BACKGROUND:The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS:In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS:In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004).CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk.
Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2 phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and phosphorylates this site in vitro. Knock-down or inhibition of the CK2α/α′ kinase subunit in epithelial cells causes de novo accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial maintenance. In malignant breast cancer cells the CK2β regulatory subunit is downregulated and FOXC2 is found in the nucleus, correlating with an increase in α-SMA expression. Restoration of CK2β expression in these cells results in cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal epithelial cells by CK2α/α′-mediated phosphorylation at serine 124 that is dependent on proper targeting of the holoenzyme via the CK2β regulatory subunit.
Bone remodeling requires osteoclast activation, resorption, and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF-κB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38(MAPK) , Akt, and p65(NF-κB). However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL-induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL-induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65(NF-κB) pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK-mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis.
A reduction in the DNA modification of cytosine methylation has been linked directly to chromosome rearrangements concomitant with retroelement amplification in several marsupial hybrid genomes. While phenotypes observed for interspecific eutherian hybrids are suggestive of methylation perturbations and retroelement instability, no link between retroelements, DNA methylation, and chromosome instability has yet been identified. Previous studies in eutherian hybrids, however, have been limited to a gross examination of methylation using methylation-sensitive restriction enzyme analysis or focused on single-copy genes and/or have avoided examination of repetitive DNA. Methylation changes and retroelements are proposed as mechanisms for double minute chromosome formation and oncogene amplification, both present in the genome of a Mus hybrid model, thus making it an ideal system to evaluate methylation status more closely. We have used the PCR-based methodologies methylation-sensitive amplicon subtraction (MS-AS) and methylation-sensitive representational difference analysis (MS-RDA) to detect differentially methylated sequences between three complex genomes and to isolate methylation perturbations in a Mus musculusxMus caroli hybrid. This novel application of MS-AS and MS-RDA resulted in the isolation of differentially methylated retroelements surrounding the locus on Chromosome 10 responsible for double minute chromosome formation within this interspecific eutherian hybrid.
Introduction: We previously reported a common intronic SCARB1 (12q24.31) variant, rs10846744, located in an enhancer region, to be significantly associated with coronary artery disease (CAD) in the Multi-Ethnic Study of Atherosclerosis (MESA). RNA-Seq showed expression of the immune checkpoint inhibitor lymphocyte activation gene 3 ( LAG3 , 12p13.31), to be 5-fold lower in carriers of the risk allele, while low plasma LAG3 protein levels were also significantly associated with increased CAD risk in MESA, after multivariate regression analysis. Hypothesis: That the SCARB1 rs10846744 variant disrupts long-range transcriptional regulation of LAG3 disturbing cardio-protective and anti-inflammatory gene networks to promote CAD. Methods and Results: Using functional genomics (HiC global chromatin capture, ChIP-Seq and RNA-Seq) in reference and risk EBV-transformed B lymphocytes to assess 3D chromatin architecture and gene-gene interactions at a 2.5kb resolution, we did not observe direct chromatin contacts between SCARB1 and LAG3. In the reference allele, an enhancer-rich intermediate contact (12q13.13) was found containing genes associated with cholesterol ( SOAT2 ) and NR2F2 signaling ( RARG ). This same 12q13.13 region was in direct contact with 22q12.3 ( APOLI) , an apoprotein associated with HDL and innate immunity. Micro-looping within the rs10846744 12q24.31 region showed direct contacts with other enhancers ( NCOR2 ) and cardiovascular loci ( TMEM132B ), while LAG3 micro-looping on 12p13.31 was associated with immune regulatory networks ( CD4 ). Loci associated with viral infection, cytokine production, heart failure and autoimmunity were also identified. NR2F2 disrupted contacts in the risk allele, implicating NR2F2 as a dysfunctional rs10846744 transcriptional repressor altering gene networks. The risk allele included contacts near PCSK9 , VLDLR and 2q33.1, a CAD locus. Conclusion: Functional genomics of the SCARB1 rs10846744 enhancer region identified a number of intra- and inter-chromosomal chromatin contacts in reference cells that were markedly disrupted in risk cells. Perturbing NR2F2 and/or genes disrupted in the SCARB1 -NR2F2 immuno-cardiovascular axis may protect against CAD in the rs10846744 risk population.
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