely to the human genome. Our analysis revealed that ∼9,000 distinct protein-coding mRNAs and ∼800 microRNAs (miRNAs) were present in the transcriptome of each of the 10 sequenced individuals. Comparison of the levels of mRNA expression across the 10 individuals showed an exceptional level of consistency with pair-wise Pearson correlation values ≥0.98. The miRNA expression profiles across the 10 individuals showed a similar consistency with pair-wise Pearson correlation values ≥0.98. Surprisingly, we found that these mRNAs and miRNAs accounted for a little over 1/2 of all of the uniquely mapped sequence reads suggesting the abundant presence of additional non-protein coding RNA (ncRNA) transcripts. Using the annotated entries of the latest release of the ENSEMBL database, we investigated the genetic make-up of these other transcripts. We found that ∼25% of each individual's uniquely mapped reads corresponded to non-protein coding transcripts from mRNA-coding loci. These reads accounted for more than 10,000 distinct such transcripts. In addition, each of the individuals in our cohort expressed an average of ∼1,500 pseudogenes and ∼200 long intergenic non-coding RNAs (lincRNAs). The short RNA profiles of the ten individuals revealed an abundance of diverse categories of ncRNAs including the signal recognition particle RNA (srpRNA), small nuclear RNA (snRNA) and small cytoplasmic RNAs (scRNA). These ncRNAs are involved in the processing of pre-mRNAs and their presence and prevalence in the anucleate platetet suggests the existence of a complex network of mRNA processing that persists after the megakaryocyte fragmentation. We also investigated the RNA-omes of the ten individuals for evidence of transcription of the pyknon category of ncRNAs. Pyknons are of particular interest because each has numerous intergenic and intronic copies whereas nearly all known human protein-coding genes contain one or more pyknons in their mRNA. Recent experimental work has shown that intergenic instances of the pyknons are transcribed in a tissue- and cell-state specific manner. An average of ∼100,000 pyknons are transcribed in each of the 10 sequenced individuals suggesting the possibility of a far-reaching network of interactions that link exonic space to distant non-exonic regions and are active in platelets. Lastly, we found that a large variety of distinct repeat element categories are expressed in the RNA-omes (both short and long) of these individuals. Among the most abundantly represented categories of repeat elements were DNA transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons such as long interspersed elements (LINEs) and short interspersed elements (SINEs). In summary, our RNA-seq analyses have revealed a spectrum of platelet transcripts that transcends protein-coding genes and miRNAs. Indeed, the transcripts that have their source in genomic features not previously discussed or analyzed in the platelet context represent a very significant portion of all platelet transcripts. This in turn suggests an unanticipated richness, and presumably commensurate complexity, for the platelet transcriptome. While the role of these novel non-protein coding RNAs is currently unknown it is expected that at least some of them may be of functional significance which will in turn permit a better understanding of the molecular mechanisms that regulate platelet physiology and may contribute to processes beyond thrombosis and hemostasis. Disclosures: No relevant conflicts of interest to declare.

Characterization of the Short RNA Transcriptome of the Anucleate Platelet

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4990-4990
Author(s):  
Eric R. Londin ◽  
Phillipe Loher ◽  
Leonard C. Edelstein ◽  
Kathy Delgrosso ◽  
Paolo M. Fortina ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Ensembl Database ◽  
Protein Coding ◽  
Repeat Elements ◽  
Short Rnas ◽  
Genomic Locations

Abstract The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. In recent years, next-generation RNA sequencing (RNA-seq) has proven very useful in shedding light on the specifics of the platelet transcriptome. For example, RNA-seq of the long RNAs in platelets has revealed many non-coding RNAs (ncRNAs) as well as a diverse set of protein-coding genes whose mRNAs are highly correlated amongst individuals but only weakly linked to the currently available platelet proteome. By comparison, the short RNA transcriptome has not been as thoroughly characterized. As a matter of fact, these studies have so far focused on the 100’s of microRNAs (miRNAs) that are present in platelets leaving large swaths of the short RNA-ome uncharacterized. To gauge the complexity of the platelet short RNA-ome we performed short RNA-seq of leukocyte-depleted platelets from 10 healthy males (5 white and 5 black). The sequencing was done on the SOLiD 5500 XL platform and generated over 1.5 billion sequenced reads. To comprehensively characterize the complete short RNA-ome we only considered sequence reads that mapped on the genome without any mismatches but allowed a read to map to as many as 10,000 locations within the genome. This approach gave us the ability to simultaneously examine both the uniquely-present and the repeat-derived expressed elements of the genome. Using this approach, we were able to map ~50% of the sequenced reads. We found that for ~55% of the mapped reads their sequences are present at multiple genomic locations whereas the remaining ~45% originated from unique locations. Of the RNAs with unique genomic origins: ~50% correspond to miRNAs (with miR-223-3p being the most abundant miRNA across all 10 individuals), ~20% originate from various classes of repeat elements, and, the remaining 30% correspond to non-annotated regions of the genome that were non-annotated a of Release 75 of the ENSEMBL database. By comparison, of the RNAs with ambiguous genomic origins: ~20% belong to miRNAs (with miR-103a-3p, a miRNA present in two locations in the genome, being the most abundant miRNA across all 10 individuals) and ~60% correspond to various classes of repeat elements (with members of the HY4 scRNA ncRNAs accounting for nearly a third of all sequence reads). These findings make it evident that the platelet transcriptome has a considerable richness in short RNAs that arise from repetitive elements. To further characterize those RNAs that map to regions of the genome that are not currently annotated, we considered the possibility that they may be novel miRNAs. Using the miRDeep2 algorithm, we sought novel miRNAs among the uncharacterized transcripts and identified 47 of them; the sequences for 18 of these 47 appear at multiple genomic locations in analogy to miR-103/107, miR-19a/19b, etc. Lastly, as our ten samples represented two races, we hypothesized that a subset of the identified sequences would be differentially expressed between the two groups. Using DESeq2, we identified over 157 sequences to be differentially expressed. The most highly differentially expressed sequences corresponded to a miRNA and a repeat element. In summary, our RNA-seq analyses have revealed a very diverse spectrum of platelet short RNAs that transcends the miRNA category. Indeed, we find that short transcripts that have their source in genomic loci that have not been previously discussed or analyzed in the platelet context represent a very significant portion of all short RNAs in platelets. This in turn highlights an unanticipated richness, and presumably commensurate complexity, for the platelet transcriptome. While the role of these novel non-protein coding short RNAs is currently unknown it is expected that at least some of them may be of functional significance. Consequently, they could contribute to processes beyond thrombosis and hemostasis and may permit a better understanding of the molecular mechanisms that regulate platelet physiology. Disclosures No relevant conflicts of interest to declare.

Ca2+-Operated Transcriptional Networks: Molecular Mechanisms and In Vivo Models

2008 ◽  
Vol 88 (2) ◽  
pp. 421-449 ◽  
Author(s):  
Britt Mellström ◽  
Magali Savignac ◽  
Rosa Gomez-Villafuertes ◽  
Jose R. Naranjo
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Transcriptional Networks ◽  
Calcium Signal ◽  
In Vivo Models ◽  
Cellular Processes ◽  
Calcium Sensors ◽  
Genes Encoding ◽  
Living Organisms

Calcium is the most universal signal used by living organisms to convey information to many different cellular processes. In this review we present well-known and recently identified proteins that sense and decode the calcium signal and are key elements in the nucleus to regulate the activity of various transcriptional networks. When possible, the review also presents in vivo models in which the genes encoding these calcium sensors-transducers have been modified, to emphasize the critical role of these Ca2+-operated mechanisms in many physiological functions.

Molecular Mechanisms of Renal Magnesium Reabsorption

2021 ◽  
pp. ASN.2021010042
Author(s):  
David Ellison ◽  
Yujiro Maeoka ◽  
James McCormick
Keyword(s):  
Molecular Mechanisms ◽  
Narrow Range ◽  
Serum Magnesium ◽  
Critical Role ◽  
Cellular Processes ◽  
Magnesium Homeostasis ◽  
Life Threatening ◽  
Transepithelial Voltage ◽  
Paracellular Pathways ◽  
Insight Into

Magnesium is an essential cofactor in many cellular processes, and aberrations in magnesium homeostasis can have life-threatening consequences. The kidney plays a central role in maintaining serum magnesium within a narrow range (0.70 to 1.10 mmol/L). Along the proximal tubule and thick ascending limbs, magnesium reabsorption occurs via paracellular pathways. Members of the claudin family form the magnesium pores in these segments, and also regulate magnesium reabsorption by adjusting the transepithelial voltage that drives it. Along the distal convoluted tubule transcellular reabsorption via heteromeric TRPM6/7 channels predominates, though paracellular reabsorption may also occur. In this segment, the NaCl cotransporter plays a critical role in determining transcellular magnesium reabsorption. While the general machinery involved in renal magnesium reabsorption has been identified by studying genetic forms of magnesium imbalance, the mechanisms regulating it are poorly understood. This review discusses pathways of renal magnesium reabsorption by different segments of the nephron, emphasizing newer findings that provide insight into regulatory process, and outlining critical unanswered questions.

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